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Method for rapidly detecting shigella by duplex scorpion primer fluorogenic quantitative PCR (Polymerase Chain Reaction)

A fluorescence quantitative and dimer technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of complex detection methods and lack of sensitivity, and achieve simple methods, high sensitivity, and easy The effect of the operation

Inactive Publication Date: 2013-12-11
NANTONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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Problems solved by technology

However, the existing detection methods are complex and lack in sensitivity and other aspects.

Method used

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  • Method for rapidly detecting shigella by duplex scorpion primer fluorogenic quantitative PCR (Polymerase Chain Reaction)
  • Method for rapidly detecting shigella by duplex scorpion primer fluorogenic quantitative PCR (Polymerase Chain Reaction)
  • Method for rapidly detecting shigella by duplex scorpion primer fluorogenic quantitative PCR (Polymerase Chain Reaction)

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Experimental program
Comparison scheme
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Embodiment Construction

[0085] (1) Preparation of pMDl8-T-ipaH standard

[0086] 1. Materials and methods

[0087] 1 material

[0088] 1.1 Plasmid vector and bacterial strain Plasmid vector 8-T-easy vector was purchased from Takara Company; Escherichia coli DH5α and Shigella flexneri were preserved by our laboratory.

[0089] 1.2 Main reagents Taq DNA polymerase (Fermenta); 10×PCR Buffer (Fermenta); 10×dNTP mix (Takara); DL-2000 DNA molecular weight standard (TaKaRa); agarose (Promega); gel recovery reagent Kit (OMEGA Company); Plasmid Extraction Kit (OMEGA Company); IPTG (Shanghai Sangon Biotechnology Engineering Co., Ltd.); X-Gal (Shanghai Sangon Biotechnology Engineering Co., Ltd.); Ampicillin (Shanghai Sangon Biotechnology Engineering Co., Ltd. Engineering Co., Ltd.); restriction enzyme EcoR V (Takara Corporation)

[0090] 1.3 Instruments and equipment Constant temperature incubator; autoclave; water bath; oscillator; ultra-clean bench; analytical balance; shaker; centrifuge; ultraviolet spect...

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Abstract

The invention discloses a method for rapidly detecting shigella by duplex scorpion primer fluorogenic quantitative PCR (Polymerase Chain Reaction). The method comprises the following steps: preparing a pMD18-T-ipaH (Polarisation Mode Dispersion 18-T-Idiopathic Pulmonary Arterial Hypertension) standard sample, and rapidly detecting the shigella by adopting fluorogenic quantitative PCR. The method is simple, convenient and easy to operate, has the advantages of high sensitivity and low cost, and lays the foundation in development of a novel pathogen test kit and scientific research application.

Description

technical field [0001] The invention relates to a method for rapidly detecting shigella by fluorescence quantitative PCR of a dimer scorpion probe. Background technique [0002] At present, there are many reports on the application of PCR technology to the detection of Shigella. Because the ipaH gene is highly specific, the copy of the ipaH gene is highly specific and has higher sensitivity than other virulence factors ipaB, ipaC, and ipaD genes. It is a specific marker of invasiveness, so this gene has been used in Shigella many times rapid detection. However, the existing detection methods are complex and lack in sensitivity and other aspects. Contents of the invention [0003] The purpose of the present invention is to provide an easy-to-operate, high-sensitivity dimer scorpion-type probe fluorescent quantitative PCR method for rapid detection of Shigella. [0004] Technical solution of the present invention is: [0005] A method for rapid detection of shigella by f...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
Inventor 陈峰吴尔翔丁锁顺邵亚军章杰唐晓宇
Owner NANTONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU OF THE PEOPLES REPUBLIC OF CHINA
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