WO2017092483A1 - 通过检测游离核酸诊断结核病的试剂盒及其应用 - Google Patents

通过检测游离核酸诊断结核病的试剂盒及其应用 Download PDF

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WO2017092483A1
WO2017092483A1 PCT/CN2016/099317 CN2016099317W WO2017092483A1 WO 2017092483 A1 WO2017092483 A1 WO 2017092483A1 CN 2016099317 W CN2016099317 W CN 2016099317W WO 2017092483 A1 WO2017092483 A1 WO 2017092483A1
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nucleic acid
free nucleic
tuberculosis
mycobacterium tuberculosis
body fluid
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PCT/CN2016/099317
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French (fr)
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车南颖
陈效友
杨新婷
张海青
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北京市结核病胸部肿瘤研究所
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the invention belongs to the field of biomedicine and relates to a kit for diagnosing tuberculosis by detecting free nucleic acid and application thereof.
  • Tuberculosis is a contagious disease that poses a serious threat to human health. Every year, 2 million people die of tuberculosis, and their mortality rate is only second to AIDS in infectious diseases. Early rapid diagnosis is an important part of controlling tuberculosis epidemic, but the current clinical diagnosis of tuberculosis faces many problems.
  • Bacteriological examination is the gold standard for the diagnosis of tuberculosis, but the sensitivity of sputum culture is only about 50%, and it can only be detected during the patient's sterilizing period. At the same time, bacteriological cultures usually produce results after one month, so there are shortcomings such as low sensitivity and long diagnostic period. Imaging studies, as another clinical routine, are an important way to detect and diagnose tuberculosis. However, the imaging results are greatly influenced by subjective factors. The diagnosis results of different doctors and different hospitals are quite different, so there is a deficiency in the diagnostic specificity. Another clinically used gamma-interferon release test can only indicate whether the subject is infected with M. tuberculosis, and cannot distinguish between active tuberculosis patients and latent infections. Therefore, it cannot be used as a basis for diagnosis of tuberculosis.
  • the amount of Mycobacterium tuberculosis in tuberculous pleural effusion specimens is usually very low, not only the culture positive rate is very low (5-10%), but the positive rate of commonly used molecular detection technology is also not ideal (30-40%).
  • the free nucleic acid is a trace amount of endogenous or exogenous nucleic acid fragments free from extracellular space in human body fluids.
  • the application provided by the invention is specifically: whether the free nucleic acid for detecting body fluid contains a knot
  • the substance for detecting a free nucleic acid of a body fluid containing a Mycobacterium tuberculosis specific gene includes: (1) a substance for extracting free nucleic acid from a body fluid; and (2) detecting whether the free nucleic acid contains A substance of a Mycobacterium tuberculosis-specific gene.
  • the product for diagnosing or assisting diagnosis of tuberculosis contains a substance for detecting whether a free nucleic acid of a body fluid contains a Mycobacterium tuberculosis specific gene.
  • the method for detecting whether a free nucleic acid of a body fluid contains a Mycobacterium tuberculosis specific gene comprises: (1) a substance for extracting free nucleic acid from a body fluid; and (2) detecting whether the free nucleic acid contains Mycobacterium tuberculosis a substance of a specific gene.
  • the product may further comprise a data processing device; the data processing device is provided with a module; the module has a function of determining whether to wait according to whether the free nucleic acid contains the detection result of the M. tuberculosis specific gene Whether the tester is a tuberculosis patient, if the free nucleic acid in the body fluid of the test subject contains the M. tuberculosis specific gene, the test subject is or candidate for a tuberculosis patient, if the test subject is The free nucleic acid in the body fluid does not contain the M. tuberculosis specific gene, and the test subject is not or the candidate is not a tuberculosis patient.
  • the substance for extracting free nucleic acid from a body fluid may be a free nucleic acid extraction kit.
  • the substance for detecting whether the free nucleic acid contains a Mycobacterium tuberculosis specific gene may be a PCR kit for detecting Mycobacterium tuberculosis.
