CN117844933B

CN117844933B - Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof

Info

Publication number: CN117844933B
Application number: CN202410258097.XA
Authority: CN
Inventors: 杨成镐; 孙建龙; 张通; 陈光辉; 丁波
Original assignee: Shanghai Fudison Life Sciences Co ltd
Current assignee: Shanghai Fudison Life Sciences Co ltd
Priority date: 2024-03-07
Filing date: 2024-03-07
Publication date: 2024-06-11
Anticipated expiration: 2044-03-07
Also published as: CN117844933A

Abstract

The invention relates to the field of molecular diagnosis, in particular to a multiplex PCR primer group for detecting lung tumor related gene variation and application thereof. The primer group provided by the invention is a primer group for detecting the following genes: ALK gene, BRAF gene, EGFR gene, ERBB2 gene, KRAS gene, MET gene, NRAS gene, PIK3CA gene, or TP53 gene. The invention can find the variation contained in the cells by carrying out gene detection on the abnormal cells in blood, further assist in judging the benign and malignant of the pulmonary nodules, also can be used for providing early warning of recurrence for patients after pulmonary tumor operation, can fill the existing unmet medical requirements to a certain extent, simplifies the complex flow of high-throughput sequencing traditional library establishment, and has great clinical and economic significance.

Description

Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof

Technical Field

The invention relates to the field of molecular diagnosis, in particular to a multiplex PCR primer group for detecting lung tumor related gene variation and application thereof.

Background

Cancer is a major public health problem worldwide, and according to the incidence rate and death rate of 2016 Chinese cancer released by 2022, 2 and 27 of the national cancer center, the new incidence rate of 2016 Chinese cancer is about 406.4 ten thousand, and the new incidence rate of 2016 Chinese cancer is about 241.35 ten thousand. Wherein, lung cancer, colorectal cancer, gastric cancer, liver cancer and female breast cancer are the first five, which account for 57.4% of the total number of new cases of cancer. Lung cancer, liver cancer, stomach cancer, colorectal cancer and esophageal cancer are the first five cancers of cancer death, accounting for 69.3% of the total number of cancer deaths (see in detail ZHENG R, ZHANG S, ZENG H,et al.Cancer incidence and mortality in China, 2016 [J].Journal of the National Cancer Center, 2022, 2(1): 1-9.).

The 5-year survival rates of tumors with better prognosis in China, such as breast cancer (82.0%), thyroid cancer (84.3%) and prostate cancer (66.4%), still vary from those in developed countries such as the United states (90.9%, 98% and 99.5%). The main reasons for this gap are the few cases in early stage of clinical diagnosis, the low rate of early diagnosis and the irregular clinical diagnosis and treatment of late stage cases.

Therefore, china should develop strength together in two aspects of enlarging the screening and early diagnosis and early treatment coverage of related tumors, standardization of clinical diagnosis and treatment of tumors and homogenization popularization and application, and the death rate of malignant tumors in China is reduced.

According to the ' blue book of health physical examination big data of the year 2022 ' of American health ', the lung nodule detection rate is 55.9% in the 9,167,999 physical examination persons except for the port australia of China, wherein the male lung nodule detection rate is 56.5% and the female lung nodule detection rate is 55.3%.

However, for some smaller pulmonary nodules, existing imaging techniques cannot accurately determine their malignancy.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a multiplex PCR primer set for detecting pulmonary tumor-related genetic variation and use thereof, which are used for solving the problems in the prior art.

In order to achieve the above and other related objects, the present invention provides a multiplex PCR primer set for detecting mutation of genes related to lung tumor, wherein the multiplex PCR primer set is a primer set for detecting any of the following genes: ALK gene, BRAF gene, EGFR gene, ERBB2 gene, KRAS gene, MET gene, NRAS gene, PIK3CA gene, or TP53 gene.

Preferably, the multiplex PCR primer set comprises a plurality of primer pairs having nucleotide sequences as shown in SEQ ID No.1-2、SEQ ID No.3-4、SEQ ID No.5-6、SEQ ID No.7-8、SEQ ID No.9-10、SEQ ID No.11-12、SEQ ID No.13-14、SEQ ID No.15-16、SEQ ID No.17-18、SEQ ID No.19-20、SEQ ID No.21-22、SEQ ID No.23-24、SEQ ID No.25-26、SEQ ID No.27-28、SEQ ID No.29-30、SEQ ID No.31-32、SEQ ID No.33-34、SEQ ID No.35-36、SEQ ID No.37-38、SEQ ID No.39-40、SEQ ID No.41-42、SEQ ID No.43-44、SEQ ID No.45-46、SEQ ID No.47-48、SEQ ID No.49-50、SEQ ID No.51-52、SEQ ID No.53-54、SEQ ID No.55-56、SEQ ID No.57-58 or SEQ ID Nos. 59-60.

The invention also provides application of the multiplex PCR primer group in preparation of detection products of the circulating tumor cell lung tumor related gene variation.

The invention also provides a detection kit for the lung tumor related gene variation of the circulating tumor cells, which comprises the multiplex PCR primer group.

