Primer probe composition for detecting gliocytoma gene and application thereof

Abstract

The invention discloses a primer probe composition for detecting eight gene mutations of human gliocytoma and application of the primer probe composition. The primer probe composition comprises primers and probes for detecting IDH1, IDH2, TERT promoters, H3F3A, H3C2, BRAF, EGFR and MET genes and a distribution mode of the primers and the probes. By screening and combining multiple primers, a primer combination with the minimum mutual interference degree is determined, the specificity and sensitivity of the primers are improved, the gene mutation low-frequency variation detection performance (VAF) reaches 0.2%, the gene fusion reaches the detection limit of 30 copies, and the detection of 6 gene mutations and 2 gene fusion can be completed at a time by applying a fluorescent PCR method. The method can be used for detecting tissue samples and cerebrospinal fluid samples.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to a primer-probe composition for detecting glioma genes and an application thereof. Background Art

[0002] Glioma, also known as glioma, is the number one tumor of the nervous system, accounting for about 40% to 50% of brain tumors. According to the characteristics of atypia, nuclear division index, endothelial cell proliferation and necrosis, WHO divides glioma into four grades; among them, grade I is mainly benign pilocytic astrocytoma, accounting for about 5% of gliomas, which can usually be cured; grade II is general astrocytoma or mixed oligoastrocytoma, accounting for about 30% to 40% of gliomas; grade III is anaplastic astrocytoma, accounting for about 15% to 25% of gliomas, generally evolved from grade I; grade IV is glioblastoma, accounting for about 1 / 3 of gliomas.

[0003] Studies have shown that among grade 2-3 gliomas, about 80% have isocitrate dehydrogenase (IDH) mutation characteristics. Isocitrate dehydrogenase (IDH) gene mutation is an important event in the early stage of glioma onset and can be used to determine the extent of glioma surgical resection and the patient's prognosis. Isocitrate dehydrogenase 1 (IDH1) mutations are common in grade 2 or 3 gliomas and secondary glioblastomas. Most IDH mutations occur at the IDH1 arginine 132 (R132) residue or the IDH2 arginine 172 (R172) residue.

[0004] Studies have shown that in addition to the IDH mutation feature, there are some other important molecular mutation features in human gliomas, such as H3, BRAF gene sites, and TERT Promoter regions. Different molecular mutation features have different effects on the occurrence, development, and treatment of tumors. Therefore, precise detection of different mutations at different sites is of great significance in clinical practice.

[0005] At present, the commonly used methods for detecting glioma gene mutations at home and abroad include immunohistochemistry, Sanger sequencing, pyrophosphate sequencing, second-generation sequencing, etc. These methods are either high-cost and long detection cycle, or low detection throughput, or low sensitivity and complex operation.

[0006] How to achieve complete inhibition of wild-type genes while detecting mutant genes without affecting the amplification of mutant genes is also a difficulty in glioma gene detection.

[0007] Therefore, the market is in urgent need of a glioma detection product with high throughput, high sensitivity, high specificity, and complete inhibition of wild-type gene amplification. Summary of the invention

[0008] In order to solve the above technical problems, the first aspect of the present invention provides a primer-probe combination for detecting glioma genes, the primer-probe combination comprising a first group of primer-probe combinations; the first group of primer-probe combinations comprising upstream primers IDH2-R172K-F, TERT-C228T-F, TERT-C250T-F, H3C2-K28M-F, downstream primers IDH2-R172K-R, TERT-R, H3C2-K28M-R, and probes IDH2-R172K-P, TERT-P, H3C2-K28M-P; the nucleotide sequences of the upstream primers are respectively as shown in SEQ ID NOs.1 to 4, wherein the first base at the 3' end of the upstream primer is complementary to the target gene, the second to sixth bases contain 1 to 3 bases that are mismatched with the target gene, and the remaining bases are completely complementary to the target gene; the nucleotide sequences of the downstream primers are respectively as shown in SEQ ID NOs.5 to 7; the nucleotide sequences of the probes are respectively as shown in SEQ ID As shown in NO.8-10, the probe sequences are modified by different fluorescent groups.

[0009] The targeted glioma genes of the first set of primer-probe combinations are shown in the table below.

[0010] Table 1 Genes corresponding to the primer-probe combinations of Group 1 and Group 2

[0011]

[0012] As a preferred embodiment, the primer-probe combination further includes a second group of primer-probe combinations; the second group of primer-probe combinations includes upstream primers IDH1-R132H-F, BRAF-V600E-F, H3F3A-K28M-F, H3F3A-G35R-F, H3F3A-G35V-F, downstream primers IDH1-R132H-R, BRAF-V600E-R, H3F3A-R, and probes IDH1-R132H-P, BRAF-V600E-P, and H3F3A-P; the nucleotide sequences of the upstream primers are respectively as shown in SEQ ID NOs.11 to 15, wherein the first base at the 3' end of the upstream primer is complementary to the target gene, the second to sixth bases contain 1 to 3 bases that are mismatched with the target gene, and the remaining bases are completely complementary to the target gene; the nucleotide sequences of the downstream primers are respectively as shown in SEQ ID The probe nucleotide sequences are shown in SEQ ID NOs. 16 to 18; the probe nucleotide sequences are shown in SEQ ID NOs. 19 to 21, respectively, and the probe sequences are modified by different fluorescent groups.

