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目的建立一种反向斑点杂交方法(reverse dot blots,RDB)以检测小鼠肝炎病毒。方法用MHV四个毒株分别感染L929细胞,收获病毒提取RNA并逆转录成cDNA;根据MHV保守区基因组序列设计引物和探针,将上游引物5’端用生物素标记。进行PCR扩增,应用PCR产物进行反向斑点杂交,优化杂交条件,进行稳定性、特异性和灵敏度实验,建立反向斑点杂交法。用此法和酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)对41只小鼠进行检测。RDB检测结果阳性样本进行T载体克隆测序。结果试剂条4℃保质期为8个月。分别检测MHV、金黄色葡萄球菌、沙门菌和乙型肝炎病毒,仅MHV为阳性,RDB特异性为100%,且MHV PCR产物最低检测限为5 ng/μL。41只小鼠经本方法检测后,有5只MHV阳性,分布于3个群,而ELISA检测结果有5只阳性,分布于2个群,阳性样本T载体克隆测序结果与RDB检测结果一致。结论该方法具有简洁明了、稳定可靠、特异性好、灵敏度高等优点,可应用于实验动物小鼠肝炎病毒的检测。
Objective To establish a reverse dot blot (RDB) method to detect mouse hepatitis virus. Methods Four strains of MHV were used to infect L929 cells. The RNA was harvested and reverse transcribed into cDNA. Primers and probes were designed according to the MHV conserved regions. The 5 ’end of the upstream primer was labeled with biotin. The PCR products were used for reverse dot blot hybridization, and the hybridization conditions were optimized. The experiments of stability, specificity and sensitivity were carried out to establish the reverse dot blot hybridization. Using this method and enzyme-linked immunosorbent assay (ELISA) on 41 mice were detected. RDB test positive samples for T vector cloning and sequencing. Results Reagent 4 ℃ shelf life of 8 months. MHV, Staphylococcus aureus, Salmonella and Hepatitis B viruses were tested for MHV-positive, MHV-positive, RDB-specific, and 5 ng / μL for MHV PCR products. Of the 41 mice tested positive for this method, 5 were positive for MHV and were distributed in 3 populations. However, 5 of the 4 positive samples were found in 2 positive samples. The results of T vector cloning and sequencing were consistent with those of RDB. Conclusion The method has the advantages of concise, reliable, good specificity and high sensitivity, which can be applied to the detection of experimental hepatitis in mice.