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WO2016106701A1 - Amorce, sonde, puce, kit de rt-pcr quantitative, application et procédé de détection du cancer du poumon non à petites cellules - Google Patents

Amorce, sonde, puce, kit de rt-pcr quantitative, application et procédé de détection du cancer du poumon non à petites cellules Download PDF

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Publication number
WO2016106701A1
WO2016106701A1 PCT/CN2014/095948 CN2014095948W WO2016106701A1 WO 2016106701 A1 WO2016106701 A1 WO 2016106701A1 CN 2014095948 W CN2014095948 W CN 2014095948W WO 2016106701 A1 WO2016106701 A1 WO 2016106701A1
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Prior art keywords
seq
primer
probe
eml4
ret12
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PCT/CN2014/095948
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English (en)
Chinese (zh)
Inventor
邵康
邵利彬
王晓倩
叶晓飞
安娜
王惠
钟国兴
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BGI Genomics Co Ltd
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BGI Genomics Co Ltd
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Priority to PCT/CN2014/095948 priority Critical patent/WO2016106701A1/fr
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    • C—CHEMISTRY; METALLURGY
    • 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

Definitions

  • 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.

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Abstract

L'invention concerne une amorce, une sonde, une puce, un kit de RT-PCR quantitative, une application et un procédé de détection d'un cancer du poumon non à petites cellules (NSCLC). L'amorce et la sonde comprennent une séquence choisie parmi SEQ ID NOs: 1 à 66, et permettent de détecter 22 sous-types de gènes de fusion, EML4-ALK, NMP1-ALK, CD74-ROS, SLC34A2-ROS, CDCC6-RET et KIF5B-RET.
PCT/CN2014/095948 2014-12-31 2014-12-31 Amorce, sonde, puce, kit de rt-pcr quantitative, application et procédé de détection du cancer du poumon non à petites cellules Ceased WO2016106701A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107574232A (zh) * 2017-08-21 2018-01-12 上海派森诺生物科技股份有限公司 人eml4‑alk融合基因检测方法
CN109022581A (zh) * 2018-08-13 2018-12-18 上海佰臻生物科技有限公司 Eml4-alk融合基因荧光定量pcr检测的试剂盒

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101466721A (zh) * 2007-04-13 2009-06-24 细胞信号技术公司 在人实体瘤中的基因缺损和突变体alk激酶
CN102575287A (zh) * 2009-05-15 2012-07-11 因赛特遗传学公司 用于诊断和治疗癌症的涉及alk融合物的方法和组合物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101466721A (zh) * 2007-04-13 2009-06-24 细胞信号技术公司 在人实体瘤中的基因缺损和突变体alk激酶
CN102575287A (zh) * 2009-05-15 2012-07-11 因赛特遗传学公司 用于诊断和治疗癌症的涉及alk融合物的方法和组合物

Non-Patent Citations (1)

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Title
WU, XIA;: "the Expression of ALK and K-Ras Protein and Somatic Gene Mutation and Its Significance in Non-small Cell Lung Cancer", CHINA MASTER'S S THESES FULL-TEXT DATABASE, 15 May 2014 (2014-05-15), pages 3 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107574232A (zh) * 2017-08-21 2018-01-12 上海派森诺生物科技股份有限公司 人eml4‑alk融合基因检测方法
CN109022581A (zh) * 2018-08-13 2018-12-18 上海佰臻生物科技有限公司 Eml4-alk融合基因荧光定量pcr检测的试剂盒

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