Development of real-time reverse transcriptase PCR assays to detect and serotype dengue viruses
Development of real-time reverse transcriptase PCR assays to detect and serotype dengue viruses
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DOI：
10.1128/jcm.44.4.1295-1304.2006
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发表时间：
2006-04-01
影响因子：
9.4
通讯作者：
Chang, GJJ
中科院分区：
文献类型：
--
作者：
Chien, LJ;Liao, TL;Chang, GJJ
从可疑人体标本中对登革病毒（DENV）进行血清分型对于在传播季节早期制定合理的流行病学控制措施和有效的患者管理至关重要。我们如先前在常规衣壳和前膜基因（C-prM）方案中所述修饰了DENV共有D1（mD 1）和病毒型特异性TS 2（mTS 2）并重新设计了病毒型特异性TS 1（rTS 1）和TS 4（rTS 4）（R. S.兰乔蒂角H. Calisher，D. J. Gubler，G.- J. Chang，A. V. Vorndam，J. Clin. Microbiol. 30：545-551，1992）。此外，我们还设计了两套新的扩增引物和探针，分别定位于登革病毒的非结构蛋白5（NS 5）和3'非编码区（3' NC）。NS 5方案利用两种黄病毒共有外部扩增物（mFU 1和CFD 2）和四种登革病毒亚型特异性TaqMan荧光探针。3 'NC方案使用两种DENV共有扩增物DC 10418和CDC 10564。传统的基于凝胶的半巢式检测方法适用于用于检测和血清分型登革病毒的C-prM方案。此外，我们开发了实时SYBR绿色I和扩增后熔解温度曲线分析的mD 1/TS和YNC协议使用相同的扩增条件。将NS 5扩增物/探针组配制为单管、多重、实时逆转录酶PCR，用于血清型鉴定。三组扩增物和探针在黄热病、日本脑炎、圣路易斯脑炎和西尼罗河病毒的测试中验证了它们的特异性;针对109种DENV毒株进行了优化;并验证了用于检测来自两组不同急性期人登革热血清标本和一组来自登革热患者血清标本的病毒分离株的血清中的病毒。两个独立实验室的临床评价表明，C-prM比NS 5（91%）或YNC（91%）方案更敏感（100%）。
Serotyping dengue virus (DENV) from suspect human specimens is crucial for developing sound epidemiological control measurements early in the transmission season and for effective patient management. We modified DENV consensus D1 (mD1) and serotype-specific TS2 (mTS2) and redesigned serotype-specific TS1 (rTS1) and TS4 (rTS4) as described previously in the conventional capsid and premembrane gene (C-prM) protocol (R. S. Lanciotti, C. H. Calisher, D. J. Gubler, G.-J. Chang, A. V. Vorndam, J. Clin. Microbiol. 30:545-551, 1992). In addition, we designed two new sets of amplimers and probes, located at nonstructural protein 5 (NS5) and the 3' noncoding region (3'NC) of DENV. The NS5 protocol utilizes two flaviviral consensus outer amplimers (mFU1 and CFD2) and four dengue virus serotype-specific TaqMan fluorogenic probes. The 3'NC protocol uses two DENV consensus amplimers, DC10418 and CDC10564. The conventional gel-based, heminested detection method was adapted for the C-prM protocol for detecting and serotyping dengue viruses. In addition, we developed the real-time SYBR green I and postamplification melting temperature curve analysis for the mD1/TS and YNC protocols using identical amplification conditions. The NS5 amplimer/probe set was formulated as a one-tube, multiplex, real-time reverse transcriptase PCR for serotype identification. Three sets of amplimers and probes were verified for their specificity in tests with yellow fever, Japanese encephalitis, St. Louis encephalitis, and West Nile viruses; optimized against 109 DENV strains; and validated for detection of the virus in sera from two different panels of acute-phase human dengue serum specimens and one panel of virus isolates from dengue patients' serum specimens. Clinical evaluation by two separate laboratories indicated that the C-prM was more sensitive (100%) than the NS5 (91%) or the YNC (91%) protocol.