  • Another object of the present invention is to provide a method of diagnosing or assisting in diagnosing whether a person to be tested has tuberculosis.
  • the method provided by the invention comprises the following steps:
  • the free nucleic acid is a trace amount of endogenous or exogenous nucleic acid fragments free from extracellular space in human body fluids.
  • the free nucleic acid extracted from the ex vivo body fluid of the human to be tested is extracted by a free nucleic acid extraction kit.
  • the detecting whether the free nucleic acid corresponding to the test subject contains a Mycobacterium tuberculosis specific gene is detected by a PCR kit for detecting Mycobacterium tuberculosis.
  • the Mycobacterium tuberculosis specific gene is the IS6110 gene of Mycobacterium tuberculosis.
  • the substance for extracting free nucleic acid from body fluid is specifically a free nucleic acid extraction kit of Qiagen No. 55114 and a QIAvac 24Plus instrument.
  • the Mycobacterium tuberculosis specific gene may specifically be the IS6110 gene of Mycobacterium tuberculosis (the nucleotide sequence is Sequence 1 in the Sequence Listing.
  • the free nucleic acid contains a Mycobacterium tuberculosis specific gene
  • the substance may be a primer pair for amplifying the IS6110 gene of the M. tuberculosis.
  • the substance for detecting whether the free nucleic acid contains a M. tuberculosis specific gene is specifically a Zhongshan University. Daan Gene Co., Ltd.
  • PCR kit for the detection of IS6110 gene of Mycobacterium tuberculosis with the product number DA-B052; the product name is "M. tuberculosis nucleic acid detection kit (PCR-fluorescence method)", food medicine Bureau registration number: National Machinery Note 20153400357.
  • the body fluid is specifically blood or pleural effusion.
  • Example 1 Detection of Mycobacterium tuberculosis infection using body fluid free nucleic acid
  • tuberculosis patients 25 cases of tuberculosis patients, 20 cases of other diseases (16 cases of lung cancer, 4 cases of other infectious lung diseases excluding tuberculosis) and 10 healthy people's peripheral blood samples were taken to extract whole blood DNA and blood free nucleic acid.
  • the tuberculosis patients tested were clinically diagnosed as tuberculosis patients by clinical symptoms, imaging, bacteriological examination and anti-tuberculosis test treatment. 20 patients with other diseases and 10 healthy people were also clinically confirmed. .
  • the test population was a voluntary consent.
  • the free nucleic acid extraction kit is a Qiagen product, item number 55114, and is operated according to the instructions. The specific process is as follows:
  • the whole blood DNA extraction kit is a Qiagen product, the article number is 51183, and is operated according to the instructions.
  • the specific process is as follows:
  • the blood free nucleic acid and whole blood DNA extracted by steps 1 and 2 are respectively the samples to be tested, and the IS6110 gene (nucleotide sequence) of M. tuberculosis is used as DA-B052 of Daan Gene Co., Ltd. of Sun Yat-sen University.
  • the kit of the sequence shown in the sequence 1 product name is "M. tuberculosis nucleic acid detection kit (PCR-fluorescence method)", Food and Drug Administration registration number: National Machinery Note 20153400357) for testing.
  • the specific process is as follows:
  • the PCR reaction solution was dispensed in proportion (40 ⁇ l of TB-PCR reaction solution/case + 3 ⁇ l/Taq enzyme), mixed, and dispensed into a PCR tube at 43 ⁇ l/tube. 2 ⁇ l of the nucleic acid sample to be tested was added. A negative, positive control was added to each experiment, the negative control was water, and the positive control was a plasmid containing the IS6110 sequence. Both negative and positive controls are supplied with the kit.
  • Fluorescence quantitative PCR reaction conditions were: 93 ° C for 3 min, (93 ° C 45 sec, 55 ° C 60 sec) ⁇ 10 cycles, (93 ° C 30 sec, 55 ° C 45 sec) ⁇ 30 cycles, and the fluorescence collection point was 55 ° C for 45 sec.
