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
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[0027] Example 1 Fluorescence quantitative RT-PCR detection of murine rotavirus
[0028] 1. Experimental samples
[0029] 246 samples of intestinal tissue samples from mice from Shanghai Experimental Animal Research Center.
[0030] 2. cDNA synthesis
[0031] Take part of the mouse’s intestinal tissue, add PBS and steel balls, and place it in a shaker for about 1 min, centrifuge at 4000 rpm for 2 min, aspirate the supernatant, and extract RNA. The extraction method refers to the instruction of TIANamp Virus RNA Kit extraction kit, 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 (40μL) is: RNA template 20μL, primer pair 2μL, 5×Reverse Transcription Buffer 8μL, dNTP (10mmol / L) 4μL, Ribonuclease Inhibitor (40U / μL) 1μL, Prime Script reverse transcriptase (200U) 1μL, DEPC water 4μL. 42℃ water bath for 1h...
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