[0013] The targeted glioma genes of the second set of primer-probe combinations are shown in Table 1 .

[0014] As a preferred embodiment, the primer-probe composition also includes a third group of primer-probe compositions; the third group of primer-probe compositions includes upstream primers EGFR-F1, EGFR-F2, EGFR-F3, EGFR-F4, MET-F1, MET-F2, MET-F3, MET-F4, MET-F5, MET-F6, downstream primers EGFR-R1, MET-R1, EGFR-R2, MET-R2, MET-R3, and probes EGFR-P1, EGFR-P2, EGFR-P3, MET-P1, MET-P2, MET-P3; the nucleotide sequences of the upstream primers are respectively shown in SEQ ID NOs.22 to 31; the nucleotide sequences of the downstream primers are respectively shown in SEQ ID NOs.32 to 36; the nucleotide sequences of the probes are respectively shown in SEQID NOs.37 to 42, and the probe sequences are modified by different fluorescent groups.

[0015] The targeted glioma genes of the third set of primer-probe combinations are shown in the table below.

[0016] Table 2 Genes corresponding to the third group of primer-probe combinations

[0017]

[0018]

[0019] As a preferred embodiment, the primer-probe composition further comprises an internal reference primer-probe composition.

[0020] As a preferred embodiment, the internal reference primer probe combination of the first or second group of primer probe combinations is GAPDH-F, GAPDH-R, GAPDH-P, and their sequences are shown in SEQ ID NOs. 43 to 45, respectively.

[0021] As a preferred embodiment, the internal reference primer probe composition of the third group of primer probe compositions is RNase PF, RNase PR, and RNase PP, and their sequences are shown in SEQ ID NOs. 46 to 48, respectively.

[0022] When the first, second, and third groups of primer-probe combinations are combined, a single test can be used to determine whether the sample belongs to the above eight gene mutations or fusions; when the sample belongs to one or more of the above eight genes, a single test can be used to characterize the sample. The above primer-probe combination information is shown in the table below, where bases represented by lowercase letters are mismatched bases.

[0023] Table 3 Primer probe composition information

[0024]

[0025]

[0026]

[0027] In a second aspect of the present invention, a kit for detecting glioma genes is provided, the kit comprising an amplification reaction system, the amplification reaction system comprising one or more groups of primer-probe combinations from Group 1 to Group 3, and the different groups are separately arranged. The probes in each group are modified with different fluorescent groups to identify the gene.

[0028] As a preferred embodiment, the amplification reaction system further includes an amplification buffer, an amplification enzyme, and an amplification enhancement buffer.

[0029] As a preferred embodiment, the kit further comprises a reverse transcription reaction system. When the kit comprises a reverse transcription reaction system, it can be used to detect RNA.

[0030] As a preferred embodiment, the kit also includes a quality control product.

[0031] As a preferred embodiment, the quality control products include negative quality control products and positive quality control products.

[0032] As a preferred embodiment, the kit further comprises an internal reference.

[0033] This kit adds an internal reference probe (ROX) to the reaction solution. Under the premise that the negative control (NC), no-template negative control (NTC), and positive control (PC) are normal, it can determine whether the gene is negative or positive in the corresponding fluorescence channel.

[0034] When the kit contains the first, second and third groups of primer-probe combinations, a single test can be used to determine whether the sample belongs to the above eight genes; when the sample belongs to one or more of the above eight genes, a single test can be used to characterize the sample.

[0035] The kit is suitable for genetic testing of fresh pathological tissue, paraffin-embedded tissue, paraffin-sectioned tissue or cerebrospinal fluid samples.

[0036] The kit composition information is shown in the table below.

[0037] Table 4 Kit composition

[0038]

[0039]

[0040] The third aspect of the present invention provides the use of the primer-probe combination in a kit for detecting glioma genes, comprising the following application steps:

[0041] S1: According to the primer-probe combination group, the primer-probe combination is mixed with an amplification buffer, an amplification enzyme, an amplification enhancement buffer, and a sample to be tested to prepare an amplification reaction system, and the amplification reaction system is added to different reaction wells;

[0042] S2: PCR amplification and collection of fluorescence signals, and judging the gene negativity or positivity of the sample based on the fluorescence signals.

[0043] As a preferred solution, the samples to be tested include DNA samples and cDNA samples. The cDNA samples are reverse transcribed from RNA.

[0044] As a preferred embodiment, the DNA sample is extracted from fresh pathological tissue, paraffin-embedded tissue, paraffin-sectioned tissue or cerebrospinal fluid sample.

[0045] As a preferred embodiment, the cDNA sample preparation method comprises the following steps:

[0046] S11: Extract sample RNA;

[0047] S12: Reverse transcription of RNA into cDNA.

[0048] As a preferred solution, the concentration of each primer in the amplification reaction system solution is 0.05-0.2 μmol / L.

[0049] As a preferred solution, the concentration of each probe in the amplification reaction system solution is 0.03-0.1 μmol / L.

