WO2016106701A1 - 检测非小细胞肺癌的qRT-PCR引物、探针、芯片、试剂盒、应用和方法 - Google Patents
检测非小细胞肺癌的qRT-PCR引物、探针、芯片、试剂盒、应用和方法 Download PDFInfo
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- the invention relates to the technical field of molecular biology, in particular to a qRT-PCR primer, a probe, a chip, a kit, an application and a method for detecting non-small cell lung cancer.
- Lung cancer is the cancer that poses the greatest threat to Chinese patients. The incidence rate is the highest among all malignant tumors, with an average of 600,000 new cases per year. Correspondingly, the number of lung cancer deaths is also the highest among all cancers.
- SCLC small cell lung cancer
- NSCLC non-small cell lung cancer
- the fusion gene is a characteristic mutation in patients with NSCLC and is significantly associated with the use of NSCLC and the prognosis of patients.
- the fusion gene was first discovered in 2007 (Soda, Manabu, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 448.7153 (2007) ): 561-566), which is a fusion of the EML4 gene and the ALK gene caused by rearrangement on chromosome 2, resulting in an abnormal EML4-ALK fusion protein.
- crizotinib is superior to traditional chemotherapy in the treatment of patients with advanced NSCLC with ALK fusion gene (Ou, Sai-Hong Ignatius, et al.Crizotinib for the treatment of ALK-rearranged non -small cell lung cancer: a success story to usher in the second decade of molecular targeted therapy in oncology. The oncologist 17.11 (2012): 1351-1375).
- crizotinib can also act on ROS fusion The gene blocks its abnormal fusion pathway and inhibits tumor cell growth.
- the targeted drugs Sorafenib, Sunitinib and Vandetanib can kill cells containing the RET fusion gene, making the RET fusion gene also become a potential drug target that is of general concern.
- ALK fusion gene has been listed as a recommended test item in the NSCLC treatment guidelines of the National Comprehensive Cancer Network (NCCN). According to the consensus of experts in China (Zhang Xuchao, et al. Chinese Syndrome Lymphoma Kinase (ALK) Positive Non-small Cell Lung Cancer Diagnostic Expert Consensus (2013 Edition). Chinese Journal of Pathology 42.006 (2013): 402-406), recommended three
- the methods for detecting fusion genes are fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and real-time quantitative PCR (qRT-PCR), wherein the qRT-PCR method has the ability to determine the fusion type and is easy to operate. With high sensitivity and accurate judgment, products approved by CFDA have been listed.
- each fusion gene has multiple subtypes.
- EML4 and ALK fusion have 21 mutants, except one of them occurs on exon 19, and all others are some explicit of EML4.
- the subunit is linked to the exon 20 of the ALK.
- mutant 1 49.6%
- mutant 3a/b 25.6%)
- mutant 2 10%).
- the invention provides a qRT-PCR primer, a probe, a chip, a kit, an application and a method for detecting non-small cell lung cancer, which have the requirement of small sample amount, can simultaneously detect a plurality of fusion genes, and is simple, rapid, sensitive and accurate. Highly characteristic.
- the present invention provides a set of qRT-PCR primers and probes for detecting non-small cell lung cancer, the primers and probes being selected from at least one of the following sequence groups (a) to (v) :
- EML4-ALKv1-F primer GCCCACACCTGGGAAAGG (SEQ ID NO: 1);
- EML4-ALKv1-R Primer CCATCTGCATGGCTTGCA (SEQ ID NO: 2);
- EML4-ALKv1-P probe R-CCTAAAGTGTACCGCCGGA-Q (SEQ ID NO: 3);
- EML4-ALKv2-F primer TCTAACTCGGGAGACTATGAAATATTGTA (SEQ ID NO: 4);
