Fluorescent quantitative PCR method for real-time detecting mononucleotide polymorphism and reagent kit thereof

A technique of single nucleotide polymorphism and fluorescence quantification, which is applied in the direction of measuring devices, biochemical equipment and methods, microbial determination/inspection, etc.

Inactive Publication Date: 2009-01-28
CHANGZHOU ANBO BIO TECH
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  • Abstract
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Problems solved by technology

To a certain extent, the detection of SNP sites can be realized, but there are also shortcomings. Some methods require several steps of PCR amplification reactions, and some require the use of multi-tube PCR reactions to determine SNPs

Method used

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  • Fluorescent quantitative PCR method for real-time detecting mononucleotide polymorphism and reagent kit thereof
  • Fluorescent quantitative PCR method for real-time detecting mononucleotide polymorphism and reagent kit thereof
  • Fluorescent quantitative PCR method for real-time detecting mononucleotide polymorphism and reagent kit thereof

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Embodiment

[0048] Embodiment-hepatitis B virus (HBV) real-time detection of 1896 mutation fluorescence quantitative PCR method and its kit

[0049] Primer design: design a sequence with a length of 16-25 bases, fix the base corresponding to the mutation site at its 3' end, the 3' end of the primer, and control the Tm value of the sequence (a complete The required temperature when half of the double-stranded DNA sequence is cleaved) meets the PCR requirements, and then the relevant primer design software is used to select another primer and probe, and two sets of positive and negative primers can be designed on the double-stranded template. Another primer and probe design-related requirements are the same as general fluorescent PCR, and the primer and probe design software are Premier primer 5.0, OLIGO 6.0, Primer Express3.0 and DNAStar, etc. The experimental results prove that the implementation of this scheme can perform SNP detection well, and at the same time, false positives in sampl...

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Abstract

The invention provides a real-time fluorescence quantitative PCR method for testing single nucleotide polymorphism. The method includes two sets of combinations of primers and probes, wherein, one set of quantitative primers and probes can determine the amount of initial nucleic acid of a sample to be tested, the other set of classifying-type primers and probes and the combination thereof can determinate whether single nucleotide polymorphism occurs or not; magnesium concentration of the system is 2 mM to 10 mM, the amount of Taq enzyme is 5U to 10U per reaction and the amount of UNG enzyme is 1U to 2U per reaction. A standard curve is made according to the Ct value of a quantitative standard; the Ct value of the sample to be tested is substituted into the standard curve to determine the copy number of an initial template; the deviation between quantitative Ct value and classifying-type Ct value of the sample is calculated, and then is compared with the wild-type and the mutant-type standards to determine whether single nucleotide mutant occurs or not. The invention also provides a kit of fluorescence quantitative PCR which adopts the method to test mutant of hepatitis B virus 1896.

Description

technical field [0001] The invention relates to the field of single nucleotide polymorphism detection, and belongs to a method for detecting gene polymorphism (such as single base polymorphism, single base insertion or deletion) in genetic material, specifically two sets of primer detection methods Needle combination, perform two-color fluorescent PCR reaction in a single tube. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. Includes substitutions, transversions, deletions and insertions. Theoretically, each SNP site can have 4 different variant forms, but usually only two occur, that is, the main SNP sites are two genotypes. SNPs appear most frequently on CG sequences, and most of them are converted from C to T. The reason is that C in CG is often methylated and becomes thymine after spontaneous deamination. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/00C12Q1/68
Inventor 龙伟红王伟彭早元
Owner CHANGZHOU ANBO BIO TECH
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