The invention also provides a method for detecting lung tumor-related genetic variation in circulating tumor cells for non-disease diagnosis purposes, the method comprising one or more of the following steps:

1) Lysing the sample to be tested, and amplifying the whole genome of the sample to be tested;

2) Amplifying the specific genes in the whole genome in the step 1) by using the multiplex PCR primer group or the detection kit, and establishing a sequencing library;

3) And (3) using the sequencing library in the step (2) to obtain the nucleotide sequence of the specific gene in the step (2), and determining the result of the genetic variation in the circulating tumor cells according to the comparison result of the nucleotide sequence and the sequence information of the standard library.

As described above, the multiplex PCR primer set for detecting the lung tumor-related genetic variation and the application thereof have the following beneficial effects: by carrying out gene detection on abnormal cells in blood, the mutation contained in the cells is found, so that the benign and malignant lung nodules can be judged in an assisted manner, the early warning of recurrence can be provided for patients after lung tumor operation, the existing unmet medical requirements can be filled to a certain extent, the complex process of constructing a traditional library for high-throughput sequencing is simplified, and the method has great clinical and economic significance.

Drawings

FIG. 1 shows the agarose gel electrophoresis results of the mixed multiplex PCR of NRAS and MET, NRAS and PIK3CA, NRAS and KRAS, BRAF and MET, BRAF and PIK3CA, BRAF and KRAS in the present invention.

FIG. 2 shows the agarose gel electrophoresis results of KRAS, BRAF and MET, KRAS, NRAS and PIK3CA mixed multiplex PCR in accordance with the present invention.

FIG. 3 shows a graph of agarose gel electrophoresis results of ALK, BRAF, EGFR, ERBB, KRAS, MET, NRAS, PIK, 3CA and TP53 multiplex PCRs in the present invention.

FIG. 4 shows the specific nucleotide sequences of the multiplex PCR primer sets of the present invention.

FIG. 5 shows the results of ALK, BRAF, EGFR, ERBB, KRAS, MET, NRAS, PIK3CA and TP53 multiplex PCR in the present invention.

FIG. 6 shows the result of sensitivity detection of multiplex PCR primer sets in the present invention.

Fig. 7 shows the expected product lengths for ALK, BRAF, EGFR, ERBB, KRAS, MET, NRAS, PIK3CA and TP53 in the present invention.

FIG. 8 shows the results of stability test for detecting genetic variation by multiplex PCR primer sets according to the present invention.

FIG. 9 shows the results of practical application of the multiplex PCR primer set of the present invention.

Detailed Description

The invention provides a multiplex PCR primer group for detecting lung tumor related gene variation, which is a primer group for detecting any of the following genes: ALK gene, BRAF gene, EGFR gene, ERBB2 gene, KRAS gene, MET gene, NRAS gene, PIK3CA gene, or TP53 gene.

In some embodiments, the multiplex PCR primer set comprises a plurality of primer pairs having nucleotide sequences as set forth in SEQ ID No.1-2、SEQ ID No.3-4、SEQ ID No.5-6、SEQ ID No.7-8、SEQ ID No.9-10、SEQ ID No.11-12、SEQ ID No.13-14、SEQ ID No.15-16、SEQ ID No.17-18、SEQ ID No.19-20、SEQ ID No.21-22、SEQ ID No.23-24、SEQ ID No.25-26、SEQ ID No.27-28、SEQ ID No.29-30、SEQ ID No.31-32、SEQ ID No.33-34、SEQ ID No.35-36、SEQ ID No.37-38、SEQ ID No.39-40、SEQ ID No.41-42、SEQ ID No.43-44、SEQ ID No.45-46、SEQ ID No.47-48、SEQ ID No.49-50、SEQ ID No.51-52、SEQ ID No.53-54、SEQ ID No.55-56、SEQ ID No.57-58 or SEQ ID Nos. 59-60. Specifically, the multiplex PCR primer group comprises primer pairs with nucleotide sequences shown as SEQ ID No.1-6, SEQ ID No.11-18, SEQ ID No.19-24 and SEQ ID No.53-60, and is used for amplifying ALK gene, EGFR gene, ERBB2 gene and TP53 gene simultaneously; and/or primer pairs with nucleotide sequences shown as SEQ ID No.7-10, SEQ ID No.25-32 and SEQ ID No.33-40, for amplifying BRAF gene, KRAS gene and MET gene simultaneously; and/or, the primer pairs with nucleotide sequences shown as SEQ ID No.41-44 and SEQ ID No.45-52 are used for amplifying the NRAS gene and the PIK3CA gene simultaneously. More specifically, the multiplex PCR primer set comprises primer pairs with nucleotide sequences shown as SEQ ID Nos. 1-60. The specific nucleotide sequence of the above primer is shown in FIG. 4.

In some embodiments, the primer pair further comprises a linker sequence fragment having a nucleotide sequence shown as SEQ ID No.61 or SEQ ID No. 62. More specifically, the nucleotide sequences of the primer pairs are shown in SEQ ID Nos. 63-122.

In some embodiments, the multiplex PCR primer set further comprises a pool-building primer comprising a nucleic acid molecule having a nucleotide sequence as set forth in any one of SEQ ID Nos. 123-134.

