Optimized SYBR green real-time PCR assay to quantify the absolute copy number of measles virus RNAs using gene specific primers.
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 log10 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.
Plumet
Sébastien
S
Immunité and Infections Virales, CNRS-Univ-Lyon1 UMR 5537, IFR Laennec, 69372 Lyon Cedex 08, France.
Gerlier
Denis
D
D000818:Animals; D052160:Benzothiazoles; D002522:Chlorocebus aethiops; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes / Q000378:metabolism; D018628:Gene Dosage; D006367:HeLa Cells; D006801:Humans; D008459:Measles virus / Q000235:genetics / Q000302:isolation & purification*; D009930:Organic Chemicals / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics*
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D014764:Viral Proteins; C098022:SYBR Green I