High-sensitivity polygene joint detection kit
By designing a highly sensitive multi-gene joint detection kit, utilizing PCR reaction to inhibit wild-type amplification, and combining it with first-generation sequencing, highly sensitive detection of multiple tumor diagnosis and treatment-related genes was achieved. This solves the problems of long detection cycles and high costs in existing technologies, and improves detection throughput and sensitivity.
Patent Information
- Application Number
- CN202510516075.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-23
- Publication Date
- 2025-11-11
AI Technical Summary
Existing technologies are difficult to detect multiple tumor-related genes simultaneously with high sensitivity, and also suffer from problems such as long detection cycles and high costs.
A highly sensitive multi-gene joint detection kit was designed. By combining eight sets of primers and probes, PCR reaction was used to inhibit wild-type amplification, and combined with first-generation sequencing, a highly sensitive detection of 37 variants of eight genes, namely BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA, was achieved.
It achieves high-throughput detection of 37 variants in 8 genes with a sensitivity of 1%-5%, requiring only 5ng of DNA for detection, avoiding sample cross-contamination and improving the accuracy of detection results.
Smart Images
Figure CN120924656A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene detection technology, and specifically relates to a highly sensitive multi-gene combined detection kit. Background Technology
[0002] A gene is a DNA sequence that carries genetic information and is the basic unit of heredity that controls traits. Gene mutations are a significant factor in causing human diseases. Genetic testing can assess an individual's health status and disease risk, and also serve as an important basis for clinical diagnosis and treatment.
[0003] Currently, the main gene testing technologies used in clinical practice include PCR, first-generation sequencing (NGS), and second-generation sequencing (NGS). PCR can only detect a few loci at a time, making it prone to missed detections; conventional first-generation sequencing has low sensitivity and is prone to false negatives; NGS is complex, time-consuming, and costly, making it difficult to implement. Therefore, there is an urgent need in the market for a new method and a new reagent kit to simultaneously detect multiple tumor diagnosis and treatment-related genes, and to achieve a good balance in terms of detection sensitivity, detection cycle, detection price, and ease of implementation.
[0004] BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA genes are common pathogenic genes for tumors such as thyroid cancer and colorectal cancer. Detection of these genes can provide important evidence for the precision diagnosis and treatment of cancer, such as helping to determine the molecular characteristics of tumors, guiding clinical treatment, and assessing prognosis. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a highly sensitive multi-gene joint detection kit, which only requires 5ng of DNA to simultaneously detect 37 variants of 8 genes: BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53 and PIK3CA with high sensitivity.
[0006] This invention provides a highly sensitive multi-gene joint detection kit, comprising reaction solution I, reaction solution A, reaction solution B, reaction solution C, reaction solution D, reaction solution E, reaction solution F, reaction solution G, and reaction solution H, as well as sequencing primers A, B, C, D, E, F, G, and H; the kit is used to detect mutations in human BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA genes.
[0007] The specific variations are shown in the table below: Preferably, the reaction solution I contains PCR master mix, thermosensitive UNG / UDG enzyme, dUTP, formamide, and primers SEQ NO.01-16.