[0050] Through the above technical solution, the present invention produces the following beneficial effects:

[0051] (1) For the DNA mutation detection part, the first base at the 3' end of the upstream primer is designed to be complementary to the target gene, the second to sixth bases contain 1 to 3 bases that are mismatched with the target gene, and the remaining bases are completely complementary to the target gene, thereby controlling the specific extension of the allele; due to the introduction of mismatched bases, the PCR primer extension speed slows down. When the mismatch reaches a certain level, the primer extension is terminated and no PCR amplification product of a specific length is obtained, thereby inhibiting the amplification of the wild-type gene.

[0052] (2) The present invention improves the specificity and sensitivity of glioma gene mutation detection primers through systematic screening, so that the detection performance of low-frequency variation of gene mutation (VAF) reaches 0.2% and the detection limit of gene fusion reaches 30 copies.

[0053] (3) The primers designed by the present invention cover 8 glioma genes. The present invention determines the primer combination with the least mutual interference through multiple primer screening, and then performs scientific and reasonable primer grouping design according to the needs of genotyping; according to the four commonly used fluorescent channels (FAM, ROX, VIC, CY5) of the fluorescent PCR instrument, different fluorescent groups are used to modify the probes, so that 8 glioma genes can be detected at the same time, with wide gene coverage and high detection throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the amplification diagram of the primer IDH1-R132H-F1 in Example 1;

[0055] Figure 2 This is the amplification diagram of the primer IDH1-R132H-F2 in Example 1;

[0056] Figure 3 This is the amplification diagram of the primer IDH1-R132H-F3 in Example 1;

[0057] Figure 4 This is the amplification diagram of the primer IDH1-R132H-F4 in Example 1;

[0058] Figure 5 This is the amplification diagram of the primer IDH1-R132H-F5 in Example 1;

[0059] Figure 6 This is the amplification diagram of the primer IDH1-R132H-F6 in Example 1;

[0060] Figure 7 This is the amplification diagram of primer IDH1-R132H-F7 in Example 1;

[0061] Figure 8 This is the amplification diagram of the primer IDH1-R132H-F8 in Example 1;

[0062] Fig. 9 This is the amplification diagram of the primer IDH1-R132H-F9 in Example 1;

[0063] Fig.10 is a sensitivity diagram of TERT-C228T of Example 10;

[0064] Fig.11 is a sensitivity diagram of TERT-C250T of Example 10;

[0065] Fig.12 is the sensitivity diagram of H3C2-K28M in Example 10;

[0066] Fig.13 is the sensitivity diagram of IDH2-R172K of Example 10;

[0067] Fig.14 is the sensitivity diagram of IDH1-R132H of Example 10;

[0068] Fig.15 is the sensitivity diagram of BRAF-V600E of Example 10;

[0069] Fig.16 is the sensitivity diagram of H3F3A-K28M of Example 10;

[0070] Fig.17 is the sensitivity diagram of H3F3A-G35R of Example 10;

[0071] Fig.18 is the sensitivity diagram of H3F3A-G35V of Example 10;

[0072] Fig.19 is a sensitivity diagram of the EGFR fusion gene EGFR VIII of Example 11;

[0073] Fig. 20 This is a sensitivity diagram of MET exon14 skipping of the MET fusion gene in Example 11;

[0074] Figures 1 to 9 The dark red curve is the 6000-copies mutant amplification curve; the green curve is the 600-copies mutant amplification curve; the blue curve is the 60-copies mutant amplification curve; the purple curve is the 6-copies mutant amplification curve; the pink curve is the 6000-copies wild-type amplification curve; Figures 10 to 18 The yellow curve in the middle is the amplification curve of GAPDH. DETAILED DESCRIPTION

[0075] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0076] This method has no special requirements for the extraction method of tissue DNA / RNA, as long as it complies with the conventional tissue DNA / RNA extraction method in the art.

[0077] Example 1 IDH1-R132H Primer Design and Screening

[0078] Through different primer designs, the inhibitory effect of primers on wild-type genes was examined, and the detection limit of primers amplifying mutant genes was examined.

[0079] 1. Primer design

[0080] In this example, a mutation site IDH1-R132H is selected to illustrate the primer screening process.

[0081] According to the IDH1-R132H mutant gene, the first base at the 3' end of the upstream primer was designed to be complementary to the target gene, the 2nd to 6th bases contained 1 to 3 bases that were mismatched with the target gene, and the remaining bases were completely complementary to the target gene (lowercase letters represent mismatched bases). According to this method, a total of 9 upstream primers were designed, and each upstream primer was combined with the downstream primer and the probe to form primer-probe combinations A to I.

[0082] Table 5 IDH1-R132H primer screening list

[0083]

[0084] 2. Test methods

[0085] (1) Preparation of samples to be tested

[0086] The IDH1-R132H mutant plasmid was diluted into 4 gradients to prepare test sample 1, test sample 2, test sample 3, and test sample 4, with concentrations of 6000 copies / ul, 600 copies / ul, 60 copies / ul, and 6 copies / ul, respectively; the concentration of the wild-type plasmid was 6000 copies / ul, which was comparison sample 5.