- EML4-ALKv2-R Primer CCATCTGCATGGCTTGCA (SEQ ID NO: 5);
- EML4-ALKv2-P probe R-ACCGCCGGAAGCA-Q (SEQ ID NO: 6);
- EML4-ALKv3-F primer GCATAAAGATGTCATCATCAACCAA (SEQ ID NO: 7);
- EML4-ALKv3-R primer GGTGCGGAGCTTGCT (SEQ ID NO: 8);
- EML4-ALKv3-P probe R-CACCAGGAGCTGCAAGCCATGC-Q (SEQ ID NO: 9);
- EML4-ALKv4-F primer ACAATCAGAGCTGTAGCAGAAGGA (SEQ ID NO: 10);
- EML4-ALKv4-R Primer GGTCGAGGTGCGGAGCTT (SEQ ID NO: 11);
- EML4-ALKv4-P probe R-AGGCAGATCAATTTT-Q (SEQ ID NO: 12);
- EML4-ALKv5-F primer GAATCCTGAAAGAGAAATAGAGATATGCT (SEQ ID NO: 13);
- EML4-ALKv5-R primer GGTCGAGGTGCGGAGCTT (SEQ ID NO: 14);
- EML4-ALKv5-P probe R-CCCTGAGTACAAGCTG-Q (SEQ ID NO: 15);
- EML4-ALKv6-F primer GGGAATGAACAGCTCTCTGTGAT (SEQ ID NO: 16);
- EML4-ALKv6-R primer GGAGCTTGCTCAGCTTGTACTCA (SEQ ID NO: 17);
- EML4-ALKv6-P probe R-ACCGCCGGAAGCA-Q (SEQ ID NO: 18);
- EML4-ALKv7-F primer CAGTGAAAAAATCAGTCTCAAGTAAAGTG (SEQ ID NO: 19);
- EML4-ALKv7-R Primer CCATCTGCATGGCTTGCA (SEQ ID NO: 20);
- EML4-ALKv7-P probe R-ACCGCCGGAAGCA-Q (SEQ ID NO: 21);
- EML4-ALKv8-R Primer GGAGCTTGCTCAGCTTGTACTCA (SEQ ID NO: 23);
- EML4-ALKv8-P probe R-ACCGCCGGAAGCA-Q (SEQ ID NO: 24);
- EML4-ALKv9-F primer AAGGTGCTGTGGTGTGTCCA (SEQ ID NO: 25);
- EML4-ALKv9-R Primer CCATCTGCATGGCTTGCA (SEQ ID NO: 26);
- EML4-ALKv9-P probe R-ACCGCCGGAAGCA-Q (SEQ ID NO: 27);
- NMP1-ALK-F primer TCAGGGCCAGTGCATATTAGTG (SEQ ID NO: 28);
- NMP1-ALK-R primer CGGAGCTTGCTCAGCTTGTA (SEQ ID NO: 29);
- NMP1-ALK-P probe R-CCGGAAGCACCAGGA-Q (SEQ ID NO: 30);
- CD74-ROS32-F primer CAGGCACTCCTTGGAGCAA (SEQ ID NO: 31);
- CD74-ROS32-R primer TTGGGAATGCCTGGTTTATTTG (SEQ ID NO: 32);
- CD74-ROS32-P probe R-CACTGACGCTCCACC-Q (SEQ ID NO: 33);
- CD74-ROS34-F primer CAGGCACTCCTTGGAGCAA (SEQ ID NO: 34);
- CD74-ROS34-R Primer GGGATTGTAACAACCAGAAATATTCC (SEQ ID NO: 35);
- CD74-ROS34-P probe R-CACTGACGCTCCACC-Q (SEQ ID NO: 36);
- SLC34A2-ROS32-F primer TTCGTGTGCTCCCTGGATATT (SEQ ID NO: 37);
- SLC34A2-ROS32-R Primer GGGAATGCCTGGTTTATTTGG (SEQ ID NO: 38);
- SLC34A2-ROS32-P probe R-CCTTCCAGCTGGTTGG-Q (SEQ ID NO: 39);
- SLC34A2-ROS34-R Primer CAAAGGTCAGTGGGATTGTAACAA (SEQ ID NO: 41);
- SLC34A2-ROS34-P probe R-TTTGGATACCAGAAACAAG-Q (SEQ ID NO: 42);
- CDCC6-RET-F primer GCCAGCGTGACCATCGA (SEQ ID NO: 43);
- CDCC6-RET-R Primer CAAATTCGCCTTCTCCTAGAGTTT (SEQ ID NO: 44);
- CDCC6-RET-P probe R-ATCCAAAGTGGGAATTC-Q (SEQ ID NO: 45);
- KIF5B15-RET11-F primer GGAATTGCTGTGGGAAATAATGA (SEQ ID NO: 46);
- KIF5B15-RET11-R Primer GACAGCGGCTGCGATCA (SEQ ID NO: 47);
- KIF5B15-RET11-P probe R-CGAGCTGTGCCGCAC-Q (SEQ ID NO: 48);
- KIF5B15-RET12-R Primer GCCTTCTCCTAGAGTTTTTCCAAGA (SEQ ID NO: 50);
- KIF5B15-RET12-P probe R-ATCCAAAGTGGGAATTC-Q (SEQ ID NO: 51);
- KIF5B16-RET12-R Primer CGCCTTCTCCTAGAGTTTTTCCA (SEQ ID NO: 53);
- KIF5B16-RET12-P probe R-ATCCAAAGTGGGAATTC-Q (SEQ ID NO: 54);
- KIF5B22-RET12-R Primer GCCTTCTCCTAGAGTTTTTCCAAGA (SEQ ID NO: 56);
- KIF5B22-RET12-P probe R-ATCCAAAGTGGGAATTC-Q (SEQ ID NO: 57);
- KIF5B23-RET12-R Primer TCGCCTTCTCCTAGAGTTTTTCC (SEQ ID NO: 59);
- KIF5B23-RET12-P probe R-ATCCAAAGTGGGAATTC-Q (SEQ ID NO: 60);
- KLF5B24-RET11-R Primer CCGAGACGATGAAGGAGAAGA (SEQ ID NO: 62);
- KIF5B24-RET11-P probe R-CGAGCTGTGCCGCAC-Q (SEQ ID NO: 63);
- KIF5B24-RET8-R Primer CACTCCAGCCGTCTCTTGCT (SEQ ID NO: 65);
- KIF5B24-RET8-P probe R-CGAGGAGGCGGGCT-Q (SEQ ID NO: 66);
- R is a fluorescent reporter group and Q is a quenching group.
- the fluorescent reporter group is a fluorescent group commonly used in qRT-PCR, and may be 6-carboxyfluorescein (FAM), hexachloro-6-methylfluorescein (HEX), etc.; quenching groups are commonly used in qRT-PCR.