In some embodiments, the pool-building primer further comprises a linker sequence fragment or a spacer sequence fragment as described above. Specifically, the nucleotide sequence of the spacer sequence fragment is shown in any one of SEQ ID No. 137-148.

In some embodiments, the nucleotide sequence of the pool-building primer is set forth in any one of SEQ ID Nos. 125-136.

The invention also provides application of the multiplex PCR primer group in preparation of a circulating tumor cell detection product.

The invention also provides a circulating tumor cell detection kit, which comprises the multiplex PCR primer set.

In some embodiments, the circulating tumor cell detection kit further comprises a PCR reaction reagent. Specifically, the PCR reaction reagent comprises one or more of PCR buffer, DNA polymerase, magnesium ion, calcium ion, dNTPs, surfactant or preservative.

In some embodiments, the PCR buffer may be a phosphate buffer. The phosphate buffer is used to provide stable enzymatic reaction conditions. The surfactant is used for solubilization, and the dispersing solute promotes enzymatic reaction. The preservative is used to control the growth of microorganisms in the PCR reaction reagents.

The phosphate in the phosphate buffer solution is selected from potassium phosphate or sodium phosphate. The potassium phosphate salt and the sodium phosphate salt are selected from any one or more of potassium phosphate, monopotassium phosphate, dipotassium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate.

The surfactant is a substance which contains hydrophilic groups and hydrophobic groups at the same time so as to obviously reduce the surface tension of the target solution. The surfactant is selected from any one or more of stearic acid, sodium dodecyl benzene sulfonate, quaternary ammonium compound, lecithin, amino acid type, betaine type, alkyl glucoside, fatty glyceride, fatty sorbitan or polysorbate.

The preservative may be self-produced or use commercial agents. Preferably, the preservative is any one or more of the commercial ProClin series of preservatives ProClin, 150, 200, 300, or 5000. More preferably, the preservative is ProClin300,300.

The invention also provides a method for detecting genetic variation related to lung tumor in circulating tumor cells for non-disease diagnosis purposes, which comprises one or more of the following steps:

In some embodiments, the amplification in step 1) is: the cleaved sample, PCR buffer, random hexamer primer, dNTP and Phi29 enzyme are mixed evenly and reacted at 25-35 ℃ for 12-20 h.

In some embodiments, the specific gene in step 2) comprises one or more of an ALK gene, a BRAF gene, an EGFR gene, an ERBB2 gene, a KRAS gene, a MET gene, an NRAS gene, a PIK3CA gene, or a TP53 gene.

In some embodiments, the amplifying in step 2) comprises one or more of the following steps:

a. the primer set shown in nucleotide sequence SEQ ID No.63-122 was synthesized.

B. configuring a Primer Mix 1: the primers shown in SEQ ID Nos. 63-68, 73-86 and 115-122 were added to nuclease-free water and mixed well.

C. Configuring a Primer Mix 2: the primers shown in SEQ ID Nos. 69-72 and 87-102 are added into the nuclease-free water and mixed uniformly.

D. Configuring a Primer Mix 3: the primers shown in SEQ ID No.103-114 are added into water without nuclease, and the mixture is uniformly mixed.

E. For each sample, the following PCR reaction system was configured

DNA polymerase and buffer, primer Mix 1 and whole genome sample obtained in step 1) above, and mixing.

(Ii) DNA polymerase and buffer, primer Mix 2 and whole genome sample obtained in the step 1) above, and mixing.

(Iii) DNA polymerase and buffer, primer Mix 3 and whole genome sample obtained in the above step 1), and mixing.

F. placing the prepared sample into a thermal cycler, and setting the temperature of a thermal cover to be 95-105 ℃. The reaction procedure was as follows: 90-105 ℃ 1-5 min, 20-40 s at 90-100 ℃, 20-40 s at 50-70 ℃ and 0.5-2 min at 65-75 ℃ are taken as one cycle, 30-40 cycles are carried out in total, and finally, 1-10min at 65-75 ℃.

In some embodiments, the creating a sequencing library in step 2) is one or more of the following steps:

a. primers shown in nucleotide sequences SEQ ID Nos. 125-136 were synthesized.

B. For each sample, one primer is selected in SEQ ID Nos. 125-130 (P7), one primer is selected in SEQ ID Nos. 131-136 (P5) as a pool-building primer pair, and the combination of pool-building primers of any two samples in the same batch cannot be the same.

C for each sample, the following PCR reaction system was configured: and (3) uniformly mixing the DNA polymerase, the buffer solution, the pool-building primer P7, the pool-building primer P5 and the amplification products in the step (2).

D. Placing the prepared sample into a thermal cycler, and setting the temperature of a thermal cover to be 95-105 ℃. The reaction procedure was as follows: 90-105 ℃ 1-5 min, 90-100 ℃ 20-40 s,50-70 ℃ 20-40 s,65-75 ℃ 0.5-2 min are used as one cycle, and the total cycle is 1-10 times, and finally 65-75 ℃ 1-10min.

G. after completion of the reaction of step d, the PCR product was purified for each sample.

H. The purified product is in a Illumina Nextera library structure, and can be sequenced on an Illumina high-throughput sequencing platform according to a normal procedure, such as Illumina Novaseq.

