TWI751439B - Use of HOXA7 and HOXA9 methylation detection reagents in preparing lung cancer diagnostic reagents, lung cancer diagnostic system and method - Google Patents

Abstract

本公開涉及一種以痰液為檢測樣本，且以HOXA7和HOXA9的甲基化為檢測對象的檢測試劑在製備肺癌診斷試劑中的用途。本公開首次在痰液中，共同以HOXA7和HOXA9作為檢測肺癌的標記，其在痰液中的敏感度高達97.1%，特異性高達90.9%，檢測敏感度高於現有的肺癌標記，尤其是對腺癌，敏感度甚至達到了100%，對於腺癌的檢測有極大的應用價值。 The present disclosure relates to the use of a detection reagent with sputum as the detection sample and the methylation of HOXA7 and HOXA9 as the detection object in the preparation of a diagnostic reagent for lung cancer. The present disclosure is the first to use HOXA7 and HOXA9 as markers for detecting lung cancer in sputum. The sensitivity in sputum is as high as 97.1% and the specificity is as high as 90.9%. The detection sensitivity is higher than the existing markers of lung cancer, especially for For adenocarcinoma, the sensitivity even reached 100%, which has great application value for the detection of adenocarcinoma.

Description

HOXA7和HOXA9甲基化檢測試劑在製備肺癌診斷試劑中 的用途、肺癌的診斷系統及方法 HOXA7 and HOXA9 methylation detection reagents in the preparation of lung cancer diagnostic reagents Uses, diagnostic systems and methods for lung cancer

本公開屬基因診斷領域，更具體地，本公開涉及一種人HOXA7和人HOXA9基因甲基化檢測試劑在製備肺癌診斷試劑中的用途，以及人HOXA7基因和HOXA9基因甲基化檢測的方法。 The present disclosure belongs to the field of gene diagnosis, and more particularly, the present disclosure relates to the use of a human HOXA7 and human HOXA9 gene methylation detection reagent in the preparation of a lung cancer diagnostic reagent, and a method for human HOXA7 gene and HOXA9 gene methylation detection.

肺癌是起源於支氣管黏膜、腺體或肺泡上皮的肺部惡性腫瘤。按照病理類型可以分為：1、小細胞肺癌(small cell lung cancer，SCLC)：一種特殊病理學類型的肺癌，有明顯的遠處轉移傾向，預後較差，但多數病人對放化療敏感。2、非小細胞肺癌(non-small cell lung cancer，NSCLC)：除小細胞肺癌以外其他病理學類型的肺癌，包括鱗狀細胞癌、腺癌、大細胞癌等。在生物學行為和臨床病程方面具有一定差異。按照發生位置又可以分為：1、中心型肺癌(central lung cancer)：生長在肺段支氣管開口及以上的肺癌。2、周圍型肺癌(peripheral lung cancer)：生長在肺段支氣管開口以遠的肺癌。 Lung cancer is a malignant tumor of the lung originating from the bronchial mucosa, glands or alveolar epithelium. According to the pathological type, it can be divided into: 1. Small cell lung cancer (SCLC): a special pathological type of lung cancer, with obvious tendency of distant metastasis and poor prognosis, but most patients are sensitive to radiotherapy and chemotherapy. 2. Non-small cell lung cancer (NSCLC): other pathological types of lung cancer other than small cell lung cancer, including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, etc. There are some differences in biological behavior and clinical course. According to the location of occurrence, it can be divided into: 1. Central lung cancer: lung cancer that grows at or above the bronchial opening of the lung segment. 2. Peripheral lung cancer: Lung cancer that grows beyond the bronchial opening of the segmental lung.

肺癌的早期診斷與早期手術是提高肺癌5年生存率、降低死亡率最有效的方法之一。 Early diagnosis and early surgery of lung cancer are one of the most effective methods to improve the 5-year survival rate and reduce the mortality rate of lung cancer.

肺癌目前的臨床輔助診斷主要有以下幾種，但是他們都不能完全做到早發現，早診斷： The current clinical auxiliary diagnosis of lung cancer mainly includes the following, but none of them can fully achieve early detection and early diagnosis:

1、血液生化檢查：對於原發性肺癌，目前無特異性血液生化檢查。肺癌病人血液鹼性磷酸酶或血鈣升高考慮骨轉移的可能，血液鹼性磷酸酶、穀草轉胺酶、乳酸脫氫酶或膽紅素升高考慮肝轉移的可能。 1. Blood biochemical examination: There is currently no specific blood biochemical examination for primary lung cancer. Lung cancer patients with elevated blood alkaline phosphatase or serum calcium should consider the possibility of bone metastasis, and elevated blood alkaline phosphatase, aspartate transaminase, lactate dehydrogenase or bilirubin should consider the possibility of liver metastasis.

2、腫瘤標記檢查：(1)CEA：30%~70%肺癌患者血清中有異常高水平的CEA，但主要見於較晚期肺癌患者。目前血清中CEA的檢查主要用於估計肺癌預後以及對治療過程的監控。(2)NSE：是小細胞肺癌首選標記，用於小細胞肺癌的診斷和監測治療反應，根據檢測方法和使用試劑的不同，參考值不同。3)CYFRA21-1：是非小細胞肺癌的首選標記，對肺鱗癌診斷的敏感性可達60%，根據檢測方法和使用試劑的不同，參考值不同。 2. Tumor marker examination: (1) CEA: 30%~70% of lung cancer patients have abnormally high levels of CEA in the serum, but it is mainly seen in patients with advanced lung cancer. At present, the detection of CEA in serum is mainly used to estimate the prognosis of lung cancer and monitor the treatment process. (2) NSE: It is the first choice marker for small cell lung cancer. It is used for the diagnosis of small cell lung cancer and monitoring the response to treatment. The reference value is different depending on the detection method and reagents used. 3) CYFRA21-1: It is the first choice marker for non-small cell lung cancer, and the sensitivity for the diagnosis of lung squamous cell carcinoma can reach 60%. The reference value is different depending on the detection method and the reagent used.

3、影像學檢查：(1)胸部X光檢查：應包括胸部正位和側位片。在基層醫院，胸部正側位片仍是肺癌初診時最基本和首選的影像診斷方法。一旦診斷或疑診肺癌，即行胸部CT檢查。(2)CT檢查：胸部CT是肺癌的最常用和最重要的檢查方法，用於肺癌的診斷與鑒別診斷、分期及治療後隨診。有條件的醫院在肺癌病人行胸部CT掃描時範圍應包括腎上腺。應儘量採用增強掃描，尤其是肺中心型病變的患者。CT是顯示腦轉移瘤的基本檢查方法，有臨床症狀者或進展期病人應行腦CT掃描，並盡可能採用增強掃描。CT引導下肺穿刺活檢是肺癌的重要診斷技術，有條件的醫院可將其用於難以定性的肺內病變的診斷，以及臨床診斷肺癌需經細胞學、組織學證實而其它方法又難以取材的病例。(3)超聲檢查：主要用於發現腹部重要器官及腹腔、腹膜後淋巴結有無轉移，也用於頸部淋巴結的檢查。對於貼鄰胸壁的肺內病變或胸壁病變，可鑒別其囊實性及進行超聲引導下穿刺活檢；超聲還常用于胸水抽取定位。(4)骨掃描：對肺癌骨轉移檢出的敏感性較高，但有一定的假陽性率。可用於以下情況：肺癌的術前檢查；伴有局部症狀的病人。 3. Imaging examination: (1) Chest X-ray examination: should include frontal and lateral chest radiographs. In primary hospitals, frontal and lateral chest radiographs are still the most basic and preferred imaging diagnostic method for the initial diagnosis of lung cancer. Once lung cancer is diagnosed or suspected, a chest CT scan is performed. (2) CT examination: chest CT is the most common and important examination method for lung cancer, and is used for the diagnosis, differential diagnosis, staging and follow-up after treatment of lung cancer. Hospitals where conditions permit should include the adrenal glands when performing chest CT scans in lung cancer patients. Contrast-enhanced scans should be used whenever possible, especially in patients with central pulmonary disease. CT is the basic examination method for showing brain metastases. Patients with clinical symptoms or advanced stage should undergo brain CT scan, and use enhanced scan as much as possible. CT-guided lung biopsy is an important diagnostic technique for lung cancer. Conditional hospitals can use it for the diagnosis of pulmonary lesions that are difficult to characterize, as well as for patients whose clinical diagnosis of lung cancer needs to be confirmed by cytology and histology, but other methods are difficult to obtain. case. (3) Ultrasound examination: It is mainly used to find out whether there is metastasis in the vital organs of the abdomen and the lymph nodes in the abdominal cavity and retroperitoneum. It is also used for the examination of lymph nodes in the neck. For intrapulmonary lesions or chest wall lesions adjacent to the chest wall, cystic and solid lesions can be identified and ultrasound-guided needle biopsy can be performed; ultrasound is also commonly used for pleural effusion extraction and localization. (4) Bone scan: It has high sensitivity in detecting bone metastases from lung cancer, but has a certain false positive rate. It can be used in the following situations: preoperative examination of lung cancer; patients with local symptoms.

4、其它檢查：(1)痰細胞學檢查：目前肺癌簡單方便的無創診斷方法，連續塗片檢查可提高陽性率約達60%，是可疑肺癌病例的常規診斷方法。(2)纖維支氣管鏡檢查：肺癌診斷中最重要的手段之一，對於肺癌的定性定位診斷和手術方案的選擇有重要的作用。對擬行手術治療的患者為必需的常規檢查項目。而經支氣管鏡穿刺活檢檢查(TBNA)，雖利於治療前分期，但因技術難度和風險較大，有需要者應轉上級醫院進一步檢查。(3)其他：如經皮肺穿刺活檢、胸腔鏡活檢、縱隔鏡活檢、胸水細胞學檢查等，在有適應症的情況下，可根據現有條件分別採用以協助診斷。4. Other examinations: (1) Sputum cytology examination: At present, a simple and convenient non-invasive method for the diagnosis of lung cancer, continuous smear examination can increase the positive rate by about 60%, which is a routine diagnosis method for suspected lung cancer cases. (2) Fiberoptic bronchoscopy: one of the most important means in the diagnosis of lung cancer, plays an important role in the qualitative diagnosis of lung cancer and the selection of surgical plans. It is a necessary routine inspection item for patients who are going to undergo surgical treatment. While transbronchial needle biopsy (TBNA) is beneficial for pre-treatment staging, it is technically difficult and risky, and those who need it should be transferred to a higher-level hospital for further examination. (3) Others: such as percutaneous lung biopsy, thoracoscopy biopsy, mediastinoscopy biopsy, pleural effusion cytology, etc., if there are indications, they can be used according to the existing conditions to assist the diagnosis.

影像學檢查中的多層螺旋CT和低劑量CT（LDCT）是發現早期肺癌和降低死亡率的有效篩查工具，全美國家肺癌篩查研究（NLST）已經表明LDCT相比胸部X光篩查可降低20%肺癌的死亡率。在臨床實踐工作中證明，任何肺癌篩查項目的成敗取決於高危人群的識別，融合多重高危因素的風險預測模型已被世界公認是識別肺癌高危人群的方法之一。風險模型通過協助臨床醫生改進干預措施或治療手段，從而進一步改善肺癌患者的療效。雖然世界已經認同針對高危人群的篩查能夠降低肺癌目前較高的死亡率，但高危人群界定仍然是難以解決的問題。為了使肺癌篩查的效益—傷害比達到最大化，關鍵的問題第一是如何界定高危患病風險的人群；第二是用什麼方法對該人群進行篩查，包括高危因素的界定，總體風險的量化匯總以及篩查效益界值的選擇。Multi-slice spiral CT and low-dose CT (LDCT) in imaging examinations are effective screening tools to detect early-stage lung cancer and reduce mortality. 20% of lung cancer mortality. It has been proved in clinical practice that the success or failure of any lung cancer screening program depends on the identification of high-risk groups. The risk prediction model integrating multiple high-risk factors has been recognized as one of the methods for identifying high-risk groups of lung cancer. Risk models further improve outcomes for patients with lung cancer by assisting clinicians in improving interventions or treatments. Although the world has recognized that screening for high-risk groups can reduce the current high mortality rate of lung cancer, the definition of high-risk groups is still an intractable problem. In order to maximize the benefit-harm ratio of lung cancer screening, the key question is how to define the high-risk population; the second is how to screen this population, including the definition of high-risk factors, the overall risk Quantitative summarization of and selection of screening benefit cut-offs.

現有的肺癌檢測技術中主要存在靈敏度低、假陽性高，有創，並且，目前常規檢測技術難以檢出早期肺癌。The existing lung cancer detection technologies mainly have low sensitivity, high false positives, and invasiveness, and the current conventional detection technologies are difficult to detect early lung cancer.

而肺癌的無創檢測，例如，痰液檢測，難度則更大。儘管也有研究者研究肺癌患者痰液中的腫瘤標記，然而，對比起其他腫瘤患者血液樣本的腫瘤標記檢測及評估，痰液樣本的成功率卻很低。這主要由於以下原因：①痰液的成分比較複雜，不同的人群在不同的疾病或者環境下痰液的成分和黏度等差異比較大；②痰液中含有較多的氣管上皮細胞和細菌，口腔黏膜細胞等非肺癌細胞的成分，一般的樣本處理方法無法有效的富集到數目充足的肺癌來源的DNA；③有很多的吸煙患者並不表現出咳痰。A J Hubers等人在《Molecular sputum analysis for the diagnosis of lung cancer》中對過去10篇文獻研究顯示，肺癌組織中標記的中位數的甲基化程度為48%，而痰液的中位數的甲基化程度為38%，結果顯示甲基化標記在組織中的檢出率明顯高於痰液。同時，Rosalia Cirincione（Methylation profile in tumor and sputum samples of lung cancer patientsdetected by spiral computed tomography: A nested case–contro）報道了RARbeta2、P16、RASSF1A在肺癌組織中檢出率分別達到65.5%、41.4%、51.7%，而在痰液中分別只有44.4%、5%、5%。Non-invasive detection of lung cancer, such as sputum detection, is even more difficult. Although researchers have also studied tumor markers in the sputum of lung cancer patients, the success rate of sputum samples is low compared to the detection and evaluation of tumor markers in blood samples from other tumor patients. This is mainly due to the following reasons: ① The composition of sputum is relatively complex, and the composition and viscosity of sputum vary greatly among different groups of people under different diseases or environments; ② There are many tracheal epithelial cells and bacteria in sputum, and oral cavity For the components of non-lung cancer cells such as mucosal cells, the general sample processing method cannot effectively enrich the DNA of sufficient lung cancer origin; ③ There are many smoking patients who do not show sputum. In "Molecular sputum analysis for the diagnosis of lung cancer" by AJ Hubers et al., a study of the past 10 literatures showed that the median methylation degree of markers in lung cancer tissue was 48%, while the median methylation degree of sputum was 48%. The degree of methylation was 38%, and the results showed that the detection rate of methylation markers in tissues was significantly higher than that in sputum. Meanwhile, Rosalia Cirincione (Methylation profile in tumor and sputum samples of lung cancer patients detected by spiral computed tomography: A nested case–contro) reported that the detection rates of RARbeta2, P16, and RASSF1A in lung cancer tissues reached 65.5%, 41.4%, and 51.7%, respectively. %, and only 44.4%, 5%, and 5% in sputum.

目前肺癌的漏檢率普遍較高，特別是，對於腺癌這種類型，痰液無創檢測更加是難上加難，檢出率極其低。這是因為，多數腺癌起源於較小的支氣管，為周圍型肺癌，肺深部的脫落細胞更加難以通過痰液咳出。因此，目前腺癌的痰液檢測手段幾乎為零。At present, the missed detection rate of lung cancer is generally high. In particular, for adenocarcinoma, non-invasive detection of sputum is even more difficult, and the detection rate is extremely low. This is because most adenocarcinomas originate in the smaller bronchi, which are peripheral lung cancers, and exfoliated cells deep in the lung are more difficult to expectorate through sputum. Therefore, the current sputum detection methods for adenocarcinoma are almost zero.

降低漏檢率在腫瘤早期篩查中是尤其重要的。如果一個腫瘤早期篩查產品無法將所有或絕大部分的病患篩查出來的話，那麼漏檢的那些將無法得到足夠的風險提示，從而延誤的治療時機，這對患者來說是一個巨大的損失。Reducing the missed detection rate is especially important in early cancer screening. If an early tumor screening product cannot screen out all or most of the patients, those missed will not be able to get enough risk indications, thus delaying the timing of treatment, which is a huge problem for patients. loss.

儘管現有技術中已經發現了一些肺癌相關的腫瘤標記，但是，受限於針對這些腫瘤標記的檢測試劑或者檢測手段，導致這些腫瘤標記的靈敏度和特異性不能滿足需求，因此，目前本領域中仍然需要進一步研究能夠切實地應用於肺癌的篩查手段。雖然無創式的篩查具有取樣方面獨到的優勢，然而，其也具有其他方面的一些局限，例如，肺癌中的腺癌這種類型，由於其肺深部的脫落細胞難以通過痰液咳出，通常說來，本領域技術人員會認為該種類型的肺癌不適宜採用無創篩查。另一方面，即使是其他類型的肺癌，目前已報道的無創篩查方法也很難達到臨床使用的要求。儘管相關研究已進展多年，但至今仍未有可以推向臨床的肺癌無創篩查方法。Although some lung cancer-related tumor markers have been found in the prior art, the sensitivity and specificity of these tumor markers cannot meet the requirements due to the limitation of detection reagents or detection methods for these tumor markers. Further research is needed on screening methods that can be practically applied to lung cancer. Although non-invasive screening has unique advantages in sampling, it also has other limitations. For example, adenocarcinoma in lung cancer, because the exfoliated cells in the deep part of the lung are difficult to expectorate through sputum, usually In other words, those skilled in the art would consider this type of lung cancer inappropriate for non-invasive screening. On the other hand, even for other types of lung cancer, the currently reported non-invasive screening methods are difficult to meet the requirements for clinical use. Although relevant research has progressed for many years, there is still no non-invasive screening method for lung cancer that can be promoted to the clinic.

本公開的目的在於提供肺腫瘤標記。The present disclosure aims to provide lung tumor markers.

本公開的另一個目的在於提供一種可進行無創檢測的肺腫瘤標記。Another object of the present disclosure is to provide a lung tumor marker that can be detected non-invasively.

本公開的另一個目的在於提供一種可降低漏檢率的肺腫瘤標記Another object of the present disclosure is to provide a lung tumor marker that can reduce the missed detection rate

本公開的另一個目的在於提供一種HOXA7和HOXA9基因或它們的核酸片段在製備腫瘤診斷試劑的用途。Another object of the present disclosure is to provide the use of HOXA7 and HOXA9 genes or their nucleic acid fragments in the preparation of tumor diagnostic reagents.

本公開的另一個目的還在於提供一種在無創中，針對腺癌具有高敏感性和特異性的腫瘤標記。Another object of the present disclosure is to provide a non-invasive tumor marker with high sensitivity and specificity for adenocarcinoma.

本公開的另一個目的還在於提供一種檢測HOXA7和HOXA9基因甲基化的試劑／試劑盒和方法。Another object of the present disclosure is to provide a reagent/kit and method for detecting the methylation of HOXA7 and HOXA9 genes.

本公開的目的還在於提供一種特異性強、敏感度高的肺腫瘤檢測試劑／試劑盒。The present disclosure also aims to provide a lung tumor detection reagent/kit with strong specificity and high sensitivity.

本公開的目的還在於提供一種對肺癌應用範圍廣的肺腫瘤檢測試劑／試劑盒。The present disclosure also aims to provide a lung tumor detection reagent/kit with wide application range for lung cancer.

本公開提供了如下方案：The present disclosure provides the following solutions:

一方面，本公開提供了HOXA7基因或其核酸片段，以及HOXA9基因或其核酸片段在製備腫瘤診斷試劑中的用途。In one aspect, the present disclosure provides the use of HOXA7 gene or nucleic acid fragment thereof, and HOXA9 gene or nucleic acid fragment thereof in the preparation of tumor diagnostic reagents.

發明人經過深入的研究，首次揭示一種同時檢測HOXA7基因和HOXA9基因或它們的核酸片段的甲基化來提高肺癌檢出率的方法。尤其是首次揭示了HOXA7基因和HOXA9基因的聯合甲基化檢測可以實現無創的、極大提高檢出率的方法。尤其是對於小細胞癌來說其漏檢率為0，這是截至目前從未有過任何技術可以比擬的效果。After in-depth research, the inventor revealed for the first time a method for simultaneously detecting the methylation of HOXA7 gene and HOXA9 gene or their nucleic acid fragments to improve the detection rate of lung cancer. In particular, it was revealed for the first time that the combined methylation detection of HOXA7 gene and HOXA9 gene can achieve a non-invasive method that greatly improves the detection rate. Especially for small cell carcinoma, the missed detection rate is 0, which has never been compared with any technology so far.

所述甲基化基因是人HOXA7基因和人HOXA9基因。發明人不僅在組織樣本中驗證了檢測HOXA7和HOXA9基因甲基化對肺癌的檢出具有較高的特異性和靈敏性，同時驗證了在痰液樣本和灌洗液樣本中具有同樣高的特異性和靈敏性。The methylated genes are the human HOXA7 gene and the human HOXA9 gene. The inventors not only verified that the detection of HOXA7 and HOXA9 gene methylation has high specificity and sensitivity in the detection of lung cancer in tissue samples, but also verified that they have the same high specificity in sputum samples and lavage samples. Sex and Sensitivity.

已知的肺癌標記有非常多種，如CHRDL1，JAK1，p‑EphB1，FABP1，p‑LCK，SOST，p‑ZAP70，BARD1，UHRF1，MiRNA‑4731‑3p，miRNA‑6729‑5p，AKAP4，ABCB1、AKT1、ALK、APC、ATIC等等。標記之間的聯合檢測效果是不可預期的。因為大部分腫瘤標記對腫瘤的診斷僅有相關性，而無特異性，所以數種腫瘤標記聯合檢測對腫瘤的診斷意義更大。目前臨床上對腫瘤的檢測基本都採用多標記聯合檢測進行輔助診斷，例如肝癌採用AFP、CEA、free-b-hCG、CA199、CA242聯合檢測；結直腸癌採用CEA、CA242、CA199等聯合檢測。There are many known lung cancer markers, such as CHRDL1, JAK1, p‑EphB1, FABP1, p‑LCK, SOST, p‑ZAP70, BARD1, UHRF1, MiRNA‑4731‑3p, miRNA‑6729‑5p, AKAP4, ABCB1, AKT1, ALK, APC, ATIC, etc. The joint detection effect between markers is unpredictable. Because most tumor markers are only relevant for the diagnosis of tumors without specificity, the combined detection of several tumor markers is more meaningful for the diagnosis of tumors. At present, the clinical detection of tumors basically uses multi-marker combined detection for auxiliary diagnosis. For example, liver cancer is detected by AFP, CEA, free-b-hCG, CA199, and CA242; colorectal cancer is detected by CEA, CA242, and CA199.

HOXA7和HOXA9基因均是HOX（homebox 同源盒）基因家族的一員，屬染色體7p15-p14上的HOXA簇基因，與其他的HOX基因一樣，均含有一段180bp的DNA片段，轉錄由60個胺基酸組成的同源結構域。HOXA7在正常造血細胞的增殖分化中發揮調節作用，目前研究比較多的是HOXA7的異常表達在白血病的發生和發展中起著重要的作用，也有部分報道HOXA7基因的甲基化與肺癌相關。HOXA9是編碼序列特異性轉錄調控因子，在胚胎的時空發育，細胞的分化、增殖與遷移，腫瘤的惡性演變和誘發凋亡都有著重要作用。目前研究比較多的是HOXA9的異常表達在白血病的發生和發展中起著重要的作用，也有部分報道HOXA9基因的肺癌、人細胞膠質瘤等癌症相關。Both HOXA7 and HOXA9 genes are members of the HOX (homebox) gene family, which belong to the HOXA cluster gene on chromosome 7p15-p14. Like other HOX genes, they both contain a 180bp DNA fragment, which is transcribed by 60 amino groups. acid-formed homology domains. HOXA7 plays a regulatory role in the proliferation and differentiation of normal hematopoietic cells. At present, many studies show that the abnormal expression of HOXA7 plays an important role in the occurrence and development of leukemia. Some reports also show that the methylation of HOXA7 gene is related to lung cancer. HOXA9 is a coding sequence-specific transcriptional regulator, which plays an important role in the spatiotemporal development of embryos, cell differentiation, proliferation and migration, malignant evolution of tumors and induction of apoptosis. At present, there are many studies that the abnormal expression of HOXA9 plays an important role in the occurrence and development of leukemia, and some reports of HOXA9 gene are related to lung cancer, human glioma and other cancers.

目前，尚未有報道將HOXA7和HOXA9基因聯合作為肺癌的腫瘤標記。本公開首次基於HOXA7和HOXA9基因，或它們核酸片段，檢測肺癌。二者聯合檢測時，極大的提高肺癌的檢出率。At present, there is no report on the combination of HOXA7 and HOXA9 genes as a tumor marker for lung cancer. The present disclosure is the first to detect lung cancer based on the HOXA7 and HOXA9 genes, or their nucleic acid fragments. When the two are combined, the detection rate of lung cancer is greatly improved.

另一方面，本公開提供了HOXA7和HOXA9基因甲基化的檢測試劑在製備肺癌診斷試劑中的用途。In another aspect, the present disclosure provides the use of HOXA7 and HOXA9 gene methylation detection reagents in the preparation of lung cancer diagnostic reagents.

作為優選的實施方式，所述甲基化的檢測試劑含有並以無創式的樣本為檢測對象，例如，痰液、肺部灌洗液、淚液或糞便等等。As a preferred embodiment, the methylation detection reagent contains and uses non-invasive samples as detection objects, such as sputum, pulmonary lavage fluid, tears or feces, and the like.

作為優選的實施方式，本公開提供的甲基化的檢測為小細胞癌或腺癌的診斷試劑；尤其地，為小細胞癌的診斷試劑。As a preferred embodiment, the detection of methylation provided by the present disclosure is a diagnostic reagent for small cell carcinoma or adenocarcinoma; in particular, a diagnostic reagent for small cell carcinoma.

進一步地，所述HOXA7基因和HOXA9基因甲基化的檢測試劑檢測HOXA7和HOXA9基因經轉化試劑修飾後的序列。其中，轉化試劑是指使DNA中胞嘧啶脫胺基成為尿嘧啶，同時使5-MeC基本上不受影響的試劑。示例性的轉化試劑包括肼鹽、重亞硫酸氫鹽（例如重亞硫酸氫鈉等）、亞硫酸氫鹽（例如偏亞硫酸氫鈉、亞硫酸氫鉀、亞硫酸氫銫、亞硫酸氫銨等）、或在適當的反應條件下可產生肼鹽、重亞硫酸氫鹽、亞硫酸氫鹽的化合物中的一種或幾種。作為一種示範性的實施方式，所述HOXA7和HOXA9基因甲基化的檢測試劑檢測經重亞硫酸氫鹽修飾後的序列。Further, the detection reagent for the methylation of the HOXA7 gene and the HOXA9 gene detects the sequences of the HOXA7 and HOXA9 genes modified by the conversion reagent. Among them, the conversion reagent refers to a reagent that deaminates cytosine in DNA to uracil while leaving 5-MeC substantially unaffected. Exemplary conversion reagents include hydrazine salts, bisulfites (eg, sodium bisulfite, etc.), bisulfites (eg, sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.) etc.), or one or more of the compounds that can produce hydrazine salt, bisulfite, and bisulfite under appropriate reaction conditions. As an exemplary embodiment, the HOXA7 and HOXA9 gene methylation detection reagents detect bisulfite-modified sequences.