- the quenching group may be a black hole quenching dye (Black Hole Quencher-1, BHQ1), 6-carboxytetramethyl rhodamine (TAMRA) or the like.
- the fluorescent reporter group is FAM; the quenching group is BHQ1.
- a chip for detecting non-small cell lung cancer the primer and probe of the first aspect being arranged on the chip.
- the invention provides the use of the primers and probes of the first aspect in the preparation of a chip for detecting non-small cell lung cancer.
- the invention provides a kit for detecting non-small cell lung cancer, the kit comprising the primer and probe of the first aspect.
- the kit further comprises a reverse transcriptase, a DNA polymerase, and a buffer thereof.
- the present invention provides the use of the primer and probe of the first aspect in the preparation of a kit for detecting non-small cell lung cancer.
- the invention provides a method for detecting non-small cell lung cancer, the method comprising: using the primers and probes of the first aspect, using the RNA of the sample to be tested as a template for real-time quantification PCR; fluorescence signals were collected and negative or positive results were determined based on fluorescence signal intensity and CT values.
- qRT-PCR primers and probes for detecting qRT-PCR of non-small cell lung cancer of the present invention it is possible to accurately and sensitively detect EML4-ALK, NMP1-ALK, CD74-ROS, SLC34A2-ROS in NSCLC patients, 22 subtypes of the CDCC6-RET and KIF5B-RET fusion genes.
- pre-spraying primers and probes onto the chip hundreds of single-q q-PCR reactions can be performed simultaneously in one experiment, improving detection efficiency and without mutual interference between primers and probes.
- it has the characteristics of requiring less sample size, automatic operation, simple operation and fast detection speed.
- Figure 1 shows qRT-PCR results of detection of EML4-ALK fusion mutant 3a/b from H2228 cell line at three template concentrations.
- FAM was attached to both ends of the probe sequence as a fluorescent reporter group.
- BHQ1 acts as a quenching group.
- the probe When the probe is intact, the fluorescence emitted by the fluorescent reporter group is absorbed by the quenching group, and no fluorescent signal is detected at this time; during the annealing of qRT-PCR, the probe specifically binds to the template, with extension
- the fluorescent reporter group is cleaved by the 5'-3' exonuclease activity of the DNA polymerase, thereby separating from the quenching group to emit fluorescence, and the intensity of the fluorescent signal can be detected by a fluorescence quantitative PCR instrument.
- the degree of qRT-PCR is performed to achieve quantitative detection.
- NCI-H2228 from the First affiliated Hospital of Guangzhou Medical University was used using the EML4-ALK fusion mutant 3a/b (corresponding to the fusion subtype EML4-ALKv3 in Table 1). Samples were tested according to the procedure.
- 22 sets of primers and corresponding probes are arranged on a chip, and 2 primers and 1 probe of each group occupy 1 micropore (accommodating 100 ⁇ L of reaction liquid), and 2 primers are sprayed 4 ⁇ 10 ⁇ 7 nmol, probe sprayed 2 ⁇ 10 -7 nmol, repeated 30 times to make a custom chip.
- RNA was subjected to quality control, a 10-fold concentration gradient was used, and 3.5 ng, 35 ng, and 350 ng of RNA were used as templates, respectively, using Takara's One Step PrimeScript RT-PCR Kit (Perfect Real Time) configuration. The mixture was mixed and the system is shown in Table 2.
- reaction solution is dispensed into the micropores of the chip by using the SmartChip TE Nanodispenser.
- the surface of the chip is sealed with a matching membrane and centrifuged to prevent the liquid from overflowing.
- the fluorescence signal intensity of FAM or ROX was measured during annealing.
- the fluorescence intensity of the FAM corresponding to each site is separately detected.
- the fluorescence intensity reaches the threshold, if the CT value is greater than 32, the site is judged to be negative; if the CT value is less than 32, the site is judged to be positive.
- the EML4-ALK fusion mutant 3a/b (corresponding to the fusion subtype EML4-ALKv3 in Table 1) was detected from the H2228 cell line at three template concentrations (3.5 ng, 35 ng, and 350 ng, respectively).
- the results of qRT-PCR are shown in Figure 1.
- Example 4 we performed a qRT-PCR test using the plasmids or samples carrying different fusion subtypes according to the experimental procedure shown in Example 1, and the results are shown in Table 4.
- the inclusion of the fusion subtype name column indicates the corresponding fusion subtype (eg, EML4-ALKv1) carried in the plasmid or sample, while the primer and probe columns indicate the corresponding primers and probes used to detect the corresponding fusion subtype (eg, SEQ ID NO: 1-3), whether the validation through column indicates whether the corresponding primer and probe can detect the corresponding fusion subtype.
- the corresponding primers and probes designed in the examples of the present invention can detect the corresponding fusion subtypes in qRT-PCR, and prove that the primers and probes of the present invention are used for detecting non-small cell lung cancer. It's useful.