  • the determination result if the amplification curve is not S-type or Ct ⁇ 30, the determination result is negative, that is, the IS6110 gene of Mycobacterium tuberculosis is not detected from the nucleic acid sample to be tested. If the amplification curve is S-type and Ct ⁇ 30, the determination is positive, that is, the IS6110 gene of Mycobacterium tuberculosis is detected from the nucleic acid sample to be tested.
  • tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, the positive rate of both bacteriology and molecular biology testing is very low. It has been reported that the bacteriological positive rate is less than 10%; the newly developed GeneXpert molecular detection positive rate this year is only about 30%. To this end, the inventors of the present invention collected pleural effusions from 56 patients with tuberculous pleurisy and 32 patients with other diseases (30 cases of lung cancer, 1 case of pleural mesothelioma, and 1 case of pulmonary schistosomiasis). Swim The method of detecting nucleic acids and the accuracy of the GeneXpert method.
  • the patients with tuberculous pleurisy tested were clinically diagnosed as patients with tuberculous pleurisy by clinical symptoms, bacteriology, biochemical indicators and anti-tuberculosis experimental treatment. 32 patients with other diseases were also clinically confirmed.
  • the test population was a voluntary consent.
  • the pleural effusion was directly drained into a 15 ml sterile centrifuge tube, centrifuged at 3500 rpm for 10 minutes, and the supernatant was taken for free nucleic acid extraction.
  • the free nucleic acid extraction kit is a product of Qiagen, which is numbered 55114 and is operated according to the instructions. See step 1 for the specific process.
  • the pleural effusion free nucleic acid extracted in step 1 is used as a sample to be tested, and the IS6110 gene for Mycobacterium tuberculosis is used as the DA-B052 of the Daan-Bao Gene Co., Ltd. of Sun Yat-sen University (nucleotide sequence such as sequence 1 in the sequence listing)
  • the kit shown (the product name is "M. tuberculosis nucleic acid detection kit (PCR-fluorescence method)", the Food and Drug Administration registration number: National Machinery Note 20153400357) for testing.
  • the product name is "M. tuberculosis nucleic acid detection kit (PCR-fluorescence method)", the Food and Drug Administration registration number: National Machinery Note 20153400357) for testing.
  • the test uses Cepheid's Xpert MTB/RIF test kit (catalog number GXMTB/RIF-CN-10) and GeneXpert instrument. follow the instructions.
  • the specific process is as follows:
  • Sensitivity true positive (A) / actual positive (G) detected by one method
  • the invention develops a new product for rapid diagnosis of tuberculosis by detecting free nucleic acid components of Mycobacterium tuberculosis in clinically common liquid samples such as blood and pleural effusion. Since the free nucleic acid does not need to collect live bacteria or intact cells of M. tuberculosis, it has better safety and sensitivity than other nucleic acid detection methods. At the same time, the product can give results within 3 hours, which is fast and simple.

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Abstract

一种通过检测游离核酸诊断结核病的试剂盒及其应用，具体为用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质在制备诊断或辅助诊断结核病的试剂盒中的应用；其中所述用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质，包括：（1）从体液中提取游离核酸的物质；（2）检测游离核酸中是否含有结核分枝杆菌特异基因的物质。通过检测游离核酸诊断结核病，由于游离核酸不需要收集结核分枝杆菌活菌或完整菌体，因此相比其他的核酸检测方法具有更好的安全性和敏感性。同时该产品可以在3小时内给出结果，具有快速、简便等特点。

Description

通过检测游离核酸诊断结核病的试剂盒及其应用 技术领域
本发明属于生物医学领域，涉及一种通过检测游离核酸诊断结核病的试剂盒及其应用。
背景技术
结核病是严重威胁人类健康的传染性疾病，每年有200万人死于结核病，其死亡率在传染性疾病中仅低于艾滋病排第二。早期快速诊断是控制结核病疫情的重要环节，但目前结核病的临床诊断面临很多问题。
细菌学检查是诊断结核病的金标准，但是痰培养敏感性只有50％左右，且只有在患者排菌期才能检测到。同时，细菌学培养一般都在1个月后才能出结果，所以存在敏感性低、诊断周期长等缺点。而影像学检查作为另一项临床常规检查是发现和诊断结核病的重要方式。但影像学结果受主观性因素影响比较大，不同医生、不同医院诊断结果差异较大，因此在诊断特异性上存在不足。而另一项临床常用的γ-干扰素释放实验只能说明受检者是否感染过结核分枝杆菌，无法区分活动性结核病患者及潜伏感染者，因此也不能作为诊断结核病的确诊依据。
近年来分子生物学技术发展迅速，TB-PCR、GeneXpert等技术为结核病的快速诊断提供了新途径。但这些方法仅限于痰标本、新鲜组织、脑脊液等标本，很难应用于血液标本。其主要原因结核分枝杆菌主要分布在发病病灶中，除了血行播散等极特殊情况，一般很少出现在血液中。另外，结核性胸腔积液标本中结核分枝杆菌荷菌量通常很低，不仅培养阳性率很低(5-10％)，常用分子检测技术的阳性率也不理想(30-40％)。
因此，结核病诊断急需一种能更加准确、快速、方便的新技术方法。
发明公开
本发明的目的是提供一种通过检测游离核酸诊断结核病的产品及其应用。
所述游离核酸为人体体液中游离于细胞外的微量内源性或外源性核酸片段。
本发明所提供的应用具体为：用于检测体液的游离核酸中是否含有结 核分枝杆菌特异基因的物质在制备诊断或辅助诊断结核病的试剂盒中的应用。
在所述应用中，所述用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质，包括：(1)从体液中提取游离核酸的物质；(2)检测游离核酸中是否含有结核分枝杆菌特异基因的物质。
本发明所提供的用于诊断或辅助诊断结核病的产品，含有用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质。
进一步，所述用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质，包括：(1)从体液中提取游离核酸的物质；(2)检测游离核酸中是否含有结核分枝杆菌特异基因的物质。
根据需要，所述产品还可包含数据处理装置；所述数据处理装置内设模块；所述模块具有如下功能：根据所述游离核酸中是否含有所述结核分枝杆菌特异基因的检测结果确定待测者是否为结核病患者，若所述待测者的体液中的游离核酸中含有所述结核分枝杆菌特异基因，则所述待测者为或候选为结核病患者，若所述待测者的体液中的游离核酸中不含有所述结核分枝杆菌特异基因，则所述待测者不为或候选不为结核病患者。
在上述应用和产品中，所述从体液中提取游离核酸的物质可为游离核酸提取试剂盒。在上述应用和产品中，所述检测游离核酸中是否含有结核分枝杆菌特异基因的物质可为用于检测结核分枝杆菌的PCR试剂盒。
本发明的另一个目的是提供一种诊断或辅助诊断待测人是否患有结核病的方法。
本发明提供的方法，包括如下步骤：
1)从待测人的离体体液中提取游离核酸；
2)检测该待测人对应的游离核酸中是否含有结核分枝杆菌特异基因，若含有，则该待测人患有或候选患有结核病，若不含有，则该待测人不患有或候选不患有结核病；
所述游离核酸为人体体液中游离于细胞外的微量内源性或外源性核酸片段。
上述方法中，所述从待测人的离体体液中提取游离核酸通过游离核酸提取试剂盒提取。
上述方法中，所述检测该待测人对应的游离核酸中是否含有结核分枝杆菌特异基因通过用于检测结核分枝杆菌的PCR试剂盒检测。
上述方法中，所述结核分枝杆菌特异基因为结核分枝杆菌的IS6110基因。
在本发明的一个实施例中，所述从体液中提取游离核酸的物质具体为Qiagen公司货号为55114的游离核酸提取试剂盒和QIAvac 24Plus仪器。
上述中，所述结核分枝杆菌特异基因具体可为结核分枝杆菌的IS6110基因(核苷酸序列如序列表中序列1。相应的，所述检测游离核酸中是否含有结核分枝杆菌特异基因的物质可为用于扩增所述结核分枝杆菌的IS6110基因的引物对。在本发明的一个实施例中，所述检测游离核酸中是否含有结核分枝杆菌特异基因的物质具体为中山大学达安基因股份有限公司的货号为DA-B052的用于检测结核分枝杆菌的IS6110基因的PCR试剂盒；产品名称是《结核分枝杆菌核酸检测试剂盒(PCR-荧光法)》，食药局注册号：国械注准20153400357。
在本发明的一个实施例中，所述体液具体为血液或胸腔积液。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、利用体液游离核酸检测结核分枝杆菌感染
一、利用血液游离核酸检测结核分枝杆菌感染
本实验以25例结核病患者、20例其他疾病(16例肺癌、4例排除结核病的其他感染性肺部疾病)患者及10例健康人的外周血为样本，分别提取全血DNA和血液游离核酸，通过检测其中是否含有结核分枝杆菌特异基因来验证两种方法对结核病的诊断准确性。其中，供试的结核病患者为临床上经过临床症状、影像学、细菌学检查及抗结核试验性治疗有效等方法确诊为结核病的患者，20例其他疾病患者以及10例健康人也是经过临床证实的。供试人群为自愿同意。
1、血液游离核苷酸的提取
用EDTA抗凝采血管采集4ml全血，3500rpm离心10分钟，分离上清用于游离核酸提取。游离核酸提取试剂盒为Qiagen公司产品，货号为55114，按照说明书进行操作。具体流程如下：
(1)将2ml待提取样品移入15ml离心管，加入200μl蛋白酶K，加入1.6ml ACL缓冲液(含1μg carrier RNA)。涡旋振荡30s后，在60℃水浴孵育30min。
(2)加入3.6ml ACB缓冲液，涡旋振荡30s后，在冰上放置5min。
(3)将DNA纯化柱固定在QIAvac 24Plus仪器上(Qiagen公司产品)，将步骤(2)中所有液体转移至纯化柱上。
(4)打开QIAvac 24Plus仪将液体全部抽干后，加入600μl ACW1缓冲液。抽干后再加入750μl ACW2缓冲液。再次抽干后加入750μl无水乙醇抽干。
(5)将纯化柱转移至新的收集管上，14000rpm离心3min去流出液。在56℃金属浴孵育10分钟。
(6)加入50μl AVE缓冲液，在室温放置3min后，14000rpm离心1min，收集流出液为纯化好的游离核酸。置于-20℃保存。
2、全血DNA的提取
全血DNA提取试剂盒为Qiagen公司产品，其货号为51183，按照说明书进行操作。具体流程如下：
(1)用EDTA抗凝管抽取2ml血液，并移至15ml离心管。
(2)加入200μl蛋白酶K，加入2.4ml AL缓冲液，颠倒混匀15次后涡旋振荡1min。
(3)70℃水浴中孵育10min，加入2ml无水乙醇，颠倒混匀10次后涡旋振荡1min。
(4)将所有液体转移至DNA离心柱上，3000rpm离心3min去除流出液。
(5)加入2ml AW1缓冲液，5000rpm离心1min，弃流出液。加入2ml AW2缓冲液，5000rpm离心15min，弃流出液。
(6)将离心柱转移至新15ml离心管，加入150μl AE缓冲液，室温孵育5min，5000rpm离心2min，收集流出液为全血DNA。置于-20℃保存。
3、荧光定量PCR法检测结核分枝杆菌特异基因序列
以步骤1和2提取的血液游离核酸和全血DNA分别为待测样品，利用中山大学达安基因股份有限公司的货号为DA-B052的针对结核分枝杆菌的IS6110基因(核苷酸序列如序列表中序列1所示)的试剂盒(产品名称是《结核分枝杆菌核酸检测试剂盒(PCR-荧光法)》，食药局注册号：国械注准20153400357)进行检测。按照说明书进行操作。具体流程如下：
(1)按比例(TB-PCR反应液40μl/例+Taq酶3μl/例)配置PCR反应液，混匀后按43μl/管分装至PCR管中。加入2μl待测核酸样品。每次实验加阴性、阳性对照，阴性对照是水，阳性对照是含有IS6110序列的质粒。阴性和阳性对照品均为试剂盒自带产品。
(2)荧光定量PCR反应条件为：93℃3min，(93℃45sec、55℃60sec)×10循环，(93℃30sec、55℃45sec)×30循环，荧光采集点在55℃45sec。
(3)按照如下判定结果：如果扩增曲线不呈S型或Ct≥30，判定结果为阴性，即从待测核酸样本中未检出结核分枝杆菌的IS6110基因。如果扩增曲线为S型且Ct<30则判定阳性，即从待测核酸样本中检出结核分枝杆菌的IS6110基因。
4、结果
结果显示：所有全血DNA样本中(25例结核病、20例其他疾病、10例健康人)均未能检测到结核分枝杆菌的IS6110基因。在血液游离核酸样本的检测中，25例结核病患者中有13例检测出结核分枝杆菌的IS6110基因；20例其他疾病患者中2例检测出结核分枝杆菌的IS6110基因；10例健康人中未检测出结核分枝杆菌的IS6110基因。从这个结果可以看出，血液游离核酸中检测结核分枝杆菌特异基因阳性率显著高于在全血DNA中的检测阳性率。
二、利用胸腔积液游离核酸检测结核分枝杆菌感染
由于结核性胸膜炎是最常见的肺外结核类型之一，但无论是细菌学还是分子生物学检测阳性率都非常低。有报道称细菌学阳性率不足10％；今年新发展出来的GeneXpert分子检测阳性率仅在30％左右。为此，本发明的发明人收集了56例结核性胸膜炎患者的胸腔积液及32例其他疾病(30例肺癌、1例胸膜间皮瘤、1例肺血吸虫病)患者的胸腔积液比较通过游 离核酸进行检测的方法与GeneXpert方法的准确性。其中，供试的结核性胸膜炎患者为临床上经过临床症状、细菌学、生化指标及抗结核试验性治疗有效等方法确诊为结核性胸膜炎患者的患者，32例其他疾病患者也是经过临床证实的。供试人群为自愿同意。
1、胸腔积液游离核苷酸的提取
胸腔积液直接引流到15ml无菌离心管，3500rpm离心10分钟，取上清用于游离核酸提取。游离核酸提取试剂盒为Qiagen公司产品，其货号为55114，按照说明书进行操作。具体流程参见步骤一1。
2、荧光定量PCR法检测结核分枝杆菌特异基因序列
以步骤1提取的胸腔积液游离核酸为待测样品，利用中山大学达安基因股份有限公司的货号为DA-B052的针对结核分枝杆菌的IS6110基因(核苷酸序列如序列表中序列1所示)的试剂盒(产品名称是《结核分枝杆菌核酸检测试剂盒(PCR-荧光法)》，食药局注册号：国械注准20153400357)进行检测。按照说明书进行操作。具体流程参见步骤一3。
3、GeneXpert检测胸腔积液结核分枝杆菌特异基因序列
该检测采用Cepheid公司的Xpert MTB/RIF检测试剂盒(货号为GXMTB/RIF-CN-10)及GeneXpert仪器。按照说明书进行操作。具体流程如下：
(1)取2ml新鲜胸腔积液样品至15ml离心管，直接加入4ml裂解液，涡旋振荡1min，室温放置15min。取2ml上清至Xpert反应器中，并放入GeneXpert仪器槽中运行仪器。
(2)反应结束后，直接记录仪器报出结果。
4、结果
结果显示，GeneXpert方法的灵敏度为44.6％(25例阳性/56例)，特异性为100％(32例阴性/32例)。相比之下，胸腔积液游离核酸的PCR检测方法灵敏度为82.1％(46例阳性/56例)，特异性为93.8％(30例阴性/32例)。其中，供试样本的胸腔积液游离核酸检测方法的检测结果如表1所示，GeneXpert方法的检测结果如表2所示。从这个结果可以看出，胸腔积液游离核酸检测结核分枝杆菌特异基因灵敏度显著高于GeneXpert(P<0.05)，且显示出良好的特异性。