[0087] (2) Amplification reaction system

[0088] The amplification reaction system includes a primer probe combination, an amplification buffer, an amplification enzyme, and a quantitative supplement, which are mixed with the sample to be tested and the comparative sample 5 for amplification. The main components and amounts thereof are shown in Table 6 below. Among them, only the primer probe combination and the sample to be tested are arranged and combined, and the other components remain unchanged. The experimental design is shown in Table 7 below.

[0089] Table 6 Main components of amplification reaction system

[0090]

[0091] Table 7 Experimental design

[0092] Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Primer Probe Combination A Test 1 Test 2 Test 3 Test 4 Test 5 Primer Probe Combination B Test 6 Test 7 Test 8 Test 9 Test 10 Primer Probe Combination C Test 11 Test 12 Test 13 Test 14 Test 15 Primer Probe Combination D Test 16 Test 17 Test 18 Test 19 Test 20 Primer Probe Combination E Test 21 Test 22 Test 23 Test 24 Test 25 Primer Probe Combination F Test 26 Test 27 Test 28 Test 29 Test 30 Primer probe combination G Test 31 Test 32 Test 33 Test 34 Test 35 Primer probe combination H Test 36 Test 37 Test 38 Test 39 Test 40 Primer Probe Combination I Test 41 Test 42 Test 43 Test 44 Test 45

[0093] (3) Amplification

[0094] Amplification was performed using the Jindian MC-1000 real-time fluorescence quantitative PCR instrument. The amplification reaction conditions were: 96°C, 5 min; (95°C, 20 s; 62°C, 40 s (collecting fluorescence)) × 45 cycles. After amplification by the fluorescence PCR amplification instrument, the detection results were determined based on the Ct value.

[0095] (4) Acceptable standards

[0096] Ct≤40 means detected, otherwise it means not detected.

[0097] 2. Results Analysis

[0098] The test results are shown in Figures 1 to 9 According to the results, although primers A, B, D, and I can detect mutant plasmids as low as 6 copies, wild-type plasmids are also detected at the same time, resulting in false positive results. Therefore, primers A, B, D, and I have poor specificity; primers H only detected 6000 copies of molecules, and their sensitivity is poor; the Ct value of wild-type results in groups C and G is greater than 40, which can be considered as no detection, and wild-type molecules in group F are also not detected, but these three primers only detected 60 copies of molecules, which did not reach the minimum detection limit of the kit (6 copies), and the sensitivity was poor; primers in group E reached the minimum detection limit and did not detect wild-type plasmids, with high sensitivity and strong specificity.

[0099] Example 2 BRAF-V600E Primer Design and Screening

[0100] The sample to be tested was prepared from a BRAF-V600E mutant plasmid. The primers designed based on the BRAF-V600E mutant gene are shown in the following table. Other test procedures and methods are the same as those in Example 1.

[0101] Table 8 BRAF-V600E primer screening list

[0102]

[0103]

[0104] The results showed that primer group I reached the lowest detection limit and did not detect the wild-type plasmid, with high sensitivity and strong specificity.

[0105] Example 3 IDH2-R172K Primer Design and Screening

[0106] The sample to be tested was prepared from the IDH2-R172K mutant plasmid. The primers designed based on the IDH2-R172K mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0107] Table 9 IDH2-R172K primer screening list

[0108] serial number Primer name Nucleotide sequence A IDH2-R172K-F1 CAAGCCCATCACCATTGGgAA B IDH2-R172K-F2 CCAAGCCCATCACCATTGGaAA C IDH2-R172K-F3 ACCAAGCCCATCACCATTGGtAA D IDH2-R172K-F4 CCAAGCCCATCACCATTGGCtA E IDH2-R172K-F5 AAGCCCATCACCATTGGCcA F IDH2-R172K-F6 AAGCCCATCACCATTGGCgA G IDH2-R172K-F7 CAAGCCCATCACCATTGGCtA H IDH2-R172K-F8 AAGCCCATCACCATTGGCtA I IDH2-R172K-F9 ACCAAGCCCATCACCATaGGaAA J IDH2-R172K-F10 CAAGCCCATCACCATcGGaAA K IDH2-R172K-F11 CAAGCCCATCACCATgGGaAA L IDH2-R172K-F12 CCAAGCCCATCACCATTcGaAA M IDH2-R172K-F13 GACCAAGCCCATCACCATTaGaAA N IDH2-R172K-F14 ACCAAGCCCATCACCATTtGaAA / IDH2-R172K-R GCGAGGAGCTCCAGTCG / IDH2-R172K-P FAM-5'-CGACCAGGTAGGCCAGGGTGGAG-3'-BHQ1

[0109] The results showed that primers in group A not only reached the minimum detection limit but also failed to detect the wild-type plasmid, with high sensitivity and strong specificity.

