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Primer probe system and kit for detection of mlh1 gene methylation

A technology for primer probes and detection primers, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of cumbersome operation, long operation time, high false positive rate, etc., and achieve elimination Effects of interference factors, guaranteed accuracy, and high sensitivity

Active Publication Date: 2020-04-17
上海达澈生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The MSP method combined with the nested PCR amplification method can improve the specificity of the single MSP method, but the operation time is long, and compared with the MSP method, there are also false positives caused by incomplete bisulfite treatment. The pH value in the modification process is relatively absolute. Accurate, all reagents require fresh configuration, and need to repeatedly explore to find the appropriate reaction time, resulting in cumbersome detection process, and can not achieve quantitative detection, there is a high false positive rate; there is also excessive sulfite treatment, resulting in amplification 3. Methylation sensitive melting curve analysis (MS-High Resolution Melting Curve, MS-HRM), which is cumbersome to operate, has a high false positive rate, and is not suitable for the detection of a large number of samples; 4. Fluorescence method (Methylight), due to the limitations of the probe, there is generally no suitable control system, and the false positive rate is high; 5, bisulfite sequencing PCR (BSP), is a PCR combined with Sanger sequencing technology, the result is accurate , but the process is cumbersome, not suitable for mass detection, and the price is too expensive to be widely used

Method used

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  • Primer probe system and kit for detection of mlh1 gene methylation
  • Primer probe system and kit for detection of mlh1 gene methylation
  • Primer probe system and kit for detection of mlh1 gene methylation

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Embodiment 1

[0050] 1. The composition of the kit.

[0051] The MLH1 gene methylation detection kit of this embodiment includes mixed solution X, mixed solution Y, mixed solution Z and hot start enzyme (Taq Hotstar). Mixed solution X, mixed solution Y, mixed solution Z and hot start enzyme (Taq Hotstar) were placed in different test tubes, as shown in Table 1 to Table 3.

[0052] Table 1 Composition list of mixed solution X

[0053] components concentration Volume (μl) PCR buffer (PCRBuffer) 10× 1 dNTPs 2mM 1 MgCl 2

15mM 1 forward primer a 4μM 1 reverse primer a 4μM 1 Probe a 4μM 0.5 Pure water (ddH 2 O)

/ 1.5 total / 7

[0054] Table 2 Composition list of mixed liquid Y

[0055] components concentration Volume (μl) PCR buffer (PCRBuffer) 10× 1 dNTPs 2mM 1 MgCl 2

15mM 1 forward primer b 4μM 1 reverse primer b 4μM 1 probe b 4μM 0.5 Pur...

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Abstract

The invention relates to a primer probe system for MLH1 (MutL homolog1) gene methylation detection and a kit adopting the primer probe system. The primer probe system comprises a primer probe set X used for determining genomic DNA quality, a primer probe set Y used for determining a methylation conversion rate and a primer probe set Z used for detecting MLH1 gene promoter methylation conditions, wherein the primer probe set X comprises a forward primer a, a reverse primer a and a probe a; the primer probe set Y comprises a forward primer b, a reverse primer b and a probe b; the primer probe set Z comprises the forward primer b, the reverse primer b, a probe c and a probe d; fluorescence report groups are arranged at 5' ends of the probe a, the probe b and the probe c, and fluorescence quenching groups are arranged at 3' ends of the probe a, the probe b, the probe c and the probe d. According to the kit, the implementing scheme is concise, and the sensitivity and the accuracy rate are high.

Description

technical field [0001] The invention relates to a gene mutation detection product, a detection primer and a detection system used in the product, and belongs to the field of biotechnology. Background technique [0002] DNA mismatch repair system (MMR) is a safety guarantee system in human cells that can repair DNA base mismatches and maintain the integrity and stability of genetic material. hMLH1, hMSH2, and hMSH3 are important mismatch repair genes in the MMR system. The reduced expression caused by the hypermethylation of DNA damage repair genes will lead to the decline of DNA damage repair ability and the subsequent gene mutation. [0003] The MLH1 gene (MutL homolog1) is located at 3p21.3 with a full length of 2439bp. Methylation of the MLH1 gene will lead to the inactivation of the expression of this gene or the inability of the expressed protein to repair the mismatched bases in time, which may lead to the inactivation of tumor suppressor genes and the activation of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/154C12Q2563/107C12Q2545/113
Inventor 唐娟娟赵新泰王明
Owner 上海达澈生物科技有限公司
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