Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates.
Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.
Ozoemena
L C
LC
Division of Virology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertfordshire, UK.
Minor
P D
PD
Afzal
M A
MA
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D008459:Measles virus / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000138:chemical synthesis / Q000235:genetics* / Q000592:standards; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral