关键词: 小鼠肝炎病毒, 病毒RNA提取, TaqMan探针, 实时荧光定量PCR

Abstract: Objective In this study, six commercial kits were used to extract mouse hepatitis virus(MHV)nucleic acid from faeces, and screen for an extraction method with higher efficiency and shorter time. Method Fecal samples infected with MHV were collected immediately and treated with liquid nitrogen grinding method , magnetic beads homogenization and PBS dissolves centrifugation method , respectively. RNA of above samples were then extracted with the same nucleic acid extraction kit to identify the best pretreatment method ; Based on which, the RNA was extracted using six kits and reverse transcribed into cDNA followed with TaqMan real-time PCR, respectively. Ct value of real-time PCR combined with sequencing and serum antibody ELISA test were used to evaluate and analyze the extraction efficiency of six kits. Result For the fecal sample pretreatment medthod, with liquid nitrogen grinding method had a significantly higher extraction efficiency than that with the magnetic bead homogenization method .For the RNA extraction efficiency among the six kits, TIANGEN DP422 concentration: (549.70±52.38) ng/μL， Ct value: (24.51±0.10) showed the highest extraction efficiency, TIANGEN SD101 concentration:(274.13±6.87)ng/μL；Ct value： (2.39±0.017) and QIAGEN 52904 concentration:(288.13±15.11)ng/μL；Ct value:(2.40±0.012) displayed the shortest extraction time, and XS VRL concentration:(348.80±15.85)ng/μL，Ct value:(24.70±0.13) turned out to be the most convenient. Conclusion Liquid nitrogen extraction method is more efficient in fecal sample pretreatment, and TIANGEN DP422 extraction kit with the highest MHV RNA extraction efficiency is recommended for the molecular biological detection of MHV in feces.

Key words: Mouse Hepatitis Virus(MHV), viral RNA extraction, TaqMan probe, Real-time fluorescent quantitative PCR