[0008] Preferably, the reaction solution A contains PCR master mix, dUTP, and primer probes SEQ NO.17-19; wherein the probe SEQ NO.19 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0009] Preferably, the reaction solution B contains PCR master mix, dUTP, and primer probes SEQ NO.20-22; wherein the probe SEQ NO.22 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0010] Preferably, the reaction solution C contains PCR master mix, dUTP, and primer probes SEQ NO.23-25; wherein the probe SEQ NO.25 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0011] Preferably, the reaction solution D contains PCR master mix, dUTP, and primer probes SEQ NO.26-28; wherein the probe SEQ NO.28 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0012] Preferably, the reaction solution E contains PCR master mix, dUTP, and primer probes SEQ NO.29-31; wherein the probe SEQ NO.31 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0013] Preferably, the reaction solution F contains PCR master mix, dUTP, and primer probes SEQ NO.32-34; wherein the probe SEQ NO.34 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0014] Preferably, the reaction solution G contains PCR master mix, dUTP, and primer probes SEQ NO.35-37; wherein the probe SEQ NO.37 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0015] Preferably, the reaction solution H contains PCR master mix, dUTP, and primer probes SEQ NO.38-40; wherein the probe SEQ NO.40 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
[0016] Preferably, the sequencing primer A sequence is SEQ NO.41, with a concentration of 2-10 μM; the sequencing primer B sequence is SEQ NO.42, with a concentration of 2-10 μM; the sequencing primer C sequence is SEQ NO.43, with a concentration of 2-10 μM; the sequencing primer D sequence is SEQ NO.44, with a concentration of 2-10 μM; the sequencing primer E sequence is SEQ NO.45, with a concentration of 2-10 μM; the sequencing primer F sequence is SEQ NO.46, with a concentration of 2-10 μM; the sequencing primer G sequence is SEQ NO.47, with a concentration of 2-10 μM; and the sequencing primer H sequence is SEQ NO.48, with a concentration of 2-10 μM.
[0017] Preferably, the kit is used to perform two-step amplification, under the following conditions: Step 1 Amplification: Second step amplification: The principle of this invention is as follows: First, target regions of eight genes (BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA) are amplified and enriched using a first-round primer amplification. Then, eight sets of inner primers and wild-type inhibitory probes are designed for the target detection sites of these eight genes, with the probes covering the target detection sites. The 3' end of the target site side primer overlaps with the 5' end of the probe by 2-10 bp, and the probe's TM value is higher than that of the primer. The probe's 3' end is blocked by one or a combination of 3' end base mismatch, phosphorylation, ddC modification, spacer modification, and MGB modification, preventing extension after binding to the template. During the PCR reaction, the probe preferentially binds to the wild-type site region, resulting in low binding efficiency of the unidirectional amplification primer or preventing extension due to steric hindrance, thereby inhibiting the amplification of wild-type samples and increasing the proportion of mutants in the sample. Subsequently, the amplified samples are detected using first-generation sequencing, which can significantly improve the sensitivity of mutation detection.
[0018] Beneficial effects (1) The present invention can simultaneously detect 37 variants of 8 genes, including BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA, with high detection throughput; (2) This invention requires only 5 ng of DNA for effective detection, which can solve the problem of difficult detection of trace samples such as puncture biopsy; (3) Conventional first-generation sequencing technology can only detect mutations with a mutation frequency of more than 20%, while this invention can detect mutations as low as 1%-5%, with high detection sensitivity; (4) The present invention adopts the UNG / UDG anti-contamination system, which can effectively avoid cross-contamination of samples caused by aerosols, etc., and further ensure the accuracy of the test results. Attached Figure Description
[0019] Figures 1-3 The results of detecting the BRAF gene using the kit of this invention are shown.
[0020] Figures 4-14 The results of detecting the KRAS gene using the kit of this invention are shown.
[0021] Figures 15-20 The results of detecting the NRAS gene using the kit of this invention are shown.
[0022] Figures 21-26 The results of detecting the HRAS gene using the kit of this invention are shown.
[0023] Figures 27-28 The results of detecting the RET gene using the kit of this invention are shown.
[0024] Figure 29The results of detecting the TERT gene using the kit of this invention are shown.
[0025] Figures 30-33 The results of detecting the TP53 gene using the kit of this invention are shown.
[0026] Figures 34-37 The results of detecting the PIK3CA gene using the kit of this invention are shown. Detailed Implementation
[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0028] Example 1 I. Reagent Kit Preparation: Reaction solution I formulation: PCR master mix 10μL, thermosensitive UNG / UDG enzyme 0.5μL, dUTP (100mM) 0.05μL, formamide 1.0μL, primer SEQ NO.01-16 (100μM) 0.05μL each, add water to 15μL.
[0029] Reaction solution A formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.17-18 (100μM) 0.05μL each, probe SEQ NO.19 (100μM) 0.5μL, add water to 9μL.
