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Human enterovirus(EV) fluorescence quantitative PCR detecting technology

A human enterovirus, fluorescence quantitative technology, applied in fluorescence/phosphorescence, sugar derivatives, organic chemistry, etc., can solve the problems of product contamination, gap, not and so on

Inactive Publication Date: 2007-08-15
河南省生物工程技术研究中心
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Problems solved by technology

However, the common RT-PCR method requires open operation of the amplified product during electrophoresis detection, and there is a risk of product contamination, so it is banned from clinical application by the state.
At the same time, this method is not a quantitative detection, and there is a big gap with clinical needs

Method used

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Examples

Experimental program
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Effect test

Embodiment Construction

[0018] Implementation: Detection of human enterovirus by fluorescent quantitative PCR detection technology.

[0019] (1) Extraction of RNA: 200ul serum + 400ul RNA lysate, shake for 15 seconds; mix in a 60°C water bath for 15 minutes; centrifuge for a few seconds; add 600ul isopropanol, room temperature for 5 minutes; wash the precipitate with 600ul 75% ethanol, wash with absorbent paper, Dry at ℃ for 10 minutes, and set aside (if RNA needs to be preserved, add DEPC-treated water, and keep at -20℃ (for storage).

[0020] (2) Synthesize cDNA by reverse transcription: add 28ul reverse transcription reaction solution (containing 0.2uM universal downstream primer, 1.8mM Mg 2+ , 50mM Tris-HCl (pH8.5), 50mM KCl) and 10UAMV enzyme, centrifuged at 12000r / min for 10s, and water bathed at 42°C for 45min.

[0021] (3) PCR amplification: Take 2.5U of Taq enzyme and add it into the fluorescence quantitative PCR reaction solution tube (fluorescence quantitative PCR reaction solution: 0.4uM...

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PUM

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Abstract

This invention relates to one human intestinal canal virus fluorescence meter test technique in the field of virus nuclear test technique, which adopts common upstream object, common downstream object and common fluorescence detector for EV each hypotype, wherein, the detector 5'end is labeled with fluorescence emission base group 6- carboxyl fluorandiol and 3'labeled fluorescence quencher base group and 6-carboxyl tetramethyl rhodamine. Due to the test agent case, it tests each EV hypotype standard blood serum for online test and to get each parameter as the following: abnormal property for 100 percent; sensitivity for 95 percent, repetitiveness CV less than 10 percent and minimum content of EV as 500 copy each ml.

Description

technical field [0001] The invention belongs to the technical field of viral nucleic acid detection, in particular to a human enterovirus (EV) fluorescent quantitative PCR detection technology, which is suitable for quantitative detection of human enterovirus, diagnosis of human enterovirus infectious diseases, and epidemiological investigation , and to evaluate the effectiveness of prevention and treatment. Background technique [0002] Human enteroviruses (enteroviruses, EVs) belong to the Picornaviridae family, including polioviruses (serotypes 1-3), coxsackieviruses A (serotypes 1-22, 24) and coxsackieviruses B (serotypes 1-6), echoviruses (serotypes 1-9, 11-27, 29-31) and new enteroviruses (serotypes 68-71). [0003] The most common symptom of enteroviral infection is nonspecific fever, sometimes accompanied by rash. The acute viral syndromes caused mainly include encephalitis, myocarditis (especially caused by Coxsackie B group virus), hemorrhagic conjunctivitis (esp...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/68C07H21/00
Inventor 董晓敏李智涛董继华王云龙
Owner 河南省生物工程技术研究中心
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