In some embodiments, the alignment of the nucleotide sequence described in step 3) with the sequence information of the standard library is:

After obtaining high throughput sequencing data for the samples, the data were first quality controlled using BBDuk to eliminate reads with library linker contamination and lower quality of sequencing by trimming. The remaining reads were then aligned to the human GRCh38 reference genome using Bowtie 2, followed by Freebayes to find sites with a mutation frequency >3%, and finally SnpEff and SnpSift to annotate the mutation, including predicting the effects of the mutation, the effects on protein production, and information on whether there are corresponding mutation sites in the NCBI Clinvar database.

Software package and database involved in data processing and analysis:

BBMap (BBDuk is part of BBMap) -Bushnell b. -sourceforge. Net/projects/bbmap-

Bowtie 2 – LANGMEAD B, SALZBERG S L. Fast gapped-read alignment with Bowtie 2 [J]. Nat Methods, 2012, 9(4): 357-359.

Freebayes – Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing.arXiv preprintarXiv:1207.3907 [q-bio.GN] 2012

SnpEff – CINGOLANI P, PLATTS A, WANG LE L,et al.A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3 [J].Fly (Austin), 2012, 6(2): 80-92.

SnpSift – CINGOLANI P, PATEL V M, COON M,et al.Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift [J].Front Genet, 2012, 3: 35.

Clinvar – LANDRUM M J, LEE J M, BENSON M,et al.ClinVar: improving access to variant interpretations and supporting evidence [J].Nucleic Acids Res, 2018, 46(D1): D1062-d1067.

We considered the variation in the sample cell genome as having a variation frequency >3%, a sequencing depth >1650, and more than 30 sites where reads supported the variation.

According to details see ：PETRACKOVA A, VASINEK M, SEDLARIKOVA L,et al.Standardization of Sequencing Coverage Depth in NGS: Recommendation for Detection of Clonal and Subclonal Mutations in Cancer Diagnostics [J].Front Oncol, 2019, 9: 851.

In the present invention, the term "circulating tumor cells" or "CTC (circulating tumor cell)" refers generally to tumor cells that shed from the primary tumor or metastasis site and spontaneously or possibly even surgically enter the peripheral blood circulation.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art, see in particular Sambrook et al MOLECULAR CLONING ：A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989 and Third edition,2001 ;Ausubel et al ,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley&Sons,New York,1987 and periodic updates ;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego ;Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998 ;METHODS IN ENZYMOLOGY,Vol.304,Chromatin (P.M.Wassarman and A.P.Wolffe,eds.),Academic Press,San Diego,1999 ; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (P.B. Becker, ed.) Humana Press, totowa,1999, et al.

The nucleotide sequence information used in the invention is as follows:

SEQ ID No.61 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG

SEQ ID No.62 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG

SEQ ID No.63 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCGGACTCTGTAGGCTGC

SEQ ID No.64 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAGGGTGTCTCTCTGTGGCTT

SEQ ID No.65 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCCAGTTTAAGATTTGCCCAGA

SEQ ID No.66 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTTGCTCCTTCCATCCTTGC

SEQ ID No.67 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAATCCTAGTGATGGCCGTTGT

SEQ ID No.68 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACACCCCATTCTTGAGGGG

SEQ ID No.69 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCCCTCTCAGGCATAAGGT

SEQ ID No.70 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTCACAATGTCACCACATTACA

SEQ ID No.71 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTTGCTCTGATAGGAAAATGAG

SEQ ID No.72 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGCATCTCAGGGCCAAAAAT

SEQ ID No.73 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGCTGAGGTGACCCTTGTCTCT

SEQ ID No.74 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACAGCTTGCAAGGACTCTGGG

SEQ ID No.75 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTGCCAGTTAACGTCTTCCTTC

SEQ ID No.76 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAAAGGTGGGCCTGAGGTT

SEQ ID No.77 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTCTGGCCACCATGCGAAG

SEQ ID No.78 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATCCTGGCTCCTTATCTCCC

SEQ ID No.79 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAGCAGGGTCTTCTCTGTTTCA

SEQ ID No.80 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATACAGCTAGTGGGAAGGCA

SEQ ID No.81 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTAGCCCTCAATCCCTGACC

SEQ ID No.82 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCGGGCTGGGAGGACTTC

SEQ ID No.83 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTCCCATACCCTCTCAGCG

SEQ ID No.84 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCATCCTAGCCCCTTGTGGAC

SEQ ID No.85 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCACAAGGGGCTAGGATGG

SEQ ID No.86 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCTAGACACCACTCCACC

SEQ ID No.87 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTGGTGGAGTATTTGATAGTGTAT

SEQ ID No.88 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGAATGGTCCTGCACCAGTAA

SEQ ID No.89 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCCAGACTGTGTTTCTCCCT

SEQ ID No.90 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGCATGGCATTAGCAAAGACTC

SEQ ID No.91 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATGACAAAAGTTGTGGACAGGT

SEQ ID No.92 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTCAGTGTTACTTACCTGTCTTGT

SEQ ID No.93 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACTCTTACCAATGCAACAGACT

SEQ ID No.94 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGTTGCCACCTTGTTACCT

SEQ ID No.95 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGCCCATGATAGCCGTCTTTA

SEQ ID No.96 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACAATGTCACAACCCACTGAG

SEQ ID No.97 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCTTCCTGTTTCAGTCCCCATTA

SEQ ID No.98 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGCAAAGGCCAAAGATAAAATGC

SEQ ID No.99 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGCAGTGCTAACCAAGTTCTTT

SEQ ID No.100 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAAACCACAAAAGTATACTCCA

SEQ ID No.101 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCTATTTCAGCCACGGGTAA

SEQ ID No.102 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGAGGAGAAACTCAGAGATAACCA

SEQ ID No.103 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTGGTTTCCAACAGGTTCTTGC

SEQ ID No.104 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGATCAGGTCAGCGGGCTA

SEQ ID No.105 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTGAACTTCCCTCCCTCCCTG

SEQ ID No.106 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAACACAAAGATCATCCTTTCAGAG

SEQ ID No.107 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACCTTCGGCTTTTTCAACCC

SEQ ID No.108 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTTAGAAAGGGACAACAGTTAAGC

SEQ ID No.109 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTCCAGACGCATTTCCACAG

SEQ ID No.110 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACATTCGGAGATTTGGATGTTCT

SEQ ID No.111 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCTGTGAATCCAGAGGGGAA

SEQ ID No.112 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAACAGAGAATCTCCATTTTAGCACT

SEQ ID No.113 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTCGAAAGACCCTAGCCTTAGA

SEQ ID No.114 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCGGTCTTTGCCTGCTGAG

SEQ ID No.115 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTTCACTTGTGCCCTGACTTTC

SEQ ID No.116 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAACCAGCCCTGTCGTCT

SEQ ID No.117 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTCTGATTCCTCACTGATTGC

SEQ ID No.118 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACAACCACCCTTAACCCCTC

SEQ ID No.119 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTCATCTTGGGCCTGTGTT

SEQ ID No.120 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGGGGATGTGATGAGAGGTG

SEQ ID No.121 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAGGGTGGTTGGGAGTAGATG

SEQ ID No.122 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGATAACTGCACCCTTGGTCTCC

SEQ ID No.123 AATGATACGGCGACCACCGAGATCTACAC

SEQ ID No.124 CAAGCAGAAGACGGCATACGAGAT

SEQ ID No.125 AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTCAGATGTGTATA

SEQ ID No.126 AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTCAGATGTGTATA

SEQ ID No.127 AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATGTGTATA

SEQ ID No.128 AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTCAGATGTGTATA

SEQ ID No.129 AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTCAGATGTGTATA

SEQ ID No.130 AATGATACGGCGACCACCGAGATCTACACAGAGTAGATCGTCGGCAGCGTCAGATGTGTATA

SEQ ID No.131 CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAGATGTGTAT

SEQ ID No.132 CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAGATGTGTAT

SEQ ID No.133 CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAGATGTGTAT

SEQ ID No.134 CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAGATGTGTAT

SEQ ID No.135 CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAGATGTGTAT

SEQ ID No.136 CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAGATGTGTAT

SEQ ID No.137 GTAAGGAG

SEQ ID No.138 ACTGCATA

SEQ ID No.139 TAGATCGC

SEQ ID No.140 CTCTCTAT

SEQ ID No.141 TATCCTCT

SEQ ID No.142 AGAGTAGA

SEQ ID No.143 TCGCCTTA

SEQ ID No.144 CTAGTACG

SEQ ID No.145 GCTCAGGA

SEQ ID No.146 TTCTGCCT

SEQ ID No.147 AGGAGTCC

SEQ ID No.148 CATGCCTA

Example 1 grouping of primer pairs in multiplex PCR

100 NCI-H1975 lung cancer cells (mimicking CTC) were spiked into 4 mL peripheral blood, and CTC was enriched to pick 9 white blood cells and 1 lung cancer cell. The sample was lysed and whole genome amplified as described in step 1). The whole genome amplified product after dilution was taken and subjected to the following experiment.

All primers (nucleotide sequences shown as SEQ ID No. 63-122) of ALK gene, BRAF gene, EGFR gene, ERBB2 gene, KRAS gene, MET gene, NRAS gene, PIK3CA gene and TP53 gene are placed in a tube for amplification and library construction, and the experimental steps are as follows.

A. primers shown in nucleotide sequences SEQ ID Nos. 63 to 122 were synthesized, and the concentration of each primer was diluted to 100. Mu.M.

B. configuration PRIMER MASTER Mix: 1 μl of each of SEQ ID Nos. 63-122 was taken and vortexed.

C, configuring a PCR reaction system as follows: to 2.6. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 2.4. Mu. L PRIMER MASTER Mix and 5. Mu.L of diluted samples of whole genome amplification product were added and vortexed.

D. The prepared sample was placed in a thermal cycler with the thermal lid temperature set at 105 ℃. The reaction procedure was as follows: 95℃3 min, with 95℃30 s,60℃30 s,72℃1 min as one cycle, a total of 35 cycles were performed, and finally 72℃5 min.

E. primers shown in nucleotide sequences SEQ ID Nos. 123 to 124 were synthesized, and the concentration of each primer was diluted to 10. Mu.M.

