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Primer pair, probe, kit and method for detecting murine rotavirus by adopting fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction)

A fluorescent quantification and primer pair technology, applied in the field of bioengineering, can solve the problems of tediousness, limitations, and low sensitivity, and achieve the effect of optimizing reaction conditions, repeatability, and specificity guarantee

Inactive Publication Date: 2017-06-20
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI +3
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Problems solved by technology

Although this method can also be used for large-scale detection, and the detection method is relatively simple, only need to detect serum samples, but this method requires the preparation of specific antibodies, and cannot distinguish antibodies produced by immunity and infection
Among them, the blood collection and serum separation of mice are particularly cumbersome and time-consuming, especially in large-scale experimental animal breeding institutions, the annual detection of experimental mice, the traditional ELISA method is too cumbersome, and the mouse wheels on the market today Virus infection detection ELISA kits, most of which are imported kits, the price is about 2000-3000 yuan, and only 96 samples can be detected. The traditional method has the disadvantages of cumbersomeness, limitations, low sensitivity and high price.

Method used

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  • Primer pair, probe, kit and method for detecting murine rotavirus by adopting fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction)
  • Primer pair, probe, kit and method for detecting murine rotavirus by adopting fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction)
  • Primer pair, probe, kit and method for detecting murine rotavirus by adopting fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction)

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Embodiment 1

[0027] Example 1 Fluorescent quantitative RT-PCR detection of mouse rotavirus

[0028] 1. Experimental samples

[0029] 246 intestinal tissue samples from Shanghai Experimental Animal Research Center.

[0030] 2. cDNA synthesis

[0031] Take some intestinal tissues of mice, add PBS and steel balls, put them in a shaker for about 1min, centrifuge at 4000rpm for 2min, suck off the supernatant, and extract RNA. The extraction method refers to the instruction manual of the TIANamp Virus RNA Kit extraction box, and the extracted nucleic acid RNA is reverse-transcribed to synthesize cDNA.

[0032] The template is extracted viral RNA, the primer pair is OligdT18, and cDNA is synthesized by reverse transcription. The reverse transcription system (40 μL) is: 20 μL RNA template, 2 μL primer pair, 8 μL 5× reverse transcription buffer, dNTP (10 mmol / L) 4 μL, Ribonuclease Inhibitor (40U / μL) 1 μL, Prime Script reverse transcriptase (200U) 1 μL, DEPC water 4 μL. Water bath at 42°C for 1 ...

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Abstract

The invention discloses a primer pair, a probe, a kit and a method for detecting the murine rotavirus by adopting fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction). According to the detection method, by taking cDNA of the brain tissue of a mouse as a template, SEQ ID No:1 and SEQ ID No:2 as primers, and SEQ ID No:3 as a probe, the fluorescence quantitative RT-PCR amplification is carried out, and fluorescence signals are collected. The fluorescence quantitative RT-PCR detection method can rapidly, accurately, conveniently, rapidly and specifically detect the murine rotavirus nucleic acid in the body of the mouse, and the sensitivity, repeatability and specificity can be guaranteed.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically, the invention relates to a primer pair, a probe, a kit and a method for detecting mouse rotavirus by fluorescence quantitative RT-PCR. Background technique [0002] Murine Rotavirus (Murine Rotavirus, MRV), a double-stranded RNA virus, is ubiquitous in mice and causes Epidemic Diarrhea of ​​mice (EDIM). Very high, seriously endangering the suckling mice of various strains of mice. EDIM virus belongs to the family Reoviridae and belongs to the genus Rotavirus. It is a double-stranded RNA virus. Its shell has a specific antigen that is different from other rotaviruses. cross-reaction, but not with group B rotavirus in suckling rats. Therefore, MRV can only cause disease in suckling mice. Mice are the only natural host of EDIM virus. The disease spreads through the digestive tract and respiratory tract and is a highly contagious infectious disease. EDIM mainly occu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/701C12Q2563/107C12Q2545/114C12Q2521/107C12Q2531/113
Inventor 魏晓锋蔡骁垚熊炜胡建华张泉其他发明人请求不公开姓名
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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