甲基化的發生是胞嘧啶上多了一個甲基，經過亞硫酸氫鹽或重亞硫酸氫鹽或肼鹽處理後，胞嘧啶會變成脲嘧啶，因為在進行PCR擴增時尿嘧啶與胸腺嘧啶相似而會被識別為胸腺嘧啶，體現在PCR擴增序列上就是沒有發生甲基化的胞嘧啶變成了胸腺嘧啶（C變成T），甲基化的胞嘧啶（C）則不會發生變化。PCR檢測甲基化基因的技術通常為甲基化特異性PCR（Methylmion Specific PCR，MSP），針對處理後的甲基化片段（即片段中未改變的C）設計引子，進行PCR擴增，如果有擴增則說明發生了甲基化，無擴增則沒有甲基化。Methylation occurs when an additional methyl group is added to cytosine. After treatment with bisulfite or bisulfite or hydrazine salt, cytosine will become uracil, because uracil and thymus during PCR amplification Pyrimidine is similar to thymine and will be recognized as thymine. It is reflected in the PCR amplification sequence that the unmethylated cytosine becomes thymine (C becomes T), and the methylated cytosine (C) does not change. . The technology of PCR detection of methylated genes is usually methylation specific PCR (Methylmion Specific PCR, MSP). The presence of amplification means that methylation has occurred, and the absence of amplification means that there is no methylation.

作為可選的實施方式，HOXA7和HOXA9基因甲基化的檢測試劑所針對H的檢測區域包含基因的CG富集區域或非CG富集區域或CTCF（CTCF-binding sites）區域。作為優選的實施方式，所述試劑的檢測區域包含基因的CG富集區域或CTCF（CTCF-binding sites）區域。As an optional embodiment, the detection region for H of the HOXA7 and HOXA9 gene methylation detection reagents includes a CG-enriched region or a non-CG-enriched region or CTCF (CTCF-binding sites) region of the gene. As a preferred embodiment, the detection region of the reagent includes a CG-enriched region or a CTCF (CTCF-binding sites) region of the gene.

或者，HOXA7和HOXA9基因甲基化的檢測試劑所針對的檢測區域包含基因體（gene body）或者其啟動子區域。Alternatively, the detection region targeted by the detection reagent for HOXA7 and HOXA9 gene methylation includes the gene body or its promoter region.

作為本公開中優選的實施方式，所述甲基化的檢測試劑針對HOXA7的檢測區域包含SEQ ID NO: 25或SEQ ID NO: 27所示的序列；所述試劑針對HOXA9的檢測區域包含SEQ ID NO: 29、SEQ ID NO: 31或SEQ ID NO: 33所示的序列。作為更優選的實施方式，所述試劑針對HOXA7的檢測區域包含SEQ ID NO: 27所示的序列，所述試劑針對HOXA9的檢測區域包含SEQ ID NO: 29所示的序列。As a preferred embodiment in the present disclosure, the detection region of the methylated detection reagent for HOXA7 comprises the sequence shown in SEQ ID NO: 25 or SEQ ID NO: 27; the detection region of the reagent for HOXA9 comprises SEQ ID NO: 29, SEQ ID NO: 31 or the sequence shown in SEQ ID NO: 33. As a more preferred embodiment, the detection region of the reagent for HOXA7 comprises the sequence shown in SEQ ID NO: 27, and the detection region of the reagent for HOXA9 comprises the sequence shown in SEQ ID NO: 29.

本公開甲基化的檢測試劑，還含有檢測HOXA7基因和HOXA9基因的引子對。作為可選的實施方式，檢測HOXA7基因的引子對選自SEQ ID NO: 1-2，SEQ ID NO: 37- 38，SEQ ID NO: 40-41， SEQ ID NO: 43-44，或SEQ ID NO: 46- 47，SEQ ID NO: 49-50，SEQ ID NO: 52-53，SEQ ID NO: 55-56，SEQ ID NO: 58-59或SEQ ID NO: 61- 62所示的引子對；檢測HOXA9基因的引子對選自SEQ ID NO: 4-5，SEQ ID NO: 64-65，SEQ ID NO: 67-68，SEQ ID NO: 70-71，SEQ ID NO: 73-74，SEQ ID NO: 76-77，SEQ ID NO: 79-80，SEQ ID NO: 82-83，SEQ ID NO: 85-86，SEQ ID NO: 88-89或SEQ ID NO: 91-92所示的引子對。The methylation detection reagent of the present disclosure also contains a primer pair for detecting the HOXA7 gene and the HOXA9 gene. As an optional embodiment, the primer pair for detecting HOXA7 gene is selected from SEQ ID NO: 1-2, SEQ ID NO: 37-38, SEQ ID NO: 40-41, SEQ ID NO: 43-44, or SEQ ID NO: 43-44 NO: 46-47, SEQ ID NO: 49-50, SEQ ID NO: 52-53, SEQ ID NO: 55-56, SEQ ID NO: 58-59 or primer pair shown in SEQ ID NO: 61-62 The primer pair of detection HOXA9 gene is selected from SEQ ID NO: 4-5, SEQ ID NO: 64-65, SEQ ID NO: 67-68, SEQ ID NO: 70-71, SEQ ID NO: 73-74, SEQ ID NO: 67-68 ID NO: 76-77, SEQ ID NO: 79-80, SEQ ID NO: 82-83, SEQ ID NO: 85-86, SEQ ID NO: 88-89 or primers shown in SEQ ID NO: 91-92 right.

作為優選的實施方式，檢測HOXA7基因的引子對選自SEQ ID NO: 1-2所示的引子對；檢測HOXA9基因的引子對選自SEQ ID NO: 4-5所示的引子對。As a preferred embodiment, the primer pair for detecting the HOXA7 gene is selected from the primer pairs shown in SEQ ID NO: 1-2; the primer pair for detecting the HOXA9 gene is selected from the primer pairs shown in SEQ ID NO: 4-5.

本公開甲基化的檢測試劑，還含有檢測HOXA7基因和HOXA9基因的探針。作為可選的實施方式，檢測HOXA7基因的探針選自SEQ ID NO: 3、SEQ ID NO: 39、SEQ ID NO: 42、SEQ ID NO: 45、SEQ ID NO: 48、SEQ ID NO: 51、SEQ ID NO: 54、SEQ ID NO: 57、SEQ ID NO: 60或SEQ ID NO: 63所示的序列；檢測HOXA9基因的探針選自SEQ ID NO: 6、SEQ ID NO: 66、SEQ ID NO: 69、SEQ ID NO: 72、SEQ ID NO: 75、SEQ ID NO: 78、SEQ ID NO: 81、SEQ ID NO: 84、SEQ ID NO: 87、SEQ ID NO: 90或SEQ ID NO: 93所示的序列。作為優選的實施方式，檢測HOXA7基因的探針選自SEQ ID NO: 3所示的序列；檢測HOXA9基因的探針選自SEQ ID NO: 6所示的序列。作為本公開的優選方式，為方便臨床使用，檢測探針標記的螢光基團可以是 VIC、ROX、FAM、Cy5、HEX、TET、JOE、NED、Texas Red 等 ；淬滅基團可以是 TAMRA、BHQ、MGB、Dabcyl等等，以適用於目前臨床檢測常用的多通道 PCR 檢測系統，實現在一個反應管中進行多色螢光檢測。The methylation detection reagent of the present disclosure also contains probes for detecting the HOXA7 gene and the HOXA9 gene. As an optional embodiment, the probe for detecting HOXA7 gene is selected from SEQ ID NO: 3, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51 , SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60 or the sequence shown in SEQ ID NO: 63; the probe for detecting HOXA9 gene is selected from SEQ ID NO: 6, SEQ ID NO: 66, SEQ ID NO: 63 ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, or SEQ ID NO : the sequence shown in 93. As a preferred embodiment, the probe for detecting the HOXA7 gene is selected from the sequence shown in SEQ ID NO: 3; the probe for detecting the HOXA9 gene is selected from the sequence shown in SEQ ID NO: 6. As a preferred mode of the present disclosure, for the convenience of clinical use, the fluorescent group labeled with the detection probe can be VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, Texas Red, etc.; the quenching group can be TAMRA , BHQ, MGB, Dabcyl, etc., are suitable for multi-channel PCR detection systems commonly used in clinical detection, and realize multi-color fluorescence detection in one reaction tube.

作為優選的實施方式，本公開甲基化的檢測試劑含有SEQ ID NO: 1和SEQ ID NO: 2所示引子對、SEQ ID NO: 4和SEQ ID NO: 5所示的引子對，以及如SEQ ID NO: 3所示和SEQ ID NO: 6所示的探針。As a preferred embodiment, the methylation detection reagent of the present disclosure contains the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, the primer pair shown in SEQ ID NO: 4 and SEQ ID NO: 5, and as shown in Probes shown in SEQ ID NO: 3 and SEQ ID NO: 6.

作為可選的實施方式，本公開甲基化的檢測還含有參考基因的檢測試劑。As an optional embodiment, the detection of methylation in the present disclosure also includes detection reagents for reference genes.

優選地，所述參考基因包含β-actin或COL2A1，除了這兩個參考基因，也可以採用現有技術的其他的甲基化檢測的參考基因。進一步的，所述參考基因的檢測試劑包含針對參考基因的引子和探針。作為優選的實施方式，所述參考基因β-actin的檢測試劑包含SEQ ID NO: 19、SEQ ID NO: 20所示的引子對，以及SEQ ID NO: 21的探針。所述參考基因COL2A1的檢測試劑含有SEQ ID NO: 94（TTTTGGATTTAAGGGGAAGATAAA）、SEQ ID NO: 95（TTTTTCCTTCTCTACATCTTTCTACC​T）所示的引子對，以及SEQ ID NO: 96（AAGGGAAATTGAGAAATGAGAGAAGGGA）所示的探針。Preferably, the reference gene comprises β-actin or COL2A1, in addition to these two reference genes, other reference genes for methylation detection in the prior art can also be used. Further, the detection reagent for the reference gene includes primers and probes for the reference gene. As a preferred embodiment, the detection reagent for the reference gene β-actin comprises the primer pair shown in SEQ ID NO: 19 and SEQ ID NO: 20, and the probe of SEQ ID NO: 21. The detection reagent for the reference gene COL2A1 contains the primer pair shown in SEQ ID NO: 94 (TTTTGGATTTAAGGGGAAGATAAA), SEQ ID NO: 95 (TTTTTCCTTCTCTACATCTTTCTACC​T), and the probe shown in SEQ ID NO: 96 (AAGGGAAATTGAGAAATGAGAGAAGGGA).

作為可選的實施方式，本公開甲基化的檢測試劑還包含亞硫酸氫鹽、重亞硫酸氫鹽或肼鹽，用於將甲基化的胞嘧啶修飾成胸腺嘧啶。當然，也可以不包含在本公開的試劑中，使用時獨立購買即可。As an optional embodiment, the methylated detection reagent of the present disclosure further comprises bisulfite, bisulfite or hydrazine salt for modifying methylated cytosine into thymine. Of course, it may not be included in the reagent of the present disclosure, and can be purchased independently when used.

作為可選的實施方式，本公開甲基化的檢測試劑還包含DNA 聚合酶、dNTPs、Mg²⁺離子、緩衝液中的一種或幾種；優選地，包含有DNA 聚合酶、dNTPs、Mg²⁺離子和緩衝液，用於對HOXA7和HOXA9基因進行擴增反應。As an optional embodiment, the methylation detection reagent of the present disclosure further comprises one or more of DNA polymerase, dNTPs, Mg²⁺ ions, and buffers; preferably, it includes DNA polymerase, dNTPs, Mg²⁺ ions and buffer for amplification of HOXA7 and HOXA9 genes.

在優選的實施方案中，所述甲基化的檢測試劑的檢測樣本包含痰液、肺部灌洗液、肺部組織、胸水，血液、血清、血漿、尿液、***液、淚液或糞便等等。作為優選的實施方式，所述檢測樣本包含痰液、肺部組織或肺部灌洗液。作為更優選的實施方式，所述檢測樣本包含痰液或肺部灌洗液。In a preferred embodiment, the detection sample of the methylation detection reagent comprises sputum, pulmonary lavage fluid, lung tissue, pleural effusion, blood, serum, plasma, urine, prostatic fluid, tears or feces, etc. Wait. As a preferred embodiment, the test sample comprises sputum, lung tissue or lung lavage fluid. As a more preferred embodiment, the test sample comprises sputum or lung lavage fluid.

另一方面，本公開還提供了一種檢測HOXA7基因和HOXA9基因的DNA甲基化的方法，包括以下步驟： （1）將檢測樣本進行亞硫酸氫鹽或重亞硫酸氫鹽或肼鹽處理，獲得經修飾的檢測樣本； （2）利用上述甲基化的檢測或試劑盒對步驟（1）經修飾的檢測樣本進行測HOXA7基因和HOXA9基因甲基化情況檢測。On the other hand, the present disclosure also provides a method for detecting DNA methylation of HOXA7 gene and HOXA9 gene, comprising the following steps: (1) Treat the test sample with bisulfite or bisulfite or hydrazine salt to obtain a modified test sample; (2) Using the above-mentioned methylation detection or kit to detect the methylation of HOXA7 gene and HOXA9 gene on the modified detection sample in step (1).

作為可選的方式，採用甲基化特異性聚合酶鏈反應（MSP）或實時螢光定量甲基化特異性聚合酶鏈反應（real-time fluorescent quantitative methylation-specific PCR，qMSP）進行檢測。其餘現有技術中報道的DNA甲基化檢測方法也可以應用於本公開中。現有技術的甲基化檢測方法可通過專利USSN62/175,916引入本公開。Alternatively, methylation-specific polymerase chain reaction (MSP) or real-time fluorescent quantitative methylation-specific PCR (qMSP) was used for detection. Other DNA methylation detection methods reported in the prior art can also be applied in the present disclosure. A prior art methylation detection method can be incorporated into the present disclosure through patent USSN 62/175,916.

另一方面，本公開還提供了一種肺癌的診斷系統，所述系統包含： HOXA7基因和HOXA9基因的DNA 甲基化檢測構件；以及 結果判斷構件。In another aspect, the present disclosure also provides a system for diagnosing lung cancer, the system comprising: DNA methylation detection components of the HOXA7 and HOXA9 genes; and The result judges the component.

在一些實施方式中，所述HOXA7基因和HOXA9基因的DNA 甲基化檢測構件包含上述甲基化的檢測試劑。In some embodiments, the DNA methylation detection components of the HOXA7 gene and HOXA9 gene comprise the above-described methylation detection reagents.

在一些實施方式中，所述甲基化檢測構件包含螢光定量PCR儀、PCR儀、測序儀中的一種或多種。In some embodiments, the methylation detection component comprises one or more of a fluorescence quantitative PCR instrument, a PCR instrument, and a sequencer.

在一些實施方式中，所述結果判斷構件用於根據檢測構件檢測HOXA7基因和HOXA9基因的DNA甲基化水平，輸出診斷結果如肺癌的患病風險和／或肺癌類型等。In some embodiments, the result judgment component is used to detect the DNA methylation levels of HOXA7 gene and HOXA9 gene according to the detection component, and output diagnostic results such as the risk of lung cancer and/or the type of lung cancer.

在一些實施方式中，所述患病風險是通過結果判斷構件比較待測樣本與正常樣本的甲基化水平，基於待測樣本與正常樣本的甲基化水平的偏離得出的。In some embodiments, the disease risk is obtained by comparing the methylation levels of the test sample and the normal sample through the result judgment component, based on the deviation of the methylation levels of the test sample and the normal sample.

在一些實施方式中，所述結果判斷構件包含數據處理機器。In some embodiments, the result determination means comprises a data processing machine.

在一些實施方式中，所述數據處理機器包括本領域技術人員可使用的任何可以進行數據處理的設備或儀器或裝置。In some embodiments, the data processing machine includes any equipment or apparatus or apparatus that can perform data processing available to those skilled in the art.

在一些實施方式中，所述數據處理機器包含計算機、電腦中的一種或多種。所述電腦中附載有本領域技術人員可使用的任何可以進行數據處理或統計分析的軟體或程序。在一些實施方式中，所述電腦包含附載有SPSS、SAS、Excel中一種或多種軟體的電腦。In some embodiments, the data processing machine comprises one or more of a computer, a computer. The computer is loaded with any software or program capable of data processing or statistical analysis that can be used by those skilled in the art. In some embodiments, the computer comprises a computer with one or more of SPSS, SAS, Excel attached.

在一些實施方式中，所述結果判斷構件還包含結果輸出器。所述輸出器包含任何可以將數據處理結果顯示為可閱讀的內容的設備或儀器或裝置。在一些實施方式中，所述結果輸出器包含屏幕、紙質報告中的一種或多種。In some embodiments, the result judgment component further includes a result outputter. The exporter includes any device or apparatus or device that can display the results of data processing as readable content. In some embodiments, the results exporter comprises one or more of a screen, a paper report.

本公開另一方面提供了一種肺癌的診斷方法，所述方法包括以下步驟： （1）檢測來源於受試者的待測樣本HOXA7基因和HOXA9基因的甲基化水平； （2）將待測樣本與正常樣本的HOXA7基因和HOXA9基因水平比較 （3）基於待測樣本與正常樣本的甲基化水平的偏離，診斷肺癌。Another aspect of the present disclosure provides a method for diagnosing lung cancer, the method comprising the steps of: (1) Detect the methylation levels of the HOXA7 gene and HOXA9 gene in the test sample derived from the subject; (2) Compare the levels of HOXA7 and HOXA9 genes in the samples to be tested and normal samples (3) Diagnose lung cancer based on the deviation of the methylation level of the test sample and the normal sample.

在一些實施方式中，當所述待測痰液樣本HOXA7基因的甲基化水平大於0.77%或HOXA9基因的甲基化水平大於2.3%時，則判定待測樣本為肺癌樣本，若待測痰液樣本HOXA7基因的甲基化水平小於等於0.77%且HOXA9基因的甲基化水平小於等於2.3%時，則判定待測樣本為非肺癌樣本。In some embodiments, when the methylation level of the HOXA7 gene in the sputum sample to be tested is greater than 0.77% or the methylation level of the HOXA9 gene is greater than 2.3%, the sample to be tested is determined to be a lung cancer sample. When the methylation level of the HOXA7 gene in the liquid sample is less than or equal to 0.77% and the methylation level of the HOXA9 gene is less than or equal to 2.3%, the sample to be tested is determined to be a non-lung cancer sample.

在一些實施方式種，當所述待測灌洗液樣本HOXA7基因的甲基化水平大於0.7%或HOXA9基因的甲基化水平大於0.5%時，則判定待測樣本為肺癌樣本。若待測灌洗液樣本HOXA7基因的甲基化水平小於等於0.7%且HOXA9基因的甲基化水平小於等於0.5%時，則判定待測樣本為非肺癌樣本。In some embodiments, when the methylation level of the HOXA7 gene in the lavage fluid sample to be tested is greater than 0.7% or the methylation level of the HOXA9 gene is greater than 0.5%, the sample to be tested is determined to be a lung cancer sample. If the methylation level of the HOXA7 gene in the lavage fluid sample to be tested is less than or equal to 0.7% and the methylation level of the HOXA9 gene is less than or equal to 0.5%, the sample to be tested is determined to be a non-lung cancer sample.

本公開的診斷方法可以在肺癌治療前後使用或者與肺癌治療聯合使用，治療後使用如評價治療的成功或者監測治療後肺癌的緩解、復發和／或進展（包括轉移）。The diagnostic methods of the present disclosure can be used before or in combination with lung cancer treatment, such as to assess the success of treatment or to monitor post-treatment remission, recurrence and/or progression (including metastasis) of lung cancer.

本公開另一方面提供了一種肺癌的治療方法，所述方法包括對經上述診斷方法診斷為肺癌的患者，施用手術、化療、放療、放化療、免疫療法、溶瘤病毒療法、或其他本領域所用的任何其他類型肺癌治療方法以及這些治療方法的組合。Another aspect of the present disclosure provides a method for treating lung cancer, the method comprising administering surgery, chemotherapy, radiotherapy, chemoradiotherapy, immunotherapy, oncolytic virus therapy, or other fields in the art to a patient diagnosed with lung cancer by the above-mentioned diagnostic method Any other types of lung cancer treatments used and combinations of these treatments.

本公開中，所述肺癌選自小細胞肺癌（small cell lung cancer，SCL）或非小細胞肺癌（non-small cell lung cancer，NSCL）；進一步地，所述非小細胞肺癌選自鱗狀細胞癌、腺癌或大細胞癌。作為優選的實施方式，所述肺癌選自小細胞肺癌或腺癌。In the present disclosure, the lung cancer is selected from small cell lung cancer (SCL) or non-small cell lung cancer (NSCL); further, the non-small cell lung cancer is selected from squamous cell lung cancer carcinoma, adenocarcinoma, or large cell carcinoma. As a preferred embodiment, the lung cancer is selected from small cell lung cancer or adenocarcinoma.

雖然，現有技術中，HOXA7和HOXA9基因的甲基化分別被報道了在肺癌中有甲基化。然而，有關肺癌的腫瘤標記的報道，不計其數，本公開在眾多被報道的，跟肺癌可能相關甲基化基因中，首次將HOXA7和HOXA9基因聯合，作為肺癌的腫瘤標記，很大程度地提高了肺癌檢出率。Although, in the prior art, the methylation of HOXA7 and HOXA9 genes has been reported to be methylated in lung cancer, respectively. However, there are countless reports on the tumor markers of lung cancer. In the present disclosure, HOXA7 and HOXA9 genes are combined for the first time as tumor markers of lung cancer among the many reported genes that may be related to lung cancer. Increased lung cancer detection rate.

針對痰液樣本，HOXA7和HOXA9基因的單獨檢測對全部肺癌的特異性達到了95%，HOXA7基因和HOXA9基因單獨檢測對肺癌的敏感性分別為88.6%和74.3%。通過聯合HOXA7和HOXA9檢測，對全部肺癌的敏感性提升到97.1%，如此高的靈敏度在現有的肺癌標記是非常少見的。而在臨床中，作為早期篩查的工具，高的靈敏度是非常關鍵的。目前肺癌早期篩查，尤其是無創篩查領域的技術嚴重缺失，最主要的原因就是缺少具有高的靈敏度的檢測方法。目前已報道的靈敏度一般在45%-75%左右。For sputum samples, the specificity of HOXA7 and HOXA9 gene detection for all lung cancers reached 95%, and the sensitivity of HOXA7 gene and HOXA9 gene detection for lung cancer was 88.6% and 74.3%, respectively. By combining HOXA7 and HOXA9 detection, the sensitivity of all lung cancers is increased to 97.1%, which is very rare in existing lung cancer markers. In the clinic, as a tool for early screening, high sensitivity is very critical. At present, the technology of early lung cancer screening, especially in the field of non-invasive screening, is seriously lacking. The main reason is the lack of detection methods with high sensitivity. The reported sensitivity is generally around 45%-75%.

此外，HOXA7基因和HOXA9基因聯合檢測針對不同類型的肺癌，包括小細胞肺癌和非小細胞肺癌中的鱗癌、大細胞癌、腺癌均具有很高的特異性和靈敏度，其適用範圍廣，基本上能作為所有肺癌的腫瘤標記。而現有的用於臨床的肺癌標記，其一般僅能適用於一類肺癌的檢測，如NSE用於小細胞肺癌的診斷和監測治療反應，而CYFRA21-1是非小細胞肺癌的首選標記。In addition, the combined detection of HOXA7 gene and HOXA9 gene has high specificity and sensitivity for different types of lung cancer, including squamous cell carcinoma, large cell carcinoma, and adenocarcinoma in small cell lung cancer and non-small cell lung cancer, and has a wide range of applications. Basically, it can be used as a tumor marker for all lung cancers. The existing clinical markers of lung cancer are generally only applicable to the detection of one type of lung cancer, such as NSE for the diagnosis of small cell lung cancer and monitoring of treatment response, while CYFRA21-1 is the preferred marker for non-small cell lung cancer.

在小細胞癌中，不管是在痰液中還是在灌洗液樣本的檢測中，採用HOXA7基因和HOXA9基因聯合檢測的無創篩查方式，竟都獲得了100%的靈敏度。這樣的0漏檢率在本領域是一個巨大的突破。In small cell carcinoma, the non-invasive screening method using the combined detection of HOXA7 and HOXA9 genes has achieved 100% sensitivity in both sputum and lavage samples. Such a zero missed detection rate is a huge breakthrough in the field.

在肺腺癌中，HOXA7基因和HOXA9基因聯合檢測比其他腫瘤標記具有尤其提高的靈敏度，達到100%的靈敏度。目前，肺腺癌的漏檢率較高。一方面，由於肺腺癌較容易發生於女性及不抽煙者，發病率比鱗癌和未分化癌低，發病年齡較小，女性相對多見；另一方面，多數腺癌起源於較小的支氣管，為周圍型肺癌，肺深部的脫落細胞更加難以通過痰液咳出；再一方面，肺腺癌早期一般沒有明顯的臨床症狀。因此，對肺腺癌的檢測更具困難性和價值性。In lung adenocarcinoma, the combined detection of HOXA7 gene and HOXA9 gene has a particularly improved sensitivity compared with other tumor markers, reaching a sensitivity of 100%. Currently, lung adenocarcinoma has a high rate of missed detection. On the one hand, because lung adenocarcinoma is more likely to occur in women and non-smokers, the incidence rate is lower than that of squamous cell carcinoma and anaplastic carcinoma, the age of onset is younger, and women are relatively more common; on the other hand, most adenocarcinomas originate from younger Bronchus is a peripheral lung cancer, and the exfoliated cells in the deep part of the lung are more difficult to cough up through sputum; on the other hand, lung adenocarcinoma generally has no obvious clinical symptoms in the early stage. Therefore, the detection of lung adenocarcinoma is more difficult and valuable.

發明人經實驗發現，在痰液中，HOXA7和HOXA9基因聯合檢測，對腺癌的檢測靈敏度達到了100%，在腺癌檢測領域已經是目前絕無僅有的極高靈敏度的水平了（作為對比，SHOX2基因對腺癌靈敏度33.3%）。現有的腫瘤標記，即便是在組織中對腺癌有很高的靈敏度，但是當以痰液為檢測樣本時，靈敏度卻大幅下降，失去腫瘤標記的作用。如SHOX2基因，在組織樣本中，對腺癌靈敏度79.0%，為本公開研究的腫瘤標記中對腺癌靈敏度最高的基因，但是在痰液樣本中，SHOX2基因對腺癌靈敏度大幅度下降到33.3%。The inventors have found through experiments that the combined detection of HOXA7 and HOXA9 genes in sputum has a detection sensitivity of 100% for adenocarcinoma, which is an extremely high sensitivity level that is unique in the field of adenocarcinoma detection (for comparison, SHOX2 gene sensitivity to adenocarcinoma 33.3%). Existing tumor markers have high sensitivity to adenocarcinoma in tissues, but when sputum is used as the detection sample, the sensitivity is greatly reduced, and the effect of tumor markers is lost. For example, SHOX2 gene, in tissue samples, has a sensitivity of 79.0% to adenocarcinoma, which is the gene with the highest sensitivity to adenocarcinoma among the tumor markers studied in the present disclosure. However, in sputum samples, the sensitivity of SHOX2 gene to adenocarcinoma dropped significantly to 33.3%. %.