F. The primers shown in SEQ ID No.123 and SEQ ID No.124 are selected as a library-building primer pair

G. the following PCR reaction system was configured: to 7.2. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 0.4. Mu.L of SEQ ID No.123, 0.4. Mu.L of SEQ ID No.124 and 2. Mu.L of the multiplex PCR product obtained in step d were added and vortexed.

H. The prepared sample was placed in a thermal cycler with the thermal lid temperature set at 105 ℃. The reaction procedure was as follows: 95℃3 min, 95℃30 s,60℃30 s,72℃1 min are used as a cycle, and 5 cycles are performed in total, and finally 72℃2 min.

I. After completion of the reaction, the PCR product was purified using Beckman Coulter AMPure XP Beads for each sample, using a magnetic bead volume of 0.9X sample volume.

The specific operation steps are as follows:

https://www.beckman.com/reagents/genomic/cleanup-and-size-selection/pcr/ampure-xp-protocol

h. the purified product was sequenced on a Illumina Novaseq high throughput sequencing platform.

J. The data obtained from sequencing were quality controlled and aligned as described in step 3), and the number of reads on each amplicon alignment was counted as shown in FIG. 5.

As can be seen from FIG. 5, ALK, EGFR, ERBB, TP53 genes still amplify efficiently in one tube, so that the four genes ALK, EGFR, ERBB and TP53 can be divided into group A, while the remaining 5 genes BRAF, KRAS, MET, NRAS and PIK3CA need to be further grouped.

Based on the detection of 2 exons of each of the BRAF and NRAS genes, and the detection of 4 exons of each of the KRAS, MET and PIK3CA genes, the genes BRAF and NRAS with the smaller number of primers are respectively placed in the B group and the C group in order to make the number of the primers in each group as small as possible and average the number.

Since the nucleic acid separation effect of the small fragment is good in 2% agarose gel, the experimental result is better observed in order to reduce the fragment size of the product, and the primers used in the following experiments do not have the linker sequence shown in SEQ ID No.61 or SEQ ID No. 62. The specific experimental scheme is as follows:

k. primers shown in nucleotide sequences SEQ ID Nos. 7-10 and 25-52 were synthesized, and the concentration of each primer was diluted to 100. Mu.M.

Configuration of BRAF Primer Mix: 4. Mu.L of each of SEQ ID Nos. 7-10 was taken and water was added to 100. Mu.L.

And m. configuring KRAS, MET, NRAS and a Primer Mix of PIK3CA according to the step b. Namely, 4. Mu.L of the primer corresponding to each gene was mixed and the mixture was made up to 100. Mu.L.

N. the following PCR reaction system was configured:

To 3. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L NRAS PRIMER Mix, 1. Mu. L MET PRIMER Mix, 5. Mu.L of diluted sample of whole genome amplification product were added and vortexed.

(Ii) to 3. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L NRAS PRIMER Mix, 1. Mu.L of PIK3CA Primer Mix, 5. Mu.L of diluted sample of whole genome amplification product were added and vortexed.

(Iii) 10. Mu.L of 2 XTaq Mix, 1. Mu. L NRAS PRIMER Mix, 1. Mu. L KRAS PRIMER Mix, 5. Mu.L of diluted sample of whole genome amplification product was added to 3. Mu.L of nuclease-free water and vortexed.

(Iv) to 3. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L BRAF Primer Mix, 1. Mu. L MET PRIMER Mix, 5. Mu.L of diluted sample of whole genome amplification product was added, and vortexed and homogenized.

(V) to 3. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L BRAF Primer Mix, 1. Mu.L of PIK3CA Primer Mix, 5. Mu.L of diluted sample of whole genome amplification product were added and vortexed.

(Vi) to 3. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L BRAF Primer Mix, 1. Mu. L KRAS PRIMER Mix, 5. Mu.L of diluted sample of whole genome amplification product were added and vortexed.

Placing the prepared sample into a thermal cycler, and setting the temperature of a thermal cover to be 105 ℃. The reaction procedure was as follows: 95℃3 min, with 95℃30 s,60℃30 s,72℃1 min as one cycle, a total of 35 cycles were performed, and finally 72℃5 min.

After the reaction, 2. Mu.L of amplified product was mixed with 1. Mu.L of DNA loading buffer and 3. Mu.L of nuclease-free water for each reaction system, and vortexed and homogenized.

The loading system was electrophoresed at 120V voltage in 5 μl of 100-1500 bp DNA Marker% 2% GelRed stained agarose gel for 50 min and photographed under uv light to give fig. 1.

As shown in fig. 1 (primer dimer is shown in the box), after combining NRAS and BRAF with KRAS, MET and PIK3CA, respectively, the combined primer dimer of BRAF and MET and NRAS and PIK3CA was minimized, thus determining group B as BRAF and MET, and group C as NRAS and PIK3CA.

KRAS was amplified with groups B (BRAF and MET) and C (NRAS and PIK3 CA) to determine whether KRAS should be placed in group B or group C. The specific experimental scheme is as follows:

and r, configuring a PCR reaction system as follows:

to 2. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L BRAF Primer Mix, 1. Mu. L MET PRIMER Mix, 1. Mu. L KRAS PRIMER Mix, 5. Mu.L of diluted sample of whole genome amplification product were added and vortexed.

