Probe and primer sensitized by locking nucleic acid and used for detecting C677T mutation of MTHFR gene, kit and detection method

Abstract

The invention discloses a probe and a primer sensitized by locking nucleic acid and used for detecting C677T mutation of an MTHFR gene, a kit and a detection method. By means of the property that the locking nucleic acid can flexibly modify a TaqMan probe, a proper position of the probe used for detecting the C677T mutation of the MTHFR gene is modified by the locking nucleic acid to increase the specificity. Fluorescence quantitative PCR amplification is carried out on a sample genome DNA obtained in step (1) by the probe and the primer sensitized by the locking nucleic acid and used for detecting the C677T mutation of the MTHFR gene, in order to measure a Ct value of a wild probe and the Ct value of a mutant probe; positive is checked when the Ct value is smaller than 35 recurring numbers; only the sample detected to be positively by the wild probe is wild homozygotic type CC; only the sample detected to be positively by the mutant probe is mutant homozygotic type CC; the sample detected to be positively by both of the wild probe and the mutant probe is CT heterozygote.

Description

Technical field: [0001] The invention belongs to the field of molecular biology, and specifically relates to a nucleic acid-locked nucleic acid-enhanced probe for detecting the C677T mutation of the MTHFR gene, a primer, a kit and a detection method, which are suitable for the qualitative detection of the C677T polymorphic site of the human MTHFR gene. Background technique: [0002] Methylenetetrahydrofolate reductase (MTHFR) is one of the key enzymes in folic acid metabolism, which can reduce 5,10-methylenetetrahydrofolate related to reduced coenzyme I (NADPH) to 5-methyl base tetrahydrofolate. And 5-methyltetrahydrofolate plays the role of coenzyme in the process of transferring "carbon groups" in vivo, including methyl (-CH3), formyl (-CHO), and methine (-CH). C677T (SNP ID: rs1801133) in the MTHFR gene is an important mutation site affecting the function of the gene. Studies have shown that after the C at the C677T site is changed to T, the encoded alanine is replaced ...

AI Technical Summary

Problems solved by technology

Advantages: accurate results, the sequencing method is the gold standard for genetic polymorphism site detection; Disadvantages: high cost, cumbersome operation, need to get together a batch of samples to be suitable for start-up detection

Advantages: low cost, more intuitive results; Disadvantages: cumbersome electrophoresis detection operation, high risk of contamination

However, in order to simultaneously distinguish wild-type, mutant and heterozygous polymorphisms in one tube of qPCR, different strategies have been derived for the design of primers and probes in the qPCR method; for example, the ARMS-PCR method: ARMS-PCR (amplification refractory mutation system) is also called allele-specific PCR. TaqDNA polymerase lacks 3'→5' exonuclease activity. To know the mutation, design the mutation primer and the wild primer separately. The two primers are mainly different in the 3' terminal base, and the purpose of distinguishing the mutant and wild-type genes can be directly achieved by qPCR method

Disadvantages: To distinguish different allelic sites of genes can only be designed by mismatching at the 3' end of the primer sequence, the specificity cannot be guaranteed, and false positives are prone to occur

But the MGB molecule can only be modified at the 3' end of the probe

One probe can only modify one MGB probe, and when detecting complex sites, the specificity cannot meet the requirements for completely distinguishing mutants and wild types

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