在肺部灌洗液中，HOXA7和HOXA9基因聯合檢測對腺癌的敏感度也高達81.8%，該靈敏度甚至高於其在組織樣本中的靈敏度，這是十分難得的。In lung lavage fluid, the combined detection of HOXA7 and HOXA9 genes has a sensitivity of 81.8% for adenocarcinoma, which is even higher than its sensitivity in tissue samples, which is very rare.

因此，對於腺癌來說，HOXA7和HOXA9基因聯合檢測具有突出的優勢，且優選的檢測樣本為痰液和肺部灌洗液。以痰液和肺部灌洗液作為檢測樣本，其具有相對比其他標記更為突出的靈敏度，尤其是以痰液作為檢測樣本時，對於鱗癌、腺癌和小細胞癌的檢測靈敏度均到了100%，有利於及時發現患者異常，進一步結合其他檢測手段確診是否為腺癌，且極大的降低了臨床上腫瘤篩查的簡便性，無需針對特定類型的肺癌使用特定的腫瘤標記，再者，肺部灌洗液和痰液樣品的獲取無創，使得HOXA7和HOXA9聯合檢測對於腺癌等多種類型的肺癌的檢測來說具有極大的應用意義。Therefore, for adenocarcinoma, the combined detection of HOXA7 and HOXA9 genes has outstanding advantages, and the preferred detection samples are sputum and lung lavage fluid. Using sputum and lung lavage fluid as detection samples, it has a relatively more prominent sensitivity than other markers, especially when sputum is used as detection sample, the detection sensitivity for squamous cell carcinoma, adenocarcinoma and small cell carcinoma is all the same. 100%, which is conducive to the timely detection of patient abnormalities, and further combined with other detection methods to confirm whether it is adenocarcinoma, and greatly reduces the simplicity of clinical tumor screening, without the need to use specific tumor markers for specific types of lung cancer. The non-invasive acquisition of lung lavage fluid and sputum samples makes the combined detection of HOXA7 and HOXA9 of great application significance for the detection of various types of lung cancers such as adenocarcinoma.

上述技術方案中的一個技術方案的有益效果為：不僅可以以組織作為檢測樣本，且突出的，其在痰液和肺部灌洗液中也具有很高的靈敏度，對於某些肺癌類型，痰液和肺部灌洗液的靈敏度甚至高於組織，因此，可以簡便地以痰液和肺部灌洗液作為檢測樣本，對肺癌進行可靠的診斷。痰液和肺部灌洗液樣品獲得非常容易，而且不會對病人造成任何的痛苦和不便。使用樣本量極少，取樣過程非常方便且對病人無任何影響。同時樣品便於郵寄或者是隨身帶到醫院做檢查。The beneficial effects of one of the above technical solutions are: not only can the tissue be used as a detection sample, but also prominently, it also has high sensitivity in sputum and lung lavage fluid, and for some types of lung cancer, sputum The sensitivity of sputum and pulmonary lavage fluid is even higher than that of tissue, so sputum and pulmonary lavage fluid can be easily used as test samples for reliable diagnosis of lung cancer. Sputum and lung lavage samples are easily obtained without any pain or inconvenience to the patient. The sample volume used is very small, the sampling process is very convenient and has no impact on the patient. At the same time, the samples are easy to be mailed or taken to the hospital for examination.

上述技術方案中的一個技術方案的有益效果為：可以檢測多種類型的肺癌，且針對難檢測的腺癌，其也具有相對其他標記更高的靈敏度。The beneficial effect of one of the above technical solutions is that various types of lung cancer can be detected, and it also has higher sensitivity than other markers for difficult-to-detect adenocarcinoma.

上述技術方案中的一個技術方案的有益效果為：無需考慮檢測的對象和年齡，適用範圍廣。The beneficial effect of one of the above technical solutions is that it does not need to consider the detected object and age, and has a wide application range.

上述技術方案中的一個技術方案的有益效果為：通過甲基化水平來檢測和診斷癌症，越來越多的研究證實甲基化改變是腫瘤發生過程中的早期事件，檢測甲基化異常更易發現早期病變。One of the above technical solutions has the beneficial effects of: detecting and diagnosing cancer through methylation levels, more and more studies have confirmed that methylation changes are early events in the process of tumorigenesis, and it is easier to detect methylation abnormalities. Early detection of lesions.

本公開中的「引子」或「探針」是指一種寡核苷酸，其包含與靶核酸分子（例如靶基因）的至少6個連續核苷酸的序列互補的區域。在一些實施方案中，所述引子或探針至少一部分序列與擴增的序列不互補。在一些實施方案中，引子或探針包含與靶分子的至少9、至少10、至少11、至少12、至少13、至少14、至少15、至少16、至少17、至少18、至少19或至少20連續核苷酸的序列互補的區域。當引子或探針包含「與靶分子的至少x 個連續核苷酸互補」的區域時，所述引子或探針與靶分子的至少x 個連續或不連續的分塊核苷酸至少95%互補。在一些實施方案中，引子或探針與靶分子至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、96%、至少97%、至少98%、至少99%或100%互補。A "primer" or "probe" in the present disclosure refers to an oligonucleotide comprising a region complementary to a sequence of at least 6 contiguous nucleotides of a target nucleic acid molecule (eg, a target gene). In some embodiments, at least a portion of the primer or probe sequence is not complementary to the amplified sequence. In some embodiments, the primer or probe comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 associations with the target molecule. A region of sequence complementarity of contiguous nucleotides. When a primer or probe comprises a region "complementary to at least x contiguous nucleotides of the target molecule", the primer or probe is at least 95% at least 95% complementary to at least x contiguous or discontinuous block nucleotides of the target molecule Complementary. In some embodiments, the primer or probe is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 87%, at least 88% 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, at least 97%, at least 98%, at least 99% or 100% complementary.

本公開中的「診斷」，除了肺癌的早期診斷，還包括肺癌中期和晚期的診斷，且也包括肺癌篩選、風險評估、預後、疾病識別、病症階段的診斷和治療性靶標的選擇。"Diagnosis" in the present disclosure, in addition to the early diagnosis of lung cancer, also includes the diagnosis of intermediate and advanced lung cancer, and also includes lung cancer screening, risk assessment, prognosis, disease identification, diagnosis of disease stage and selection of therapeutic targets.

肺癌標記HOXA7和HOXA9的應用使得肺癌的早期診斷成為可能。當確定在癌症細胞中甲基化的基因在臨床上或形態學上正常表像的細胞中甲基化時，這就表明該正常表像的細胞向癌症發展。這樣，肺癌可在早期通過在正常表像的細胞中的肺癌特異性基因HOXA7和HOXA9的甲基化而診斷。The application of lung cancer markers HOXA7 and HOXA9 makes the early diagnosis of lung cancer possible. When a gene that is methylated in cancer cells is determined to be methylated in a clinically or morphologically normal-looking cell, this indicates that the normal-looking cell is progressing toward cancer. In this way, lung cancer can be diagnosed at an early stage by methylation of the lung cancer-specific genes HOXA7 and HOXA9 in normal-appearing cells.

其中，早期診斷指的是在轉移之前發現癌症的可能性，優選在可觀察到組織或者細胞的形態學變化之前。Among them, early diagnosis refers to the possibility of finding cancer before metastasis, preferably before morphological changes in tissue or cells can be observed.

作為病症階段可選的實施方式，可在肺癌在不同階段或時期的進展，通過從樣品中獲取的HOXA7和HOXA9的甲基化程度的測量進行診斷。通過比較從肺癌的每個階段的樣品中分離出的核酸的HOXA7基因和HOXA9基因甲基化程度與從沒有細胞增殖性異常的肺部組織中的樣品中分離出的一個或多個核酸的HOXA7基因和HOXA9基因甲基化程度，可檢測樣品中肺癌的具體階段。As an alternative to the stage of the condition, the progression of lung cancer at different stages or periods can be diagnosed by measuring the degree of methylation of HOXA7 and HOXA9 obtained from the sample. By comparing the degree of methylation of HOXA7 and HOXA9 genes of nucleic acids isolated from samples of each stage of lung cancer with HOXA7 of one or more nucleic acids isolated from samples of lung tissue without cellular proliferative abnormalities The degree of methylation of the gene and HOXA9 gene, which can detect the specific stage of lung cancer in the sample.

本公開中，「正常」樣本指分離自已知無所述癌症或腫瘤的個體的相同類型的樣本。In the present disclosure, a "normal" sample refers to a sample of the same type isolated from an individual known to be free of the cancer or tumor.

本公開中，所述「受試者」是哺乳動物，例如是人。In the present disclosure, the "subject" is a mammal, such as a human.

本公開甲基化檢測的樣本包括但不限於DNA，或RNA，或含mRNA的DNA和RNA樣品、或DNA-RNA雜交體。其中DNA或者 RNA可為單鏈或雙鏈。Samples for methylation detection of the present disclosure include, but are not limited to, DNA, or RNA, or mRNA-containing DNA and RNA samples, or DNA-RNA hybrids. The DNA or RNA can be single-stranded or double-stranded.

用於甲基化檢測的方法是公知的，如甲基化特異性聚合酶鏈反應、實時螢光定量甲基化特異性聚合酶鏈反應、焦磷酸測序、使用甲基化DNA特異性結合蛋白的PCR，定量PCR，以及DNA芯片、差別化甲基化檢測—甲基化敏感的限制性內切酶、差別化甲基化檢測—亞硫酸鹽測序法等等，除此之外，其他的甲基化檢測方法可以通過專利US62007687引入。Methods for methylation detection are well known, such as methylation-specific polymerase chain reaction, real-time fluorescence quantitative methylation-specific polymerase chain reaction, pyrosequencing, the use of methylated DNA-specific binding proteins PCR, quantitative PCR, and DNA chips, differential methylation detection-methylation-sensitive restriction enzymes, differential methylation detection-sulfite sequencing, etc., in addition, other The methylation detection method can be introduced by patent US62007687.

本公開中，「甲基化水平」同「甲基化程度」，通常可以表示為甲基化胞嘧啶的百分比，其為甲基化的胞嘧啶數量除以甲基化胞嘧啶的數量與未甲基化胞嘧啶數量的總和；以及目前普遍採用甲基化靶向基因數量除以參考基因數量的方法來表示甲基化水平；以及其他現有技術中甲基化水平表示方法。In the present disclosure, "methylation level" is the same as "methylation degree", which can usually be expressed as the percentage of methylated cytosines, which is the number of methylated cytosines divided by the number of methylated cytosines and the number of unmethylated cytosines. The sum of the number of methylated cytosines; and the method of dividing the number of methylation target genes by the number of reference genes is generally used to express the methylation level; and other methods of expressing the methylation level in the prior art.

以下通過具體的實施例進一步說明本公開的技術方案，具體實施例不代表對本公開保護範圍的限制。其他人根據本公開理念所做出的一些非本質的修改和調整仍屬本公開的保護範圍。The technical solutions of the present disclosure are further described below through specific embodiments, which do not represent limitations on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others based on the concepts of the present disclosure still fall within the protection scope of the present disclosure.

實施例1：檢測靶基因的選擇Example 1: Selection of detection target genes

甲基化 DNA 作為檢測靶標具有明顯的優點，相比蛋白質類標記，DNA 是可以擴增的，並且很容易檢測到 ；與突變類標記相比，DNA 甲基化的部位都位於基因的特定部位，一般在啟動子區，使檢測變得更容易和方便。為了完成本公開，發明人篩選了大量基因，選擇有代表性的 HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3 作為候選的檢測基因，β-actin基因作為參考基因，研究各個基因甲基化位點分佈情況，設計檢測的引子探針分別用於實時螢光定量甲基化特異性聚合酶鏈反應（real-time fluorescent quantitative methylation-specific PCR，qMSP）檢測。各基因檢測引子探針如下：Methylated DNA has obvious advantages as a detection target. Compared with protein markers, DNA can be amplified and easily detected. Compared with mutant markers, DNA methylation sites are located in specific parts of genes. , generally in the promoter region, making detection easier and more convenient. In order to complete the present disclosure, the inventors screened a large number of genes, selected representative HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3 as candidate detection genes, β-actin gene as a reference gene, and studied the methylation sites of each gene According to the distribution, the primer probes designed for detection were respectively used for real-time fluorescent quantitative methylation-specific PCR (qMSP) detection. The primer probes for each gene detection are as follows:

HOXA7的檢測引子和探針為： SEQ ID NO: 1  HOXA7-F2 引子F：TAAAGGCGTTTGCGATAAGAC SEQ ID NO: 2  HOXA7-R2 引子R：TAACCCGCCTAACGACTACG SEQ ID NO: 3  HOXA7-P2 探針：FAM-AGGGCGCGTTGTATGGCGC-BQ1The detection primers and probes for HOXA7 are: SEQ ID NO: 1 HOXA7-F2 Primer F: TAAAGGCGTTTGCGATAAGAC SEQ ID NO: 2 HOXA7-R2 Primer R: TAACCCGCCTAACGACTACG SEQ ID NO: 3 HOXA7-P2 probe: FAM-AGGGCGCGTTGTATGGCGC-BQ1

HOXA9的檢測引子和探針為： SEQ ID NO: 4  HOXA9-F2 引子F：TTAGTTTTTTCGGTAGGCGGC SEQ ID NO: 5  HOXA9-R2 引子R：AAACGCCAAACACCGTCG SEQ ID NO: 6  HOXA9-P2 探針：FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1The detection primers and probes for HOXA9 are: SEQ ID NO: 4 HOXA9-F2 Primer F: TTAGTTTTTTCGGTAGGCGGC SEQ ID NO: 5 HOXA9-R2 Primer R: AAACGCCAAACACCGTCG SEQ ID NO: 6 HOXA9-P2 probe: FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1

SHOX2的檢測引子和探針為： SEQ ID NO: 7  SHOX2 引子F：TTTAAAGGGTTCGTCGTTTAAGTC SEQ ID NO: 8  SHOX2 引子R：AAACGATTACTTTCGCCCG SEQ ID NO: 9  SHOX2 探針：FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1The detection primers and probes for SHOX2 are: SEQ ID NO: 7 SHOX2 Primer F: TTTAAAGGGTTCGTCGTTTAAGTC SEQ ID NO: 8 SHOX2 Primer R: AAACGATTACTTTCGCCCG SEQ ID NO: 9 SHOX2 probe: FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1

PCDHGA12的檢測引子和探針為： SEQ ID NO: 10  PCDHGA12 引子F：TTGGTTTTTACGGTTTTCGAC SEQ ID NO: 11  PCDHGA12 引子R：AAATTCTCCGAAACGCTCG SEQ ID NO: 12  PCDHGA12 探針：FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1The detection primers and probes for PCDHGA12 are: SEQ ID NO: 10 PCDHGA12 Primer F: TGGTTTTTACGGTTTTCGAC SEQ ID NO: 11 PCDHGA12 Primer R: AAATTCTCCGAAACGCTCG SEQ ID NO: 12 PCDHGA12 probe: FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1

HOXD8的檢測引子和探針為： SEQ ID NO: 13  HOXD8 引子F：TTAGTTTCGGCGCGTAGC SEQ ID NO: 14  HOXD8 引子R：CCTAAAACCGACGCGATCTA SEQ ID NO: 15  HOXD8 探針：FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1The detection primers and probes for HOXD8 are: SEQ ID NO: 13 HOXD8 Primer F: TTAGTTTCGGCGCGTAGC SEQ ID NO: 14 HOXD8 Primer R: CCTAAAACCGACGCGATCTA SEQ ID NO: 15 HOXD8 probe: FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1

GATA3的檢測引子和探針為： SEQ ID NO: 16  GATA3 引子F：TTTCGGTAGCGGGTATTGC SEQ ID NO: 17  GATA3 引子R：AAAATAACGACGAACCAACCG SEQ ID NO: 18  GATA3 探針：FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1The detection primers and probes for GATA3 are: SEQ ID NO: 16 GATA3 Primer F: TTTCGGTAGCGGGTATTGC SEQ ID NO: 17 GATA3 Primer R: AAAATAACGACGAACCAACCG SEQ ID NO: 18 GATA3 probe: FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1

β-actin的檢測引子和探針為： SEQ ID NO: 19  β-actin 引子F：TTTTGGATTGTGAATTTGTG SEQ ID NO: 20  β-actin 引子R：AAAACCTACTCCTCCCTTAAA SEQ ID NO: 21  β-actin 探針：FAM-TTGTGTGTTGGGTGGTGGTT-BQ1The detection primers and probes for β-actin are: SEQ ID NO: 19 β-actin Primer F: TTTTGGATTGTGAATTTGTG SEQ ID NO: 20 β-actin Primer R: AAAACCTACTCCTCCCTTAAA SEQ ID NO: 21 β-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1

實驗過程：experiment procedure:

1、提取DNA1. DNA extraction

收集確診肺癌患者的標本和非腫瘤患者標本，分別包括石蠟組織標本、痰液標本、灌洗液標本，樣品預處理及分離細胞，按美基生物公司試劑盒HiPure FFPE DNA Kit(D3126-03) 說明書進行DNA提取。Collect specimens from patients with confirmed lung cancer and non-tumor patients, including paraffin tissue specimens, sputum specimens, lavage fluid specimens, sample pretreatment and cell separation, according to the HiPure FFPE DNA Kit (D3126-03) of Mekey Biotech. Instructions for DNA extraction.

2、DNA 修飾2. DNA modification

以 ZYMO RESEARCH 生物公司試劑盒 EZ DNA Methylation™ KIT(D5002)說明書進行重亞硫酸氫鹽修飾。Bisulfite modification was carried out according to the instructions of ZYMO RESEARCH Bio Inc. kit EZ DNA Methylation™ KIT (D5002).

3、擴增與檢測3. Amplification and detection

配液體系： [表1] 配液體系   HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin 反應組分 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 上游引子 (100 µM) 0.125 0.125 0.05 0.125 0.05 0.125 0.125 下游引子 (100 µM) 0.125 0.125 0.125 0.125 0.125 0.125 0.125 探針(100 µM) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 鎂離子(25 mM) 5 4 5 5 5 3 5 dNTPs(10 mM) 1 1 1 1 1 1 1 Taq聚合酶(5 unit/µl) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 5×緩衝液 5 5 5 5 5 5 5 滅菌水 12.2 13.2 12.275 12.2 12.275 14.2 12.2 模板DNA 1 1 1 1 1 1 1 總體積 25 25 25 25 25 25 25 Dosing system: [Table 1] Dosing system HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin reactive components Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Upstream Primer (100 µM) 0.125 0.125 0.05 0.125 0.05 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 0.125 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Magnesium (25 mM) 5 4 5 5 5 3 5 dNTPs (10 mM) 1 1 1 1 1 1 1 Taq polymerase (5 unit/µl) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 5x buffer 5 5 5 5 5 5 5 Sterilized water 12.2 13.2 12.275 12.2 12.275 14.2 12.2 template DNA 1 1 1 1 1 1 1 total capacity 25 25 25 25 25 25 25

擴增體系： [表2] PCR反應過程   HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin   步驟 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 循環數目 預變性 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 1 擴增1 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 10 63°C 30秒 63°C 30秒 60°C 30秒 60°C 30秒 60°C 30秒 60°C 30秒 62°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 擴增2 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 45 60°C 30秒 60°C 30秒 55°C 60秒 55°C 60秒 55°C 60秒 55°C 60秒 54°C 60秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 冷卻 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 1 Amplification system: [Table 2] PCR reaction process HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin step temperature and time temperature and time temperature and time temperature and time temperature and time temperature and time temperature and time Number of cycles predenaturation 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 63°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 60°C for 30 seconds 55°C for 60 seconds 55°C for 60 seconds 55°C for 60 seconds 55°C for 60 seconds 54°C for 60 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 1

4、檢測結果4. Test results

4.1、石蠟組織中的檢測結果4.1. Detection results in paraffin tissue

樣本信息：肺組織樣本共計185例，其中正常組織樣本87例，癌組織樣本98例，98例癌症組樣本中有鱗癌15例，腺癌81例，未明確分類的肺癌2例，其中癌和癌旁對照樣本73對。Sample information: A total of 185 lung tissue samples, including 87 normal tissue samples, 98 cancer tissue samples, 15 squamous cell carcinomas, 81 adenocarcinomas, and 2 unclassified lung cancers among the 98 cancer group samples. and 73 pairs of paracancerous control samples.

HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3、以及HOXA7和HOXA9組合在所有組織標本中檢測的ROC曲線圖1所示。各基因在組織中檢測的統計結果表3所示。 [表3] 組織中的檢測結果 分析組別 指標 HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 正常組和全部癌症組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 靈敏度 63.3% 78.6% 80.6% 69.4% 40.8% 67.3% 正常組和全部鱗癌組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 靈敏度 60% 93.3% 93.3% 53.3% 60.0% 73.3% 正常組和全部腺癌組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 靈敏度 63.0% 75.3% 79.0% 71.6% 38.3% 65.4% [續表3] 組織中的檢測結果 分析組別 指標 HOXA7&HOXA9 HOXA7&HOXD8 HOXA9&HOXD8 正常組和全部癌症組比較 特異性 89.7% 88.5% 88.5% 靈敏度 84.7% 70.4% 78.6% 正常組和全部鱗癌組比較 特異性 89.7% 88.5% 88.5% 靈敏度 93.3% 73.3% 93.3% 正常組和全部腺癌組比較 特異性 89.7% 88.5% 88.5% 靈敏度 82.7% 69.1% 75.3% The ROC curves of HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3, and the combination of HOXA7 and HOXA9 detected in all tissue samples are shown in Figure 1. The statistical results of the detection of each gene in the tissue are shown in Table 3. [Table 3] Detection results in tissues Analysis group index HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% Sensitivity 63.3% 78.6% 80.6% 69.4% 40.8% 67.3% Comparison of normal group and all squamous cell carcinoma groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% Sensitivity 60% 93.3% 93.3% 53.3% 60.0% 73.3% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% Sensitivity 63.0% 75.3% 79.0% 71.6% 38.3% 65.4% [Continued from Table 3] Test results in tissues Analysis group index HOXA7&HOXA9 HOXA7&HOXD8 HOXA9&HOXD8 Comparison of normal group and all cancer groups specificity 89.7% 88.5% 88.5% Sensitivity 84.7% 70.4% 78.6% Comparison of normal group and all squamous cell carcinoma groups specificity 89.7% 88.5% 88.5% Sensitivity 93.3% 73.3% 93.3% Comparison of normal group and all adenocarcinoma group specificity 89.7% 88.5% 88.5% Sensitivity 82.7% 69.1% 75.3%

從以上結果可以看出，在組織樣本中，無論是將肺癌作為一個整體進行比較分析，還是按照肺癌的亞型進行比較分析，SHOX2和HOXA9的檢測效果最好，HOXA7、PCDHGA12和GATA3的檢測效果次之，HOXD8的檢測效果最差，靈敏度只達到40.8%。It can be seen from the above results that in the tissue samples, whether the lung cancer as a whole is compared or analyzed according to the subtype of lung cancer, SHOX2 and HOXA9 have the best detection effect, and HOXA7, PCDHGA12 and GATA3 have the best detection effect. Next, HOXD8 has the worst detection effect, with a sensitivity of only 40.8%.

儘管聯合檢測已經在現有技術中有所應用，然而，並非聯合檢測就具有協同性，如Hubers等人（DNA hypermethylation analysis in sputum forthe diagnosis of lung cancer: training validation set approach）研究RASSF1A、3OST2及PHACTR3單獨在痰液中檢測肺癌的靈敏度分別為42.5%、31.5%和28.8%，三者聯合檢測靈敏度只能達到67.1%，特異性由96.5%降至89.5%，結果表明肺癌標記的組合檢測，效果並沒有很協同性的增強。同樣地，本公開中，同為HOXA家族的HOXA7、HOXA9、HOXD8三者兩兩組合，僅HOXA7&HOXA9的組合，無論是在全部癌症中，還是在鱗癌中或者是較難檢測的肺癌中，具有比其他組合更高的靈敏度，而其他HOXA家族的聯合則無顯著增強效果。Although combined detection has been used in the existing technology, however, not combined detection is synergistic. For example, Hubers et al. (DNA hypermethylation analysis in sputum for the diagnosis of lung cancer: training validation set approach) studied RASSF1A, 3OST2 and PHACTR3 alone The sensitivity of detecting lung cancer in sputum was 42.5%, 31.5% and 28.8% respectively. The combined detection sensitivity of the three could only reach 67.1%, and the specificity decreased from 96.5% to 89.5%. The results show that the combined detection of lung cancer markers has the same effect There are no very synergistic enhancements. Similarly, in the present disclosure, HOXA7, HOXA9, and HOXD8, which are both HOXA families, are combined in pairs, and only the combination of HOXA7 & HOXA9, whether in all cancers, in squamous cell carcinomas, or in difficult-to-detect lung cancers, has Higher sensitivity than other combinations, while other HOXA family combinations have no significant enhancement effect.

根據以上結果，為了研究痰液中的不同基因的檢測情況，發明人對HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8和GATA3這6個標記在痰液中進行進一步的篩選。Based on the above results, in order to study the detection of different genes in sputum, the inventors further screened the 6 markers HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3 in sputum.

實施例2：HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8和GATA3、以及HOXA7和HOXA9組合在痰液中的檢測Example 2: Detection of HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3, and the combination of HOXA7 and HOXA9 in sputum

樣本信息：測試痰樣本共計90例，其中正常對照組樣本55例，癌症組對照樣本35例，35例癌症組樣本中有鱗癌12例，小細胞癌6例，腺癌9例，大細胞癌2例，未明確分類的肺癌6例。Sample information: A total of 90 sputum samples were tested, including 55 normal control samples, 35 cancer control samples, 12 squamous cell carcinomas, 6 small cell carcinomas, 9 adenocarcinomas, and large cell carcinomas among the 35 cancer samples. There were 2 cases of cancer and 6 cases of unclassified lung cancer.

