六种核酸提取试剂盒对粪样中小鼠肝炎病毒核酸提取效率的比较

Comparation of Six Nucleic Acid Extraction Kits in the Extraction Efficiecy of Hepatitis Virus Nucleic Acid in Fecal Samples

目的比较六种商品化试剂盒在小鼠粪便中提取小鼠肝炎病毒(MHV)RNA的效率,筛选出效率高、耗时短的核酸提取方法。方法采集感染MHV小鼠的新鲜粪便样本,分别用液氮研磨法、磁珠匀浆法以及PBS溶解离心法进行处理,通过同一核酸提取试剂盒比较病毒核酸提取效率,明确最佳样本前处理方法;之后,用6种试剂盒进行RNA提取,经反转录后进行TaqMan实时定量PCR检测,以Ct值评价6种试剂盒的提取效率,结合产物测序和血清抗体ELISA结果,筛选MHV的最佳核酸提取方法。结果对于粪便样本的前处理,液氮研磨法的RNA提取效率显著高于磁珠匀浆法。对于6种试剂盒的提取效果,TIANGEN DP422[总RNA浓度:(549.70±52.38) ng/μL,Ct值:(24.51±0.10)]核酸提取效率最高;TIANGEN SD101[总RNA浓度:(274.13±6.87) ng/μL,Ct值:(2.39±0.017)]和QIAGEN 52904[总RNA浓度:(288.13±15.11) ng/μL,Ct值:(2.40±0.012)]用时最短;XS VRL[总RNA浓度:(348.80±15.85) ng/μL,Ct值:(24.70±0.13)]操作最为方便。结论粪便样本前处理建议采用液氮研磨法。针对粪便中MHV RNA的提取,TIANGEN DP422提取效率最高,推荐用于小鼠肝炎病毒的分子生物学检测。

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.