(Ii) to 2. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 1. Mu. L NRAS PRIMER Mix, 1. Mu.L of PIK3CA Primer Mix, 1. Mu. L KRAS PRIMER Mix, 5. Mu.L of diluted sample of whole genome amplification product were added and vortexed.

And S, placing the prepared sample into a thermal cycler, wherein the temperature of a thermal cover is set to be 105 ℃. The reaction procedure was as follows: 95℃3 min, with 95℃30 s,60℃30 s,72℃1 min as one cycle, a total of 35 cycles were performed, and finally 72℃5 min.

After completion of the reaction, 2. Mu.L of the amplified product was mixed with 1. Mu.L of the DNA loading buffer and 3. Mu.L of nuclease-free water for each reaction system, and vortexed and homogenized.

U. the loading system was electrophoresed 50 min with 5 μl of 100-1500 bp DNA Marker in a 2% GelRed stained agarose gel at 120V voltage and photographed under uv light to give fig. 2.

As shown in FIG. 2, KRAS amplified with group B (BRAF and MET) performed significantly less than group C (NRAS and PIK3 CA) primer dimer.

In summary, the primer pairs in multiplex PCR were grouped as follows: a primer pair for amplifying ALK gene, EGFR gene, ERBB2 gene and TP53 gene; a primer pair for amplifying BRAF gene, KRAS gene and MET gene; a set of primer pairs for amplifying the NRAS gene and the PIK3CA gene.

Example 2 sensitive detection of primer sets

100 NCI-H1975 lung cancer cells (mimicking CTC) were spiked into 4 mL peripheral blood, and CTC was enriched to pick up 9 white blood cells and 1 lung cancer cell as one fraction, together with 4 fractions. These 4 samples were subjected to lysis, whole genome amplification, multiplex PCR and pooling, sequencing in accordance with steps 1) and 2). And (3) performing quality control and comparison on the data obtained by sequencing according to the step (3), and counting the numbers of reads on the comparison of each amplicon region, wherein the obtained results are shown in FIG. 6.

As can be seen from FIG. 6, in4 samples, the reads number was greater than 2000 for all amplicon alignments, indicating that all the regions to be detected were sequenced more than 2000 times, sufficient for subsequent variation discovery and analysis, i.e., the sensitivity of the primer pairs met the downstream analysis requirements.

Example 3 specific detection of primer sets

The 9 genes were subjected to multiplex PCR, respectively, and the template for the PCR reaction was the whole genome amplification product of #3 in the sample of example 2. The resulting product was electrophoresed in a 2% GelRed stained agarose gel to evaluate whether other non-specific bands were produced in addition to the expected bands. The specific experimental scheme is as follows:

a. primers shown in nucleotide sequences SEQ ID Nos. 63 to 122 were synthesized, and the concentration of each primer was diluted to 10. Mu.M.

B. Configuration ALK PRIMER Mix: 10. Mu.L of each of the primers shown in SEQ ID Nos. 63 to 68 was mixed, and the nuclease-free water was added to 80. Mu.L.

C. Referring to the method in step b, the Primer Mix of BRAF, EGFR, ERBB, KRAS, MET, NRAS, PIK3CA, and TP53 are respectively disposed. 10 mu L of primer corresponding to each gene is mixed, and less than 80 mu L of water without nuclease is supplemented to 80 mu L.

D. The following PCR reaction system was configured: to 1.8. Mu.L of nuclease-free water, 10. Mu.L of 2 XTaq Mix, 3.2. Mu.L of ALK/BRAF/EGFR/ERBB2/KRAS/MET/NRAS/PIK3CA/TP53 Primer Mix and 5. Mu.L of the whole genome amplification product of #3 in the example 2 sample were added and vortexed. A total of 9 reaction systems.

E. The prepared sample was placed in a thermal cycler with the thermal lid temperature set at 105 ℃. The reaction procedure was as follows: 95℃3 min, with 95℃30 s,60℃30 s,72℃1 min as one cycle, a total of 35 cycles were performed, and finally 72℃5 min.

G. After completion of the reaction, 2. Mu.L of the amplified product was mixed with 1. Mu.L of the DNA loading buffer and 3. Mu.L of nuclease-free water for each reaction system, and vortexed and homogenized.

H. The loading system was electrophoresed at 120V voltage in 5 μl of 50-700 bp DNA Marker% agarose gel stained with 2% GelRed for 50 min and photographed under uv light to give fig. 3. The expected band length of each gene is shown in FIG. 7.

As shown in fig. 3: the multiplex PCR products of 9 genes have no obvious impurity bands except the expected bands, which shows that the specificity of each primer pair is better.

Example 4 stability test for detecting Gene variation by primer set

Referring to example 2, 100 NCI-H1975 lung cancer cells (mimicking CTCs) were spiked into 4 mL peripheral blood, CTCs were enriched, 9 white blood cells were picked and 1 lung cancer cell was taken as one portion, and a total of 4 portions were picked. The 4 samples were subjected to lysis, whole genome amplification, multiplex PCR, library construction, sequencing and data analysis according to steps 1) -3).