試驗過程：Experimental procedure:

a. 收集確診為肺癌患者和非肺癌患者的痰液標本，使用DTT解稠後，離心取沉澱分離細胞，使用PBS洗滌2遍，然後使用美基生物公司（Magen）的DNA提取試劑盒（HiPure FFPE DNA Kit，D3126-03）提取DNA。a. Collect sputum samples from patients diagnosed with lung cancer and patients with non-lung cancer. After thickening with DTT, centrifuge to separate cells, wash twice with PBS, and then use Magen DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.

b. 使用ZYMO RESEARCH生物公司的DNA轉化試劑盒（EZ DNA Methylation Kit，D5002）進行DNA的亞硫酸氫鹽修飾。b. Use the DNA transformation kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH to carry out the bisulfite modification of DNA.

c. 配液體系如下： [表4] 配液體系   HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin 反應組分 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 上游引子 (100 µM) 0.125 0.125 0.05 0.125 0.05 0.125 0.125 下游引子 (100 µM) 0.125 0.125 0.125 0.125 0.125 0.125 0.125 探針(100 µM) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 鎂離子(25 mM) 5 4 5 5 5 3 5 dNTPs(10 mM) 1 1 1 1 1 1 1 Taq聚合酶(5 unit/µl) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 5×緩衝液 5 5 5 5 5 5 5 滅菌水 12.2 13.2 12.275 12.2 12.275 14.2 12.2 模板DNA 1 1 1 1 1 1 1 總體積 25 25 25 25 25 25 25 c. The liquid distribution system is as follows: [Table 4] Liquid distribution system HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin reactive components Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Upstream Primer (100 µM) 0.125 0.125 0.05 0.125 0.05 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 0.125 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 Magnesium (25 mM) 5 4 5 5 5 3 5 dNTPs (10 mM) 1 1 1 1 1 1 1 Taq polymerase (5 unit/µl) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 5x buffer 5 5 5 5 5 5 5 Sterilized water 12.2 13.2 12.275 12.2 12.275 14.2 12.2 template DNA 1 1 1 1 1 1 1 total capacity 25 25 25 25 25 25 25

擴增體系如下： [表5] 擴增體系   PCR反應過程   HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin   步驟 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 溫度 和時間 循環數目 預變性 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 1 擴增1 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 10 63°C 30秒 63°C 30秒 60°C 30秒 60°C 30秒 60°C 30秒 60°C 30秒 62°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 擴增2 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 95°C 20秒 45 60°C 30秒 60°C 30秒 55°C 60秒 55°C 60秒 55°C 60秒 55°C 60秒 54°C 60秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 72°C 30秒 冷卻 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 40°C 30秒 1 The amplification system is as follows: [Table 5] Amplification system PCR reaction process HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin step temperature and time temperature and time temperature and time temperature and time temperature and time temperature and time temperature and time Number of cycles predenaturation 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 63°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 60°C for 30 seconds 55°C for 60 seconds 55°C for 60 seconds 55°C for 60 seconds 55°C for 60 seconds 54°C for 60 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 1

檢測結果如下： [表6] 痰液中的檢測結果 分析組別 指標 HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 HOXA7&HOXA9組合 正常組和全部癌症組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% 靈敏度 88.6% 74.3% 51.4% 20.0% 42.9% 17.1% 97.1% 正常組和全部鱗癌組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% 靈敏度 100.0% 75.0% 66.7% 25.0% 66.7% 25.0% 100% 正常組和全部腺癌組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% 靈敏度 88.9% 55.6% 11.1% 33.3% 11.1% 0.0% 100% 正常組和全部小細胞癌組比較 特異性 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% 靈敏度 83.3% 100% 83.3% 0.0% 50.0% 33.3% 100% 續表6痰液中的檢測結果 分析組別 指標 HOXA7&HOXA9組合 HOXA7&HOXD8 HOXA9&HOXD8 正常組和全部癌症組比較 特異性 90.9% 90.9% 90.9% 靈敏度 97.1% 91.4% 80.0% 正常組和全部鱗癌組比較 特異性 90.9% 90.9% 90.9% 靈敏度 100% 100% 91.7% 正常組和全部腺癌組比較 特異性 90.9% 90.9% 90.9% 靈敏度 100% 88.9% 55.6% 正常組和全部小細胞癌組比較 特異性 90.9% 90.9% 90.9% 靈敏度 100% 83.3% 100% The test results are as follows: [Table 6] Test results in sputum Analysis group index HOXA7 HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 HOXA7&HOXA9 combination Comparison of normal group and all cancer groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% Sensitivity 88.6% 74.3% 51.4% 20.0% 42.9% 17.1% 97.1% Comparison of normal group and all squamous cell carcinoma groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% Sensitivity 100.0% 75.0% 66.7% 25.0% 66.7% 25.0% 100% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% Sensitivity 88.9% 55.6% 11.1% 33.3% 11.1% 0.0% 100% Comparison between normal group and all small cell carcinoma groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% 95.0% 90.9% Sensitivity 83.3% 100% 83.3% 0.0% 50.0% 33.3% 100% Continued table 6 detection results in sputum Analysis group index HOXA7&HOXA9 combination HOXA7&HOXD8 HOXA9&HOXD8 Comparison of normal group and all cancer groups specificity 90.9% 90.9% 90.9% Sensitivity 97.1% 91.4% 80.0% Comparison of normal group and all squamous cell carcinoma groups specificity 90.9% 90.9% 90.9% Sensitivity 100% 100% 91.7% Comparison of normal group and all adenocarcinoma group specificity 90.9% 90.9% 90.9% Sensitivity 100% 88.9% 55.6% Comparison between normal group and all small cell carcinoma groups specificity 90.9% 90.9% 90.9% Sensitivity 100% 83.3% 100%

f. HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3以及HOXA7和HOXA9組合在所有痰液標本中檢測的ROC曲線見圖2，統計結果見表6，從以上結果可以看出，在痰液樣本中，同時檢測這6個基因進行比較，無論是將肺癌作為一個整體進行比較分析，還是按照肺癌的亞型進行比較分析，HOXA7和HOXA9的聯合檢測效果都要優於其他4個基因或者HOXA7和HOXA9分別單獨檢測的效果；同時也優於HOXA7&HOXD8和HOXA9&HOXD8的組合。f. The ROC curves of HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3 and the combination of HOXA7 and HOXA9 detected in all sputum samples are shown in Figure 2, and the statistical results are shown in Table 6. It can be seen from the above results that in the sputum samples , Detecting these 6 genes at the same time for comparison, whether the lung cancer as a whole is compared or analyzed according to the subtype of lung cancer, the combined detection effect of HOXA7 and HOXA9 is better than the other 4 genes or HOXA7 and HOXA9 The effect of detection separately; also better than the combination of HOXA7&HOXD8 and HOXA9&HOXD8.

特別是對腺癌的檢測效果，HOXA7和HOXA9的組合相比其他基因的檢出率更加好，敏感度甚至達到100%，如此高的靈敏度基本上可實現對腺癌早期篩查的0漏檢率，從而使患者可以得到準確的風險提示，及早治療，降低腺癌的死亡率。而現有的肺癌中的腫瘤標記其對腺癌的敏感性僅為14.3%，而HOXA7或HOXA9單獨檢測時，對腺癌的敏感性也僅有88.9%，二者聯合檢測時，對肺癌的靈敏度提升到100%，二者具有協同性。Especially for the detection of adenocarcinoma, the combination of HOXA7 and HOXA9 has a better detection rate than other genes, and the sensitivity even reaches 100%. Such a high sensitivity can basically achieve 0 missed detection of early screening of adenocarcinoma Therefore, patients can get accurate risk prompts, early treatment, and reduce the mortality of adenocarcinoma. The sensitivity of the existing tumor markers in lung cancer to adenocarcinoma is only 14.3%, while the sensitivity of HOXA7 or HOXA9 to adenocarcinoma is only 88.9% when detected alone. Raised to 100%, the two are synergistic.

比較各基因的檢測結果，發現最優的兩個基因HOXA7和HOXA9對提高體系檢測陽性率具有較明顯的協同作用，具體表現為明顯提高了腺癌的檢出率。腺癌一般為周圍型，由於支氣管的樹狀生理結構，肺深部的脫落細胞更加難以通過痰液咳出，因此對這部分的檢測更加困難和有意義。Comparing the detection results of each gene, it was found that the two optimal genes, HOXA7 and HOXA9, had obvious synergistic effects on improving the detection rate of the system, which was manifested as significantly improving the detection rate of adenocarcinoma. Adenocarcinomas are generally of peripheral type. Due to the dendritic physiological structure of the bronchi, the exfoliated cells in the deep part of the lung are more difficult to expectorate through sputum, so the detection of this part is more difficult and meaningful.

實施例3：HOXA7、HOXA9以及SHOX2基因在痰液中的檢測Example 3: Detection of HOXA7, HOXA9 and SHOX2 genes in sputum

大量文獻顯示SHOX2可用作檢測肺癌的標記，並且有專利顯示[CN201510203539診斷人SHOX2基因和人RASSF1A基因甲基化的方法和試劑盒-申請公開]，SHOX2在肺泡灌洗液、病變部位組織、胸水、痰液等樣本中具有較高的檢出率。為了驗證HOXA7和HOXA9的檢測效果，發明人同時檢測HOXA7、HOXA9 SHOX2_n3基因。在該實施例中，SHOX2基因的檢出效率採用專利CN201510203539中公開的引子和探針序列，並將SHOX2基因表述為SHOX2_n3，以區別於本公開實施例1和2中採用自行設計的引子和探針檢測的SHOX2基因。A large number of literatures show that SHOX2 can be used as a marker for detecting lung cancer, and there are patents showing that [CN201510203539 Method and kit for diagnosing methylation of human SHOX2 gene and human RASSF1A gene-application publication], SHOX2 is in bronchoalveolar lavage fluid, lesion tissue, It has a high detection rate in pleural effusion, sputum and other samples. In order to verify the detection effect of HOXA7 and HOXA9, the inventors simultaneously detected HOXA7 and HOXA9 SHOX2_n3 genes. In this example, the detection efficiency of the SHOX2 gene adopts the primer and probe sequences disclosed in the patent CN201510203539, and the SHOX2 gene is expressed as SHOX2_n3, in order to distinguish it from the self-designed primers and probes in Examples 1 and 2 of the present disclosure. Needle detection of SHOX2 gene.

的檢出結果。detection result.

各基因檢測引子探針如下：The primer probes for each gene detection are as follows:

HOXA7的檢測引子和探針為： SEQ ID NO: 1  HOXA7-F2 引子F：TAAAGGCGTTTGCGATAAGAC SEQ ID NO: 2  HOXA7-R2 引子R：TAACCCGCCTAACGACTACG SEQ ID NO: 3  HOXA7-P2 探針：FAM-AGGGCGCGTTGTATGGCGC-BQ1The detection primers and probes for HOXA7 are: SEQ ID NO: 1 HOXA7-F2 Primer F: TAAAGGCGTTTGCGATAAGAC SEQ ID NO: 2 HOXA7-R2 Primer R: TAACCCGCCTAACGACTACG SEQ ID NO: 3 HOXA7-P2 probe: FAM-AGGGCGCGTTGTATGGCGC-BQ1

HOXA9的檢測引子和探針為： SEQ ID NO: 4  HOXA9-F2 引子F：TTAGTTTTTTCGGTAGGCGGC SEQ ID NO: 5  HOXA9-R2 引子R：AAACGCCAAACACCGTCG SEQ ID NO: 6  HOXA9-P2 探針：FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1The detection primers and probes for HOXA9 are: SEQ ID NO: 4 HOXA9-F2 Primer F: TTAGTTTTTTCGGTAGGCGGC SEQ ID NO: 5 HOXA9-R2 Primer R: AAACGCCAAACACCGTCG SEQ ID NO: 6 HOXA9-P2 probe: FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1

SHOX2_n3的檢測引子和探針為： SEQ ID NO: 22  SHOX2_n3 引子F：TTTGGATAGTTAGGTAATTTTCG SEQ ID NO: 23  SHOX2_n3 引子R：CGTACACGCCTATACTCGTACG SEQ ID NO: 24  SHOX2_n3.2 探針：FAM-CCCCGATCGAACAAACGAAAC-BQ1The detection primers and probes for SHOX2_n3 are: SEQ ID NO: 22 SHOX2_n3 Primer F: TTTGGATAGTTAGGTAATTTTCG SEQ ID NO: 23 SHOX2_n3 Primer R: CGTACACGCCTATACTCGTACG SEQ ID NO: 24 SHOX2_n3.2 probe: FAM-CCCCGATCGAACAAACGAAAC-BQ1

a. 配液體系如下： [表7] 配液體系   HOXA7 HOXA9 SHOX2_n3 β-actin 反應組分 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 上游引子 (100 µM) 0.125 0.125 0.125 0.125 下游引子 (100 µM) 0.125 0.125 0.125 0.125 探針(100 µM) 0.05 0.05 0.05 0.05 鎂離子(25 mM) 5 4 5 5 dNTPs(10 mM) 1 1 1 1 Taq聚合酶(5 unit/µl) 0.5 0.5 0.5 0.5 5×緩衝液 5 5 5 5 滅菌水 12.2 13.2 12.2 12.2 模板DNA 1 1 1 1 總體積 25 25 25 25 a. The dosing system is as follows: [Table 7] Dosing system HOXA7 HOXA9 SHOX2_n3 β-actin reactive components Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Upstream Primer (100 µM) 0.125 0.125 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 0.05 Magnesium (25 mM) 5 4 5 5 dNTPs (10 mM) 1 1 1 1 Taq polymerase (5 unit/µl) 0.5 0.5 0.5 0.5 5x buffer 5 5 5 5 Sterilized water 12.2 13.2 12.2 12.2 template DNA 1 1 1 1 total capacity 25 25 25 25

b. 擴增體系如下： [表8] 擴增體系   HOXA7 HOXA9 β-actin   步驟 溫度和時間 溫度和時間 溫度和時間 循環數目 預變性 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 1 擴增1 95°C 20秒 95°C 20秒 95°C 20秒 10 63°C 30秒 63°C 30秒 62°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 擴增2 95°C 20秒 95°C 20秒 95°C 20秒 45 60°C 30秒 60°C 30秒 54°C 60秒 72°C 30秒 72°C 30秒 72°C 30秒 冷卻 40°C 30秒 40°C 30秒 40°C 30秒 1 [表9] SHOX2_n3反應程序 步驟 溫度和時間 循環數目 預變性 95°C 5分鐘 1 擴增1 95°C 20秒 45 60°C 30秒 72°C 30秒 冷卻 40°C 30秒 1 b. The amplification system is as follows: [Table 8] Amplification system HOXA7 HOXA9 β-actin step temperature and time temperature and time temperature and time Number of cycles predenaturation 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 63°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 60°C for 30 seconds 54°C for 60 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 1 [Table 9] SHOX2_n3 reaction program step temperature and time Number of cycles predenaturation 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 45 60°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 1

c. 檢測結果如下：c. The test results are as follows:

利用標準曲線計算各基因在標本中的甲基化拷貝數，採用比值=靶向基因拷貝數/ACTB拷貝數*100來進行判斷兩組樣本的甲基化程度，最後選取HOXA7的閾值為0.77，HOXA9的閾值為2.3，SHOX2_n3的閾值為1.3，作為判斷癌症組和對照組的標準，換算後的比值超過設定閾值可判斷為陽性「+」，等於或小於設定閾值可判斷為陰性「−」。根據此標準，90例痰液標本的檢測結果如下： [表10] 檢測結果     HOXA7 HOXA9 SHOX2_n3 HOXA7 HOXA9 SHOX2_n3 HOXA7& HOXA9組合 序號 樣本類型 甲基化率 甲基化率 甲基化率 檢出情況 檢出情況 檢出情況 檢出情況 1 非肺癌對照 0.0 0.1 0.2 − − − − 2 非肺癌對照 0.0 0.5 0.0 − − − − 3 非肺癌對照 0.0 0.5 0.0 − − − − 4 非肺癌對照 0.7 0.8 0.2 − − − − 5 非肺癌對照 0.0 0.5 0.1 − − − − 6 非肺癌對照 0.0 0.6 0.3 − − − − 7 非肺癌對照 0.6 1.6 0.2 − − − − 8 非肺癌對照 0.4 0.6 0.3 − − − − 9 非肺癌對照 0.3 0.3 0.0 − − − − 10 非肺癌對照 0.4 7.1 4.0 − + + + 11 非肺癌對照 0.6 0.9 0.4 − − − − 12 非肺癌對照 0.6 0.0 0.2 − − − − 13 非肺癌對照 0.0 0.3 0.0 − − − − 14 非肺癌對照 0.2 0.4 0.1 − − − − 15 非肺癌對照 0.0 0.0 0.0 − − − − 16 非肺癌對照 0.6 0.3 0.3 − − − − 17 非肺癌對照 0.0 0.4 0.0 − − − − 18 非肺癌對照 0.0 0.4 0.0 − − − − 19 非肺癌對照 0.0 0.0 0.0 − − − − 20 非肺癌對照 0.8 2.3 1.1 + − − + 21 非肺癌對照 0.5 1.3 0.4 − − − − 22 非肺癌對照 0.7 0.8 2.0 − − + − 23 非肺癌對照 0.76 0.3 0.1 − − − − 24 非肺癌對照 0.0 0.5 0.0 − − − − 25 非肺癌對照 0.1 1.2 0.5 − − − − 26 非肺癌對照 0.4 0.5 0.3 − − − − 27 非肺癌對照 0.2 0.2 0.0 − − − − 28 非肺癌對照 0.0 0.4 0.0 − − − − 29 非肺癌對照 0.0 0.3 0.0 − − − − 30 非肺癌對照 0.0 0.0 0.0 − − − − 31 非肺癌對照 0.0 0.0 0.4 − − − − 32 非肺癌對照 0.0 0.0 0.0 − − − − 33 非肺癌對照 0.0 0.3 0.5 − − − − 34 非肺癌對照 0.4 0.0 0.7 − − − − 35 非肺癌對照 0.7 0.2 0.3 − − − − 36 非肺癌對照 0.4 0.7 0.8 − − − − 37 非肺癌對照 0.0 0.0 0.1 − − − − 38 非肺癌對照 2.2 0.8 0.6 + − − + 39 非肺癌對照 0.3 0.2 0.3 − − − − 40 非肺癌對照 0.0 0.0 0.2 − − − − 41 非肺癌對照 0.6 3.7 1.7 − + + + 42 非肺癌對照 0.0 0.4 0.0 − − − − 43 非肺癌對照 0.0 0.6 0.6 − − − − 44 非肺癌對照 0.1 0.0 1.1 − − − − 45 非肺癌對照 0.2 0.0 0.2 − − − − 46 非肺癌對照 0.2 0.1 0.2 − − − − 47 非肺癌對照 0.0 0.0 0.2 − − − − 48 非肺癌對照 0.3 0.1 0.0 − − − − 49 非肺癌對照 0.6 0.1 0.4 − − − − 50 非肺癌對照 0.0 0.1 0.2 − − − − 51 非肺癌對照 0.2 0.0 0.4 − − − − 52 非肺癌對照 0.0 0.0 0.2 − − − − 53 非肺癌對照 0.5 0.5 0.6 − − − − 54 非肺癌對照 0.0 0.0 0.0 − − − − 55 非肺癌對照 3.1 2.3 0.7 + + − + 56 鱗癌 2.1 4.9 3.5 + + + + 57 鱗癌 14.6 46.5 22.7 + + + + 58 鱗癌 14.7 27.4 8.3 + + + + 59 鱗癌 29.2 73.8 23.6 + + + + 60 鱗癌 2.1 2.2 9.5 + − + + 61 鱗癌 3.2 4.7 1.2 + + − + 62 鱗癌 12.1 7.2 7.1 + + + + 63 鱗癌 8.3 98.0 96.8 + + + + 64 鱗癌 1.4 2.1 0.9 + − − + 65 鱗癌 25.9 22.3 23.0 + + + + 66 鱗癌 4.1 66.7 94.2 + + + + 67 鱗癌 1.3 0.9 0.0 + − − + 68 腺癌 4.6 2.5 2.9 + + + + 69 腺癌 0.9 1.0 0.2 + − − + 70 腺癌 2.6 0.7 0.0 + − − + 71 腺癌 2.5 1.7 1.6 + − + + 72 腺癌 0.9 6.0 0.0 + + − + 73 腺癌 0.0 2.8 0.4 − + − + 74 腺癌 4.4 3.5 2.7 + + + + 75 腺癌 6.0 0.8 0.1 + − − + 76 腺癌 3.3 2.5 0.0 + + − + 77 小細胞癌 10.0 29.3 17.3 + + + + 78 小細胞癌 21.7 34.1 16.8 + + + + 79 小細胞癌 6.5 29.9 20.4 + + + + 80 小細胞癌 10.1 14.8 16.5 + + + + 81 小細胞癌 37.3 21.3 40.1 + + + + 82 小細胞癌 0.2 5.9 0.8 − + − + 83 大細胞癌 4.4 9.9 7.7 + + + + 84 大細胞癌 1.0 1.1 0.1 + − − + 85 低分化癌 0.3 0.3 0.5 − − − − 86 低分化癌 0.2 9.0 11.6 − + + + 87 肺癌 15.7 29.9 6.3 + + + + 88 肺癌 46.9 49.4 88.7 + + + + 89 肺癌 77.0 48.6 48.6 + + + + 90 肺癌 2.3 4.4 1.1 + + − + The standard curve was used to calculate the methylated copy number of each gene in the sample, and the ratio=target gene copy number/ACTB copy number*100 was used to judge the methylation degree of the two groups of samples. Finally, the threshold of HOXA7 was selected as 0.77. The threshold of HOXA9 is 2.3, and the threshold of SHOX2_n3 is 1.3. As the standard for judging the cancer group and the control group, the converted ratio can be judged as positive “+” when the ratio exceeds the set threshold, and it can be judged as negative “−” when it is equal to or less than the set threshold. According to this standard, the test results of 90 sputum samples are as follows: [Table 10] Test results HOXA7 HOXA9 SHOX2_n3 HOXA7 HOXA9 SHOX2_n3 HOXA7 & HOXA9 combination serial number sample type methylation rate methylation rate methylation rate detection detection detection detection 1 Non-lung cancer control 0.0 0.1 0.2 − − − − 2 Non-lung cancer control 0.0 0.5 0.0 − − − − 3 Non-lung cancer control 0.0 0.5 0.0 − − − − 4 Non-lung cancer control 0.7 0.8 0.2 − − − − 5 Non-lung cancer control 0.0 0.5 0.1 − − − − 6 Non-lung cancer control 0.0 0.6 0.3 − − − − 7 Non-lung cancer control 0.6 1.6 0.2 − − − − 8 Non-lung cancer control 0.4 0.6 0.3 − − − − 9 Non-lung cancer control 0.3 0.3 0.0 − − − − 10 Non-lung cancer control 0.4 7.1 4.0 − + + + 11 Non-lung cancer control 0.6 0.9 0.4 − − − − 12 Non-lung cancer control 0.6 0.0 0.2 − − − − 13 Non-lung cancer control 0.0 0.3 0.0 − − − − 14 Non-lung cancer control 0.2 0.4 0.1 − − − − 15 Non-lung cancer control 0.0 0.0 0.0 − − − − 16 Non-lung cancer control 0.6 0.3 0.3 − − − − 17 Non-lung cancer control 0.0 0.4 0.0 − − − − 18 Non-lung cancer control 0.0 0.4 0.0 − − − − 19 Non-lung cancer control 0.0 0.0 0.0 − − − − 20 Non-lung cancer control 0.8 2.3 1.1 + − − + twenty one Non-lung cancer control 0.5 1.3 0.4 − − − − twenty two Non-lung cancer control 0.7 0.8 2.0 − − + − twenty three Non-lung cancer control 0.76 0.3 0.1 − − − − twenty four Non-lung cancer control 0.0 0.5 0.0 − − − − 25 Non-lung cancer control 0.1 1.2 0.5 − − − − 26 Non-lung cancer control 0.4 0.5 0.3 − − − − 27 Non-lung cancer control 0.2 0.2 0.0 − − − − 28 Non-lung cancer control 0.0 0.4 0.0 − − − − 29 Non-lung cancer control 0.0 0.3 0.0 − − − − 30 Non-lung cancer control 0.0 0.0 0.0 − − − − 31 Non-lung cancer control 0.0 0.0 0.4 − − − − 32 Non-lung cancer control 0.0 0.0 0.0 − − − − 33 Non-lung cancer control 0.0 0.3 0.5 − − − − 34 Non-lung cancer control 0.4 0.0 0.7 − − − − 35 Non-lung cancer control 0.7 0.2 0.3 − − − − 36 Non-lung cancer control 0.4 0.7 0.8 − − − − 37 Non-lung cancer control 0.0 0.0 0.1 − − − − 38 Non-lung cancer control 2.2 0.8 0.6 + − − + 39 Non-lung cancer control 0.3 0.2 0.3 − − − − 40 Non-lung cancer control 0.0 0.0 0.2 − − − − 41 Non-lung cancer control 0.6 3.7 1.7 − + + + 42 Non-lung cancer control 0.0 0.4 0.0 − − − − 43 Non-lung cancer control 0.0 0.6 0.6 − − − − 44 Non-lung cancer control 0.1 0.0 1.1 − − − − 45 Non-lung cancer control 0.2 0.0 0.2 − − − − 46 Non-lung cancer control 0.2 0.1 0.2 − − − − 47 Non-lung cancer control 0.0 0.0 0.2 − − − − 48 Non-lung cancer control 0.3 0.1 0.0 − − − − 49 Non-lung cancer control 0.6 0.1 0.4 − − − − 50 Non-lung cancer control 0.0 0.1 0.2 − − − − 51 Non-lung cancer control 0.2 0.0 0.4 − − − − 52 Non-lung cancer control 0.0 0.0 0.2 − − − − 53 Non-lung cancer control 0.5 0.5 0.6 − − − − 54 non-lung cancer control 0.0 0.0 0.0 − − − − 55 Non-lung cancer control 3.1 2.3 0.7 + + − + 56 squamous cell carcinoma 2.1 4.9 3.5 + + + + 57 squamous cell carcinoma 14.6 46.5 22.7 + + + + 58 squamous cell carcinoma 14.7 27.4 8.3 + + + + 59 squamous cell carcinoma 29.2 73.8 23.6 + + + + 60 squamous cell carcinoma 2.1 2.2 9.5 + − + + 61 squamous cell carcinoma 3.2 4.7 1.2 + + − + 62 squamous cell carcinoma 12.1 7.2 7.1 + + + + 63 squamous cell carcinoma 8.3 98.0 96.8 + + + + 64 squamous cell carcinoma 1.4 2.1 0.9 + − − + 65 squamous cell carcinoma 25.9 22.3 23.0 + + + + 66 squamous cell carcinoma 4.1 66.7 94.2 + + + + 67 squamous cell carcinoma 1.3 0.9 0.0 + − − + 68 adenocarcinoma 4.6 2.5 2.9 + + + + 69 adenocarcinoma 0.9 1.0 0.2 + − − + 70 adenocarcinoma 2.6 0.7 0.0 + − − + 71 adenocarcinoma 2.5 1.7 1.6 + − + + 72 adenocarcinoma 0.9 6.0 0.0 + + − + 73 adenocarcinoma 0.0 2.8 0.4 − + − + 74 adenocarcinoma 4.4 3.5 2.7 + + + + 75 adenocarcinoma 6.0 0.8 0.1 + − − + 76 adenocarcinoma 3.3 2.5 0.0 + + − + 77 small cell carcinoma 10.0 29.3 17.3 + + + + 78 small cell carcinoma 21.7 34.1 16.8 + + + + 79 small cell carcinoma 6.5 29.9 20.4 + + + + 80 small cell carcinoma 10.1 14.8 16.5 + + + + 81 small cell carcinoma 37.3 21.3 40.1 + + + + 82 small cell carcinoma 0.2 5.9 0.8 − + − + 83 large cell carcinoma 4.4 9.9 7.7 + + + + 84 large cell carcinoma 1.0 1.1 0.1 + − − + 85 poorly differentiated carcinoma 0.3 0.3 0.5 − − − − 86 poorly differentiated carcinoma 0.2 9.0 11.6 − + + + 87 lung cancer 15.7 29.9 6.3 + + + + 88 lung cancer 46.9 49.4 88.7 + + + + 89 lung cancer 77.0 48.6 48.6 + + + + 90 lung cancer 2.3 4.4 1.1 + + − +

