Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers

A real-time fluorescence and probe technology, used in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., which can solve the problem that weak positive templates are not easy to be missed.

Inactive Publication Date: 2012-03-14
BEIJING TAG ARRAY MOLECULAR TEST
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Problems solved by technology

What's more serious is that probes such as TaqMan and excess primers will also hybridize, complement and extend. When no template is added, the Ct value of SYBR Green I real-time PCR with only a pair of primers and TaqMan probes is advanced to about 25 cycles. It shows tha...

Method used

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  • Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers
  • Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers
  • Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers

Examples

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Effect test

example 1

[0047] Example 1: Real-time fluorescent PCR detection of human hepatitis B virus:

[0048] 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.

[0049] Select the sequence of the X region (1545-1887) of hepatitis B virus (Hepatitis B virus, HBV) as follows:

[0050]...

example 2

[0081] Example 2: Real-time fluorescent PCR detection of human enterovirus pathogenic strains:

[0082] 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 EV is further divided into five types: A, B, C, D, and PolioVirus. Among them, 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 segments of all strains homologous conserved region , the published EV universal primers all select this conserved region, so they misdetect non-pa...

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Abstract

The invention provides a Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers. The Taqman real-time fluorescence PCR is improved Taqman and real-time fluorescence PCR which is in a hybrid stem structure combining a panhandle shape at the tail end of a molecular beacon and improves self combination. The Taqman real-time fluorescence PCR is characterized in that: fluorescein is labeled conventionally at 5'-terminal of a template probe sequence, complementary sequences of 5'-terminals of 5 to 6 probes are added to 3'-terminal of the template probe sequence, and a quencher is labeled at the tail terminal so as to form a stem structure similar to the tail terminal of the molecular beacon by hybridizing with the sequence at 5'-terminal of the probe. Two terminals of the probe are self-hybridized and combined, so that the quenching effect is increased, and the background is reduced; the probe is difficultly polymerized with a PCR primer due to self combination, so that primer-probe nonspecific polymerization amplification is eliminated, the detection sensitivity of the Taqman real-time fluorescence PCR is improved, and especially the relevance ratio of a weak-positive specimen is increased.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology and nucleic acid amplification PCR, and specifically relates to a real-time fluorescent PCR technology of a hydrolysis probe that reduces or even does not polymerize with primers. Background technique: [0002] Nucleic acid amplification PCR technology is developed and matured together with the progress of modern molecular biology. As early as 1971, Korana proposed the idea of ​​nucleic acid amplification in vitro: "After DNA denaturation, hybridize with suitable primers, use DNA The polymerase extends the primer, and the process is repeated to clone the tRNA gene." 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: to simulate the natural DNA in vivo replication process in test tubes. Provide a suitable condition---template DNA, oligonucleotide p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 江洪江必胜周勇刘涛张宝林林昊宇廖同兵
Owner BEIJING TAG ARRAY MOLECULAR TEST
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