Primer middle sequence interference PCR (Polymerase Chain Reaction) technology

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

Inactive Publication Date: 2013-05-22
珠海市坤元科技有限公司
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  • 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 back...

Method used

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  • Primer middle sequence interference PCR (Polymerase Chain Reaction) technology
  • Primer middle sequence interference PCR (Polymerase Chain Reaction) technology
  • Primer middle sequence interference PCR (Polymerase Chain Reaction) technology

Examples

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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

The invention relates to a primer middle sequence interference PCR (Polymerase Chain Reaction) technology. The improved PCR technology is characterized in that one segment of relatively non-complemented or same-sequence basic group primer molecules in the intermediate domain of primers perform the antisense interference inside and outside so as to competitively destroy the polymerization among the primers to selectively inhibit the primer dimer (PD) from being amplified. For the interference of the intermediate domain of the primers, based on the primers optimally selected by the conventional design principle, the technology that the intermediate domain (ID) of a pair of the primers are in parallel but are not complemented with each other or are in the same sequence or/and the technology that ID antisense oligonucleotides (Oligo) are added into the primers to perform the interference action or/and the Oligo antonymy is carried out in the primer molecules via the ID so as to perform the interference action are adopted, or the combined technology of the three types of the technologies is adopted. As a result, only the ID of the primers is interfered while the target specific amplification is not influenced; the combining force acting on the minority of base-group pairing hydrogen bonds at the tail end and the base-group hydrogen bonds outside the tail end due to the action of the primers is dispersed to a maximum extent, so that the PD is selectively inhibited. Therefore, the PD accumulation in the PCR system is avoided. If the mineral oil is additionally used, the sealed primers can slowly release the hot starting and the UDG pretreatment so as to prevent aerosol glue as a byproduct of the PCR system from causing the pollution. Consequently, the nucleic acid is amplified reliably and the real-time fluorescence PCR is quantified accurately.

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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IPC IPC(8): C12Q1/68
CPCC12Q1/68C12Q1/686C12Q1/6848C12Q2525/185C12Q2549/126
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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