[0030] Reaction solution B formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.20-21 (100μM) 0.05μL each, probe SEQ NO.22 (100μM) 0.5μL, add water to 9μL.
[0031] Reaction solution C formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.23-24 (100μM) 0.05μL each, probe SEQ NO.25 (100μM) 0.5μL, add water to 9μL.
[0032] Reaction solution D formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.26-27 (100μM) 0.05μL each, probe SEQ NO.28 (100μM) 0.5μL, add water to 9μL.
[0033] Reaction solution E formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.29-30 (100μM) 0.05μL each, probe SEQ NO.31 (100μM) 0.5μL, add water to 9μL.
[0034] Reaction solution F formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.32-33 (100μM) 0.05μL each, probe SEQ NO.34 (100μM) 0.5μL, add water to 9μL.
[0035] Reaction solution G formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.35-36 (100μM) 0.05μL each, probe SEQ NO.37 (100μM) 0.5μL, add water to 9μL.
[0036] Reaction solution H formula: PCR master mix 5μL, dUTP (100mM) 0.025μL, primers SEQ NO.38-39 (100μM) 0.05μL each, probe SEQ NO.40 (100μM) 0.5μL, add water to 9μL.
[0037] Sequencing primer A formulation: Primer SEQ NO.41 (100 μM), diluted to 5 μM with ultrapure water.
[0038] Sequencing primer B formulation: Primer SEQ NO.42 (100 μM), diluted to 5 μM with ultrapure water.
[0039] Sequencing primer C formulation: Primer SEQ NO.43 (100 μM), diluted to 5 μM with ultrapure water.
[0040] Sequencing primer D formulation: Primer SEQ NO.44 (100 μM), diluted to 5 μM with ultrapure water.
[0041] Sequencing primer E formulation: Primer SEQ NO.45 (100 μM), diluted to 5 μM with ultrapure water.
[0042] Sequencing primer F formulation: Primer SEQ NO.46 (100 μM), diluted to 5 μM with ultrapure water.
[0043] Sequencing primer G formulation: Primer SEQ NO.47 (100 μM), diluted to 5 μM with ultrapure water.
[0044] Sequencing primer H formulation: Primer SEQ NO.48 (100 μM), diluted to 5 μM with ultrapure water.
[0045] The primer and probe sequences are as follows: The probe is blocked at its 3' end by modifications such as base mismatch, phosphorylation, ddC, MGB, and spacer. The modifications can be any one or a combination of modifications.
[0046] SEQ NO.1-16 are used for amplification and enrichment of target regions of the BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA genes; SEQ NO.17, SEQ NO.18, and SEQ NO.19 are used in combination for BRAF gene amplification, and SEQ NO.41 is a BRAF sequencing primer; SEQ NO.20, SEQ NO.21, and SEQ NO.22 are used in combination for KRAS gene amplification, and SEQ NO.42 is a KRAS sequencing primer; SEQ NO.23, SEQ NO.24, and SEQ NO.25 are used in combination for NRAS gene amplification, and SEQ NO.43 is an NRAS sequencing primer; SEQ NO.26, SEQ NO.27, and SEQ NO.28 are used in combination for HRAS gene amplification, and SEQ NO.44 is the HRAS sequencing primer; SEQ NO.29, SEQ NO.30, and SEQ NO.31 are used in combination for RET gene amplification, and SEQ NO.45 is a RET sequencing primer; SEQ NO.32, SEQ NO.33, and SEQ NO.34 are used in combination for TERT gene amplification, and SEQ NO.46 is a TERT sequencing primer; SEQ NO.35, SEQ NO.36, and SEQ NO.37 are used in combination for TP53 gene amplification, and SEQ NO.47 is the TP53 sequencing primer; SEQ NO.38, SEQ NO.39, and SEQ NO.40 are used in combination for PIK3CA gene amplification, and SEQ NO.48 is a PIK3CA sequencing primer.
[0047] II. Testing methods: 1. Sample preparation The following samples were prepared using human wild-type genomic DNA and mutant genomic DNA for testing and verification.