[0110] Example 4 TERT-228CT primer design and screening

[0111] The sample to be tested was prepared from the TERT-228CT mutant plasmid. The primers designed based on the TERT-228CT mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0112] Table 10 TERT-228CT primer screening list

[0113] serial number Primer name Nucleotide sequence A TERT-228CT-F1 TCCCCGGCCCAGCCgCT B TERT-228CT-F2 TCCCCGGCCCAGCCaCT C TERT-228CT-F3 GTCCCCGGCCCAGCCtCT D TERT-228CT-F4 TCCCCGGCCCAGCCCgT E TERT-228CT-F5 TCCCCGGCCCAGCCCaT F TERT-228CT-F6 TCCCCGGCCCAGCCCtT G TERT-228CT-F7 TCCCCGGCCCAGCaCCT H TERT-228CT-F8 TCCCCGGCCCAGCtCCT I TERT-228CT-F9 GTCCCCGGCCCAGCgCCT / TERT-R CGCAGCGCTGCCTGAAACT / TERT-P VIC-5’-CTTCCTTTCCGCGGCCCCGCCCT-3’-BHQ2

[0114] The results showed that primers in group E not only reached the minimum detection limit but also failed to detect the wild-type plasmid, with high sensitivity and strong specificity.

[0115] Example 5 TERT-250CT Primer Design and Screening

[0116] The sample to be tested was prepared from the TERT-250CT mutant plasmid. The primers designed based on the TERT-250CT mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0117] Table 11 TERT-250CT primer screening list

[0118]

[0119]

[0120] The results showed that primers in group F not only reached the minimum detection limit but also failed to detect the wild-type plasmid, with high sensitivity and strong specificity.

[0121] Example 6 H3F3A-K28M primer design and screening

[0122] The sample to be tested was prepared from the H3F3A-K28M mutant plasmid. The primers designed based on the H3F3A-K28M mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0123] Table 12H3F3A-K28M primer screening list

[0124] Number Primer Name Nucleotide Sequence A H3F3A-K28M-F1 CCAGTAGAGGGCGCtCgCA B H3F3A-K28M-F2 CCAGTAGAGGGCGCAggCA C H3F3A-K28M-F3 CCTCCAGTAGAGGGCGCACcgA D H3F3A-K28M-F4 CCTCCAGTAGAGGGCGCAgTgA E H3F3A-K28M-F5 CCCTCCAGTAGAGGGCGCAgTaA F H3F3A-K28M-F6 CCCTCCAGTAGAGGGCGCtCTtA G H3F3A-K28M-F7 CCCTCCAGTAGAGGGCGCAgTtA / H3F3A-R AGACTGCCCGCAAATCGAC / H3F3A-P Cy5-5’-CGGCTTTTGTAGCCAGTTGCTTCC-3’-BHQ3

[0125] The results showed that primers in group F not only reached the minimum detection limit but also failed to detect the wild-type plasmid, with high sensitivity and strong specificity.

[0126] Example 7 H3F3A-G35R Primer Design and Screening

[0127] The sample to be tested was prepared from the H3F3A-G35R mutant plasmid. The primers designed based on the H3F3A-G35R mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0128] Table 13H3F3A-G35R primer screening list

[0129] Number Primer Name Nucleotide Sequence A H3F3A-G35R-F1 CTGTAACGATGAGGTTTCTTCACggT B H3F3A-G35R-F2 CCTGTAACGATGAGGTTTCTTCAgCtT C H3F3A-G35R-F3 CTGTAACGATGAGGTTTCTTgACCgT D H3F3A-G35R-F4 CCTGTAACGATGAGGTTTCTTtACCgT E H3F3A-G35R-F5 CTGTAACGATGAGGTTTCTTCAaCgT F H3F3A-G35R-F6 CTGTAACGATGAGGTTTCTTCgCCgT G H3F3A-G35R-F7 AATACCTGTAACGATGAGGTTTCTTtACtCT H H3F3A-G35R-F8 AATACCTGTAACGATGAGGTTTCTTgACtCT I H3F3A-G35R-F9 GTAACGATGAGGTTTCTTgCCgCT G H3F3A-G35R-F10 CTGTAACGATGAGGTTTCTTaCCgCT K H3F3A-G35R-F11 ATACCTGTAACGATGAGGTTTCTTCtCtCT L H3F3A-G35R-F12 CCTGTAACGATGAGGTTTCTTCcCtCT M H3F3A-G35R-F13 CTGTAACGATGAGGTTTCTTCgCtCT / H3F3A-R AGACTGCCCGCAAATCGAC / H3F3A-P Cy5-5'-CGGCTTTTGTAGCCAGTTGCTTCC-3'-BHQ3

[0130] The results showed that primers in group C reached the minimum detection limit and did not detect the wild-type plasmid, with high sensitivity and strong specificity.

[0131] Example 8 H3F3A-G35V Primer Design and Screening

[0132] The sample to be tested was prepared from the H3F3A-G35V mutant plasmid. The primers designed based on the H3F3A-G35V mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0133] Table 14H3F3A-G35V primer screening list

[0134]

[0135]

[0136] The results showed that primer group I reached the lowest detection limit and did not detect the wild-type plasmid, with high sensitivity and strong specificity.

[0137] Example 9: H3C2-K28M primer design and screening

[0138] The sample to be tested was prepared from the H3C2-K28M mutant plasmid. The primers designed based on the H3C2-K28M mutant gene are shown in the following table. The other test procedures and methods are the same as those in Example 1.