注意：「+」為甲基化DNA檢測陽性，「−」為甲基化DNA檢測陰性；在協同檢測標本時，兩者為陰性則判為陰性，一陰一陽或兩者均為陽性則判為陽性。Note: "+" means positive for methylated DNA test, and "-" means negative for methylated DNA test; in the case of co-testing specimens, if both are negative, it will be judged as negative, and if one yin and one yang or both are positive, it will judge is positive.

d. 結果分析 [表11] 統計結果 分析組別 指標 HOXA7 HOXA9 HOXA7&HOXA9組合 SHOX2_n3 正常組和全部癌症組比較 特異性 95.0% 95.0% 90.9% 95.0% 靈敏度 88.6% 74.3% 97.1% 62.9% 正常組和全部鱗癌組比較 特異性 95.0% 95.0% 90.9% 95.0% 靈敏度 100% 75.0% 100% 75.0% 正常組和全部腺癌組比較 特異性 95.0% 95.0% 90.9% 95.0% 靈敏度 88.9% 55.6% 100% 33.3% 正常組和全部小細胞癌組比較 特異性 95.0% 95.0% 90.9% 95.0% 靈敏度 83.3% 100% 100% 83.3% d. Analysis of results [Table 11] Statistical results Analysis group index HOXA7 HOXA9 HOXA7&HOXA9 combination SHOX2_n3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% 90.9% 95.0% Sensitivity 88.6% 74.3% 97.1% 62.9% Comparison of normal group and all squamous cell carcinoma groups specificity 95.0% 95.0% 90.9% 95.0% Sensitivity 100% 75.0% 100% 75.0% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% 90.9% 95.0% Sensitivity 88.9% 55.6% 100% 33.3% Comparison between normal group and all small cell carcinoma groups specificity 95.0% 95.0% 90.9% 95.0% Sensitivity 83.3% 100% 100% 83.3%

e. HOXA7與HOXA9組合與SHOX2_n3在所有痰液標本中檢測的ROC曲線見圖3，HOXA7、HOXA9與SHOX2_n3在痰液標本中的擴增曲線見圖4，統計結果見表11，從以上結果可以看出，在痰液樣本中，將肺癌作為一個整體進行比較分析，還是按照肺癌的亞型進行比較分析，HOXA7和HOXA9的檢測效果都要優於SHOX2基因的檢測效果，通過HOXA7和HOXA9組合檢測樣本，可以有效提高陽性檢出率，在所有樣本中，陽性檢出率為97.1%，其中對於鱗癌、腺癌和小細胞癌，檢測靈敏度均達到了100%。在多種類型的肺癌中，均能達到如此高的靈敏度，當用於臨床檢測時，無疑具有更大的通用性。特別是對腺癌的檢測效果，HOXA7和HOXA9聯合檢出腺癌的靈敏性高達100%，而現有的針對SHOX2檢測的引子和探針對腺癌的敏感度僅為33.3%。腺癌一般為周圍型，由於支氣管的樹狀生理結構，肺深部的脫落細胞更加難以通過痰液咳出，而本公開首次發現了一種能以痰液作為樣本檢測腺癌，並可將靈敏度大幅提升至100%的標記，這一突破對腺癌的檢測具有重要意義。e. The ROC curves of the combination of HOXA7 and HOXA9 and SHOX2_n3 in all sputum samples are shown in Figure 3, and the amplification curves of HOXA7, HOXA9 and SHOX2_n3 in sputum samples are shown in Figure 4, and the statistical results are shown in Table 11. It can be seen that in the sputum samples, the comparison and analysis of lung cancer as a whole or according to the subtype of lung cancer, the detection effect of HOXA7 and HOXA9 is better than the detection effect of SHOX2 gene, and the combined detection of HOXA7 and HOXA9 In all samples, the positive detection rate was 97.1%, and for squamous cell carcinoma, adenocarcinoma and small cell carcinoma, the detection sensitivity reached 100%. Such high sensitivity can be achieved in various types of lung cancers, and when used for clinical detection, it will undoubtedly have greater versatility. Especially for the detection of adenocarcinoma, the combined sensitivity of HOXA7 and HOXA9 to detect adenocarcinoma is as high as 100%, while the sensitivity of the existing primers and probes for SHOX2 detection is only 33.3% for adenocarcinoma. Adenocarcinomas are generally of peripheral type. Due to the dendritic physiological structure of the bronchi, the exfoliated cells in the deep part of the lung are more difficult to expectorate through sputum. The present disclosure is the first to discover a method that can detect adenocarcinoma with sputum as a sample, and can greatly improve the sensitivity. Up to 100% markup, this breakthrough has important implications for the detection of adenocarcinoma.

實施例4：HOXA7、HOXA9以及SHOX2基因在灌洗液中的檢測Example 4: Detection of HOXA7, HOXA9 and SHOX2 genes in lavage fluid

樣本信息：測試肺泡灌洗液樣本共計79例，其中正常對照組樣本58例，癌症組對照樣本21例，21例癌症組樣本中有鱗癌6例，小細胞癌4例，腺癌11例。Sample information: A total of 79 samples of bronchoalveolar lavage fluid were tested, including 58 samples from the normal control group, 21 samples from the cancer group, 6 samples from the 21 samples from the cancer group, 6 samples from squamous cell carcinoma, 4 samples from small cell carcinoma, and 11 samples from adenocarcinoma. .

試驗過程：Experimental procedure:

a. 收集確診為肺癌患者和非肺癌患者的肺泡灌洗液標本，離心分離細胞，然後使用Magen的DNA提取試劑盒（HiPure FFPE DNA Kit，D3126-03）提取DNA。a. Collect bronchoalveolar lavage fluid samples from patients diagnosed with lung cancer and patients without lung cancer, centrifuge to separate cells, and then use Magen's DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.

b. 使用ZYMO RESEARCH生物公司的DNA轉化試劑盒（EZ DNA Methylation Kit，D5002）進行DNA的重亞硫酸氫鹽修飾。b. Bisulfite modification of DNA was performed using the DNA transformation kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH.

c. 擴增檢測體系如下： [表12] 擴增體系   HOXA7 HOXA9 SHOX2_n3 β-actin 反應組分 加入量(µl) 加入量(µl) 加入量(µl) 加入量(µl) 上游引子 (100 µM) 0.125 0.125 0.125 0.125 下游引子 (100 µM) 0.125 0.125 0.125 0.125 探針(100 µM) 0.05 0.05 0.05 0.05 鎂離子(25 mM) 5 4 5 5 dNTPs(10 mM) 1 1 1 1 Taq聚合酶(5 unit/µl) 0.5 0.5 0.5 0.5 5×緩衝液 5 5 5 5 滅菌水 12.2 13.2 12.2 12.2 模板DNA 1 1 1 1 總體積 25 25 25 25 c. The amplification detection system is as follows: [Table 12] Amplification system HOXA7 HOXA9 SHOX2_n3 β-actin reactive components Addition amount (µl) Addition amount (µl) Addition amount (µl) Addition amount (µl) Upstream Primer (100 µM) 0.125 0.125 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 0.05 Magnesium (25 mM) 5 4 5 5 dNTPs (10 mM) 1 1 1 1 Taq polymerase (5 unit/µl) 0.5 0.5 0.5 0.5 5x buffer 5 5 5 5 Sterilized water 12.2 13.2 12.2 12.2 template DNA 1 1 1 1 total capacity 25 25 25 25

d. 檢測體系如下： [表13] HOXA7和HOXA9的反應程序   HOXA7 HOXA9 β-actin   步驟 溫度和時間 溫度和時間 溫度和時間 循環數目 預變性 95°C 5分鐘 95°C 5分鐘 95°C 5分鐘 1 擴增1 95°C 20秒 95°C 20秒 95°C 20秒 10 63°C 30秒 63°C 30秒 62°C 30秒 70°C 30秒 70°C 30秒 70°C 30秒 擴增2 95°C 20秒 95°C 20秒 95°C 20秒 45 60°C 30秒 60°C 30秒 54°C 60秒 72°C 30秒 72°C 30秒 72°C 30秒 冷卻 40°C 30秒 40°C 30秒 40°C 30秒 1 [表14] SHOX2_n3反應程序 步驟 溫度和時間 循環數目 預變性 95°C 5分鐘 1 擴增1 95°C 20秒 45 60°C 30秒 72°C 30秒 冷卻 40°C 30秒 1 d. The detection system is as follows: [Table 13] Reaction procedure of HOXA7 and HOXA9 HOXA7 HOXA9 β-actin step temperature and time temperature and time temperature and time Number of cycles predenaturation 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 63°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 60°C for 30 seconds 54°C for 60 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 1 [Table 14] SHOX2_n3 reaction program step temperature and time Number of cycles predenaturation 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 45 60°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 1

e. 檢測結果如下：e. The test results are as follows:

利用標準曲線計算各基因在標本中的甲基化拷貝數，採用比值=靶向基因拷貝數/ACTB拷貝數*100來進行判斷兩組樣本的甲基化程度，最後選取HOXA7的閾值為0.7，HOXA9的閾值為0.5，SHOX2_n3的閾值為0.6，作為判斷癌症組和對照組的標準，換算後的比值超過設定閾值可判斷為陽性「+」，等於或小於設定閾值可判斷為陰性「−」。根據此標準，79例灌洗液標本的檢測結果如下： [表15] 檢測結果     HOXA7 HOXA9 SHOX2_n3 HOXA7 HOXA9 HOXA7& HOXA9組合 SHOX2_n3 序號 組織類型 甲基化率 甲基化率 甲基化率 檢出情況 檢出情況 檢出情況 檢出情況 1 非肺癌對照 0.3 0.2 0.3 − − − − 2 非肺癌對照 0.6 0.1 0.7 − − − + 3 非肺癌對照 0.3 0.2 0.1 − − − − 4 非肺癌對照 0.3 0.5 0.6 − − − − 5 非肺癌對照 0.0 0.1 0.2 − − − − 6 非肺癌對照 0.3 0.2 0.1 − − − − 7 非肺癌對照 0.0 0.0 0.0 − − − − 8 非肺癌對照 0.2 0.1 0.0 − − − − 9 非肺癌對照 0.0 0.0 0.0 − − − − 10 非肺癌對照 0.0 0.0 0.0 − − − − 11 非肺癌對照 0.0 0.0 0.1 − − − − 12 非肺癌對照 0.3 0.0 0.1 − − − − 13 非肺癌對照 0.3 0.0 0.1 − − − − 14 非肺癌對照 0.0 0.3 0.1 − − − − 15 非肺癌對照 0.70 0.3 0.5 + − + − 16 非肺癌對照 0.2 0.1 0.1 − − − − 17 非肺癌對照 0.1 0.0 0.0 − − − − 18 非肺癌對照 0.2 0.4 0.2 − − − − 19 非肺癌對照 0.2 0.1 0.2 − − − − 20 非肺癌對照 0.0 0.0 0.3 − − − − 21 非肺癌對照 0.0 0.0 0.0 − − − − 22 非肺癌對照 0.0 0.0 0.2 − − − − 23 非肺癌對照 0.0 0.0 0.1 − − − − 24 非肺癌對照 0.3 0.0 0.1 − − − − 25 非肺癌對照 0.0 0.0 0.0 − − − − 26 非肺癌對照 0.6 0.1 0.2 − − − − 27 非肺癌對照 0.0 0.1 0.1 − − − − 28 非肺癌對照 0.3 0.1 0.3 − − − − 29 非肺癌對照 0.3 0.1 0.1 − − − − 30 非肺癌對照 0.67 0.5 0.5 + − + − 31 非肺癌對照 0.0 0.0 0.2 − − − − 32 非肺癌對照 0.0 0.0 0.4 − − − − 33 非肺癌對照 0.1 0.1 0.1 − − − − 34 非肺癌對照 0.3 0.5 0.5 − − − − 35 非肺癌對照 0.2 0.1 0.2 − − − − 36 非肺癌對照 0.4 0.8 0.8 − + + + 37 非肺癌對照 0.0 0.1 0.4 − − − − 38 非肺癌對照 0.2 0.1 0.1 − − − − 39 非肺癌對照 0.3 0.2 0.2 − − − − 40 非肺癌對照 0.5 0.3 0.3 − − − − 41 非肺癌對照 0.3 0.4 0.3 − − − − 42 非肺癌對照 0.2 0.1 0.1 − − − − 43 非肺癌對照 0.3 0.8 0.6 − + + + 44 非肺癌對照 0.3 0.0 0.1 − − − − 45 非肺癌對照 0.7 0.2 0.2 − − − − 46 非肺癌對照 0.2 0.0 0.1 − − − − 47 非肺癌對照 0.2 0.1 0.1 − − − − 48 非肺癌對照 0.2 0.3 0.2 − − − − 49 非肺癌對照 0.2 0.1 0.4 − − − − 50 非肺癌對照 0.0 0.0 0.0 − − − − 51 非肺癌對照 0.2 0.2 0.0 − − − − 52 非肺癌對照 0.4 0.5 0.5 − − − − 53 非肺癌對照 0.0 0.0 0.0 − − − − 54 非肺癌對照 0.4 1.1 0.1 − + + − 55 非肺癌對照 0.2 0.4 0.0 − − − − 56 非肺癌對照 0.0 0.0 0.0 − − − − 57 非肺癌對照 2.6 0.2 0.0 + − + − 58 非肺癌對照 0.0 0.0 0.0 − − − − 59 鱗癌 0.2 0.5 0.3 − − − − 60 鱗癌 42.2 76.9 321.9 + + + + 61 鱗癌 21.9 60.9 56.7 + + + + 62 鱗癌 11.3 0.3 0.0 + − + − 63 鱗癌 14.5 17.7 7.6 + + + + 64 鱗癌 1.6 1.5 0.6 + + + + 65 腺癌 0.7 0.5 0.6 + − + − 66 腺癌 1.2 0.3 0.0 + − + − 67 腺癌 0.8 0.3 0.4 + − + − 68 腺癌 34.8 3.7 33.5 + + + + 69 腺癌 2.8 2.9 2.3 + + + + 70 腺癌 0.3 0.1 0.0 − − − − 71 腺癌 0.3 0.3 0.3 − − − − 72 腺癌 1.9 0.0 1.4 + + + + 73 腺癌 0.0 20.8 8.6 − + + + 74 腺癌 4.2 5.3 0.3 + + + − 75 腺癌 0.9 0.9 0.2 + + + − 76 小細胞癌 0.6 0.8 0.3 − + + − 77 小細胞癌 24.2 26.4 97.7 + + + + 78 小細胞癌 0.8 1.8 7.6 + + + + 79 小細胞癌 1.7 0.6 2.4 + + + + The standard curve was used to calculate the methylated copy number of each gene in the sample, and the ratio=target gene copy number/ACTB copy number*100 was used to judge the methylation degree of the two groups of samples. Finally, the threshold of HOXA7 was selected as 0.7. The threshold of HOXA9 is 0.5, and the threshold of SHOX2_n3 is 0.6. As the standard for judging the cancer group and the control group, if the converted ratio exceeds the set threshold, it can be judged as positive "+", and if it is equal to or less than the set threshold, it can be judged as negative "-". According to this standard, the test results of 79 lavage fluid samples are as follows: [Table 15] Test results HOXA7 HOXA9 SHOX2_n3 HOXA7 HOXA9 HOXA7 & HOXA9 combination SHOX2_n3 serial number Organization type methylation rate methylation rate methylation rate detection detection detection detection 1 Non-lung cancer control 0.3 0.2 0.3 − − − − 2 Non-lung cancer control 0.6 0.1 0.7 − − − + 3 Non-lung cancer control 0.3 0.2 0.1 − − − − 4 Non-lung cancer control 0.3 0.5 0.6 − − − − 5 Non-lung cancer control 0.0 0.1 0.2 − − − − 6 Non-lung cancer control 0.3 0.2 0.1 − − − − 7 Non-lung cancer control 0.0 0.0 0.0 − − − − 8 Non-lung cancer control 0.2 0.1 0.0 − − − − 9 Non-lung cancer control 0.0 0.0 0.0 − − − − 10 Non-lung cancer control 0.0 0.0 0.0 − − − − 11 Non-lung cancer control 0.0 0.0 0.1 − − − − 12 Non-lung cancer control 0.3 0.0 0.1 − − − − 13 Non-lung cancer control 0.3 0.0 0.1 − − − − 14 Non-lung cancer control 0.0 0.3 0.1 − − − − 15 Non-lung cancer control 0.70 0.3 0.5 + − + − 16 Non-lung cancer control 0.2 0.1 0.1 − − − − 17 Non-lung cancer control 0.1 0.0 0.0 − − − − 18 Non-lung cancer control 0.2 0.4 0.2 − − − − 19 Non-lung cancer control 0.2 0.1 0.2 − − − − 20 Non-lung cancer control 0.0 0.0 0.3 − − − − twenty one Non-lung cancer control 0.0 0.0 0.0 − − − − twenty two Non-lung cancer control 0.0 0.0 0.2 − − − − twenty three Non-lung cancer control 0.0 0.0 0.1 − − − − twenty four Non-lung cancer control 0.3 0.0 0.1 − − − − 25 Non-lung cancer control 0.0 0.0 0.0 − − − − 26 Non-lung cancer control 0.6 0.1 0.2 − − − − 27 Non-lung cancer control 0.0 0.1 0.1 − − − − 28 Non-lung cancer control 0.3 0.1 0.3 − − − − 29 Non-lung cancer control 0.3 0.1 0.1 − − − − 30 Non-lung cancer control 0.67 0.5 0.5 + − + − 31 Non-lung cancer control 0.0 0.0 0.2 − − − − 32 Non-lung cancer control 0.0 0.0 0.4 − − − − 33 Non-lung cancer control 0.1 0.1 0.1 − − − − 34 Non-lung cancer control 0.3 0.5 0.5 − − − − 35 Non-lung cancer control 0.2 0.1 0.2 − − − − 36 Non-lung cancer control 0.4 0.8 0.8 − + + + 37 Non-lung cancer control 0.0 0.1 0.4 − − − − 38 Non-lung cancer control 0.2 0.1 0.1 − − − − 39 Non-lung cancer control 0.3 0.2 0.2 − − − − 40 Non-lung cancer control 0.5 0.3 0.3 − − − − 41 Non-lung cancer control 0.3 0.4 0.3 − − − − 42 Non-lung cancer control 0.2 0.1 0.1 − − − − 43 Non-lung cancer control 0.3 0.8 0.6 − + + + 44 Non-lung cancer control 0.3 0.0 0.1 − − − − 45 Non-lung cancer control 0.7 0.2 0.2 − − − − 46 Non-lung cancer control 0.2 0.0 0.1 − − − − 47 Non-lung cancer control 0.2 0.1 0.1 − − − − 48 Non-lung cancer control 0.2 0.3 0.2 − − − − 49 Non-lung cancer control 0.2 0.1 0.4 − − − − 50 Non-lung cancer control 0.0 0.0 0.0 − − − − 51 Non-lung cancer control 0.2 0.2 0.0 − − − − 52 Non-lung cancer control 0.4 0.5 0.5 − − − − 53 Non-lung cancer control 0.0 0.0 0.0 − − − − 54 Non-lung cancer control 0.4 1.1 0.1 − + + − 55 Non-lung cancer control 0.2 0.4 0.0 − − − − 56 Non-lung cancer control 0.0 0.0 0.0 − − − − 57 Non-lung cancer control 2.6 0.2 0.0 + − + − 58 Non-lung cancer control 0.0 0.0 0.0 − − − − 59 squamous cell carcinoma 0.2 0.5 0.3 − − − − 60 squamous cell carcinoma 42.2 76.9 321.9 + + + + 61 squamous cell carcinoma 21.9 60.9 56.7 + + + + 62 squamous cell carcinoma 11.3 0.3 0.0 + − + − 63 squamous cell carcinoma 14.5 17.7 7.6 + + + + 64 squamous cell carcinoma 1.6 1.5 0.6 + + + + 65 adenocarcinoma 0.7 0.5 0.6 + − + − 66 adenocarcinoma 1.2 0.3 0.0 + − + − 67 adenocarcinoma 0.8 0.3 0.4 + − + − 68 adenocarcinoma 34.8 3.7 33.5 + + + + 69 adenocarcinoma 2.8 2.9 2.3 + + + + 70 adenocarcinoma 0.3 0.1 0.0 − − − − 71 adenocarcinoma 0.3 0.3 0.3 − − − − 72 adenocarcinoma 1.9 0.0 1.4 + + + + 73 adenocarcinoma 0.0 20.8 8.6 − + + + 74 adenocarcinoma 4.2 5.3 0.3 + + + − 75 adenocarcinoma 0.9 0.9 0.2 + + + − 76 small cell carcinoma 0.6 0.8 0.3 − + + − 77 small cell carcinoma 24.2 26.4 97.7 + + + + 78 small cell carcinoma 0.8 1.8 7.6 + + + + 79 small cell carcinoma 1.7 0.6 2.4 + + + +

注意：「+」為甲基化DNA檢測陽性，「−」為甲基化DNA檢測陰性；在協同檢測標本時，兩者為陰性則判為陰性，一陰一陽或兩者均為陽性則判為陽性。 [表16] 統計結果 分析組別 指標 HOXA7 HOXA9 HOXA7&HOXA9組合 SHOX2_n3 正常組和全部癌症組比較 特異性 95.0% 95.0% 89.7% 95.0% 靈敏度 76.2% 61.9% 85.7% 52.4% 正常組和全部鱗癌組比較 特異性 95.0% 95.0% 89.7% 95.0% 靈敏度 83.3% 66.7% 83.3% 66.7% 正常組和全部腺癌組比較 特異性 95.0% 95.0% 89.7% 95.0% 靈敏度 72.7% 45.5% 81.8% 36.4% 正常組和全部小細胞癌組比較 特異性 95.0% 95.0% 89.7% 95.0% 靈敏度 75.0% 100% 100% 75.0% Note: "+" means positive for methylated DNA test, and "-" means negative for methylated DNA test; in the case of co-testing specimens, if both are negative, it will be judged as negative, and if one yin and one yang or both are positive, it will be judged is positive. [Table 16] Statistical results Analysis group index HOXA7 HOXA9 HOXA7&HOXA9 combination SHOX2_n3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% 89.7% 95.0% Sensitivity 76.2% 61.9% 85.7% 52.4% Comparison of normal group and all squamous cell carcinoma groups specificity 95.0% 95.0% 89.7% 95.0% Sensitivity 83.3% 66.7% 83.3% 66.7% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% 89.7% 95.0% Sensitivity 72.7% 45.5% 81.8% 36.4% Comparison between normal group and all small cell carcinoma groups specificity 95.0% 95.0% 89.7% 95.0% Sensitivity 75.0% 100% 100% 75.0%

f. HOXA7與HOXA9組合與SHOX2_n3在所有灌洗液標本中檢測的ROC曲線見圖5，HOXA7、HOXA9與SHOX2_n3在灌洗液標本中的擴增曲線見圖6，統計結果見表16，從以上結果可以看出，在灌洗液樣本中，無論將肺癌作為一個整體進行比較分析，還是按照肺癌的亞型進行比較分析，HOXA7和HOXA9的檢測效果都要優於SHOX2基因的檢測效果，通過HOXA7和HOXA9組合檢測樣本，可以有效提高陽性檢出率，在所有樣本中，陽性檢出率為85.7%。特別是對腺癌的檢測效果，HOXA7和HOXA9聯合檢出腺癌的靈敏性高達81.8%，遠高於SHOX2的36.4%，也高於HOXA7和HOXA9基因在組織中對腺癌的靈敏度，這是十分難得且罕見的。腺癌一般為周圍型，由於支氣管的樹狀生理結構，肺深部的脫落細胞更加難以通過痰液咳出，因此對這部分的檢測更加困難和有意義。f. The ROC curves of the combination of HOXA7 and HOXA9 and SHOX2_n3 in all lavage samples are shown in Figure 5, and the amplification curves of HOXA7, HOXA9 and SHOX2_n3 in the lavage samples are shown in Figure 6, and the statistical results are shown in Table 16. From the above It can be seen from the results that in the lavage fluid samples, whether the lung cancer as a whole or the subtypes of lung cancer is compared and analyzed, the detection effect of HOXA7 and HOXA9 is better than that of SHOX2 gene. Combined with HOXA9 to detect samples, the positive detection rate can be effectively improved. Among all samples, the positive detection rate was 85.7%. Especially for the detection of adenocarcinoma, the combined sensitivity of HOXA7 and HOXA9 in detecting adenocarcinoma is as high as 81.8%, which is much higher than 36.4% of SHOX2, and also higher than the sensitivity of HOXA7 and HOXA9 genes to adenocarcinoma in tissues. Very rare and rare. Adenocarcinomas are generally of peripheral type. Due to the dendritic physiological structure of the bronchi, the exfoliated cells in the deep part of the lung are more difficult to expectorate through sputum, so the detection of this part is more difficult and meaningful.

此外，在灌洗液中，针对小细胞癌，其敏感性达到了100%，而无论是HOXA7或HOXA9，或SHOX2，其对小细胞癌的灵敏度仅为75%。這樣的0漏檢率對小細胞癌的早期篩查具有重要的意義。In addition, in the lavage fluid, the sensitivity for small cell carcinoma reached 100%, while the sensitivity for small cell carcinoma was only 75% for either HOXA7 or HOXA9, or SHOX2. Such a 0 missed detection rate is of great significance for the early screening of small cell carcinoma.

綜合上述的4個實施例，能夠充分的說明HOXA7與HOXA9組合在對肺癌檢測診斷，尤其是應用痰液，肺泡灌洗液等生物樣本上具有更好的檢測效果。能夠更加容易的應用于大規模的人群篩查。具有更加優越的社會經濟學價值。Combining the above four examples, it can be fully demonstrated that the combination of HOXA7 and HOXA9 has a better detection effect in the detection and diagnosis of lung cancer, especially in biological samples such as sputum and bronchoalveolar lavage fluid. It can be more easily applied to large-scale population screening. It has more superior socioeconomic value.

