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A PCR technique of interfering sequence in the middle of primer

A technology of sequence and comprehensive interference, applied in the field of selective inhibition of PCR non-specific amplification

Inactive Publication Date: 2018-10-02
珠海市坤元科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Real-time fluorescent PCR amplification does not interfere with the specific amplification of the target molecule, and its background is basically a straight line within 45 cycles of the PCR reaction, without interference from non-specific amplification values

Method used

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  • A PCR technique of interfering sequence in the middle of primer
  • A PCR technique of interfering sequence in the middle of primer
  • A PCR technique of interfering sequence in the middle of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Example 1: Real-time fluorescent PCR detection of human enterovirus pathogenic strains:

[0178] In recent years, hand, foot and mouth disease has become prevalent among young children in my country, and the case fatality rate is high. The nucleic acid real-time fluorescent PCR detection of its pathogenic enterovirus (EnteroVirus, EV) has become an important technical means to monitor the prevalence of its infection. EV is an RNA virus, which was originally divided into more than 60 different serotypes, including enterovirus 68-71 . Based on its nucleic acid sequence classification, human EVs are classified into five types: A, B, C, D and PolioVirus, among which the main pathogenic strains CoxsackieA16 (CA16) and EnteroVirus 71 (EV71) are classified as human enterovirus A. The EV gene of enteroviruses varies greatly, only the 5'UTR is conserved, and there are three segments common to all strains homologous conserved region (underlined), the published EV universal pri...

Embodiment 2

[0200] Embodiment two: human hepatitis B virus SYBR Green I real-time fluorescent PCR:

[0201] Hepatitis B virus (referred to as hepatitis B) is a worldwide infectious disease caused by hepatitis B virus (Hepatitis B virus, HBV). The infection rate of hepatitis B in our country is very high, which greatly endangers people's health. At present, the detection methods of hepatitis B mainly include enzyme immunoassay, radioimmunoassay, chemiluminescence, immunofluorescence, nucleic acid amplification (PCR) fluorescence quantitative method, etc. Enzyme immunoassay is widely used, but real-time fluorescent PCR analysis can accurately measure the viral gene content of hepatitis B patients, and plays an irreplaceable important role in judging the virus replication level of infected patients, monitoring the infectivity of the disease and the efficacy of antiviral drugs. The HBV real-time fluorescent PCR in this embodiment is further divided into A. hepatitis B HBV load determination ...

Embodiment 3

[0252] Embodiment three: HBV improved TaqMan probe method real-time fluorescent PCR:

[0253] Probe-based real-time fluorescent PCR is mainly represented by TaqMan probes, and also includes MGB probes with increased binding force and locked nucleic acid LNA base probes. The increased signal of the amplified product is detected by a fluorescent probe with a quencher group. Generally, the 5' end of TaqMan probes is labeled with fluorescent groups such as FAM, VIC, NED, and the 3' end is labeled with quenching groups such as TAMRA, DABCYL&BHQ, etc. Free fluorophores generate fluorescence. TaqMan probe design respects the following general principles: 1) T of the probe m Value ratio of primer T m The value should be higher than 10°C; 2) The 5' end of the probe cannot be a G base, and the degraded G still has the effect of quenching reporter fluorescence; 3) The G in the probe cannot be more than C; 4) Avoid Single nucleotides in strings, especially G; 5) AT-rich sequences shou...

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Abstract

Provided is a primer middle sequence interference PCR method, and the method uses one segment of a non-complemented or same-sequence base of the middle sequence of primers to perform antisense interference inside and outside the primer molecules, so as to competitively destroy the polymerization among the primers to selectively inhibit amplification of the primer dimer (PD).

Description

Technical field: [0001] The invention belongs to the technical field of nucleic acid amplification in the field of molecular biology and molecular testing, and specifically relates to the technical field of selectively inhibiting PCR non-specific amplification by competitively destroying polymerization between primers by interfering with the middle sequence of primers. Background technique: [0002] The idea of ​​nucleic acid amplification originated in 1971. Khorana, who discovered the genetic code, once proposed the idea of ​​nucleic acid amplification in vitro: "After DNA denaturation, hybridization with suitable primers, extension of primers with DNA polymerase, and repeated this process can clone tRNA gene" (Kleppe, 1971, J. Molec. Biol., 56:341), however, its development was limited by the lack of oligonucleotide synthesis, thermostable polymerase, and thermal cycler at that time. Until 1983, Kary Mullis of the Human Genetics Laboratory of PE-Cetus Company in the Unite...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686C12Q1/6848C12Q2525/185C12Q2549/126
Inventor 江洪岳素文廖同兵江必胜曲越江雨康
Owner 珠海市坤元科技有限公司
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