EMA-PCR技术快速鉴别死活腺病毒的初探

发布时间：2018-04-13 18:45

本文选题：EMA + PCR　；参考：《四川大学学报(医学版)》2017年01期

【摘要】：目的探讨采用叠氮溴化乙锭(EMA)联合聚合酶链反应(PCR)快速鉴别死活腺病毒的方法。方法将不同稀释度的病毒接种至生长良好的单层细胞上,通过细胞病变效应(CPE)的发生情况计算病毒滴度;将3种腺病毒和新城疫鸡瘟病毒分别制备成106 PFU/mL的母液并梯度稀释备用,提取DNA后用PCR扩增后进行凝胶电泳,观察目的片段的扩增结果;分别用0μg/mL、70μg/mL、120μg/mL和150μg/mL的EMA处理灭活后的腺病毒,提取DNA进行PCR,观察目的片段的电泳结果;用120μg/mL的EMA处理107 PFU/mL、106 PFU/mL、105PFU/mL、104 PFU/mL、103 PFU/mL的腺病毒,观察PCR和凝胶电泳结果。结果 104 PFU/mL及以上滴度的病毒DNA PCR后得到阳性条带,其他滴度的病毒DNA PCR后未检测到阳性条带;3个型别的腺病毒(共8个分离株)的DNA均扩增出目的条带,新城疫鸡瘟病毒的DNA未扩增出目的条带;120μg/mL及150μg/mL的EMA抑制灭活腺病毒DNA扩增,未得到阳性条带;120μg/mL EMA不影响107 PFU/mL、106 PFU/mL、105 PFU/mL的腺病毒活病毒DNA扩增,得到阳性条带。结论本研究证实EMA-PCR方法可快速鉴别死活腺病毒,能有效避免单纯PCR检测腺病毒产生的假阳性结果。
[Abstract]:Objective to study the method of rapid identification of dead and living adenovirus by EMA combined with polymerase chain reaction (PCR).Methods the virus with different dilution was inoculated into the monolayer cells with good growth, and the titer of the virus was calculated by CPE (cytopathic effect).Three kinds of adenovirus and Newcastle disease chicken plague virus were prepared into 106 PFU/mL mother liquor and diluted by gradient dilution. The DNA was extracted and amplified by PCR. The result of the amplification of the target fragment was observed.The inactivated adenovirus was treated with 0 渭 g / mL 70 渭 g / mL EMA and 150 渭 g/mL respectively. The DNA was extracted to observe the electrophoresis results of the target fragment, and the 107PFU / mL105PFUmL105PFUmL104PFUmL103pmL10 PFU/mL adenovirus was treated with 120 渭 g/mL EMA, and the results of PCR and gel electrophoresis were observed.Results positive bands were obtained after DNA PCR of 104 PFU/mL and above, but no positive bands were detected after DNA PCR of other titers, and the target bands were amplified from the DNA of 3 types of adenovirus (8 isolates).The DNA of Newcastle disease chicken plague virus did not amplify the target band of 120 渭 g/mL and 150 渭 g/mL EMA to inhibit the DNA amplification of inactivated adenovirus. The positive band of 120 渭 g/mL EMA did not affect the DNA amplification of 107PFU / mL 106PFU / mL 105 PFU/mL adenovirus live virus, and the positive band was obtained.Conclusion EMA-PCR method can be used to identify adenovirus rapidly and avoid the false positive results of PCR alone.
【作者单位】：四川大学华西公共卫生学院卫生检验与检疫系;
【基金】：大学生创新训练计划(No.201410610119)资助
【分类号】：R440

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