Abstract
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 µl 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. J. Med. Virol. 79:1761–1767, 2007. © 2007 Wiley‐Liss, Inc.