Abstract
本发明公开了一种检测非小细胞肺癌的qRT-PCR引物、探针、芯片、试剂盒、应用和方法,所述引物和探针具有选自SEQ ID NO:1-66的序列,可检测EML4-ALK、NMP1-ALK、CD74-ROS、SLC34A2-ROS、CDCC6-RET和KIF5B-RET融合基因的22种亚型。
Description
本发明涉及分子生物学技术领域,尤其涉及一种检测非小细胞肺癌的qRT-PCR引物、探针、芯片、试剂盒、应用和方法。
当今社会,肿瘤已经成为对人类健康的一大威胁,2012年全球有1410万肿瘤新发病例,820万死亡病例,根据中国肿瘤登记中心2012年报,现在我国2010年新增肿瘤患者高达309万,死亡病例196万,平均每分钟有6人被确诊为肿瘤患者,按平均寿命74岁计算,每人的一生中有22%的几率患上肿瘤。而肺癌则是其中对中国患者威胁最大的癌症,发病率居所有恶性肿瘤的首位,平均每年有60万新发病例。相应的,肺癌死亡数也高居所有癌症之首,全国共有49万死亡病例,且死亡率在近些年快速上升,根据抽样调查,肺癌的死亡率从2005年的31/10万人,增至2010年的78/10万人,比排名第二的肝癌高出50%,对肺癌的检测与治疗已成为医学界最关注的问题之一。
从组织学上,肺癌分为小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC),两种癌症在恶性程度,肿瘤特点和治疗方法上均有较大的区别。NSCLC患者占病人总数的比例较多,为80%-85%。
融合基因则是NSCLC患者中存在的一种特征性突变,与NSCLC的用药和患者的预后显著相关。
以EML4-ALK这一融合基因为例,该融合基因首先于2007年被发现(Soda,Manabu,et al.Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.Nature 448.7153(2007):561-566),是发生在2号染色体上的重排所致的EML4基因与ALK基因相互融合,产生异常的EML4-ALK融合蛋白。根据临床实验,克唑替尼(Crizotinib)针对具有ALK融合基因的晚期NSCLC患者,产生的疗效显著优于传统化疗手段(Ou,Sai-Hong Ignatius,et al.Crizotinib for the treatment of ALK-rearranged non-small cell lung cancer:a success story to usher in the second decade of molecular targeted therapy in oncology.The oncologist 17.11(2012):1351-1375)。除此之外,克唑替尼也可作用于ROS融合
基因,阻断其异常的融合通路,抑制肿瘤细胞生长。还有科学研究发现,靶向药物索拉非尼(Sorafenib)、舒尼替尼(Sunitinib)和凡德他尼(Vandetanib)可以对含RET融合基因的细胞产生杀灭作用,使得RET融合基因也成为被普遍关注的潜在药物靶点。
目前美国国立综合癌症网络(NCCN)的NSCLC治疗指南中,已将ALK融合基因的检测列为推荐检测项目。而我国专家根据共识(张绪超,等人.中国间变性淋巴瘤激酶(ALK)阳性非小细胞肺癌诊断专家共识(2013版).中华病理学杂志42.006(2013):402-406),推荐了三种用于检测融合基因的方法,分别是荧光原位杂交(FISH)、免疫组化(IHC)和实时定量PCR(qRT-PCR),其中qRT-PCR的方法具有可确定融合型、操作简便、敏感性高、判定准确等特点,目前已有被CFDA批准的产品上市。
但是每种融合基因都有多种亚型,根据现有文献报道,EML4与ALK融合共有21种突变体,除其中1种发生在外显子19上之外,其余全部为EML4的某个外显子与ALK的外显子20相连。在所有发表的突变体中,出现频率最高的依次为:突变体1(49.6%),突变体3a/b(25.6%)和突变体2(10%)。也存在ALK与其他基因的融合,但是其临床影响尚无法确定,即使仅选取出现频率较高的亚型,在一个单一的qRT-PCR结果中也难以同时检测多种不同的融合基因。
发明内容
本发明提供一种检测非小细胞肺癌的qRT-PCR引物、探针、芯片、试剂盒、应用和方法,具有需要样本量少、可同时检测多种融合基因、操作简便快速及敏感性和准确性高的特点。
根据本发明的第一方面,本发明提供一组检测非小细胞肺癌的qRT-PCR引物和探针,所述引物和探针选自如下序列组(a)~(v)中的至少一组:
(a)EML4-ALKv1-F引物:GCCCACACCTGGGAAAGG(SEQ ID NO:1);
EML4-ALKv1-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:2);
EML4-ALKv1-P探针:R-CCTAAAGTGTACCGCCGGA-Q(SEQ ID NO:3);
(b)EML4-ALKv2-F引物:TCTAACTCGGGAGACTATGAAATATTGTA(SEQ ID NO:4);
EML4-ALKv2-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:5);
EML4-ALKv2-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:6);
(c)EML4-ALKv3-F引物:GCATAAAGATGTCATCATCAACCAA(SEQ ID NO:7);
EML4-ALKv3-R引物:GGTGCGGAGCTTGCT(SEQ ID NO:8);
EML4-ALKv3-P探针:R-CACCAGGAGCTGCAAGCCATGC-Q(SEQ ID NO:9);
(d)EML4-ALKv4-F引物:ACAATCAGAGCTGTAGCAGAAGGA(SEQ ID NO:10);
EML4-ALKv4-R引物:GGTCGAGGTGCGGAGCTT(SEQ ID NO:11);
