Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus by real-time reverse transcription-PCR
Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus by real-time reverse transcription-PCR
复制标题
DOI：
10.1128/jcm.40.7.2323-2330.2002
复制
发表时间：
2002-07-01
影响因子：
9.4
通讯作者：
Günther, S
中科院分区：
文献类型：
--
作者：
Drosten, C;Göttig, S;Günther, S
病毒性出血热（VHF）是一种急性感染，病死率很高。重要的VHF病原体是埃博拉病毒和马尔堡病毒（MBGV/EBOV）、拉沙病毒（LASV）、克里米亚-刚果出血热病毒（CCHFV）、裂谷热病毒（RVFV）、登革热病毒（DENV）和黄热病病毒（YFV）。VHF在临床上难以诊断和区分;在疑似病例中需要快速可靠的实验室诊断。我们已经建立了六个一步，实时逆转录PCR检测这些病原体的基础上的Superscript逆转录酶-铂Taq聚合酶混合物。通过使用最新的DNA数据库条目，针对RVFV、DENV、YFV和CCHFV设计了新的引物和/或5 '-核酸酶检测探针。PCR产物在LightCycler仪器上通过使用5 '-核酸酶技术（RVFV、DENV和YFV）或SybrGreen染料嵌入（MBGV/EBOV、LASV和CCHFV）进行真实的检测。SybrGreen对逆转录的抑制作用通过将染料初始固定在反应毛细管中来克服。建立了SybrGreen和5 ′-核酸酶探针检测的通用循环条件。因此，最多可平行进行三次检测，便于快速检测多种病原体。通过使用体外转录的RNA，对所有检测方法进行了彻底优化和分析灵敏度验证。通过概率单位回归分析确定的大于或等于95%的检测限范围为1，545至2，835病毒基因组当量/ml血清（每次测定8.6至16个RNA拷贝）。通过检测和定量VHF患者血清样品中的病毒RNA来举例说明该测定法的适用性。
Viral hemorrhagic fevers (VHFs) are acute infections with high case fatality rates. Important VHF agents are Ebola and Marburg viruses (MBGV/EBOV), Lassa virus (LASV), Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), dengue virus (DENV), and yellow fever virus (YFV). VHFs are clinically difficult to diagnose and to distinguish; a rapid and reliable laboratory diagnosis is required in suspected cases. We have established six one-step, real-time reverse transcription-PCR assays for these pathogens based on the Superscript reverse transcriptase-Platinum Taq polymerase enzyme mixture. Novel primers and/or 5'-nuclease detection probes were designed for RVFV, DENV, YFV, and CCHFV by using the latest DNA database entries. PCR products were detected in real time on a LightCycler instrument by using 5'-nuclease technology (RVFV, DENV, and YFV) or SybrGreen dye intercalation (MBGV/EBOV, LASV, and CCHFV). The inhibitory effect of SybrGreen on reverse transcription was overcome by initial immobilization of the dye in the reaction capillaries. Universal cycling conditions for SybrGreen and 5'-nuclease probe detection were established. Thus, up to three assays could be performed in parallel, facilitating rapid testing for several pathogens. All assays were thoroughly optimized and validated in terms of analytical sensitivity by using in vitro-transcribed RNA. The greater than or equal to95% detection limits as determined by probit regression analysis ranged from 1,545 to 2,835 viral genome equivalents/ml of serum (8.6 to 16 RNA copies per assay). The suitability of the assays was exemplified by detection and quantification of viral RNA in serum samples of VHF patients.