實施例5：HOXA7和HOXA9的引子設計和優化Example 5: Primer design and optimization of HOXA7 and HOXA9

一、檢測區域對檢測效果的影響1. The influence of the detection area on the detection effect

各種研究資料表明，同一個基因的甲基化狀態和分佈並不均勻，因此對於同一個基因來說，選擇不同的區域設計的甲基化引子、探針檢測體系對同一樣本，同一腫瘤的診斷檢測效能並不一樣，甚至有時候選擇的區域不合適造成對腫瘤完全沒有診斷效果，發明人經過反復的研究和比較後，部分HOXA7和HOXA9的區域序列如下： [表17] HOXA7待檢測序列 序列名稱 序列 SEQ ID NO: 25 HOXA7區域1參考序列 ACCACATGGCTCCAGTTTGCGGTGGCAATCTCTCTGCAGCTGCAAGAGATGCTGCGCCTTCCCCGTCTGGATCCGAGTCTAAGTCCGGCCTGTCGCCCACTGGACCTGGGTGAGAGAAGACTTGGGCAGAGTCGATCTGCTCATAGCTGAGTCCTGCCCACAAGGCCACCGCGGGGCAGGCTGTTGCGGGGGACAGAGACCCTTCCAGGGTCTGGGCAGGCGGACAGGAGAGGGATGGGGAGGATCCCAAGCTTGGTCCAGGGCTCACTAGCAGGAGTCGGCGGGGGGGCGGGGTGGGGGGTGCTGCGTGGGGCCGGGCCGCCTGGCGTCCGCAGACCCCAGTGCGGAGGTTGGCCGCCAGCTGGGCGCTCCCGCGGAGCCTCCAGGTCTTTTTCCGCGGGACGCGCCAGGCCCGCCGGGCGCGGGCGGATTCTTTGGCCGCATATTTGAGCCTCTTGCCCTTCCATTCTAGGCGGCTGCGGGCCCTGCGGAGCGAGACCACCTGTGAGGACTGCTGAGATTGGCGGAGGCGGTCATGTGGGCGGTCACGTGCTGCGGCGAGCTCCGTCCAAAAGAAAATGGGGTTTGGTGTAAATCTGGGGGTGTAATGTTATCATATATCACTCTACCTCGTAAAACCGACACTGAAAGCTGCCGGACAACAAATCACAGGTCAAAATTATGAGTTCTTCGTATTATGTGAACGCGCTTTTTAGCAAATATACGGCGGGGGCTTCTCTGTTCCAAAATGCCGAGCCGACTTCTTGCTCCTTTGCTCCCAACTCACAGAGAAGCGGCTACGGGGCGGGCGCCGGCGCCTTCGCCTCGACCGTTCCGGGCTTATACAATGTCAACAGCCCCCTTTATCAGAGCCCCTTTG SEQ ID NO: 26 HOXA7區域1序列經重亞硫酸氫鹽處理過後轉化的序列 ATTATATGGTTTTAGTTTGCGGTGGTAATTTTTTTGTAGTTGTAAGAGATGTTGCGTTTTTTTCGTTTGGATTCGAGTTTAAGTTCGGTTTGTCGTTTATTGGATTTGGGTGAGAGAAGATTTGGGTAGAGTCGATTTGTTTATAGTTGAGTTTTGTTTATAAGGTTATCGCGGGGTAGGTTGTTGCGGGGGATAGAGATTTTTTTAGGGTTTGGGTAGGCGGATAGGAGAGGGATGGGGAGGATTTTAAGTTTGGTTTAGGGTTTATTAGTAGGAGTCGGCGGGGGGGCGGGGTGGGGGGTGTTGCGTGGGGTCGGGTCGTTTGGCGTTCGTAGATTTTAGTGCGGAGGTTGGTCGTTAGTTGGGCGTTTTCGCGGAGTTTTTAGGTTTTTTTTCGCGGGACGCGTTAGGTTCGTCGGGCGCGGGCGGATTTTTTGGTCGTATATTTGAGTTTTTTGTTTTTTTATTTTAGGCGGTTGCGGGTTTTGCGGAGCGAGATTATTTGTGAGGATTGTTGAGATTGGCGGAGGCGGTTATGTGGGCGGTTACGTGTTGCGGCGAGTTTCGTTTAAAAGAAAATGGGGTTTGGTGTAAATTTGGGGGTGTAATGTTATTATATATTATTTTATTTCGTAAAATCGATATTGAAAGTTGTCGGATAATAAATTATAGGTTAAAATTATGAGTTTTTCGTATTATGTGAACGCGTTTTTTAGTAAATATACGGCGGGGGTTTTTTTGTTTTAAAATGTCGAGTCGATTTTTTGTTTTTTTGTTTTTAATTTATAGAGAAGCGGTTACGGGGCGGGCGTCGGCGTTTTCGTTTCGATCGTTTCGGGTTTATATAATGTTAATAGTTTTTTTTATTAGAGTTTTTTTG SEQ ID NO: 27 HOXA7區域2參考序列 CGTCCGGCTACGGCCTGGGCGCCGACGCCTACGGCAACCTGCCCTGCGCCTCCTACGACCAAAACATCCCCGGGCTCTGCAGTGACCTCGCCAAAGGCGCCTGCGACAAGACGGACGAGGGCGCGCTGCATGGCGCGGCTGAGGCCAATTTCCGCATCTACCCCTGGATGCGGTCTTCAGGTAGGCGCAGTCGCTAGGCGGGCCAGGCTGGCGGAGCGGGACCGGGAGCGGGGAGCGCAGCGCTGGGGAGCGCGGAGCGCGGGGCGCGGGGCCGGAAGAGCGGAGCCAGGCTGTTGCGAGCCGGTAGCCCCGTGACTCCCGGCGCA SEQ ID NO: 28 HOXA7區域2參考序列經重亞硫酸氫鹽處理過後轉化的序列 CGTTCGGTTACGGTTTGGGCGTCGACGTTTACGGTAATTTGTTTTGCGTTTTTTACGATTAAAATATTTTCGGGTTTTGTAGTGATTTCGTTAAAGGCGTTTGCGATAAGACGGACGAGGGCGCGTTGTATGGCGCGGTTGAGGTTAATTTTCGTATTTATTTTTGGATGCGGTTTTTAGGTAGGCGTAGTCGTTAGGCGGGTTAGGTTGGCGGAGCGGGATCGGGAGCGGGGAGCGTAGCGTTGGGGAGCGCGGAGCGCGGGGCGCGGGGTCGGAAGAGCGGAGTTAGGTTGTTGCGAGTCGGTAGTTTCGTGATTTTCGGCGTA [續表17] HOXA9待檢測序列 序列名稱 序列 SEQ ID NO: 29 HOXA9區域1參考序列 AATTTCCGTGGGTCGGGCCGGGCGGGCCAGGCGCTGGGCACGGTGATGGCCACCACTGGGGCCCTGGGCAACTACTACGTGGACTCGTTCCTGCTGGGCGCCGACGCCGCGGATGAGCTGAGCGTTGGCCGCTATGCGCCGGGGACCCTGGGCCAGCCTCCCCGGCAGGCGGCGACGCTGGCCGAGCACCCCGACTTCAGCCCGTGCAGCTTCCAGTCCAAGGCGACGGTGTTTGGCGCCTCGTGGAACCCAGTGCACGCGGCGGGCGCCAACGCTGTACCCGCTGCGGTGTACCACCACCATCACCACCACCCCTACGTGCACCCCCAGGCGCCCGTGGCGGCG SEQ ID NO: 30 HOXA9區域1序列經重亞硫酸氫鹽處理過後轉化的序列 AATTTTCGTGGGTCGGGTCGGGCGGGTTAGGCGTTGGGTACGGTGATGGTTATTATTGGGGTTTTGGGTAATTATTACGTGGATTCGTTTTTGTTGGGCGTCGACGTCGCGGATGAGTTGAGCGTTGGTCGTTATGCGTCGGGGATTTTGGGTTAGTTTTTTCGGTAGGCGGCGACGTTGGTCGAGTATTTCGATTTTAGTTCGTGTAGTTTTTAGTTTAAGGCGACGGTGTTTGGCGTTTCGTGGAATTTAGTGTACGCGGCGGGCGTTAACGTTGTATTCGTTGCGGTGTATTATTATTATTATTATTATTTTTACGTGTATTTTTAGGCGTTCGTGGCGGCG SEQ ID NO: 31 HOXA9區域2參考序列 GACAGGACGGGGCCATTTCGGAGTTCATTGTGTCGGCCACTTCCCTCTTCCAGGCGCGGGTGCAGGAAGGGGCACCCAGTCGGTATCCGCGCGGCTTGGCAGCCTCGCTGGTATTTGGGAGTCCCAGCCGGAAGTGTGTCAGGGTGTTTGAGGGGGGGATTACTGGAACTGCTGGTGAGGATGAAGGCAAAAGAGAGAGAGAGAGATGGAAGCGCCCGAGGCCGCCAGCCTCGCCGCCAGGGAAGTGGGCTAATGAAAAACACACTGTTGCAGGCACAGTATCCACACGTGAATTTGATTACCCCTGTTCTAGGAGTCGCTGCTTTCTGTTAGGAATTGGGGGCAGGGGGAGTTTCCTTCCAATTAACGGAGTGGCGGCGACCTTTTAATTTACCCCCAACGGGTGAGAAATAAACTTCCCCAACGTGGCCAGGCCCAGGAATGGGACTGGAGTCGATGCCCTTTTACCCCTCCCCGTTCTAATTTCCAGCCCTGGCCTTGAGCTGTGGCTGCCTCTCTTTGGGCCTTGTACCTCTCCGCCGAGTCTCCGGGCCCCGTAGGTAACCAAGGCGAGGCCCGGAGTAGCAGCTGGAAAGGGAGGAAGGAGCCCTGAAAGGCTCACGCGGCCCCGGGACAGGCCACATCGGTGCGGGCCTCCCAGGTTCCGGAGCTGCGGGGTCTCTTAGGCGAGGCTGCCTTTTCCCAAACCGAACTTGCCTTCCATTCATGCCACTTGTAGTTTTTTCCCCAGCTGGGATTCACGGAGCGCAACCAGGCTTGCAGCGCTCATGGTTAGAGCCTCTGAGGCTGGAGCACAGGGCTGGGTCGCCAGCCGCCTGCGCCTGGGAATCCTGATTGCCAGCTGATGAGAAAGGCGGGCTGGGCGCGCGTGTGCGTGGGGTCGAGGGCCGGGGACCGAGCGCGCCGCACAACCAACCAGGCCCTCAAAACCTTCGCCCTGGTGGCGGCTGGCCGCTCCCTCCTGGCCAGCTCCTCCGTGGGGTCCTCGTAGCAAAGGCGAATTTAAGGGTTGCCCGGGCGCCCCTCGCTCCAGGCGGGTAGCTGTGGGGACCTACACCCGCGGTACTCCCTGAGCGGCCGGTCCCTGCCTGGAGTGCCCTGGTAGGGCCGGCGGCGGCTCCGTTTGGGACGGATCCTGCGTTGAATTTGACTTTTCGAGGGCGGCCGCGGGTAAACTCGCCTCTCCCGGGGACCGCAGGGATTATTTACAGGGAGCTCGCCAACCAAACACAACAGTCTAACCTTTCCAAGTCCTCGTAAATTTTTACAGCTGGGAGCCACGGCGAGGCAAACGAATCTGTTGGTCGTTTCCGACTTCCCGCCAGCCTGTGTGGCTTCTGAAACAATAACTCCTTATGAAATATCATAAATATAGATTTAAATACAGTAGAGCGACAATGCGATTTGGCTGCTTTTTTATGGCTTCAATTATTGTCTAATTTTATGTGAGGGGCTCCGCTGGCCGCACTCGCACGCGGGACCCGCGCCTTCTTGATGGCGTGATTAATTGTGATATAAAATAGTCCGCTTAAGAAGTGTGTGTATGGGGGGGGAGACGGGAGAGTACAGAGACAAGGCTAGATTTGATCTTTTAATCGTCGTTGGCCACAATTAAAACAAACCCCATCGTAGAGCGGCACGATCCCTTTACATAAAAACATATGGCTTTTGCTATAAAAATTATGACTGCAAAACATCGGACCATTAATAGCGTGCGGAGTGATTTACGCGTTATTGTTCTGCTGGACGGGCACGTGACGCGCACGGCCAATGGGGGCGCGGGCGCCGGCAACTTATTAGGTGACTGTACTTCCCCCCCGGTGCCACCAAGTTGTTACATGAAATCTGCAGTTTCATAATTTCCGTGGGTCGGGCCG SEQ ID NO: 32 HOXA9區域2參考序列經重亞硫酸鹽處理過後轉化的序列 GATAGGACGGGGTTATTTCGGAGTTTATTGTGTCGGTTATTTTTTTTTTTTAGGCGCGGGTGTAGGAAGGGGTATTTAGTCGGTATTCGCGCGGTTTGGTAGTTTCGTTGGTATTTGGGAGTTTTAGTCGGAAGTGTGTTAGGGTGTTTGAGGGGGGGATTATTGGAATTGTTGGTGAGGATGAAGGTAAAAGAGAGAGAGAGAGATGGAAGCGTTCGAGGTCGTTAGTTTCGTCGTTAGGGAAGTGGGTTAATGAAAAATATATTGTTGTAGGTATAGTATTTATACGTGAATTTGATTATTTTTGTTTTAGGAGTCGTTGTTTTTTGTTAGGAATTGGGGGTAGGGGGAGTTTTTTTTTAATTAACGGAGTGGCGGCGATTTTTTAATTTATTTTTAACGGGTGAGAAATAAATTTTTTTAACGTGGTTAGGTTTAGGAATGGGATTGGAGTCGATGTTTTTTTATTTTTTTTCGTTTTAATTTTTAGTTTTGGTTTTGAGTTGTGGTTGTTTTTTTTTGGGTTTTGTATTTTTTCGTCGAGTTTTCGGGTTTCGTAGGTAATTAAGGCGAGGTTCGGAGTAGTAGTTGGAAAGGGAGGAAGGAGTTTTGAAAGGTTTACGCGGTTTCGGGATAGGTTATATCGGTGCGGGTTTTTTAGGTTTCGGAGTTGCGGGGTTTTTTAGGCGAGGTTGTTTTTTTTTAAATCGAATTTGTTTTTTATTTATGTTATTTGTAGTTTTTTTTTTAGTTGGGATTTACGGAGCGTAATTAGGTTTGTAGCGTTTATGGTTAGAGTTTTTGAGGTTGGAGTATAGGGTTGGGTCGTTAGTCGTTTGCGTTTGGGAATTTTGATTGTTAGTTGATGAGAAAGGCGGGTTGGGCGCGCGTGTGCGTGGGGTCGAGGGTCGGGGATCGAGCGCGTCGTATAATTAATTAGGTTTTTAAAATTTTCGTTTTGGTGGCGGTTGGTCGTTTTTTTTTGGTTAGTTTTTTCGTGGGGTTTTCGTAGTAAAGGCGAATTTAAGGGTTGTTCGGGCGTTTTTCGTTTTAGGCGGGTAGTTGTGGGGATTTATATTCGCGGTATTTTTTGAGCGGTCGGTTTTTGTTTGGAGTGTTTTGGTAGGGTCGGCGGCGGTTTCGTTTGGGACGGATTTTGCGTTGAATTTGATTTTTCGAGGGCGGTCGCGGGTAAATTCGTTTTTTTCGGGGATCGTAGGGATTATTTATAGGGAGTTCGTTAATTAAATATAATAGTTTAATTTTTTTAAGTTTTCGTAAATTTTTATAGTTGGGAGTTACGGCGAGGTAAACGAATTTGTTGGTCGTTTTCGATTTTTCGTTAGTTTGTGTGGTTTTTGAAATAATAATTTTTTATGAAATATTATAAATATAGATTTAAATATAGTAGAGCGATAATGCGATTTGGTTGTTTTTTTATGGTTTTAATTATTGTTTAATTTTATGTGAGGGGTTTCGTTGGTCGTATTCGTACGCGGGATTCGCGTTTTTTTGATGGCGTGATTAATTGTGATATAAAATAGTTCGTTTAAGAAGTGTGTGTATGGGGGGGGAGACGGGAGAGTATAGAGATAAGGTTAGATTTGATTTTTTAATCGTCGTTGGTTATAATTAAAATAAATTTTATCGTAGAGCGGTACGATTTTTTTATATAAAAATATATGGTTTTTGTTATAAAAATTATGATTGTAAAATATCGGATTATTAATAGCGTGCGGAGTGATTTACGCGTTATTGTTTTGTTGGACGGGTACGTGACGCGTACGGTTAATGGGGGCGCGGGCGTCGGTAATTTATTAGGTGATTGTATTTTTTTTTCGGTGTTATTAAGTTGTTATATGAAATTTGTAGTTTTATAATTTTCGTGGGTCGGGTCG SEQ ID NO: 33 HOXA9區域3參考序列 CCCAGGCGCCCGTGGCGGCGGCGGCGCCGGACGGCAGGTACATGCGCTCCTGGCTGGAGCCCACGCCCGGTGCGCTCTCCTTCGCGGGCTTGCCCTCCAGCCGGCCTTATGGCATTAAACCTGAACCGCTGTCGGCCAGAAGGGGTGACTGTCCCACGCTTGACACTCACACTTTGTCCCTGACTGACTATGCTTGTGGTTCTCCTCCAGTTGATAGAGAAAAACAACCCAGCGAAGGCGCCTTCTCTGAAAACAATGCTGAGAATGAGAGCGGCGGAGACAAGCCCCCCATCGATCCCAGTAAGTGTCTCCTCCCTTCAAATCCGCCGCCGCCTCCACGCCGGCCTCCCGGATCTGCTGGCCCGCCAGGTTTCTCTCGAGCCTGCCTTCGTCCTCGCTGGAAGCCTCTCGAGTTGGGGCCAGGAGCCAGAAGTTGGTGTTTGGGACGCCTCAGATAGGGCCCCAAGTCTGGAGAGCAGTGAAGAGCGGCCCGCAGGGCTACGGGAGAGGAGGCGGCTGCTGCAGCGAGAGGGGGCGGGGCGGGCACTTCGGGACGAGCCAAGACTGGCCGCCCCTCTCCTTGGCTGCCCAGGCCCAGGACCGAGATACTTTGGGCCGTTCTTCGAAAGCAGTGCAGCCCAGAGAGCCTTTTGTACAACTAGATTGTCCGTGAGCGGCGGCAGCCAGGGCAGCCGGAGCTGGGACGCTGGGGGAGACGGCCGATTCCTTCCACTTCTTGCCTTCGGCCAGTGGCGGCGTAAATCCTGCCAAGATGAGGCTGCGGGCGACCCGGGCCACAAGGGTCCCCATGACAGATTATTCAAATAAGCCACAGACGTGATCAGCGGCCTTAGGGCGCCCTGACGGCTTGCCCAGCTCCGAAGGCCTTCCAGGAAGGTTAAATAAGGAGTGGGGGGCGTAGAGGGACAGGTTGGGAAAGAAAGACG SEQ ID NO: 34 HOXA9區域3參考序列經重亞硫酸鹽處理過後轉化的序列 TTTAGGCGTTCGTGGCGGCGGCGGCGTCGGACGGTAGGTATATGCGTTTTTGGTTGGAGTTTACGTTCGGTGCGTTTTTTTTCGCGGGTTTGTTTTTTAGTCGGTTTTATGGTATTAAATTTGAATCGTTGTCGGTTAGAAGGGGTGATTGTTTTACGTTTGATATTTATATTTTGTTTTTGATTGATTATGTTTGTGGTTTTTTTTTAGTTGATAGAGAAAAATAATTTAGCGAAGGCGTTTTTTTTGAAAATAATGTTGAGAATGAGAGCGGCGGAGATAAGTTTTTTATCGATTTTAGTAAGTGTTTTTTTTTTTTAAATTCGTCGTCGTTTTTACGTCGGTTTTTCGGATTTGTTGGTTCGTTAGGTTTTTTTCGAGTTTGTTTTCGTTTTCGTTGGAAGTTTTTCGAGTTGGGGTTAGGAGTTAGAAGTTGGTGTTTGGGACGTTTTAGATAGGGTTTTAAGTTTGGAGAGTAGTGAAGAGCGGTTCGTAGGGTTACGGGAGAGGAGGCGGTTGTTGTAGCGAGAGGGGGCGGGGCGGGTATTTCGGGACGAGTTAAGATTGGTCGTTTTTTTTTTTGGTTGTTTAGGTTTAGGATCGAGATATTTTGGGTCGTTTTTCGAAAGTAGTGTAGTTTAGAGAGTTTTTTGTATAATTAGATTGTTCGTGAGCGGCGGTAGTTAGGGTAGTCGGAGTTGGGACGTTGGGGGAGACGGTCGATTTTTTTTATTTTTTGTTTTCGGTTAGTGGCGGCGTAAATTTTGTTAAGATGAGGTTGCGGGCGATTCGGGTTATAAGGGTTTTTATGATAGATTATTTAAATAAGTTATAGACGTGATTAGCGGTTTTAGGGCGTTTTGACGGTTTGTTTAGTTTCGAAGGTTTTTTAGGAAGGTTAAATAAGGAGTGGGGGGCGTAGAGGGATAGGTTGGGAAAGAAAGACG SEQ ID NO: 35 β-actin區域參考序列 AGCCCGGGGCGGGGTGGGGCTGGAGCTCCTGTCTCTTGGCCAGCTGAATGGAGGCCCAGTGGCAACACAGGTCCTGCCTGGGGATCAGGTCTGCTCTGCACCCCACCTTGCTGCCTGGAGCCGCCCACCTGACAACCTCTCATCCCTGCTCTGCAGATCCGGTCCCATCCCCACTGCCCACCCCACCCCCCCAGCACTCCACCCAGTTCAACGTTCCACGAACCCCCAGAACCAGCCCTCATCAACAGGCAGCAAGAAGGGCCCCCCGCCCATCGCCCCACAACGCCAGCCGGGTGAACGTTGGCAGGTCCTGAGGCAGCTGGCAAGACGCCTGCAGCTGAAAGATACAAGGCCAGGGACAGGACAGTCCCATCCCCAGGAGGCAGGGAGTATACAGGCTGGGGAAGTTTGCCCTTGCGTGGGGTGGTGATGGAGGAGGCTCAGCAAGTCTTCTGGACTGTGAACCTGTGTCTGCCACTGTGTGCTGGGTGGTGGTCATCTTTCCCACCAGGCTGTGGCCTCTGCAACCTTCAAGGGAGGAGCAGGTCCCATTGGCTGAGCACAGCCTTGTACCGTGAACTGGAACAAGCAGCCTCCTTCCTGGCCACAGGTTCCATGTCCTTATATGGACTCATCTTTGCCTATTGCGACACACACTCAGTGAACACCTACTACGCGCTGCAAAGAGCCCCGCAGGCCTGAGGTGCCCCCACCTCACCACTCTTCCTATTTTTGTGTAAAAATCCAGCTTCTTGTCACCACCTCCAAGGAGGGGGAGGAGGAGGAAGGCAGGTTCCTCTAGGCTGAGCCGAATGCCCCTCTGTGGTCCCACGCCACTGATCGCTGCATGCCCACCACCTGGGTACACACAGTCTGTGATTCCCGGAGCAGAACGGACCCTGCCCACCCGGTCTTGTGTGCTACTCAGTGGACAGACCCAAGGCAAGAAAGGGTGACAAGGACAGGGTCTTC SEQ ID NO: 36 β-actin序列經重亞硫酸氫鹽處理過後轉化的序列 AGTTCGGGGCGGGGTGGGGTTGGAGTTTTTGTTTTTTGGTTAGTTGAATGGAGGTTTAGTGGTAATATAGGTTTTGTTTGGGGATTAGGTTTGTTTTGTATTTTATTTTGTTGTTTGGAGTCGTTTATTTGATAATTTTTTATTTTTGTTTTGTAGATTCGGTTTTATTTTTATTGTTTATTTTATTTTTTTAGTATTTTATTTAGTTTAACGTTTTACGAATTTTTAGAATTAGTTTTTATTAATAGGTAGTAAGAAGGGTTTTTCGTTTATCGTTTTATAACGTTAGTCGGGTGAACGTTGGTAGGTTTTGAGGTAGTTGGTAAGACGTTTGTAGTTGAAAGATATAAGGTTAGGGATAGGATAGTTTTATTTTTAGGAGGTAGGGAGTATATAGGTTGGGGAAGTTTGTTTTTGCGTGGGGTGGTGATGGAGGAGGTTTAGTAAGTTTTTTGGATTGTGAATTTGTGTTTGTTATTGTGTGTTGGGTGGTGGTTATTTTTTTTATTAGGTTGTGGTTTTTGTAATTTTTAAGGGAGGAGTAGGTTTTATTGGTTGAGTATAGTTTTGTATCGTGAATTGGAATAAGTAGTTTTTTTTTTGGTTATAGGTTTTATGTTTTTATATGGATTTATTTTTGTTTATTGCGATATATATTTAGTGAATATTTATTACGCGTTGTAAAGAGTTTCGTAGGTTTGAGGTGTTTTTATTTTATTATTTTTTTTATTTTTGTGTAAAAATTTAGTTTTTTGTTATTATTTTTAAGGAGGGGGAGGAGGAGGAAGGTAGGTTTTTTTAGGTTGAGTCGAATGTTTTTTTGTGGTTTTACGTTATTGATCGTTGTATGTTTATTATTTGGGTATATATAGTTTGTGATTTTCGGAGTAGAACGGATTTTGTTTATTCGGTTTTGTGTGTTATTTAGTGGATAGATTTAAGGTAAGAAAGGGTGATAAGGATAGGGTTTTT Various research data show that the methylation status and distribution of the same gene are not uniform. Therefore, for the same gene, methylation primers and probe detection systems designed in different regions are selected to diagnose the same sample and the same tumor. The detection efficiency is not the same, and sometimes the selected region is not suitable, resulting in no diagnostic effect on the tumor at all. After repeated research and comparison by the inventor, the regional sequences of some HOXA7 and HOXA9 are as follows: [Table 17] HOXA7 sequences to be detected sequence name sequence SEQ ID NO: 25 HOXA7 Region 1 Reference Sequence SEQ ID NO: 26 HOXA7 region 1 sequence converted after bisulfite treatment SEQ ID NO: 27 HOXA7 Region 2 Reference Sequence CGTCCGGCTACGGCCTGGGCGCCGACGCCTACGGCAACCTGCCCTGCGCCTCCTACGACCAAAACATCCCCGGGCTCTGCAGTGACCTCGCCAAAGGCGCCTGCGACAAGACGGACGAGGGCGCGCTGCATGGCGCGGCTGAGGCCAATTTCCGCATCTACCCCTGGATGCGGTCTTCAGGTAGGCGCAGTCGCTAGGCGGGCCAGGCTGGCGGAGCGGGACCGGGAGCGGGGAGCGCAGCGCTGGGGAGCGCGGAGCGCGGGGCGCGGGGCCGGAAGAGCGGAGCCAGGCTGTTGCGAGCCGGTAGCCCCGTGACTCCCGGCGCA SEQ ID NO: 28 HOXA7 region 2 reference sequence converted after bisulfite treatment CGTTCGGTTACGGTTTGGGCGTCGACGTTTACGGTAATTTGTTTTGCGTTTTTTACGATTAAAATATTTTCGGGTTTTGTAGTGATTTCGTTAAAGGCGTTTGCGATAAGACGGACGAGGGCGCGTTGTATGGCGCGGTTGAGGTTAATTTTCGTATTTATTTTTGGATGCGGTTTTTAGGTAGGCGTAGTCGTTAGGCGGGTTAGGTTGGCGGAGCGGGATCGGGAGCGGGGAGCGTAGCGTTGGGGAGCGCGGAGCGCGGGGCGCGGGGTCGGAAGAGCGGAGTTAGGTTGTTGCGAGTCGGTAGTTTCGTGATTTTCGGCGTA [Continued Table 17] HOXA9 sequences to be detected sequence name sequence SEQ ID NO: 29 HOXA9 Region 1 Reference Sequence AATTTCCGTGGGTCGGGCCGGGCGGGCCAGGCGCTGGGCACGGTGATGGCCACCACTGGGGCCCTGGGCAACTACTACGTGGACTCGTTCCTGCTGGGCGCCGACGCCGCGGATGAGCTGAGCGTTGGCCGCTATGCGCCGGGGACCCTGGGCCAGCCTCCCCGGCAGGCGGCGACGCTGGCCGAGCACCCCGACTTCAGCCCGTGCAGCTTCCAGTCCAAGGCGACGGTGTTTGGCGCCTCGTGGAACCCAGTGCACGCGGCGGGCGCCAACGCTGTACCCGCTGCGGTGTACCACCACCATCACCACCACCCCTACGTGCACCCCCAGGCGCCCGTGGCGGCG SEQ ID NO: 30 HOXA9 region 1 sequence converted after bisulfite treatment AATTTTCGTGGGTCGGGTCGGGCGGGTTAGGCGTTGGGTACGGTGATGGTTATTATTGGGGTTTTGGGTAATTATTACGTGGATTCGTTTTTGTTGGGCGTCGACGTCGCGGATGAGTTGAGCGTTGGTCGTTATGCGTCGGGGATTTTGGGTTAGTTTTTTCGGTAGGCGGCGACGTTGGTCGAGTATTTCGATTTTAGTTCGTGTAGTTTTTAGTTTAAGGCGACGGTGTTTGGCGTTTCGTGGAATTTAGTGTACGCGGCGGGCGTTAACGTTGTATTCGTTGCGGTGTATTATTATTATTATTATTATTTTTACGTGTATTTTTAGGCGTTCGTGGCGGCG SEQ ID NO: 31 HOXA9 Region 2 Reference Sequence SEQ ID NO: 32 HOXA9 region 2 reference sequence converted after bisulfite treatment SEQ ID NO: 33 HOXA9 region 3 reference sequence SEQ ID NO: 34 HOXA9 region 3 reference sequence converted after bisulfite treatment SEQ ID NO: 35 β-actin region reference sequence SEQ ID NO: 36 β-actin sequence converted after bisulfite treatment

我們根據HOXA7序列的區域1、區域2，以及HOXA9序列區域1、區域2和區域3設計不同的甲基化引子和探針，各引子探針信息見表19。We designed different methylation primers and probes according to Region 1, Region 2 of HOXA7 sequence, and Region 1, Region 2 and Region 3 of HOXA9 sequence. See Table 19 for the information of each primer and probe.

