EMA-PCR技术快速鉴别死活腺病毒的初探

Study on Application of EMA-PCR Rapid Identification of Infectious and Non-infectious Adenovirus

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。

Objective To investigate the application of cytopathic effect （EMA）-PCR to rapid identification of infectious and non-infectious adenovirus. Methods The different dilutions of virus were inoculated to well-grown monolayer cells and then calculated the concentration of virus through the cytopathic effect （CPE）. The adenovirus and New Castle disease virus were prepared in a fixed concentration of 106 PFU/mL then diluted in ten-time gradient and the DNA of virus were extracted and PCR were applied, then the target DNA fragments were observed through gel electrophoresis. The non-infectious adenovirus were treated with different concentrations of EMA （0 μg/mL, 70 μg/mL, 120 μg/mL and 150 μg/mL） and then detected the target DNA fragment in the same way. The different concentrations of infectious adenovirus （107 PFU/mL, 106 PFU/mL, 105 PFU/mL, 104 PFU/mL and 103 PFU/mL） were treated with 120 μg/mL EMA and then detected target DNA fragment in the same way. Results The target DNA fragments were detected in adenovirus of 104 PFU/mL and above. The target DNA fragments were detected in all 3 types of adenovirus while not detected in New Castle disease virus. The DNA amplification of non-infectious adenovirus were suppressed by EMA at 120 μg/mL and 150 μg/mL concentration, while the DNA amplification of infectious adenovirus in concentration of 105 PFU/mL and above were not affected by 120 μg/mL EMA. Conclusion EMA-PCR was proved to be feasible to identify the infectious and non-infectious adenovirus, in which false positive results of PCR were effectively avoid at the same time.