[0139] Table 15H3C2-K28M primer screening list

[0140] serial number Primer name Nucleotide sequence A H3C2-K28M-F1 CTGGCTACCAAGGCTGCTCaCtT B H3C2-K28M-F2 CTGGCTACCAAGGCTGCTCcCtT C H3C2-K28M-F3 CTGGCTACCAAGGCTGCTCagAT D H3C2-K28M-F4 CTGGCTACCAAGGCTGCTCcaAT E H3C2-K28M-F5 CTGGCTACCAAGGCTGCTCatAT F H3C2-K28M-F6 CTGGCTACCAAGGCTGCaCGgAT G H3C2-K28M-F7 CTGGCTACCAAGGCTGCTgGaAT H H3C2-K28M-F8 CTGGCTACCAAGGCTGCTtGtAT / H3C2-K28M-R ATCTCGCGCAGAGCCACA / H3C2-K28M-P Cy5-5'-CTACCGGCGGCGTGAAAAAGC-3'-BHQ3

[0141] The results showed that primers in group A not only reached the minimum detection limit but also failed to detect the wild-type plasmid, with high sensitivity and strong specificity.

[0142] Example 10 Gene mutation low frequency variation detection performance (VAF)

[0143] The mutant plasmid was mixed with the wild-type plasmid to examine the low-frequency variation detection performance of the primer-probe combination.

[0144] 1. Sample preparation

[0145] The mutant plasmids IDH2-R172K, TERT-C228T, TERT-C250T, and H3C2-K28M were mixed with the corresponding wild-type plasmids so that the ratio of each mutant plasmid to the wild-type plasmid reached 0.2% to prepare weak positive samples (0.2% VAF), which were samples 6 to 9 to be tested, corresponding to the above four mutant plasmids.

[0146] The mutant plasmids IDH1-R132H, BRAF-V600E, H3F3A-K28M, H3F3A-G35R, and H3F3A-G35V were mixed with the corresponding wild-type plasmids so that the quantity ratio of each mutant plasmid to the wild-type plasmid reached 0.2% to prepare weak positive samples (0.2% VAF), which were samples 10 to 14 to be tested, corresponding to the above-mentioned 5 mutant plasmids.

[0147] The first group of primer probe compositions includes IDH2-R172K-F, IDH2-R172K-FR, IDH2-R172K-FP, TERT-C228T-F, TERT-C250T-F, TERT-R, TERT-P, H3C2-K28M-F, H3C2-K28M-R, and H3C2-K28M-P.

[0148] The second group of primer probe compositions includes IDH1-R132H-F, IDH1-R132H-R, IDH1-R132H-P, BRAF-V600E-F, BRAF-V600E-R, BRAF-V600E-P, H3F3A-K28M-F, H3F3A-G35R-F, H3F3A-G35V-F, H3F3A-R, and H3F3A-P.

[0149] 2. Test methods

[0150] (1) Preparation of amplification reaction system

[0151] The amplification reaction system includes a primer-probe combination, an amplification buffer, an amplification enzyme, an internal reference solution, and a quantitative supplement solution. The main components and amounts thereof are shown in the table below.

[0152] The first group of primer probe combination, amplification buffer, amplification enzyme, internal reference solution, quantitative supplement solution and one of the samples 6 to 9 to be tested are mixed to prepare a reaction solution, so that each wild plasmid contains 3000 copies in the reaction solution, each mutant plasmid contains 6 copies in the reaction solution, and the GAPDH plasmid contains 3000 copies in the reaction solution. The reaction solution is placed in well 1 of a PCR instrument.

[0153] Table 16 Main components of the reaction solution

[0154] Components Main ingredients Dosage Primer probe combination Group 1 1μl Amplification Buffer <![CDATA[Mg 2+ 、dNTP]]> 4μl Amplification enzyme Taq DNA polymerase 0.4μl Internal reference solution GAPDH Plasmid 1μl Samples to be tested One of the samples 6 to 9 to be tested 1μl Quantitative supplement solution Purified water 12.6μl total 20μl

[0155] The second set of primer probe combination, amplification buffer, amplification enzyme, internal reference solution, quantitative supplement solution and 10-14 samples to be tested are mixed to prepare a reaction solution, so that each wild plasmid contains 3000 copies in the reaction solution, each mutant plasmid contains 6 copies in the reaction solution, and the GAPDH plasmid contains 3000 copies in the reaction solution. The reaction solution is placed in well 2 of a PCR instrument.

[0156] Table 17 Main components of the reaction solution

[0157] Components Main ingredients Dosage Primer probe combination Group 2 1μl Amplification Buffer <![CDATA[Mg 2+ 、dNTP]]> 4μl Amplification enzyme Taq DNA polymerase 0.4μl Internal reference solution GAPDH Plasmid 1μl Samples to be tested One of the samples 10 to 14 to be tested 1μl Quantitative supplement solution Purified water 12.6μl total 20μl

[0158] (2) Amplification

[0159] Amplification was performed using a Jindian MC-1000 real-time fluorescence quantitative PCR instrument. The amplification reaction conditions were: 96°C, 5 min; (95°C, 20 s; 62°C, 40 s (collecting fluorescence)) × 45 cycles. After amplification by the fluorescence PCR amplification instrument, the detection result was determined based on the displayed Ct value.