針對HOXA7序列，其中組8、組9、組10是根據區域1設計的甲基化引子和探針；組1、組2、組3、組4、組5、組6、組7是根據區域2設計的甲基化引子和探針。For HOXA7 sequence, group 8, group 9, group 10 are methylated primers and probes designed according to region 1; group 1, group 2, group 3, group 4, group 5, group 6, group 7 are based on region 2 Designed methylation primers and probes.

針對HOXA9序列其中組1、組2、組3、組4和組5是根據區域1設計的甲基化引子和探針；組6、組7、組8是根據區域2設計的甲基化引子和探針；組9、組10、組11是根據區域3設計的甲基化引子和探針。For HOXA9 sequence, group 1, group 2, group 3, group 4 and group 5 are methylated primers and probes designed according to region 1; group 6, group 7, and group 8 are methylated primers designed according to region 2 and probes; set 9, set 10, set 11 are methylated primers and probes designed according to region 3.

在36例肺組織樣本檢測以下引子探針組合，其中正常組織樣本11例，癌組織樣本25例，25例癌症組樣本中有鱗癌4例，腺癌21例。檢測結果如下表18。 [表18] HOXA7的引子在組織中的檢測結果 所處區域 組別 引子探針組合 特異性 靈敏性 區域2 組1 H7-F2, H7-R2, H7-P2 100% 80% 區域2 組2 H7-F3, H7-R3, H7-P3 100% 76% 區域2 組3 H7-F4, H7-R4, H7-P4 100% 56% 區域2 組4 H7-F5, H7-R5, H7-P5 100% 72% 區域2 組5 H7-F6, H7-R6, H7-P6 100% 80% 區域2 組6 H7-F7, H7-R7, H7-P7 100% 72% 區域2 組7 H7-F8, H7-R8, H7-P8 100% 56% 區域1 組8 H7-F9, H7-R9, H7-P9 100% 48% 區域1 組9 H7-F10, H7-R10, H7-P10 100% 16% 區域1 組10 H7-F11, H7-R11, H7-P11 100% 24% [續表18]  HOXA9的引子在組織中的檢測結果 所處區域 組別 引子探針組合 特異性 靈敏性 區域1 組1 H9-F2, H9-R2, H9-P2 100% 76% 區域1 組2 H9-F3, H9-R3, H9-P3 100% 76% 區域1 組3 H9-F4, H9-R4, H9-P4 100% 40% 區域1 組4 H9-F5, H9-R5, H9-P5 100% 60% 區域1 組5 H9-F6, H9-R6, H9-P6 100% 68% 區域2 組6 H9-F7, H9-R7, H9-P7 100% 32% 區域2 組7 H9-F8, H9-R8, H9-P8 100% 20% 區域2 組8 H9-F9, H9-R9, H9-P9 100% 12% 區域3 組9 H9-F10, H9-R10, H9-P10 100% 16% 區域3 組10 H9-F11, H9-R11, H9-P11 100% 24% 區域3 組11 H9-F12, H9-R12, H9-P12 100% 24% The following primer-probe combinations were detected in 36 lung tissue samples, including 11 normal tissue samples, 25 cancer tissue samples, 4 squamous cell carcinomas and 21 adenocarcinomas in the 25 cancer group samples. The test results are shown in Table 18 below. [Table 18] Detection results of primers for HOXA7 in tissues In the area group primer probe combination specificity Sensitivity Zone 2 Group 1 H7-F2, H7-R2, H7-P2 100% 80% Zone 2 group 2 H7-F3, H7-R3, H7-P3 100% 76% Zone 2 group 3 H7-F4, H7-R4, H7-P4 100% 56% Zone 2 Group 4 H7-F5, H7-R5, H7-P5 100% 72% Zone 2 Group 5 H7-F6, H7-R6, H7-P6 100% 80% Zone 2 Group 6 H7-F7, H7-R7, H7-P7 100% 72% Zone 2 Group 7 H7-F8, H7-R8, H7-P8 100% 56% Area 1 Group 8 H7-F9, H7-R9, H7-P9 100% 48% Area 1 Group 9 H7-F10, H7-R10, H7-P10 100% 16% Area 1 group 10 H7-F11, H7-R11, H7-P11 100% twenty four% [Continued from Table 18] Detection results of HOXA9 primers in tissues In the area group primer probe combination specificity Sensitivity Area 1 Group 1 H9-F2, H9-R2, H9-P2 100% 76% Area 1 group 2 H9-F3, H9-R3, H9-P3 100% 76% Area 1 group 3 H9-F4, H9-R4, H9-P4 100% 40% Area 1 Group 4 H9-F5, H9-R5, H9-P5 100% 60% Area 1 Group 5 H9-F6, H9-R6, H9-P6 100% 68% Zone 2 Group 6 H9-F7, H9-R7, H9-P7 100% 32% Zone 2 Group 7 H9-F8, H9-R8, H9-P8 100% 20% Zone 2 Group 8 H9-F9, H9-R9, H9-P9 100% 12% Zone 3 Group 9 H9-F10, H9-R10, H9-P10 100% 16% Zone 3 group 10 H9-F11, H9-R11, H9-P11 100% twenty four% Zone 3 Group 11 H9-F12, H9-R12, H9-P12 100% twenty four%

結果顯示，針對HOXA7，無論區域1如何設計引子和探針，針對區域1的檢測靈敏度最高僅能達到48%，而無論採用本公開設計的何種引子和探針，區域2的檢測靈敏度最低也可達到56%，最高達到80%。因此，HOXA7的區域2的檢出率明顯較區域1高（見表18）。The results show that for HOXA7, no matter how primers and probes are designed in region 1, the highest detection sensitivity for region 1 can only reach 48%, and regardless of the primers and probes designed in the present disclosure, region 2 has the lowest detection sensitivity. Up to 56%, up to 80%. Therefore, the detection rate of region 2 of HOXA7 was significantly higher than that of region 1 (see Table 18).

針對HOXA9，無論區域2和3如何設計引子和探針，針對這兩個區域的檢測靈敏度最高僅能達到32%，而無論採用本公開設計的何種引子和探針，區域1的檢測靈敏度最低也可達到40%，最高達到76%。因此，HOXA7區域1的檢出率明顯較區域2和3高（見表18）。For HOXA9, no matter how primers and probes are designed for regions 2 and 3, the detection sensitivity for these two regions can only reach 32% at the highest, and regardless of the primers and probes designed in this disclosure, region 1 has the lowest detection sensitivity It can also reach 40%, up to 76%. Therefore, the detection rate of HOXA7 region 1 was significantly higher than that of regions 2 and 3 (see Table 18).

二、引子和探針對檢測效果的影響2. The influence of primers and probes on the detection effect

除了檢測區域會影響檢測效果，引子和探針也對腫瘤標記的檢測效果有極大的影響，發明人在研究過程中，設計了多對引子及其對應的探針，以尋找到盡可能提高檢測靈敏度和特異性的探針和引子，以使本公開的檢測試劑能夠實際應用到臨床檢測中。部分引子和探針如下表19所示，檢測結果如表20所示。所有的引子和探針均由英濰捷基（上海）貿易有限公司合成。 [表19] HOXA7的引子和探針 名稱 序列編號 序列 作用 H7-F2 SEQ ID NO: 1 TAAAGGCGTTTGCGATAAGAC HOXA7基因上游引子 H7-R2 SEQ ID NO: 2 TAACCCGCCTAACGACTACG HOXA7基因下游引子 H7-P2 SEQ ID NO: 3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7基因檢測探針 H7-F3 SEQ ID NO: 37 GCGTTTGCGATAAGACGGAC HOXA7基因上游引子 H7-R3 SEQ ID NO: 38 CCAACCTAACCCGCCTAACG HOXA7基因下游引子 H7-P3 SEQ ID NO: 39 FAM-CGTTGTATGGCGCGGTTGAGG-BQ1 HOXA7基因檢測探針 H7-F4 SEQ ID NO: 40 CGTTCGGTTACGGTTTGGGC HOXA7基因上游引子 H7-R4 SEQ ID NO: 41 GCCCTCGTCCGTCTTATCGC HOXA7基因下游引子 H7-P4 SEQ ID NO: 42 FAM-TCGACGTTTACGGTAATTTGTTTTGCG-BQ1 HOXA7基因檢測探針 H7-F5 SEQ ID NO: 43 ATTTCGTTAAAGGCGTTTGC HOXA7基因上游引子 H7-R5 SEQ ID NO: 44 TCCGCCAACCTAACCCG HOXA7基因下游引子 H7-P5 SEQ ID NO: 45 FAM-CGGACGAGGGCGCGTTGTAT-BQ1 HOXA7基因檢測探針 H7-F6 SEQ ID NO: 46 CGTTAAAGGCGTTTGCGATAAGAC HOXA7基因上游引子 H7-R6 SEQ ID NO: 47 TACGCTCCCCGCTCCCGAT HOXA7基因下游引子 H7-P6 SEQ ID NO: 48 FAM-GACGGACGAGGGCGCGTTGTATG-BQ1 HOXA7基因檢測探針 H7-F7 SEQ ID NO: 49 CAACGCTACGCTCCCCGCT HOXA7基因下游引子 H7-R7 SEQ ID NO: 50 GCGATAAGACGGACGAGGGC HOXA7基因上游引子 H7-P7 SEQ ID NO: 51 FAM-CCGATCCCGCTCCGCCAACC-BQ1 HOXA7基因檢測探針 H7-F8 SEQ ID NO: 52 CGTTAGGCGGGTTAGGTTGGC HOXA7基因上游引子 H7-R8 SEQ ID NO: 53 GCCGAAAATCACGAAACTACCG HOXA7基因下游引子 H7-P8 SEQ ID NO: 54 FAM-AGCGGGATCGGGAGCGGG-BQ1 HOXA7基因檢測探針 H7-F9 SEQ ID NO: 55 TCGGGTCGTTTGGCGTTC HOXA7基因上游引子 H7-R9 SEQ ID NO: 56 AACCTAACGCGTCCCGCG HOXA7基因下游引子 H7-P9 SEQ ID NO: 57 FAM-TTGGTCGTTAGTTGGGCGTTTTCGC-BQ1 HOXA7基因檢測探針 H7-F10 SEQ ID NO: 58 GGCGTTCGTAGATTTTAGTGC HOXA7基因上游引子 H7-R10 SEQ ID NO: 59 CAAATAATCTCGCTCCGCA HOXA7基因下游引子 H7-P10 SEQ ID NO: 60 FAM-GGTCGTTAGTTGGGCGTTTTCG-BQ1 HOXA7基因檢測探針 H7-F11 SEQ ID NO: 61 GCGTTAGGTTCGTCGGGC HOXA7基因上游引子 H7-R11 SEQ ID NO: 62 AAACTCGCCGCAACACGTAA HOXA7基因下游引子 H7-P11 SEQ ID NO: 63 FAM-CGGATTTTTTGGTCGTATATTTGAGT-BQ1 HOXA7基因檢測探針 [續表19] HOXA9的引子和探針 名稱 序列編號 序列 作用 H9-F2 SEQ ID NO: 1 TTAGTTTTTTCGGTAGGCGGC HOXA9基因上游引子 H9-R2 SEQ ID NO: 2 AAACGCCAAACACCGTCG HOXA9基因下游引子 H9-P2 SEQ ID NO: 3 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9基因檢測探針 H9-F3 SEQ ID NO: 64 AATTTTCG TGGGTCG GGTC HOXA9基因上游引子 H9-R3 SEQ ID NO: 65 CCAAACACCGTCGCCTTAA HOXA9基因下游引子 H9-P3 SEQ ID NO: 66 FAM-ACGTGGATTCGTTTTTGTTGGGC-BQ1 HOXA9基因檢測探針 H9-F4 SEQ ID NO: 67 CGTCGCGGATGAGTTGAGC HOXA9基因上游引子 H9-R4 SEQ ID NO: 68 CACGAACGCCTAAAAATACACG HOXA9基因下游引子 H9-P4 SEQ ID NO: 69 FAM-TGGTCGTTATGCGTCGGGGATT-BQ1 HOXA9基因檢測探針 H9-F5 SEQ ID NO: 70 AGCGTTGGTCGTTATGCGTC HOXA9基因上游引子 H9-R5 SEQ ID NO: 71 CCAAACACCGTCGCCTTAAAC HOXA9基因下游引子 H9-P5 SEQ ID NO: 72 FAM-GACGTTGGTCGAGTATTTCGATTTTAG-BQ1 HOXA9基因檢測探針 H9-F6 SEQ ID NO: 73 CGACGTTGGTCGAGTATTTC HOXA9基因上游引子 H9-R6 SEQ ID NO: 74 GCCACGAACGCCTAAAAAT HOXA9基因下游引子 H9-P6 SEQ ID NO: 75 FAM-TTAGTTTAAGGCGACGGTGTTTGG-BQ1 HOXA9基因檢測探針 H9-F7 SEQ ID NO: 76 CGTTTTGGTGGCGGTTGGTC HOXA9基因上游引子 H9-R7 SEQ ID NO: 77 AATAATCCCTACGATCCCCGA HOXA9基因下游引子 H9-P7 SEQ ID NO: 78 FAM-GGGCGTTTTTCGTTTTAGGCGG-BQ1 HOXA9基因檢測探針 H9-F8 SEQ ID NO: 79 TCGTTTGGGACGGATTTTGC HOXA9基因上游引子 H9-R8 SEQ ID NO: 80 ACAAATTCGTTTACCTCGCCG HOXA9基因下游引子 H9-P8 SEQ ID NO: 81 FAM-ATTCGTTTTTTTCGGGGATCGTAGG-BQ1 HOXA9基因檢測探針 H9-F9 SEQ ID NO: 82 AATAGCG TGCG GAGTGATTTAC HOXA9基因上游引子 H9-R9 SEQ ID NO: 83 CGACCCGACCCACGAAAAT HOXA9基因下游引子 H9-P9 SEQ ID NO: 84 FAM-CGTTATTGTTTTGTTGGACGGGTACG-BQ1 HOXA9基因檢測探針 H9-F10 SEQ ID NO: 85 TTTAGGCGTTCGTGGCGGC HOXA9基因上游引子 H9-R10 SEQ ID NO: 86 CCCTTCTAACCGACAACGATTC HOXA9基因下游引子 H9-P10 SEQ ID NO: 87 FAM-CGGACGGTAGGTATATGCGTTTTTGG-BQ1 HOXA9基因檢測探針 H9-F11 SEQ ID NO: 88 AATTCGTCGTCGTTTTTACGTC HOXA9基因上游引子 H9-R11 SEQ ID NO: 89 TCCCGTAACCCTACGAACCG HOXA9基因下游引子 H9-P11 SEQ ID NO: 90 FAM-TCGGATTTGTTGGTTCGTTAGGTTTTTTTC-BQ1 HOXA9基因檢測探針 H9-F12 SEQ ID NO: 91 TTAGATTGTTCGTGAGCGGC HOXA9基因上游引子 H9-R12 SEQ ID NO: 92 TTATAACCCGAATCGCCCG HOXA9基因下游引子 H9-P12 SEQ ID NO: 93 FAM-TTGGGACGTTGGGGGAGACGGT-BQ1 HOXA9基因檢測探針 [表20] HOXA7在組織中的檢測結果 組別 引子探針組合 特異性 靈敏性 組1 H7-F2, H7-R2, H7-P2 100% 80% 組2 H7-F3, H7-R3, H7-P3 100% 76% 組3 H7-F4, H7-R4, H7-P4 100% 56% 組4 H7-F5, H7-R5, H7-P5 100% 72% 組5 H7-F6, H7-R6, H7-P6 100% 80% 組6 H7-F7, H7-R7, H7-P7 100% 72% 組7 H7-F8, H7-R8, H7-P8 100% 56% 組8 H7-F9, H7-R9, H7-P9 100% 48% 組9 H7-F10, H7-R10, H7-P10 100% 16% 組10 H7-F11, H7-R11, H7-P11 100% 24% [續表20] HOXA9在組織中的檢測結果 組別 引子探針組合 特異性 靈敏性 組1 H9-F2, H9-R2, H9-P2 100% 76% 組2 H9-F3, H9-R3, H9-P3 100% 76% 組3 H9-F4, H9-R4, H9-P4 100% 40% 組4 H9-F5, H9-R5, H9-P5 100% 60% 組5 H9-F6, H9-R6, H9-P6 100% 68% 組6 H9-F7, H9-R7, H9-P7 100% 32% 組7 H9-F8, H9-R8, H9-P8 100% 20% 組8 H9-F9, H9-R9, H9-P9 100% 12% 組9 H9-F10, H9-R10, H9-P10 100% 16% 組10 H9-F11, H9-R11, H9-P11 100% 24% 組11 H9-F12, H9-R12, H9-P12 100% 24% In addition to the detection area affecting the detection effect, primers and probes also have a great impact on the detection effect of tumor markers. Sensitivity and specificity of probes and primers, so that the detection reagent of the present disclosure can be practically applied to clinical detection. Some primers and probes are shown in Table 19 below, and the detection results are shown in Table 20. All primers and probes were synthesized by Yingweijieji (Shanghai) Trading Co., Ltd. [Table 19] Primers and probes for HOXA7 name serial number sequence effect H7-F2 SEQ ID NO: 1 TAAAGGCGTTTGCGATAAGAC Primer upstream of HOXA7 gene H7-R2 SEQ ID NO: 2 TAACCCGCCTAACGACTACG Primer downstream of HOXA7 gene H7-P2 SEQ ID NO: 3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7 gene detection probe H7-F3 SEQ ID NO: 37 GCGTTTGCGATAAGACGGAC Primer upstream of HOXA7 gene H7-R3 SEQ ID NO: 38 CCAACCTAACCCGCCTAACG Primer downstream of HOXA7 gene H7-P3 SEQ ID NO: 39 FAM-CGTTGTATGGCGCGGTTGAGG-BQ1 HOXA7 gene detection probe H7-F4 SEQ ID NO: 40 CGTTCGGTTACGGTTTGGGC Primer upstream of HOXA7 gene H7-R4 SEQ ID NO: 41 GCCCTCGTCCGTCTTATCGC Primer downstream of HOXA7 gene H7-P4 SEQ ID NO: 42 FAM-TCGACGTTTACGGTAATTTGTTTTGCG-BQ1 HOXA7 gene detection probe H7-F5 SEQ ID NO: 43 ATTTCGTTAAAGGCGTTTGC Primer upstream of HOXA7 gene H7-R5 SEQ ID NO: 44 TCCGCCAACCTAACCCG Primer downstream of HOXA7 gene H7-P5 SEQ ID NO: 45 FAM-CGGACGAGGGCGCGTTGTAT-BQ1 HOXA7 gene detection probe H7-F6 SEQ ID NO: 46 CGTTAAAGGCGTTTGCGATAAGAC Primer upstream of HOXA7 gene H7-R6 SEQ ID NO: 47 TACGCTCCCCGCTCCCGAT Primer downstream of HOXA7 gene H7-P6 SEQ ID NO: 48 FAM-GACGGACGAGGGCGCGTTGTATG-BQ1 HOXA7 gene detection probe H7-F7 SEQ ID NO: 49 CAACGCTACGCTCCCCGCT Primer downstream of HOXA7 gene H7-R7 SEQ ID NO: 50 GCGATAAGACGGACGAGGGC Primer upstream of HOXA7 gene H7-P7 SEQ ID NO: 51 FAM-CCGATCCCGCTCCGCCAACC-BQ1 HOXA7 gene detection probe H7-F8 SEQ ID NO: 52 CGTTAGGCGGGTTAGGTTGGC Primer upstream of HOXA7 gene H7-R8 SEQ ID NO: 53 GCCGAAAATCACGAAACTACCG Primer downstream of HOXA7 gene H7-P8 SEQ ID NO: 54 FAM-AGCGGGATCGGGAGCGGG-BQ1 HOXA7 gene detection probe H7-F9 SEQ ID NO: 55 TCGGGTCGTTTGGCGTTC Primer upstream of HOXA7 gene H7-R9 SEQ ID NO: 56 AACCTAACGCGTCCCGCG Primer downstream of HOXA7 gene H7-P9 SEQ ID NO: 57 FAM-TTGGTCGTTAGTTGGGCGTTTTCGC-BQ1 HOXA7 gene detection probe H7-F10 SEQ ID NO: 58 GGCGTTCGTAGATTTTAGTGC Primer upstream of HOXA7 gene H7-R10 SEQ ID NO: 59 CAAATAATCTCGCTCCGCA Primer downstream of HOXA7 gene H7-P10 SEQ ID NO: 60 FAM-GGTCGTTAGTTGGGCGTTTTCG-BQ1 HOXA7 gene detection probe H7-F11 SEQ ID NO: 61 GCGTTAGGTTCGTCGGGC Primer upstream of HOXA7 gene H7-R11 SEQ ID NO: 62 AAACTCGCCGCAACACGTAA Primer downstream of HOXA7 gene H7-P11 SEQ ID NO: 63 FAM-CGGATTTTTTGGTCGTATATTTGAGT-BQ1 HOXA7 gene detection probe [Continued from Table 19] Primers and probes for HOXA9 name serial number sequence effect H9-F2 SEQ ID NO: 1 TTAGTTTTTTTCGGTAGGCGGC Primer upstream of HOXA9 gene H9-R2 SEQ ID NO: 2 AAACGCCAAACACCGTCG Primers downstream of HOXA9 gene H9-P2 SEQ ID NO: 3 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9 gene detection probe H9-F3 SEQ ID NO: 64 AATTTT CG TGGGT CG GGT C Primer upstream of HOXA9 gene H9-R3 SEQ ID NO: 65 CCAAACACCGTCGCCTTAA Primers downstream of HOXA9 gene H9-P3 SEQ ID NO: 66 FAM-ACGTGGATTCGTTTTTGTTGGGC-BQ1 HOXA9 gene detection probe H9-F4 SEQ ID NO: 67 CGTCGCGGATGAGTTGAGC Primer upstream of HOXA9 gene H9-R4 SEQ ID NO: 68 CACGAACGCCTAAAAATACACG Primers downstream of HOXA9 gene H9-P4 SEQ ID NO: 69 FAM-TGGTCGTTATGCGTCGGGGATT-BQ1 HOXA9 gene detection probe H9-F5 SEQ ID NO: 70 AGCGTTGGTCGTTATGCGTC Primer upstream of HOXA9 gene H9-R5 SEQ ID NO: 71 CCAAACACCGTCGCCTTAAAC Primers downstream of HOXA9 gene H9-P5 SEQ ID NO: 72 FAM-GACGTTGGTCGAGTATTTCGATTTTAG-BQ1 HOXA9 gene detection probe H9-F6 SEQ ID NO: 73 CGACGTTGGTCGAGTATTTC Primer upstream of HOXA9 gene H9-R6 SEQ ID NO: 74 GCCACGAACGCCTAAAAAT Primers downstream of HOXA9 gene H9-P6 SEQ ID NO: 75 FAM-TTAGTTTAAGGCGACGGTGTTTGG-BQ1 HOXA9 gene detection probe H9-F7 SEQ ID NO: 76 CGTTTTGGTGGCGGTTGGTC Primer upstream of HOXA9 gene H9-R7 SEQ ID NO: 77 AATAATCCCTACGATCCCCGA Primers downstream of HOXA9 gene H9-P7 SEQ ID NO: 78 FAM-GGGCGTTTTTTCGTTTTAGGCGG-BQ1 HOXA9 gene detection probe H9-F8 SEQ ID NO: 79 TCGTTTGGGACGGATTTTGC Primer upstream of HOXA9 gene H9-R8 SEQ ID NO: 80 ACAAATTCGTTTACCTCGCCG Primers downstream of HOXA9 gene H9-P8 SEQ ID NO: 81 FAM-ATTCGTTTTTTTCGGGGATCGTAGG-BQ1 HOXA9 gene detection probe H9-F9 SEQ ID NO: 82 AATAG CG TG CG GAGTGATTTA C Primer upstream of HOXA9 gene H9-R9 SEQ ID NO: 83 CGACCCGACCCACGAAAAT Primers downstream of HOXA9 gene H9-P9 SEQ ID NO: 84 FAM-CGTTATTGTTTTGTTGGACGGGTACG-BQ1 HOXA9 gene detection probe H9-F10 SEQ ID NO: 85 TTTAGGCGTTCGTGGCGGC Primer upstream of HOXA9 gene H9-R10 SEQ ID NO: 86 CCCTTCTAACCGACAACGATTC Primers downstream of HOXA9 gene H9-P10 SEQ ID NO: 87 FAM-CGGACGGTAGGTATATGCGTTTTTGG-BQ1 HOXA9 gene detection probe H9-F11 SEQ ID NO: 88 AATTCGTCGTCGTTTTTACGTC Primer upstream of HOXA9 gene H9-R11 SEQ ID NO: 89 TCCCGTAACCCTACGAACCG Primers downstream of HOXA9 gene H9-P11 SEQ ID NO: 90 FAM-TCGGATTTGTTGGTTCGTTAGGTTTTTTTTC-BQ1 HOXA9 gene detection probe H9-F12 SEQ ID NO: 91 TTAGATTGTTCGTGAGCGGC Primer upstream of HOXA9 gene H9-R12 SEQ ID NO: 92 TTATAACCCGAATCGCCCG Primers downstream of HOXA9 gene H9-P12 SEQ ID NO: 93 FAM-TTGGGACGTTGGGGGAGACGGT-BQ1 HOXA9 gene detection probe [Table 20] Detection results of HOXA7 in tissues group primer probe combination specificity Sensitivity Group 1 H7-F2, H7-R2, H7-P2 100% 80% group 2 H7-F3, H7-R3, H7-P3 100% 76% group 3 H7-F4, H7-R4, H7-P4 100% 56% Group 4 H7-F5, H7-R5, H7-P5 100% 72% Group 5 H7-F6, H7-R6, H7-P6 100% 80% Group 6 H7-F7, H7-R7, H7-P7 100% 72% Group 7 H7-F8, H7-R8, H7-P8 100% 56% Group 8 H7-F9, H7-R9, H7-P9 100% 48% Group 9 H7-F10, H7-R10, H7-P10 100% 16% group 10 H7-F11, H7-R11, H7-P11 100% twenty four% [Continued Table 20] Detection results of HOXA9 in tissues group primer probe combination specificity Sensitivity Group 1 H9-F2, H9-R2, H9-P2 100% 76% group 2 H9-F3, H9-R3, H9-P3 100% 76% group 3 H9-F4, H9-R4, H9-P4 100% 40% Group 4 H9-F5, H9-R5, H9-P5 100% 60% Group 5 H9-F6, H9-R6, H9-P6 100% 68% Group 6 H9-F7, H9-R7, H9-P7 100% 32% Group 7 H9-F8, H9-R8, H9-P8 100% 20% Group 8 H9-F9, H9-R9, H9-P9 100% 12% Group 9 H9-F10, H9-R10, H9-P10 100% 16% group 10 H9-F11, H9-R11, H9-P11 100% twenty four% Group 11 H9-F12, H9-R12, H9-P12 100% twenty four%

本公開中，採用表19中的引子和探針對組織樣本的進行了驗證。在36例肺組織樣本檢測以上10組引子探針組合，其中正常組織樣本11例，癌組織樣本25例，25例癌症組樣本中有鱗癌4例，腺癌21例。檢測結果如上表20。In this disclosure, the primers and probes in Table 19 were used for validation of tissue samples. The above 10 primer-probe combinations were detected in 36 lung tissue samples, including 11 normal tissue samples, 25 cancer tissue samples, 4 squamous cell carcinomas and 21 adenocarcinoma samples in the 25 cancer samples. The test results are shown in Table 20 above.

