两种检测尼帕病毒的实时荧光定量PCR方法的比较与效果验证  

Comparison and validation of two TaqMan real-time RT-PCR methods for detection and quantification of Nipah virus

作　　者：肖舒奇 师永霞 夏菡[1] 危宏平[1] 袁志明[1,3] 黄弋 XIAO Shu-qi;SHI Yong-xia;XIA Han;WEI Hong-ping;YUAN Zhi-ming;HUANG Yi(Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071,China;Guangzhou Customs Technology Center,Guangzhou 510623,China;Wuhan National Biosafety Laboratory,Wuhan 430020,China)

机构地区：[1]中国科学院武汉病毒研究所,武汉430071 [2]广州海关技术中心,广州510623 [3]武汉国家生物安全实验室,武汉430020

出　　处：《中国人兽共患病学报》2022年第4期303-308,共6页Chinese Journal of Zoonoses

基　　金：国家科技重大专项(No.2018ZX10711001-006)。

摘　　要：目的 建立稳定可靠的尼帕病毒特异性检测技术和方法,对该病毒进行监测和检测、预警预报。方法 本研究对已建立的两种尼帕病毒的实时荧光定量PCR检测方法的特异性与敏感性进行了比较和验证。结果 研究结果显示两种方法检测体外转录RNA时灵敏度为10 copies/μL,检测活病毒的灵敏度为10 TCID_(50)/mL。特异性检测结果表明,两种检测方法均与登革病毒、日本乙型脑炎病毒、云南版纳病毒、艾比湖病毒、寨卡病毒、西藏环状病毒、克里米亚-刚果出血热病毒和埃博拉病毒样本无交叉扩增,特异性良好。结论 本研究是国内首次利用尼帕病毒细胞感染样品对两种尼帕病毒的实时荧光定量PCR方法进行评估验证和比较验证,证明两种检测方法均具有较好的灵敏度和特异度,为建立稳定可靠的尼帕病毒实验室诊断方法提供参考。Nipah virus(NiV)is a single-stranded negative strand RNA virus belonging to the Henipavirus genus in the Paramyxoviridae family.The virus,which has a wide range of hosts,can infect many animals and cause serious illness and death.It has become a major public health concern,owing to its long-term prevalence in parts of Southeast Asia,and high morbidity and mortality.Establishing stable and reliable detection techniques and methods for monitoring,detecting and predicting NiV is highly important.This study compared and validated the specificity and sensitivity of two TaqMan real-time RT-PCR detection methods for the detection and quantification of NiV in the laboratory.The sensitivity of the two assays was 10 copies/μL in detecting in vitro transcribed RNA,and 10 TCID_(50)/mL in detecting viral stock.Specificity analysis indicated no cross amplification with viral RNA extracted from Dengue virus,Japanese encephalitis virus,Banna virus,Ebinur Lake Virus,Zika virus,Tibet orbivirus,Crimean-Congo hemorrhagic fever virus and Ebola virus.Both real-time RT-PCR methods had good specificity.We evaluated and verified these real-time RT-PCR methods by using NiV virus for the first time in China.Our study should contribute to establishment of good,effective and stable laboratory diagnostic assays.

关 键 词：尼帕病毒 实时荧光定量PCR 效果验证和比较 生物安全四级实验室 

分 类 号：R373.9[医药卫生—病原生物学] S855.2[医药卫生—基础医学]