[0160] 2. Results Analysis

[0161] The amplification results of each mutant gene and internal reference are shown in Figures 10 to 18 .

[0162] Depend on Figures 10 to 18 It can be seen that each mutant gene can reach a detection limit of 0.2% VAF, the wild-type gene has no interference with the detection of the mutant gene, and the primers designed by the present invention have high detection specificity and high sensitivity.

[0163] Example 11 Gene fusion sensitivity detection

[0164] 1. Sample preparation

[0165] The RNA fusion standard EGFR VⅢFusion was reverse transcribed into cDNA using a reverse transcription system and prepared into samples with concentrations of 5000 copies / ul, 500 copies / ul, 50 copies / ul, and 5 copies / ul, which were samples 15 to 18 to be tested.

[0166] The RNA fusion standard MET exon 14 Skipping was reverse transcribed into cDNA using a reverse transcription system and prepared into samples with concentrations of 5000 copies / ul, 500 copies / ul, 50 copies / ul, and 5 copies / ul, which were samples 19 to 22 to be tested.

[0167] Two RNA standards, EGFR VⅢFusion (Cat. No. CBP20179R) and MET exon 14Skipping (Cat. No. CBP20171R), were purchased from Nanjing Kebai Biotechnology.

[0168] The third group of primer probe compositions used include upstream primers EGFR-F1, EGFR-F2, EGFR-F3, EGFR-F4, MET-F1, MET-F2, MET-F3, MET-F4, MET-F5, and MET-F6; downstream primers EGFR-R1, MET-R1, EGFR-R2, MET-R2, and MET-R3; and probes EGFR-P1, EGFR-P2, EGFR-P3, MET-P1, MET-P2, and MET-P3.

[0169] 2. Test methods

[0170] (1) cDNA preparation

[0171] a. Pipette 1 μl of RNA standard and 0.5 μl of PCR fusion denaturation buffer, add water to 8.5 μl, run PCR at 70°C, and place on ice immediately after 1.5 minutes.

[0172] b. Add 1 μl PCR fusion cleavage buffer and 0.5 μl PCR fusion cleavage enzyme to the above reaction system, a total of 10 μl reaction system, PCR run at 30°C, after 5 minutes immediately placed on ice.

[0173] c. Add 3 μl of PCR fusion reverse transcription buffer, 2 μl of PCR fusion reverse transcriptase, 1 μl of PCR fusion reverse transcription primer mix, 1 μl of PCR fusion reverse transcription binding solution, 2 μl of PCR fusion cutting buffer to the above reaction system, make up to 30 μl with water, run PCR at 25℃, 5 min; 30℃, 10 min; 37℃, 30 min; 50℃, 5 min; 85℃, 5 min; store the obtained cDNA at 4℃.

[0174] (2) Preparation of amplification reaction system

[0175] The amplification reaction system includes a primer probe combination, an amplification buffer, an amplification enzyme, a fusion amplification enhancement buffer, a quantitative supplement, and a sample to be tested for amplification. The main components and dosages are shown in the table below, where only the sample to be tested is arranged and combined, and other components remain unchanged.

[0176] Table 18 Main components of amplification reaction system

[0177]

[0178] (3) Amplification

[0179] Amplification was performed using a Jindian MC-1000 real-time fluorescence quantitative PCR instrument. The amplification reaction conditions were: 96°C, 5 min; (95°C, 20 s; 62°C, 40 s (collecting fluorescence)) × 45 cycles. After amplification by the fluorescence PCR amplification instrument, the detection result was determined based on the displayed Ct value.

[0180] 2. Results Analysis

[0181] The amplification curve of EGFR VⅢFusion is shown in Fig.19 ; The amplification curve of MET exon 14 Skipping is shown in Fig. 20 .Depend on Fig.19 and Fig. 20 It can be seen that each fusion mutation can reach a detection limit of 30 copies (sample concentration is 5 copies / ul, 6ul sample is added), and the primers designed by the present invention have high sensitivity.

[0182] Example 12 Clinical tissue sample detection

[0183] This example uses 15 clinical tissue samples, and DNA and RNA are extracted for DNA mutation detection and RNA fusion mutation detection. The tissue samples were tested by NGS, and the mutation type and fusion type were clearly identified. The specific information is shown in Table 21 and Table 22.

[0184] 1. Sample preparation

[0185] DNAs of 15 clinical tissue samples were extracted respectively as DNA test samples for the first and second groups of primer-probe combinations;

[0186] RNA was extracted from 15 clinical tissue samples, and the RNA was reverse transcribed into cDNA to serve as cDNA test samples for the third set of primer-probe combinations.

[0187] 2. Test methods

[0188] (1) Preparation of amplification reaction system

[0189] The first and second groups of primer probe combinations, amplification buffer, amplification enzyme, internal reference solution, and quantitative supplement solution were respectively mixed with 15 DNA samples to be tested to prepare reaction solutions, and the reaction solutions were placed in well 1 of a PCR instrument. The main components and amounts of the reaction solutions are shown in the table below.

[0190] Table 19 Main components of the reaction solution

[0191]

[0192]

[0193] The third set of primer probe combination, amplification buffer, amplification enzyme, internal reference solution, and quantitative supplement solution were mixed with 15 cDNA samples to be tested to prepare reaction solutions, and the reaction solutions were placed in well 2 of a PCR instrument. The main components and amounts of the reaction solutions are shown in the table below.

