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目的测试病毒RNA提取试剂(硅胶柱法和改良异硫氰酸胍法)灭活甲型流感病毒的能力,以评估在低防护区操作甲型流感病毒核酸提取过程的可能性。方法根据试剂盒操作指导分别用两种提取试剂的裂解液Buffer AVL(德国QIAGEN)和MGTC溶液(广州华银)处理一甲型流感2009年大流行株(105.67TCID50/ml);系列对数稀释病毒处理液后接种于MDCK细胞单层进行分离培养;倒置显微镜每日观察细胞生长状态,出现75%以上病变后收获培养物进行血凝实验测定。同时设置试剂对照以了解试剂的细胞毒性,病毒对照以确定病毒的感染能力。结果未稀释和10倍稀释的两种裂解液都可引起MDCK大范围死亡,两病毒对照感染细胞的终点稀释度都为104,而所有102稀释度以上的病毒处理液培养孔细胞生长良好,血凝实验阴性。结论两种病毒RNA提取试剂可以完全灭活高滴度的甲型流感病毒,标本裂解处理后可在低防护区域操作。
Objective To test the ability of virus RNA extraction reagent (silica gel column method and modified guanidium isothiocyanate method) to inactivate influenza A virus in order to evaluate the possibility of nucleic acid extraction of influenza A virus in low protection area. Methods The influenza A 2009 pandemic strain (105.67 TCID50 / ml) was treated with two reagent lysis buffers Buffer AVL (Germany QIAGEN) and MGTC solution (Guangzhou Huayin) according to the instructions of the kit. Serial dilution series Virus treatment solution was inoculated on MDCK cell monolayer for isolation and culture; inverted microscope to observe the cell growth state daily, more than 75% lesions were harvested after the culture test for the determination of hemagglutination. At the same time set up a reagent control to understand the reagent cytotoxicity, virus control to determine the virus’s ability to infect. RESULTS Both undiluted and 10-fold dilutions of the lysate caused extensive MDCK deaths, with an endpoint dilution of 104 for both virus-infected cells and all well above 102 dilutions of virus-treated cells grew well with blood Condensation experiment negative. Conclusion Both virus RNA extraction reagents can completely inactivate high-titer influenza A virus, and the samples can be manipulated in low-protection area after being lysed.