| 查看: 144 | 回复: 1 | |||
| 当前主题已经存档。 | |||
gaoshandian1金虫 (小有名气)
|
[交流]
Development of Real-Time Reverse Transcriptase PCR Assays
|
||
|
Li-Jung Chien,1 Tsai-Ling Liao,1 Pei-Yun Shu,1 Jyh-Hsiung Huang,1 Duane J. Gubler,2, and Gwong-Jen J. Chang2* Center for Disease Control—Taiwan, Taipei, Taiwan, Republic of China,1 Division of Vector-Borne Infectious Diseases, National Center for Infectious Disease, Centers for Disease Control and Prevention, Fort Collins, Colorado2 Received 9 September 2005/ Returned for modification 28 October 2005/ Accepted 25 January 2006 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 3'NC 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 3'NC (91%) protocol. |
回复此楼» 猜你喜欢
《中国药典》2020年版电子版分享
已经有13人回复
-
调剂求助
已经有0人回复
-
康复医学论文润色/翻译怎么收费?
已经有55人回复
-
博士后面上项目申请书生成后，最后的签字页在哪里提交？
已经有5人回复
-
求助：执业中药师视频备考资料
已经有0人回复
-
求助：执业中药师视频备考资料
已经有0人回复
-
Phytomedicine的作者同意声明
已经有0人回复
-
插文献
已经有1人回复
1
 |
2楼2006-05-17 09:04:15