表1 利用胸腔积液游离核酸样本辅助诊断结核性胸膜炎的情况
Figure PCTCN2016099317-appb-000001
表2 利用GeneXpert方法辅助诊断结核性胸膜炎的情况
Figure PCTCN2016099317-appb-000002
其中，灵敏度、特异性是按照如下方法计算得到的：
表3
Figure PCTCN2016099317-appb-000003
灵敏度＝一种方法检测的真阳性(A)/实际阳性(G)；
特异性＝一种方法检测的真阴性(D)/实际阴性(H)。
工业应用
本发明开发了一种通过检测血液、胸腔积液等临床常见液体标本中结核分枝杆菌游离核酸成分进行结核病快速诊断的新产品。由于游离核酸不需要收集结核分枝杆菌活菌或完整菌体，因此相比其他的核酸检测方法具有更好的安全性和敏感性。同时该产品可以在3小时内给出结果，具有快速、简便等特点。

Claims (14)

  1. 用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质在制备诊断或辅助诊断结核病的试剂盒中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质，包括：
    (1)从体液中提取游离核酸的物质；
    (2)检测游离核酸中是否含有结核分枝杆菌特异基因的物质。
  3. 一种用于诊断或辅助诊断结核病的产品，含有用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质。
  4. 根据权利要求3所述的产品，其特征在于：所述用于检测体液的游离核酸中是否含有结核分枝杆菌特异基因的物质，包括：
    (1)从体液中提取游离核酸的物质；
    (2)检测游离核酸中是否含有结核分枝杆菌特异基因的物质。
  5. 根据权利要求3或4所述的产品，其特征在于：所述产品还包含数据处理装置；所述数据处理装置内设模块；所述模块具有如下功能：根据所述游离核酸中是否含有所述结核分枝杆菌特异基因的检测结果确定待测者是否为结核病患者，若所述待测者的体液中的游离核酸中含有所述结核分枝杆菌特异基因，则所述待测者为或候选为结核病患者，若所述待测者的体液中的游离核酸中不含有所述结核分枝杆菌特异基因，则所述待测者不为或候选不为结核病患者。
  6. 根据权利要求1-5中任一所述的应用或产品，其特征在于：所述从体液中提取游离核酸的物质为游离核酸提取试剂盒。
  7. 根据权利要求1-6中任一所述的应用或产品，其特征在于：所述检测游离核酸中是否含有结核分枝杆菌特异基因的物质为用于检测结核分枝杆菌的PCR试剂盒。
  8. 根据权利要求1-7中任一所述的应用或产品，其特征在于：所述结核分枝杆菌特异基因为结核分枝杆菌的IS6110基因。
  9. 根据权利要求1-8中任一所述的应用或产品，其特征在于：所述体液为血液或胸腔积液。
  10. 一种诊断或辅助诊断待测人是否患有结核病的方法，包括如下步骤：
    1)从待测人的离体体液中提取游离核酸；
    2)检测该待测人对应的游离核酸中是否含有结核分枝杆菌特异基因，若含有，则该待测人患有或候选患有结核病，若不含有，则该待测人不患有或候选不患有结核病；
    所述游离核酸为人体体液中游离于细胞外的微量内源性或外源性核酸片段。
  11. 根据权利要求10所述的方法，其特征在于：
    所述从待测人的离体体液中提取游离核酸通过游离核酸提取试剂盒提取。
  12. 根据权利要求10或11所述的方法，其特征在于：
    所述检测该待测人对应的游离核酸中是否含有结核分枝杆菌特异基因通过用于检测结核分枝杆菌的PCR试剂盒检测。
  13. 根据权利要求10-12中任一所述的方法，其特征在于：所述结核分枝杆菌特异基因为结核分枝杆菌的IS6110基因。
  14. 根据权利要求10-13中任一所述的方法，其特征在于：所述体液为血液或胸腔积液。
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