[0048] Step 1 Amplification: Take 15 μL of reaction solution I for each sample, add 5 ng of the corresponding DNA, and add water to a final volume of 20 μL. Vortex to mix, centrifuge, and then place on a PCR instrument for reaction under the following conditions: After the first step of amplification is completed, the amplification product is diluted 20 times with ultrapure water for later use.
[0049] Step 2 Amplification: Take 9 μL each of reaction solutions A, B, C, D, E, F, G, and H, and add 1 μL of the diluted product from Step 1 to each solution. Vortex to mix, centrifuge, and then place on a PCR instrument for reaction under the following conditions: After the second amplification step, the amplified products are purified. The purified products are then subjected to sequencing reactions. The sequencing primers corresponding to the amplified products from reaction solutions A, B, C, D, E, F, G, and H are primers A, B, C, D, E, F, G, and H, respectively. After the sequencing reaction, the purified products are purified again and sequenced using a first-generation sequencer.
[0050] After sequencing was completed, the variation of the target sequence was assessed based on the sequencing results. The detection results are as follows: The results show that, under the 5ng DNA starting condition, all the above-mentioned mutation abundance variations were detected, while no variations were detected in the wild-type sample, indicating 100% detection accuracy.
[0051] Conclusion: This invention can detect the above-mentioned variant sites in human BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA genes with high sensitivity and accuracy.
[0052] The sequencing results peak diagram is as follows:
Claims
1. A highly sensitive multi-gene combined detection kit, characterized in that: The kit includes reaction solution I, reaction solution A, reaction solution B, reaction solution C, reaction solution D, reaction solution E, reaction solution F, reaction solution G, reaction solution H, and sequencing primers A, B, C, D, E, F, G, and H. The kit is used to detect mutations in human BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53, and PIK3CA genes.
2. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution I contains PCR master mix, thermosensitive UNG / UDG enzyme, dUTP, formamide, and primers SEQ NO.01-16.
3. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution A contains PCR master mix, dUTP, and primer probes SEQ NO.17-19; wherein the probe SEQ NO.19 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
4. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution B contains PCR master mix, dUTP, and primer probes SEQ NO.20-22; wherein the probe SEQ NO.22 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
5. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution C contains PCR master mix, dUTP, and primer probes SEQ NO.23-25; wherein the probe SEQ NO.25 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
6. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution D contains PCR master mix, dUTP, and primer probes SEQ NO.26-28; wherein the probe SEQ NO.28 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
7. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution E contains PCR master mix, dUTP, and primer probes SEQ NO.29-31; wherein the probe SEQ NO.31 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
8. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution F contains PCR master mix, dUTP, and primer probes SEQ NO.32-34; wherein the probe SEQ NO.34 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, stericon modification, and MGB modification, so that it will not extend after binding to the template.
9. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The reaction solution G contains PCR master mix, dUTP, and primers / probes SEQ NO.35-37; wherein probe SEQ NO.37 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, sterigma modification, and MGB modification, so that it will not extend after binding to the template; the reaction solution H contains PCR master mix, dUTP, and primers / probes SEQ NO.38-40; wherein probe SEQ NO.40 is 3' end blocked by one or more combinations of 3' end base mismatch, phosphorylation, ddC modification, sterigma modification, and MGB modification, so that it will not extend after binding to the template.
10. The high-sensitivity multi-gene combined detection kit according to claim 1, characterized in that: The sequencing primer A sequence is SEQ NO.41, with a concentration of 2-10 μM; the sequencing primer B sequence is SEQ NO.42, with a concentration of 2-10 μM; the sequencing primer C sequence is SEQ NO.43, with a concentration of 2-10 μM; the sequencing primer D sequence is SEQ NO.44, with a concentration of 2-10 μM; the sequencing primer E sequence is SEQ NO.45, with a concentration of 2-10 μM; the sequencing primer F sequence is SEQ NO.46, with a concentration of 2-10 μM; the sequencing primer G sequence is SEQ NO.47, with a concentration of 2-10 μM; and the sequencing primer H sequence is SEQ NO.48, with a concentration of 2-10 μM.