Same sequence primer transpositional nucleic acid amplification technology

A technology of nucleic acid and technology, applied in the field of nucleic acid amplification, which can solve the problems of delay, inability to eliminate, and limited degradation

Inactive Publication Date: 2012-02-15
北京万达因生物医学技术有限责任公司
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Problems solved by technology

Generally, dUTP is used to replace the dTTP product for aerosol recontamination of the amplified product, combined with uracil-DNA-glycosylase (UDG/UNG, US Patent 6,090,553) to selectively degrade the contaminated product. UDG/UNG is subsequently thermally denatur

Method used

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  • Same sequence primer transpositional nucleic acid amplification technology
  • Same sequence primer transpositional nucleic acid amplification technology
  • Same sequence primer transpositional nucleic acid amplification technology

Examples

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example 1

[0093] Example 1: Human hepatitis B virus homogeneous primer displacement fluorescent PCR detection:

[0094] 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 and so on. Enzyme immunoassay is widely used, but real-time fluorescent PCR analysis can accurately detect the virus gene 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.

[0095] Hepatitis B virus (HBV) is a partially double-stranded DNA virus. It mainly has three type-speci...

example 2

[0126] Example 2: Double fluorescent PCR detection of human enterovirus pathogenic strains:

[0127] 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 a key means to control its epidemic. Enterovirus EV is initially divided into more than 60 different serotypes, including enterovirus 68-71. Based on its nucleic acid sequence classification, human EVs are further divided into five categories: A, B, C, D and PolioVirus, among which the main pathogenic strains Coxsackie A16 (CA16) and EnteroVirus 71 (EV71) are classified as human enterovirus A. The EV gene of enterovirus is highly variable, only the 5'UTR is conserved, and there are three homologous conserved regions of all strains. The published EV universal primers all select this conserved region, so they misdetect non-pathogenic...

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Abstract

The invention provides a same sequence primer transpositional nucleic acid amplification technology, which comprises the steps of: (1) linking the artificial sequence to the upstream of the target specific primer 5', and forming a chimeric primer; (2) taking the chimeric primer as an amplification target template to produce a secondary template with the two ends having artificial sequences; and (3) taking the artificial sequences with the same sequence of 65%-75% as the transpositional primer amplification secondary template. The technology is applicable to clinic molecular diagnosis, basically can utilize the primer design to prevent the false positive result from generating, and is particularly applicable to communicable disease nucleic acid detection and accurate diagnosis. The technology is particularly suitable to multiple PRC amplification technology, is nearly designed by aiming at the multiple PRC amplification technology, and the redundant and extraordinarily complicated primers of the multiple PRC amplification technology can only adopt the primer transpositional design to eliminate the nonspecific amplification of the primer polymer.

Description

Technical field: [0001] The invention belongs to the technical field of nucleic acid amplification in the fields of molecular biology and molecular diagnosis, and in particular relates to a nucleic acid amplification technology using a pair of primers with most of the same intermediate sequence in order to reduce the polymerization non-specific amplification between primers. Background technique: [0002] Nucleic acid amplification technology originated in 1971. Korana proposed the concept of in vitro nucleic acid amplification: "after DNA denaturation, hybridization with suitable primers, extension of primers with DNA polymerase, and continuous repetition of this process, the tRNA gene can be cloned." It also developed and matured together with the progress of modern molecular biology. In 1983, Kary Mullis of the Human Genetics Laboratory of PE-Cetus Company in the United States had the inspiration of nucleic acid amplification when studying nucleic acid sequencing methods:...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 江洪江必胜岳素文阮志强曲越
Owner 北京万达因生物医学技术有限责任公司
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