Strand substitute polymerase chain reaction

A technology of strand displacement and polymerase, applied in the field of molecular biology, can solve problems such as cross-contamination and difficult precise quantification

Inactive Publication Date: 2010-02-03
BEIJING TAG ARRAY MOLECULAR TEST
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Problems solved by technology

However, cross-contamination between samples and adding samples is still easy to cause, and RNA needs t

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Examples

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Embodiment 1

[0083] Example 1 Multiple amplification of replacement reporter gene of HFMD enterovirus:

[0084] At present, the source of infectious diseases, including enteroviruses, is generally diagnosed by isolation and culture methods, gene cloning and sequencing methods, immunoserological and other diagnostic methods, and real-time fluorescent PCR methods for molecular detection. The detection results of the first two methods are the most reliable, but it takes a long time and depends on certain experimental technical conditions. The latter two are clinical diagnostic methods widely used in clinical testing.

[0085] Human enteroviruses are pathogenic strains of Picornaviridae, including enterovirus (EV) A, B, Coxsack virus (COX) A16, echovirus (echorirus30) and EVA new serotype EV71, EV71 includes BrCr, B1, 2, 3, 4, 5 and C1, 2, 3, 4 subtypes. The epidemic is sporadic in Asia all the year round, peaking in June and July, and the virus is prone to highly mutated, small epidemics in...

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Abstract

The invention relates to a strand substitute polymerase chain reaction, which is characterized in that a target gene chain to be detected is substituted by a probe containing reporter gene chain having a different sequence for indirect amplification, the probe sequence is complementary sequences selecting two small segments of conserved or specific sequences adjacent to target genes as double probes, different sequences of the reporter gene chain refers to a gene sequence which has minimum homology with the target gene chain or is in farther germ line, and the head and the tail of the reportergene chain are added with the given double-probe sequences to serve as linear band probe reporter genes. The target genes to be detected and the given head and tail probe sequences of the reporter genes are complementally hybridized so that the head and the tail of linear reporter genes are closed and linked, single-strand gaps of a hybrid are connected by ligase, and the target link is substituted by a reporter gene ring, namely a reverse PCR amplified reporter gene ring. By monitoring different sizes (at two ends) of reverse PCR amplification or the reporter genes with different codes, multi-target molecules can be indirectly indicated. More than one set of reporter genes can be alternately used.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to an in vitro gene amplification-polymerase chain reaction (Polymerase Chain Reaction) and an application thereof. Background technique [0002] One night in 1983, Cetus scientist Kary Mullis was driving on the highway in Northern California. His thoughts were still on the research of synthetic oligonucleotide sequencing from time to time, which gave birth to the inspiration of PCR technology. PCR is to simulate the natural DNA replication process in a test tube, and amplify a DNA molecule with a known sequence at both ends in a geometric progression. Using the DNA to be amplified as a template, using a pair of oligonucleotide fragments complementary to both ends of the template as primers, under the action of DNA polymerase, it extends along the template chain according to the mechanism of semi-conservative replication until a new DNA is completed. chain synthesis. Repea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34C12N15/11
Inventor 江洪江必胜张辉
Owner BEIJING TAG ARRAY MOLECULAR TEST
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