EML4-ALKv4-P探针:R-AGGCAGATCAATTTT-Q(SEQ ID NO:12);
(e)EML4-ALKv5-F引物:GAATCCTGAAAGAGAAATAGAGATATGCT(SEQ ID NO:13);
EML4-ALKv5-R引物:GGTCGAGGTGCGGAGCTT(SEQ ID NO:14);
EML4-ALKv5-P探针:R-CCCTGAGTACAAGCTG-Q(SEQ ID NO:15);
(f)EML4-ALKv6-F引物:GGGAATGAACAGCTCTCTGTGAT(SEQ ID NO:16);
EML4-ALKv6-R引物:GGAGCTTGCTCAGCTTGTACTCA(SEQ ID NO:17);
EML4-ALKv6-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:18);
(g)EML4-ALKv7-F引物:CAGTGAAAAAATCAGTCTCAAGTAAAGTG(SEQ ID NO:19);
EML4-ALKv7-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:20);
EML4-ALKv7-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:21);
(h)EML4-ALKv8-F引物:GGAGTGCAGTGGCAATTTACAC(SEQ ID NO:22);
EML4-ALKv8-R引物:GGAGCTTGCTCAGCTTGTACTCA(SEQ ID NO:23);
EML4-ALKv8-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:24);
(i)EML4-ALKv9-F引物:AAGTGGCCTGTGTAGTGCTTCA(SEQ ID NO:25);
EML4-ALKv9-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:26);
EML4-ALKv9-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:27);
(j)NMP1-ALK-F引物:TCAGGGCCAGTGCATATTAGTG(SEQ ID NO:28);
NMP1-ALK-R引物:CGGAGCTTGCTCAGCTTGTA(SEQ ID NO:29);
NMP1-ALK-P探针:R-CCGGAAGCACCAGGA-Q(SEQ ID NO:30);
(k)CD74-ROS32-F引物:CAGGCACTCCTTGGAGCAA(SEQ ID NO:31);
CD74-ROS32-R引物:TTGGGAATGCCTGGTTTATTTG(SEQ ID NO:32);
CD74-ROS32-P探针:R-CACTGACGCTCCACC-Q(SEQ ID NO:33);
(1)CD74-ROS34-F引物:CAGGCACTCCTTGGAGCAA(SEQ ID NO:34);
CD74-ROS34-R引物:GGGATTGTAACAACCAGAAATATTCC(SEQ ID NO:35);
CD74-ROS34-P探针:R-CACTGACGCTCCACC-Q(SEQ ID NO:36);
(m)SLC34A2-ROS32-F引物:TTCGTGTGCTCCCTGGATATT(SEQ ID NO:37);
SLC34A2-ROS32-R引物:GGGAATGCCTGGTTTATTTGG(SEQ ID NO:38);
SLC34A2-ROS32-P探针:R-CCTTCCAGCTGGTTGG-Q(SEQ ID NO:39);
(n)SLC34A2-ROS34-F引物:GCGCCTTCCAGCTGGTT(SEQ ID NO:40);
SLC34A2-ROS34-R引物:CAAAGGTCAGTGGGATTGTAACAA(SEQ ID NO:41);
SLC34A2-ROS34-P探针:R-TTTGGATACCAGAAACAAG-Q(SEQ ID NO:42);
(o)CDCC6-RET-F引物:GCCAGCGTGACCATCGA(SEQ ID NO:43);
CDCC6-RET-R引物:CAAATTCGCCTTCTCCTAGAGTTT(SEQ ID NO:44);
CDCC6-RET-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:45);
(p)KIF5B15-RET11-F引物:GGAATTGCTGTGGGAAATAATGA(SEQ ID NO:46);
KIF5B15-RET11-R引物:GACAGCGGCTGCGATCA(SEQ ID NO:47);
KIF5B15-RET11-P探针:R-CGAGCTGTGCCGCAC-Q(SEQ ID NO:48);
(q)KIF5B15-RET12-F引物:GAATTGCTGTGGGAAATAATGATG(SEQ ID NO:49);
KIF5B15-RET12-R引物:GCCTTCTCCTAGAGTTTTTCCAAGA(SEQ ID NO:50);
KIF5B15-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:51);
(r)KIF5B16-RET12-F引物:GCAGCATGTCAGCTTCGTATCT(SEQ ID NO:52);
KIF5B16-RET12-R引物:CGCCTTCTCCTAGAGTTTTTCCA(SEQ ID NO:53);
KIF5B16-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:54);
(s)KIF5B22-RET12-F引物:TTCAGGACCTGGCTACAAGAGTT(SEQ ID NO:55);
KIF5B22-RET12-R引物:GCCTTCTCCTAGAGTTTTTCCAAGA(SEQ ID NO:56);
KIF5B22-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:57);
