基于PMA-RT-qPCR的新型冠状病毒活性检测方法的建立  

Establishment of 2019-nCoV activity detection method based on PMA-RT-qPCR

作　　者：刘丹丹 罗君红 葛以跃[3] 朱小娟[3] 吴涛[3] 赵康辰[3] 王祥喜[4] 朱凤才[3] 朱宝立[3] 崔仑标[3] Liu Dandan;Luo Junhong;Ge Yiyue;Zhu Xiaojuan;Wu Tao;Zhao Kangchen;Wang Xiangxi;Zhu Fengcai;Zhu Baoli;Cui Lunbiao(College of Pharmacy,Nankai University,Tianjin 300000,China;Nanjing Medical University,Nanjing 210009,China;NHC Key laboratory of Enteric Pathogenic Microbiology,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China;Institute of Biophysics,Chinese Academy of Sciences,Beijing 100000,China)

机构地区：[1]南开大学药学院,天津300000 [2]南京医科大学,210009 [3]江苏省疾病预防控制中心,国家卫生健康委员会肠道病原微生物重点实验室,南京210009 [4]中国科学院生物物理研究所,北京100000

出　　处：《中华实验和临床病毒学杂志》2021年第6期658-663,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：江苏省自然科学基金(BK20191489,BK20211373);江苏省六大人才高峰项目(WSN-024);江苏省卫健委重点科研项目(ZD2021060)。

摘　　要：目的建立一种可以快速检测新型冠状病毒活性的方法。方法将热灭活新型冠状病毒样本叠氮溴化丙锭(propidium monoazide,PMA)处理和曝光后对RT-qPCR检测体系进行筛选,优化PMA预处理条件,建立新型冠状病毒PMA-RT-qPCR检测方法。以建立的检测方法对不同温度和含氯消毒剂灭活的病毒进行检测,评估其检测病毒活性的效果。结果PMA-RT-qPCR检测方法的PMA浓度确定为200μmol/L,孵育时间为10 min,曝光时间为15 min,以CDC ORF1ab检测体系进行扩增检测;活病毒PMA-RT-qPCR和直接进行RT-qPCR检测结果差异无统计学意义;95℃热灭活和含氯消毒剂灭活病毒在不同稀释度下PMA-RT-qPCR检测Ct值显著高于对照组;70℃和56℃热灭活病毒只有部分稀释度PMA-RT-qPCR检测Ct值高于对照组。结论建立的PMA-RT-qPCR新型冠状病毒活性检测方法对95℃热灭活和含氯消毒剂灭活病毒具有良好的检测效果,为判断样本中病毒的感染性大小提供了辅助手段。Objective To establish a rapid method for detecting the activity of 2019-nCoV2.Methods The reverse transcription-quantitative polymerase chain reaction(RT-qPCR)detection system was screened after propidium monoazide(PMA)treatment and exposure to heat-inactivated 2019-nCoV samples,and the PMA pretreatment conditions were optimized to establish the 2019-nCoV PMA-RT-qPCR detection method.The established method was used to detect virus inactivated by different temperatures and chlorine-containing disinfectant,to evaluate its effect in detecting virus activity.Results For the PMA-RT-qPCR assay,200μmol/L of PMA,10 min of incubation time,15 min of exposure time,and the CDC ORF1ab detection system were selected;there was no significant difference in the result of PMA-RT-qPCR and direct RT-qPCR for the active virus;the Ct values of PMA-RT-qPCR for virus inactivated by 95℃and chlorine-containing disinfectant were higher than that of control groups at different dilutions;only partial dilutions of 70℃and 56℃heat-inactivated virus had higher Ct values than control groups.Conclusions The established PMA-RT-qPCR for 2019-nCoV activity detection method has a good detection effect on the virus inactivated by 95℃heat and chlorine disinfectant,and provides an auxiliary means for judging the infectivity of the virus in the sample.

关 键 词：新型冠状病毒 叠氮溴化丙锭 RT-QPCR 活病毒 灭活病毒 

分 类 号：R563.1[医药卫生—呼吸系统] R440[医药卫生—内科学]