Excluding genetic variation, the variation shown in fig. 8 was found to be detected in all 4 samples.

In FIG. 8, the detected variations are consistent with the variations of the EGFR and TP53 genes of the NCI-H1975 cell line recorded in the depmap database. See in detail: https:// depmap. Org/portal/cell_line/NCIH _ LUNGtab = mutations.

Therefore, the primer group can stably detect the mutation contained in single CTC cells, and has higher sensitivity.

Example 5 use of primer set in examining clinical samples

Collecting blood of a 45-year-old male suffering from lung adenocarcinoma, carrying out CTC enrichment on the blood, picking 10 morphologically abnormal large cells, and carrying out lysis, whole genome amplification, multiplex PCR, library establishment, sequencing and data analysis according to the steps.

Variations as shown in fig. 9 were detected in the samples.

The results of FIG. 9 demonstrate that it is feasible to detect variations in primer sets in clinically authentic samples.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. A multiplex PCR primer set for detecting lung tumor related gene variation, which is characterized in that the multiplex PCR primer set is a primer set for detecting the following genes: ALK genes, BRAF genes, EGFR genes, ERBB2 genes, KRAS genes, MET genes, NRAS genes, PIK3CA genes, and TP53 genes;

The multiplex PCR primer group consists of primer pairs with nucleotide sequences shown as SEQ ID No.1-2、SEQ ID No.3-4、SEQ ID No.5-6、SEQ ID No.7-8、SEQ ID No.9-10、SEQ ID No.11-12、SEQ ID No.13-14、SEQ ID No.15-16、SEQ ID No.17-18、SEQ ID No.19-20、SEQ ID No.21-22、SEQ ID No.23-24、SEQ ID No.25-26、SEQ ID No.27-28、SEQ ID No.29-30、SEQ ID No.31-32、SEQ ID No.33-34、SEQ ID No.35-36、SEQ ID No.37-38、SEQ ID No.39-40、SEQ ID No.41-42、SEQ ID No.43-44、SEQ ID No.45-46、SEQ ID No.47-48、SEQ ID No.49-50、SEQ ID No.51-52、SEQ ID No.53-54、SEQ ID No.55-56、SEQ ID No.57-58 and SEQ ID No. 59-60;

The nucleotide sequences of the multiplex PCR primer group are primer pairs shown as SEQ ID No.1-6, SEQ ID No.11-18, SEQ ID No.19-24 and SEQ ID No.53-60, and the primer pairs are used for amplifying ALK gene, EGFR gene, ERBB2 gene and TP53 gene simultaneously; primer pairs with nucleotide sequences shown as SEQ ID No.7-10, SEQ ID No.25-32 and SEQ ID No.33-40 are used for amplifying BRAF gene, KRAS gene and MET gene simultaneously; primer pairs with nucleotide sequences shown as SEQ ID No.41-44 and SEQ ID No.45-52 are used for amplifying NRAS genes and PIK3CA genes simultaneously;

The multiplex PCR primer group is a primer group for detecting ALK gene, BRAF gene, EGFR gene, ERBB2 gene, KRAS gene, MET gene, NRAS gene, PIK3CA gene and TP53 gene in circulating tumor cells.

2. The multiplex PCR primer set as claimed in claim 1 further comprising a linker sequence fragment having a nucleotide sequence as set forth in SEQ ID No.61 or SEQ ID No. 62.

3. The multiplex PCR primer set as claimed in claim 2 wherein the nucleotide sequences of the primer pairs are set forth in SEQ ID nos. 63-122.

4. The multiplex PCR primer set as defined in claim 1 wherein the multiplex PCR primer set comprises a pool-building primer comprising a nucleic acid molecule having a nucleotide sequence as set forth in any one of SEQ ID nos. 123-124.

5. The multiplex PCR primer set as defined in claim 4 wherein the pool-building primer further comprises a linker sequence fragment or a spacer sequence fragment of the multiplex PCR primer set as defined in claim 2, the nucleotide sequence of the spacer sequence fragment being set forth in any one of SEQ ID nos. 137-148.

6. The multiplex PCR primer set according to claim 5, wherein the nucleotide sequence of the pool-building primer is shown in any one of SEQ ID Nos. 125 to 136.

7. Use of the multiplex PCR primer set as defined in any one of claims 1 to 6 for the preparation of a detection product for lung tumor-related genetic variation of circulating tumor cells.

8. A kit for detecting a circulating tumor cell lung tumor-associated genetic variation, comprising the multiplex PCR primer set according to any one of claims 1-6.

9. The test kit of claim 8, wherein the test kit further comprises PCR reaction reagents.

10. The test kit of claim 9, wherein the PCR reaction reagents comprise one or more of PCR buffers, DNA polymerase, magnesium ions, calcium ions, dNTPs, surfactants, or preservatives.

11. A method for detecting a lung tumor-associated genetic variation in circulating tumor cells for non-disease diagnostic purposes, said method comprising one or more of the following steps:

2) Amplifying the specific genes in the whole genome in step 1) with the multiplex PCR primer set according to any one of claims 1 to 6 or the detection kit according to any one of claims 8 to 10 to create a sequencing library;