期刊
JOURNAL OF VIROLOGICAL METHODS
卷 128, 期 1-2, 页码 79-87出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jviromet.2005.03.020
关键词
SYBR green; gene specific primer; measles virus
A sensitive and specific RT-QPCR based on real-time analysis of PCR products stained with SYBR green, was designed and carefully optimised to quantify individual measles virus RNA species. Pairs of specific primers were designed to detect N, P, M, F, H, or L sequences. To detect the genome and/or antigenome, two primers were chosen so as to amplify a 221 nt fragment (L-Tr) encompassing L gene end and trailer. Every gene-specific PCR assay was able to detect = 10 copies/sample, with a dynamic range of 4-5 log(10) copies. No significant fluorescent signal was detected from non-infected cell cDNA template. When measles virus microccocal nuclease resistant genomic RNA was reverse transcribed, a 1:1 ratio was observed between single gene amplicons except for L-Tr which displayed a 2.6-fold excess over the other genes. This likely reflects the presence of some shorter abortive genome since the use of a plasmid encoding the entire virus genome resulted in 1:1 ratio for L-Tr segment when compared to others amplicons. Thus, this RT-QPCR assay appears suitable for follow-up studies of viral RNA populations during infection and may also be useful for reliable detection of measles virus in clinical samples. (c) 2005 Elsevier B.V. All rights reserved.
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