Real-time PCR and its application to mumps rapid diagnosis.
A real-time polymerase chain reaction assay was initially developed in China to detect mumps genome. The primers and TaqMan-MGB probe were selected from regions of the hemagglutinin gene of mumps virus. The primers and probe for the real-time PCR were evaluated by both laboratories in China and in the UK using three different pieces of equipment, LightCycler (Roche), MJ DNA Engine Option 2 (BIO-RAD) and TaqMan (ABI Prism) on different samples. The reaction was performed with either a one-step (China) or two-step (UK) process. The sensitivity (10 copies) was estimated using a serial dilution of constructed mumps-plasmid DNA and a linear standard curve was obtained between 10 and 10(7) DNA copies/reaction, which can be used to quantify viral loads. The detection limit on cell culture-grown virus was approximately 2 pfu/ml with a two-step assay on TaqMan, which was equivalent to the sensitivity of the nested PCR routinely used in the UK. The specificity was proved by testing a range of respiratory viruses and several genotypes of mumps strains. The concentration of primers and probe is 22 pmol and 6.25 or 7 pmol respectively for a 25 microl reaction. The assay took 3 hr from viral RNA extraction to complete the detection using any of the three pieces of equipment. Three hundred forty-one (35 in China and 306 in the UK) clinical specimens were tested, the results showing that this real-time PCR assay is suitable for rapid and accurate detection of mumps virus RNA in various types of clinical specimens.
Jin
L
L
Virus Reference Department, Centre for Infections, Health Protection Agency, London, UK. li.jin@hpa.org.uk
Feng
Y
Y
Parry
R
R
Cui
A
A
Lu
Y
Y
D002681:China; D017931:DNA Primers; D004196:Disease Outbreaks; D006801:Humans; D009107:Mumps / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D009109:Mumps virus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D006113:United Kingdom
D017931:DNA Primers; D012367:RNA, Viral