(t)KIF5B23-RET12-F引物:TGAACAGCTCACTAAAGTGCACAA(SEQ ID NO:58);
KIF5B23-RET12-R引物:TCGCCTTCTCCTAGAGTTTTTCC(SEQ ID NO:59);
KIF5B23-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:60);
(u)KIF5B24-RET11-F引物:GAGGGCATTCTGCACAGATTG(SEQ ID NO:61);
KLF5B24-RET11-R引物:CCGAGACGATGAAGGAGAAGA(SEQ ID NO:62);
KIF5B24-RET11-P探针:R-CGAGCTGTGCCGCAC-Q(SEQ ID NO:63);
(v)KIF5B24-RET8-F引物:AAGAGGGCATTCTGCACAGATT(SEQ ID NO:64);
KIF5B24-RET8-R引物:CACTCCAGCCGTCTCTTGCT(SEQ ID NO:65);
KIF5B24-RET8-P探针:R-CGAGGAGGCGGGCT-Q(SEQ ID NO:66);
其中,R为荧光报告基团,Q为淬灭基团。
荧光报告基团为qRT-PCR中常用的荧光基团,可以是6-羧基荧光素(FAM)、六氯-6-甲基荧光素(HEX)等;淬灭基团为qRT-PCR中常用的淬灭基团,可以是黑洞淬灭染料(Black Hole Quencher-1,BHQ1)、6-羧基四甲基若丹明(TAMRA)等。
优选地,所述荧光报告基团为FAM;所述淬灭基团为BHQ1。
根据本发明的第二方面,本发明提供一种检测非小细胞肺癌的芯片,所述芯片上布置有第一方面所述的引物和探针。
根据本发明的第三方面,本发明提供第一方面所述的引物和探针在制备检测非小细胞肺癌的芯片中的应用。
根据本发明的第四方面,本发明提供一种检测非小细胞肺癌的试剂盒,所述试剂盒包含第一方面所述的引物和探针。
优选地,所述试剂盒还包含逆转录酶、DNA聚合酶及其缓冲液。
根据本发明的第五方面,本发明提供第一方面所述的引物和探针在制备检测非小细胞肺癌的试剂盒中的应用。
根据本发明的第六方面,本发明提供一种检测非小细胞肺癌的方法,所述方法包括:使用第一方面所述的引物和探针,以待检样品的RNA为模板,进行实时定量PCR;收集荧光信号,根据荧光信号强度和CT值判断阴性或阳性结果。
使用本发明的检测非小细胞肺癌的qRT-PCR的引物和探针进行qRT-PCR,能够准确而灵敏地检测出NSCLC患者体内的EML4-ALK、NMP1-ALK、CD74-ROS、SLC34A2-ROS、CDCC6-RET和KIF5B-RET融合基因的22种亚型。此外,通过将引物和探针预先喷涂到芯片上的方法,能在一次实验中同时进行数百个单重qRT-PCR反应,提升了检测效率,并且无引物和探针之间的相互干扰,而且具有需要样本量少、自动化操作、操作简单、检测速度快的特点。
图1为3种模板浓度下,从H2228细胞系检测EML4-ALK融合突变体3a/b的qRT-PCR结果。
下面通过具体实施例对本发明作进一步详细说明。除非特别说明,下面实施例中所使用的技术均为本领域内的技术人员已知的常规技术;所使用的仪器设备和试剂等,均为本领域内的技术人员可以通过公共途径如商购等获得的。
下面的实施例中,探针序列两端分别连有FAM作为作为荧光报告基团,
BHQ1作为淬灭基团。在探针完整的情况下,荧光报告基团发出的荧光被淬灭基团吸收,此时检测不到荧光信号;在qRT-PCR的退火过程中,探针与模板特异性结合,随着延伸反应的进行,荧光报告基团被DNA聚合酶的5’-3’核酸外切酶活性切除,从而与淬灭基团分离而发出荧光,通过荧光定量PCR仪检测荧光信号的强度,即可跟踪qRT-PCR的进行程度,实现定量检测。
配合使用WaferGen公司的SmartChipTM TE平台和SmartChip Cycler real-time PCR仪进行下面实施例。
实施例1
为检验本发明引物和探针的检测能力,使用含有EML4-ALK融合突变体3a/b(对应表1中的融合亚型EML4-ALKv3)的来自广州医科大学第一附属医院的NCI-H2228做样品,依流程进行实验。
1、针对融合亚型设计特异性引物及探针
通过查阅文献,对非小细胞肺癌用药相关的6个基因22个融合亚型设计特异性引物和探针,我们使用Primer express和Primer premier5针对这些位点设计引物和探针并进行比对。设计时保证上下游引物横跨融合位点,最后选择了22组,具体序列见表1。
表1融合亚型名称、引物及探针序列以及检测的拼接基因及外显子
2、定制芯片
本发明将22组引物和对应的探针布置在芯片上,每组的2条引物和1条探针占据1个微孔(可容纳100μL的反应液),2条引物各喷涂4×10-7nmol,探针喷涂2×10-7nmol,重复30次,制成定制芯片。
3、从样品组织提取RNA
使用Life technologies公司的Trizol LS,从细胞系H2228中提取RNA,用安捷伦2100生物分析仪(agilent 2100bioanalyzer)测定RNA的片段长度及浓度,结果显示:片段长度集中在1000bp~4000bp,且RNA总量在0.35μg-2μg。
4、配置一步法qRT-PCR体系,将样品和反应液加入芯片
在样本RNA通过质控后,取以10倍为浓度梯度,分别取3.5ng、35ng和350ng的RNA作为模板,使用Takara公司的一步法试剂盒One Step PrimeScript RT-PCR Kit(Perfect Real Time)配置混合液,体系如表2所示。
表2一步法qRT-PCR体系
配置完成后使用SmartChip TE Nanodispenser将反应液分装至芯片的微孔中,分液完成后,用配套的膜封好芯片表面,并离心使液体不溢出。
5、使用qRT-PCR仪进行扩增反应
反应程序如表3所示。
表3 qRT-PCR反应程序
在退火时检测FAM或ROX的荧光信号强度。
6、分析结果
分别检测每个位点对应的FAM的荧光强度,当荧光强度达阈值时,若CT值大于32,则该位点判定为阴性;若CT值小于32,则该位点判断为阳性。
该实施例中,在3种模板浓度(用量分别3.5ng、35ng和350ng)下,从H2228细胞系检测EML4-ALK融合突变体3a/b(对应表1中的融合亚型EML4-ALKv3)的qRT-PCR结果如图1所示。
检测结果表明,3种模板浓度下,CT值均小于32。说明该细胞系中存在EML4-ALK融合突变体3a/b,与实际结果相符。
实施例2
接下来我们又使用携带不同融合亚型的质粒或样本,按照实施例1所示的实验过程进行qRT-PCR测试,结果如表4所示。表4中,包含融合亚型名称列表示质粒或样本中携带的相应融合亚型(如EML4-ALKv1),而引物和探针列表示检测相应融合亚型使用的相应引物和探针(如SEQ ID NO:1-3),是否验证通过列表示相应引物和探针能否检测到相应融合亚型。
表4其它融合亚型验证结果
包含融合亚型名称 | 引物和探针 | 是否验证通过 |
SEQ ID NO | ||
EML4-ALKv1 | 1-3 | 是 |
EML4-ALKv2 | 4-6 | 是 |
EML4-ALKv4 | 10-12 | 是 |
EML4-ALKv5 | 13-15 | 是 |
EML4-ALKv6 | 16-18 | 是 |
EML4-ALKv7 | 19-21 | 是 |
EML4-ALKv8 | 22-24 | 是 |
NMP1-ALK | 28-30 | 是 |
CD74-ROS32 | 31-33 | 是 |
CD74-ROS34 | 34-36 | 是 |
SLC34A2-ROS32 | 37-39 | 是 |
SLC34A2-ROS34 | 40-42 | 是 |
CDCC6-RET | 43-45 | 是 |
KIF5B15-RET11 | 46-48 | 是 |
KIF5B22-RET12 | 55-57 | 是 |
KIF5B23-RET12 | 58-60 | 是 |
KIF5B24-RET11 | 61-63 | 是 |
KIF5B24-RET8 | 64-66 | 是 |
由表4所示的结果可知,本发明实施例中设计的相应引物和探针均能在qRT-PCR中检测到相应融合亚型,证明本发明的引物和探针在检测非小细胞肺癌中是可用的。
以上内容是结合具体的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干简单推演或替换。
Claims (8)
- 一组检测非小细胞肺癌的qRT-PCR引物和探针,其特征在于,所述引物和探针选自如下序列组(a)~(v)中的至少一组:(a)EML4-ALKv1-F引物:GCCCACACCTGGGAAAGG(SEQ ID NO:1);EML4-ALKv1-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:2);EML4-ALKv1-P探针:R-CCTAAAGTGTACCGCCGGA-Q(SEQ ID NO:3);(b)EML4-ALKv2-F引物:TCTAACTCGGGAGACTATGAAATATTGTA(SEQ ID NO:4);EML4-ALKv2-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:5);EML4-ALKv2-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:6);(c)EML4-ALKv3-F引物:GCATAAAGATGTCATCATCAACCAA(SEQ ID NO:7);EML4-ALKv3-R引物:GGTGCGGAGCTTGCT(SEQ ID NO:8);EML4-ALKv3-P探针:R-CACCAGGAGCTGCAAGCCATGC-Q(SEQ ID NO:9);(d)EML4-ALKv4-F引物:ACAATCAGAGCTGTAGCAGAAGGA(SEQ ID NO:10);EML4-ALKv4-R引物:GGTCGAGGTGCGGAGCTT(SEQ ID NO:11);EML4-ALKv4-P探针:R-AGGCAGATCAATTTT-Q(SEQ ID NO:12);(e)EML4-ALKv5-F引物:GAATCCTGAAAGAGAAATAGAGATATGCT(SEQ ID NO:13);EML4-ALKv5-R引物:GGTCGAGGTGCGGAGCTT(SEQ ID NO:14);EML4-ALKv5-P探针:R-CCCTGAGTACAAGCTG-Q(SEQ ID NO:15);(f)EML4-ALKv6-F引物:GGGAATGAACAGCTCTCTGTGAT(SEQ ID NO:16);EML4-ALKv6-R引物:GGAGCTTGCTCAGCTTGTACTCA(SEQ ID NO:17);EML4-ALKv6-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:18);(g)EML4-ALKv7-F引物:CAGTGAAAAAATCAGTCTCAAGTAAAGTG(SEQ ID NO:19);EML4-ALKv7-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:20);EML4-ALKv7-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:21);(h)EML4-ALKv8-F引物:GGAGTGCAGTGGCAATTTACAC(SEQ ID NO:22);EML4-ALKv8-R引物:GGAGCTTGCTCAGCTTGTACTCA(SEQ ID NO:23);EML4-ALKv8-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:24);(i)EML4-ALKv9-F引物:AAGTGGCCTGTGTAGTGCTTCA(SEQ ID NO:25);EML4-ALKv9-R引物:CCATCTGCATGGCTTGCA(SEQ ID NO:26);EML4-ALKv9-P探针:R-ACCGCCGGAAGCA-Q(SEQ ID NO:27);(j)NMP1-ALK-F引物:TCAGGGCCAGTGCATATTAGTG(SEQ ID NO:28);NMP1-ALK-R引物:CGGAGCTTGCTCAGCTTGTA(SEQ ID NO:29);NMP1-ALK-P探针:R-CCGGAAGCACCAGGA-Q(SEQ ID NO:30);(k)CD74-ROS32-F引物:CAGGCACTCCTTGGAGCAA(SEQ ID NO:31);CD74-ROS32-R引物:TTGGGAATGCCTGGTTTATTTG(SEQ ID NO:32);CD74-ROS32-P探针:R-CACTGACGCTCCACC-Q(SEQ ID NO:33);(1)CD74-ROS34-F引物:CAGGCACTCCTTGGAGCAA(SEQ ID NO:34);CD74-ROS34-R引物:GGGATTGTAACAACCAGAAATATTCC(SEQ ID NO:35);CD74-ROS34-P探针:R-CACTGACGCTCCACC-Q(SEQ ID NO:36);(m)SLC34A2-ROS32-F引物:TTCGTGTGCTCCCTGGATATT(SEQ ID NO:37);SLC34A2-ROS32-R引物:GGGAATGCCTGGTTTATTTGG(SEQ ID NO:38);SLC34A2-ROS32-P探针:R-CCTTCCAGCTGGTTGG-Q(SEQ ID NO:39);(n)SLC34A2-ROS34-F引物:GCGCCTTCCAGCTGGTT(SEQ ID NO:40);SLC34A2-ROS34-R引物:CAAAGGTCAGTGGGATTGTAACAA(SEQ ID NO:41);SLC34A2-ROS34-P探针:R-TTTGGATACCAGAAACAAG-Q(SEQ ID NO:42);(o)CDCC6-RET-F引物:GCCAGCGTGACCATCGA(SEQ ID NO:43);CDCC6-RET-R引物:CAAATTCGCCTTCTCCTAGAGTTT(SEQ ID NO:44);CDCC6-RET-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:45);(p)KIF5B15-RET11-F引物:GGAATTGCTGTGGGAAATAATGA(SEQ ID NO:46);KIF5B15-RET11-R引物:GACAGCGGCTGCGATCA(SEQ ID NO:47);KIF5B15-RET11-P探针:R-CGAGCTGTGCCGCAC-Q(SEQ ID NO:48);(q)KIF5B15-RET12-F引物:GAATTGCTGTGGGAAATAATGATG(SEQ ID NO:49);KIF5B15-RET12-R引物:GCCTTCTCCTAGAGTTTTTCCAAGA(SEQ ID NO:50);KIF5B15-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:51);(r)KIF5B16-RET12-F引物:GCAGCATGTCAGCTTCGTATCT(SEQ ID NO:52);KIF5B16-RET12-R引物:CGCCTTCTCCTAGAGTTTTTCCA(SEQ ID NO:53);KIF5B16-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:54);(s)KIF5B22-RET12-F引物:TTCAGGACCTGGCTACAAGAGTT(SEQ ID NO:55);KIF5B22-RET12-R引物:GCCTTCTCCTAGAGTTTTTCCAAGA(SEQ ID NO:56);KIF5B22-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:57);(t)KIF5B23-RET12-F引物:TGAACAGCTCACTAAAGTGCACAA(SEQ ID NO:58);KIF5B23-RET12-R引物:TCGCCTTCTCCTAGAGTTTTTCC(SEQ ID NO:59);KIF5B23-RET12-P探针:R-ATCCAAAGTGGGAATTC-Q(SEQ ID NO:60);(u)KIF5B24-RET11-F引物:GAGGGCATTCTGCACAGATTG(SEQ ID NO:61);KIF5B24-RET11-R引物:CCGAGACGATGAAGGAGAAGA(SEQ ID NO:62);KIF5B24-RET11-P探针:R-CGAGCTGTGCCGCAC-Q(SEQ ID NO:63);(v)KIF5B24-RET8-F引物:AAGAGGGCATTCTGCACAGATT(SEQ ID NO:64);KIF5B24-RET8-R引物:CACTCCAGCCGTCTCTTGCT(SEQ ID NO:65);KIF5B24-RET8-P探针:R-CGAGGAGGCGGGCT-Q(SEQ ID NO:66);其中,R为荧光报告基团,Q为淬灭基团。
- 根据权利要求1所述的引物和探针,其特征在于,所述荧光报告基团为FAM;所述淬灭基团为BHQ1。
- 一种检测非小细胞肺癌的芯片,其特征在于,所述芯片上布置有权利要求1或2所述的引物和探针。
- 权利要求1或2所述的引物和探针在制备检测非小细胞肺癌的芯片中的应用。
- 一种检测非小细胞肺癌的试剂盒,其特征在于,所述试剂盒包含权利要求1或2所述的引物和探针。
- 根据权利要求5所述的试剂盒,其特征在于,所述试剂盒还包含逆转录酶、DNA聚合酶及其缓冲液。
- 权利要求1或2所述的引物和探针在制备检测非小细胞肺癌的试剂盒中的应用。
- 一种检测非小细胞肺癌的方法,其特征在于,所述方法包括:使用权利要求1或2所述的引物和探针,以待检样品的RNA为模板,进行实时定量PCR;收集荧光信号,根据荧光信号强度和CT值判断阴性或阳性结果。
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