結果顯示HOXA7的組1、組2、組4、組5、組6均有較好的檢出率。HOXA9的組1、組2、組4、組5均有較好的檢出率。The results showed that HOXA7 group 1, group 2, group 4, group 5, and group 6 all had good detection rates. HOXA9 group 1, group 2, group 4 and group 5 all had good detection rates.

為了進一步驗證其在痰液中的檢出率，我們選取了22例痰液標本，採用表19中的引子和探針進行驗證，其中包括7例正常對照，15例肺癌對照，15例肺癌中有鱗癌7例，腺癌7例，大細胞癌1例，檢測結果如下表21。 [表21] HOXA7在痰液中的檢測結果 組別 引子探針組合 特異性 靈敏性 組1 H7-F2, H7-R2, H7-P2 100% 73.3% 組2 H7-F3, H7-R3, H7-P3 100% 53.3% 組4 H7-F5, H7-R5, H7-P5 100% 60.0% 組5 H7-F6, H7-R6, H7-P6 100% 53.3% 組6 H7-F7, H7-R7, H7-P7 100% 46.7% [續表21] HOXA9在痰液中的檢測結果 組別 引子探針組合 特異性 靈敏性 組1 H9-F2, H9-R2, H9-P2 100% 66.7% 組2 H9-F3, H9-R3, H9-P3 100% 44.6% 組4 H9-F5, H9-R5, H9-P5 100% 33.3% 組5 H9-F6, H9-R6, H9-P6 100% 40.0% In order to further verify its detection rate in sputum, we selected 22 sputum samples and used the primers and probes in Table 19 for verification, including 7 normal controls, 15 lung cancer controls, and 15 lung cancer patients. There were 7 cases of squamous cell carcinoma, 7 cases of adenocarcinoma, and 1 case of large cell carcinoma. The test results are shown in Table 21. [Table 21] Detection results of HOXA7 in sputum group primer probe combination specificity Sensitivity Group 1 H7-F2, H7-R2, H7-P2 100% 73.3% group 2 H7-F3, H7-R3, H7-P3 100% 53.3% Group 4 H7-F5, H7-R5, H7-P5 100% 60.0% Group 5 H7-F6, H7-R6, H7-P6 100% 53.3% Group 6 H7-F7, H7-R7, H7-P7 100% 46.7% [Continued table 21] The detection result of HOXA9 in sputum group primer probe combination specificity Sensitivity Group 1 H9-F2, H9-R2, H9-P2 100% 66.7% group 2 H9-F3, H9-R3, H9-P3 100% 44.6% Group 4 H9-F5, H9-R5, H9-P5 100% 33.3% Group 5 H9-F6, H9-R6, H9-P6 100% 40.0%

從22例痰液標本的檢測結果顯示，HOXA7的組1：H7-F2,H7-R2,H7-P2的檢出率最高，達到73.3%。雖然在組織樣本中，組1和組5的靈敏度均能達到80%，但針對痰液檢測樣本，組5的靈敏度卻大幅下降至53.3%。The detection results of 22 sputum samples showed that HOXA7 group 1: H7-F2, H7-R2, H7-P2 had the highest detection rate, reaching 73.3%. Although both groups 1 and 5 achieved 80% sensitivity in tissue samples, for sputum samples, the sensitivity of group 5 dropped significantly to 53.3%.

HOXA9的組1：H9-F2,H9-R2,H9-P2的檢出率最高，達到66.7%。雖然在組織樣本中，組1和組2的靈敏度均能達到76%，但針對痰液檢測樣本，組2的靈敏度卻大幅下降至44.6%。Group 1 of HOXA9: H9-F2, H9-R2, H9-P2 had the highest detection rate, reaching 66.7%. Although both groups 1 and 2 achieved 76% sensitivity in tissue samples, for sputum samples, the sensitivity of group 2 dropped significantly to 44.6%.

最終優選的引子如下表22所示。 [表22] 優化後的引子和探針 名稱 序列編號 序列 作用 H7-F2 SEQ ID NO: 1 TAAAGGCGTTTGCGATAAGAC HOXA7基因上游引子 H7-R2 SEQ ID NO: 2 TAACCCGCCTAACGACTACG HOXA7基因下游引子 H7-P2 SEQ ID NO: 3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7基因檢測探針 H9-F2 SEQ ID NO: 4 TTAGTTTTTTCGGTAGGCGGC HOXA9基因上游引子 H9-R2 SEQ ID NO: 5 AAACGCCAAACACCGTCG HOXA9基因下游引子 H9-P2 SEQ ID NO: 6 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9基因檢測探針 A3-TqMF SEQ ID NO: 19 TTTTGGATTGTGAATTTGTG β-actin基因上游引子 A3-TqMR SEQ ID NO: 20 AAAACCTACTCCTCCCTTAAA β-actin基因下游引子 A3-TqP SEQ ID NO: 21 FAM-TTGTGTGTTGGGTGGTGGTT-BQ1 β-actin基因檢測探針 The final preferred primers are shown in Table 22 below. [Table 22] Optimized primers and probes name serial number sequence effect H7-F2 SEQ ID NO: 1 TAAAGGCGTTTGCGATAAGAC Primer upstream of HOXA7 gene H7-R2 SEQ ID NO: 2 TAACCCGCCTAACGACTACG Primer downstream of HOXA7 gene H7-P2 SEQ ID NO: 3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7 gene detection probe H9-F2 SEQ ID NO: 4 TTAGTTTTTTTCGGTAGGCGGC Primer upstream of HOXA9 gene H9-R2 SEQ ID NO: 5 AAACGCCAAACACCGTCG Primers downstream of HOXA9 gene H9-P2 SEQ ID NO: 6 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9 gene detection probe A3-TqMF SEQ ID NO: 19 TTTTGGATTGTGAATTTGTG β-actin gene upstream primer A3-TqMR SEQ ID NO: 20 AAAACCTACTCCTCCCTTAAA β-actin gene downstream primer A3-TqP SEQ ID NO: 21 FAM-TTGTGTGTTGGGTGGTGGTT-BQ1 β-actin gene detection probe

無。none.

[圖1] HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3、以及HOXA7和HOXA9組合在所有組織標本中檢測的ROC曲線； [圖2] HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3、以及HOXA7和HOXA9組合在所有痰液標本中檢測的ROC曲線； [圖3] HOXA7、HOXA9、與SHOX2_n3、以及HOXA7和HOXA9組合在所有痰液標本中檢測的ROC曲線； [圖4] HOXA7、HOXA9與SHOX2_n3在痰液標本中的擴增曲線（A為HOXA7的擴增圖，B為HOXA9的擴增圖，C為SHOX2_n3的擴增圖）； [圖5] HOXA7、HOXA9、SHOX2_n3、以及HOXA7和HOXA9組合在所有灌洗液標本中檢測的ROC曲線； [圖6] HOXA7、HOXA9與SHOX2_n3在灌洗液標本中的擴增曲線（A為HOXA7的擴增圖，B為HOXA9的擴增圖，C為SHOX2_n3的擴增圖）。[Fig. 1] ROC curves of HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3, and the combination of HOXA7 and HOXA9 detected in all tissue samples; [Fig. 2] The ROC curves of HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3, and the combination of HOXA7 and HOXA9 in all sputum samples; [Fig. 3] ROC curves of HOXA7, HOXA9, combined with SHOX2_n3, and HOXA7 and HOXA9 in all sputum samples; [Figure 4] Amplification curves of HOXA7, HOXA9 and SHOX2_n3 in sputum samples (A is the amplification map of HOXA7, B is the amplification map of HOXA9, and C is the amplification map of SHOX2_n3); [Fig. 5] ROC curves of HOXA7, HOXA9, SHOX2_n3, and the combination of HOXA7 and HOXA9 detected in all lavage fluid samples; [Fig. 6] Amplification curves of HOXA7, HOXA9 and SHOX2_n3 in lavage samples (A is the amplification map of HOXA7, B is the amplification map of HOXA9, and C is the amplification map of SHOX2_n3).

Claims (28)

一種引子對，包含如SEQ ID NO：1-2，SEQ ID NO：37-38，SEQ ID NO：40-41，SEQ ID NO：43-44，或SEQ ID NO：46-47，SEQ ID NO：49-50，SEQ ID NO：52-53，SEQ ID NO：55-56，SEQ ID NO：58-59或SEQ ID NO：61-62所示的引子對，以及，包含如SEQ ID NO：4-5，SEQ ID NO：64-65，SEQ ID NO：67-68，SEQ ID NO：70-71，SEQ ID NO：73-74，SEQ ID NO：76-77，SEQ ID NO：79-80，SEQ ID NO：82-83，SEQ ID NO：85-86，SEQ ID NO：88-89或SEQ ID NO：91-92所示的引子對。 A primer pair comprising, for example, SEQ ID NO: 1-2, SEQ ID NO: 37-38, SEQ ID NO: 40-41, SEQ ID NO: 43-44, or SEQ ID NO: 46-47, SEQ ID NO : 49-50, SEQ ID NO: 52-53, SEQ ID NO: 55-56, SEQ ID NO: 58-59 or the primer pair shown in SEQ ID NO: 61-62, and, comprising as SEQ ID NO: 4-5, SEQ ID NO: 64-65, SEQ ID NO: 67-68, SEQ ID NO: 70-71, SEQ ID NO: 73-74, SEQ ID NO: 76-77, SEQ ID NO: 79- 80, the primer pair shown in SEQ ID NO: 82-83, SEQ ID NO: 85-86, SEQ ID NO: 88-89 or SEQ ID NO: 91-92. 一種HOXA7基因和HOXA9基因甲基化的檢測試劑在製備肺癌診斷試劑中的用途，其中該甲基化的檢測試劑的一檢測樣本為痰液，該肺癌為肺腺癌；其中該甲基化的檢測試劑包含檢測HOXA7基因的引子對和探針的組合；所述檢測HOXA7基因的引子對和探針的組合包含如SEQ ID NO：1-2所示的引子對和如SEQ ID NO：3所示的探針，或如SEQ ID NO：37-38所示的引子對和如SEQ ID NO：39所示的探針，或如SEQ ID NO：40-41所示的引子對和如SEQ ID NO：42所示的探針，或如SEQ ID NO：43-44所示的引子對和如SEQ ID NO：45所示的探針，或如SEQ ID NO：46-47所示的引子對和如SEQ ID NO：48所示的探針，或如SEQ ID NO：49-50所示的引子對和如SEQ ID NO：51所示的探針，或如SEQ ID NO：52-53所示的引子對和如SEQ ID NO：54所示的探針，或如SEQ ID NO：55-56所示的探針和如SEQ ID NO：57所示的探針，或如SEQ ID NO：58-59所示的引子對和如SEQ ID NO：60所示的探針或如SEQ ID NO：61-62所示的引子對和如SEQ ID NO：63所示的探針。 Use of a detection reagent for methylation of HOXA7 gene and HOXA9 gene in the preparation of a diagnostic reagent for lung cancer, wherein a detection sample of the methylated detection reagent is sputum, and the lung cancer is lung adenocarcinoma; wherein the methylated The detection reagent comprises a combination of primer pairs and probes for detecting HOXA7 gene; the combination of primer pairs and probes for detecting HOXA7 gene comprises a primer pair as shown in SEQ ID NO: 1-2 and a combination as shown in SEQ ID NO: 3. The probes shown, or the primer pairs shown in SEQ ID NOs: 37-38 and the probes shown in SEQ ID NO: 39, or the primer pairs shown in SEQ ID NOs: 40-41 and the probes shown in SEQ ID NOs: 40-41 The probe set forth in NO: 42, or the primer pair set forth in SEQ ID NO: 43-44 and the probe set forth in SEQ ID NO: 45, or the primer pair set forth in SEQ ID NO: 46-47 and a probe as set forth in SEQ ID NO: 48, or a primer pair as set forth in SEQ ID NO: 49-50 and a probe as set forth in SEQ ID NO: 51, or as set forth in SEQ ID NO: 52-53 The primer pair shown and the probe shown in SEQ ID NO: 54, or the probe shown in SEQ ID NO: 55-56 and the probe shown in SEQ ID NO: 57, or the probe shown in SEQ ID NO: The primer pair shown in 58-59 and the probe shown in SEQ ID NO:60 or the primer pair shown in SEQ ID NO:61-62 and the probe shown in SEQ ID NO:63. 根據請求項2所述之用途，其中該HOXA7和HOXA9基因甲基化的檢測試劑檢測HOXA7基因和HOXA9基因經一轉化試劑修飾後的序列。 The use according to claim 2, wherein the HOXA7 and HOXA9 gene methylation detection reagent detects the sequences of the HOXA7 gene and the HOXA9 gene modified by a conversion reagent. 根據請求項3所述之用途，該轉化試劑選自肼鹽、重亞硫酸氫鹽和亞硫酸氫鹽中的一種或幾種。 According to the use described in claim 3, the conversion reagent is selected from one or more of hydrazine salts, bisulfites and bisulfites. 根據請求項3所述之用途，該轉化試劑選自重亞硫酸氫鹽。 According to the use according to claim 3, the conversion reagent is selected from bisulfite. 根據請求項2所述之用途，其中該甲基化的檢測試劑的檢測區域包含HOXA7基因和HOXA9基因的基因體或啟動子區域。 The use according to claim 2, wherein the detection region of the methylation detection reagent comprises the gene bodies or promoter regions of the HOXA7 gene and the HOXA9 gene. 根據請求項6所述之用途，該試劑針對HOXA7的檢測區域包含SEQ ID NO：25或SEQ ID NO：27所示的序列；該試劑針對HOXA9的檢測區域包含SEQ ID NO：29、SEQ ID NO：31或SEQ ID NO：33所示的序列。 According to the use described in claim 6, the detection region of the reagent for HOXA7 comprises the sequence shown in SEQ ID NO: 25 or SEQ ID NO: 27; the detection region of the reagent for HOXA9 comprises SEQ ID NO: 29, SEQ ID NO: 27 : 31 or the sequence shown in SEQ ID NO: 33. 根據請求項6所述之用途，該試劑針對HOXA7的檢測區域包含SEQ ID NO：27所示的序列，該試劑針對HOXA9的檢測區域包含SEQ ID NO：29所示的序列。 According to the use described in claim 6, the detection region of the reagent for HOXA7 comprises the sequence shown in SEQ ID NO: 27, and the detection region of the reagent for HOXA9 comprises the sequence shown in SEQ ID NO: 29. 根據請求項2所述之用途，其中該檢測HOXA7基因的引子對包含SEQ ID NO：1-2所示的引子對。 The use according to claim 2, wherein the primer pair for detecting the HOXA7 gene comprises the primer pair shown in SEQ ID NOs: 1-2. 根據請求項2所述之用途，其中該甲基化的檢測試劑還包含檢測HOXA9基因的引子對，該檢測HOXA9基因的引子對包含SEQ ID NO：4-5，SEQ ID NO：64-65，SEQ ID NO：67-68，SEQ ID NO：70-71，SEQ ID NO：73-74，SEQ ID NO：76-77，SEQ ID NO：79-80，SEQ ID NO：82-83，SEQ ID NO：85-86，SEQ ID NO：88-89或SEQ ID NO：91-92所示的引子對。 The use according to claim 2, wherein the methylation detection reagent further comprises a primer pair for detecting HOXA9 gene, and the primer pair for detecting HOXA9 gene comprises SEQ ID NO: 4-5, SEQ ID NO: 64-65, SEQ ID NO:67-68, SEQ ID NO:70-71, SEQ ID NO:73-74, SEQ ID NO:76-77, SEQ ID NO:79-80, SEQ ID NO:82-83, SEQ ID The primer pairs shown in NO: 85-86, SEQ ID NO: 88-89 or SEQ ID NO: 91-92. 根據請求項10所述之用途，其中該檢測HOXA9基因的引子對包含SEQ ID NO：4-5所示的引子對。 The use according to claim 10, wherein the primer pair for detecting the HOXA9 gene comprises the primer pair shown in SEQ ID NOs: 4-5. 根據請求項2所述之用途，其中該檢測HOXA7基因的探針包含SEQ ID NO：3所示的序列。 The use according to claim 2, wherein the probe for detecting the HOXA7 gene comprises the sequence shown in SEQ ID NO:3. 根據請求項2所述之用途，其中該甲基化的檢測試劑還包含檢測HOXA9基因的探針，該檢測HOXA9基因的探針包含SEQ ID NO：6、SEQ ID NO：66、SEQ ID NO：69、SEQ ID NO：72、SEQ ID NO：75、SEQ ID NO：78、SEQ ID NO：81、SEQ ID NO：84、SEQ ID NO：87、SEQ ID NO：90或SEQ ID NO：93所示的序列。 The use according to claim 2, wherein the methylation detection reagent further comprises a probe for detecting HOXA9 gene, and the probe for detecting HOXA9 gene comprises SEQ ID NO: 6, SEQ ID NO: 66, SEQ ID NO: 69. SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, or SEQ ID NO: 93 the sequence shown. 根據請求項13所述之用途，其中該檢測HOXA9基因的探針包含SEQ ID NO：6所示的序列。 The use according to claim 13, wherein the probe for detecting the HOXA9 gene comprises the sequence shown in SEQ ID NO:6. 根據請求項2所述之用途，其中該甲基化的檢測試劑更包含一參考基因的檢測試劑。 The use according to claim 2, wherein the methylation detection reagent further comprises a reference gene detection reagent. 根據請求項15所述之用途，該參考基因包含β-actin或COL2A1。 According to the use of claim 15, the reference gene comprises β-actin or COL2A1. 根據請求項16所述之用途，該參考基因的檢測試劑含包含參考基因的引子和探針。 According to the use described in claim 16, the detection reagent for the reference gene comprises primers and probes comprising the reference gene. 根據請求項16所述之用途，該參考基因β-actin的檢測試劑包含SEQ ID NO：19和SEQ ID NO：20所示引子對，以及SEQ ID NO：21所示探針。 According to the use described in claim 16, the detection reagent for the reference gene β-actin comprises a primer pair shown in SEQ ID NO: 19 and SEQ ID NO: 20, and a probe shown in SEQ ID NO: 21. 根據請求項16所述之用途，該參考基因COL2A1的檢測試劑包含SEQ ID NO：94和SEQ ID NO：95所示引子對，以及SEQ ID NO：96所示探針。 According to the use described in claim 16, the detection reagent for the reference gene COL2A1 comprises a primer pair shown in SEQ ID NO: 94 and SEQ ID NO: 95, and a probe shown in SEQ ID NO: 96. 根據請求項2所述之用途，其中該甲基化的檢測試劑更包含亞硫酸氫鹽、重亞硫酸氫鹽或肼鹽。 The use according to claim 2, wherein the methylated detection reagent further comprises bisulfite, bisulfite or hydrazine salt. 根據請求項2所述之用途，其中該甲基化的檢測試劑更包含DNA聚合酶、dNTPs、Mg²⁺離子和緩衝液中的一種或幾種。 The use according to claim 2, wherein the methylation detection reagent further comprises ^{one or more of DNA polymerase, dNTPs, Mg 2+} ions and buffer. 根據請求項2所述之用途，其中該甲基化的檢測試劑更包含DNA聚合酶、dNTPs、Mg²⁺離子和緩衝液。 The use according to claim 2, wherein the methylation detection reagent further comprises DNA polymerase, dNTPs, Mg ²⁺ ions and a buffer. 一種肺癌的診斷系統，包含：i.一HOXA7基因和HOXA9基因的DNA甲基化檢測構件；以及，ii.一結果判斷構件；該HOXA7基因和HOXA9基因的DNA甲基化檢測構件包含請求項1-25任一所述之用途的甲基化的檢測試劑；其中該甲基化的檢測試劑的一檢測樣本為痰液，該肺癌為肺腺癌。 A diagnostic system for lung cancer, comprising: i. a DNA methylation detection component of HOXA7 gene and HOXA9 gene; and, ii. a result judgment component; the DNA methylation detection component of this HOXA7 gene and HOXA9 gene comprises claim 1 -25 The methylation detection reagent according to any one of the uses; wherein a detection sample of the methylation detection reagent is sputum, and the lung cancer is lung adenocarcinoma. 根據請求項23所述之診斷系統，該甲基化檢測構件包含螢光定量PCR儀、PCR儀、測序儀中的一種或多種。 According to the diagnostic system of claim 23, the methylation detection component comprises one or more of a fluorescence quantitative PCR instrument, a PCR instrument, and a sequencer. 根據請求項23所述之診斷系統，該結果判斷構件包含資料處理機器。 According to the diagnosing system of claim 23, the result judging means includes a data processing machine. 根據請求項23所述之診斷系統，該結果判斷構件用於根據檢測構件檢測的HOXA7基因和HOXA9基因的DNA甲基化水準，輸出一診斷結果。 According to the diagnostic system of claim 23, the result determination means is configured to output a diagnosis result based on the DNA methylation levels of the HOXA7 gene and the HOXA9 gene detected by the detection means. 根據請求項26所述之診斷系統，該診斷結果通過結果判斷構件比較待測樣本與正常樣本的甲基化水準，基於待測樣本與正常樣本的甲基化水準的偏離得出的。 According to the diagnostic system of claim 26, the diagnostic result is obtained by comparing the methylation levels of the test sample and the normal sample by the result judgment means, based on the deviation of the methylation levels of the test sample and the normal sample. 一種肺癌的診斷方法，包括以下步驟：a)檢測來源於受試者的待測樣本HOXA7基因和HOXA9基因的甲基化水平；b)將待測樣本與正常樣本的HOXA7基因和HOXA9基因水平比較；c)基於待測樣本與正常樣本的甲基化水平的偏離，診斷肺癌；其中該待測樣本為痰液，該肺癌為肺腺癌；所述步驟a)中的檢測所使用的試劑包含檢測HOXA7基因的引子對和探針的組合；所述檢測HOXA7基因的引子對和探針的組合包含如SEQ ID NO：1-2所示的引子對和如SEQ ID NO：3所示的探針，或如SEQ ID NO：37-38所示的引子對和如SEQ ID NO：39所示的探針，或如SEQ ID NO：40-41所示的引子對和如SEQ ID NO：42所示的探針，或如SEQ ID NO：43-44所示的引子對和如SEQ ID NO：45所示的探針，或如SEQ ID NO：46-47所示的引子對和如SEQ ID NO：48所示的探針，或如SEQ ID NO：49-50所示的引子對和如SEQ ID NO：51所示的探針，或如SEQ ID NO：52-53所示的引子對和如SEQ ID NO：54所示的探針，或如SEQ ID NO：55-56所示的探針和如SEQ ID NO：57所示的探針，或如SEQ ID NO：58-59所示的引子對和如SEQ ID NO：60所示的探針或如SEQ ID NO：61-62所示的引子對和如SEQ ID NO：63所示的探針。 A method for diagnosing lung cancer, comprising the steps of: a) detecting the methylation levels of HOXA7 gene and HOXA9 gene in a test sample derived from a subject; b) comparing the HOXA7 gene and HOXA9 gene levels of the test sample with a normal sample c) based on the deviation of the methylation level of the sample to be tested and the normal sample, diagnose lung cancer; wherein the sample to be tested is sputum, and this lung cancer is lung adenocarcinoma; the reagent used in the detection in the step a) comprises: The combination of primer pair and probe for detecting HOXA7 gene; the combination of primer pair and probe for detecting HOXA7 gene comprises the primer pair shown in SEQ ID NO: 1-2 and the probe shown in SEQ ID NO: 3 needle, or a primer pair as shown in SEQ ID NO:37-38 and a probe as shown in SEQ ID NO:39, or a primer pair as shown in SEQ ID NO:40-41 and as SEQ ID NO:42 Probes as shown, or primer pairs as shown in SEQ ID NOs: 43-44 and probes as shown in SEQ ID NO: 45, or primer pairs as shown in SEQ ID NOs: 46-47 and primer pairs as shown in SEQ ID NO: 47 The probe set forth in ID NO: 48, or the primer pair set forth in SEQ ID NO: 49-50 and the probe set forth in SEQ ID NO: 51, or the primer set forth in SEQ ID NO: 52-53 pair and the probe as shown in SEQ ID NO: 54, or the probe as shown in SEQ ID NO: 55-56 and the probe as shown in SEQ ID NO: 57, or as SEQ ID NO: 58-59 The primer pair shown and the probe shown as SEQ ID NO:60 or the primer pair shown as SEQ ID NO:61-62 and the probe shown as SEQ ID NO:63.

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