[0194] Table 20 Main components of the reaction solution

[0195] Components Main ingredients Dosage Primer probe combination Group 3 1μl Amplification Buffer <![CDATA[Mg 2+ 、dNTP]]> 4μl Amplification enzyme Taq DNA polymerase 0.4μl Fusion Amplification Enhancement Buffer Betaine 1μl Samples to be tested cDNA samples to be tested 6μl Quantitative supplement solution Purified water 7.6μl total 20μl

[0196] (2) Amplification

[0197] Amplification was performed using a Jindian MC-1000 real-time fluorescence quantitative PCR instrument. The amplification reaction conditions were: 96°C, 5 min; (95°C, 20 s; 62°C, 40 s (collecting fluorescence)) × 45 cycles. After amplification by the fluorescence PCR amplification instrument, the detection result was determined based on the displayed Ct value.

[0198] 3. Results Analysis

[0199] The comparison of the detection results using the primers of the present invention and the results of NGS is shown in Table 21. The detection results show that the detection results of the tissue samples by the kit of the present invention are completely consistent with the NGS detection results, and the accuracy rate is high.

[0200] Table 21 Clinical tissue sample test results

[0201]

[0202]

[0203] Example 13 Clinical cerebrospinal fluid sample detection

[0204] The 15 clinical cerebrospinal fluid samples in this example are from the same source as the samples in Example 12, and are all from the same group of 15 patients.

[0205] 1. Sample preparation

[0206] Qiagen Circulating Nucleic Acid Kit (Cat. No. 55114) was used to extract cfDNA from 15 clinical cerebrospinal fluid samples;

[0207] Using Quick-cfRNA from ZYMO RESEARCH Biotech TM Serum&Plasma Kit (Cat. No. R1059) was used to extract cfRNA from 15 clinical cerebrospinal fluid samples.

[0208] 2. Test methods

[0209] Same as Example 12.

[0210] 3. Results Analysis

[0211] The results of gene mutation and gene fusion detection of cerebrospinal fluid samples and the comparison results with NGS are shown in Table 22. The test results of the kit were the same as those of NGS in 13 cases and different in 2 cases. The reasons were analyzed as follows: the cfDNA extracted from the cerebrospinal fluid of the second case was only 5ng, which was lower than the minimum input of 10ng of the kit, so a false negative was detected. The cerebrospinal fluid sample of the 12th case was hemolyzed, resulting in abnormal cfRNA extraction, so the result was not detected. Therefore, when using this kit, strict quality control of the extracted nucleic acid should be performed.

[0212] Table 22 Clinical cerebrospinal fluid sample test results

[0213]

[0214]

[0215] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A primer-probe combination for detecting glioma genes, It is characterized in that The primer-probe combination includes 3 groups of primer-probe combinations, wherein the primer and probe nucleotide sequences are shown in SEQ ID NOs. 1 to 42 respectively; the probe sequences in each group are modified by different fluorescent groups; wherein the first base at the 3' end of the upstream primers of the first group and the second group is complementary to the target gene, the second to sixth bases contain 1 to 3 bases that are mismatched with the target gene, and the remaining bases are completely complementary to the target gene; the primer-probe combination is grouped as follows:

2. The primer-probe combination according to claim 1, It is characterized in that The primer-probe composition also includes an internal reference primer-probe composition.

3. The primer-probe composition according to claim 2, It is characterized in that The internal reference primer probe compositions of the first and second groups of primer probe compositions are GAPDH-F, GAPDH-R, and GAPDH-P, and their sequences are shown in SEQ ID NOs. 43 to 45, respectively.

4. The primer-probe composition according to claim 2, It is characterized in that The internal reference primer-probe combination of the third group of primer-probe combinations is RNase PF, RNase PR, and RNase PP, and their sequences are shown in SEQ ID NOs. 46 to 48, respectively.

5. A kit for detecting glioma genes, It is characterized in that The kit comprises an amplification reaction system, and the amplification reaction system comprises a primer-probe combination according to any one of claims 1 to 4, wherein the primer-probe combination is arranged in separate groups.

6. The kit according to claim 5, It is characterized in that The kit also includes a reverse transcription reaction system.

7. The kit according to claim 5, It is characterized in that The concentration of each primer in the amplification reaction system is 0.05 μmol / L to 0.2 μmol / L.

8. The kit according to claim 5, It is characterized in that The concentration of each probe in the amplification reaction system solution is 0.03 μmol / L to 0.1 μmol / L.

9. Use of any one of the primer-probe combinations according to claims 1 to 4 in a kit for detecting glioma genes.

10. The use according to claim 9, It is characterized in that The steps include: S1: According to the groups of the primer-probe combination, the primer-probe combination is mixed with an amplification buffer, an amplification enzyme, an amplification enhancement buffer, and a sample to be tested to prepare an amplification reaction system, and the amplification reaction system is added to different reaction wells; S2: PCR amplification and collection of fluorescence signals, and judging the negative or positive of the sample gene based on the fluorescence signals.

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