Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCR

Abstract

A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis.

Molecular Epidemiological Evaluation of the Recent Resurgence in Mumps Virus Infections in Ireland

“…MuV RNA was detected in ϳ44% of CSF specimens tested, and no herpesvirus DNA (HSV-1, HSV-2, VZV, or HHV-6) or enterovirus RNA was found in these specimens from patients with neurological symptoms. A large study from the United Kingdom (19) analyzed 88 CSF specimens and detected five cases of meningitis (5.7%) by using the real-time method of Uchida and coworkers (26) which targets the highly conserved F gene in the MuV genome. The assay employed in the present study targets the SH gene (4), and the MuV CSF positivity of 44% compares well to the findings described above.…”

An assessment of mumps vaccine effectiveness by dose during an outbreak in Canada

“…Respiratory tract, urine and cerebrospinal fluid samples were tested by viral culture and inhouse real-time reverse-transcriptase polymerase chain reaction adapted from previously published methods. 7,8 Confirmed cases were designated as confirmed in iPHIS or cases satisfying the outbreak case definition (Box 1). 9 Case and coverage data by dose were used to calculate vaccine effectiveness using the screening method (Box 2).…”

Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens

“…The primer and probe sets were also tested under the same cycling conditions using reagents within the TaqMan PCR core reagents kit (Applied Biosystems), as follows: 5.5 mM MgCl 2 , 300 M dATP, 300 M dCTP, 300 M dGTP, 600 M dUTP, 0.025 U/l AmpliTaq Gold, plus 0.25 U/l MultiScribe reverse transcriptase and 0.4 U/l RNase inhibitor (Applied Biosystems). The real-time PCR primers F1073 and R1151 and the probe developed by Uchida et al were used for the detection of RNA from the mumps F gene (22). Optimization of RT-PCR was performed by various primer and probe concentrations and PCR cycling conditions to obtain a one-step real-time RT-PCR with the TaqMan One-Step RT-PCR Master Mix reagents kit (Applied Biosystems, Foster City, CA) in a 25-l total volume, using 5 l of patient specimen nucleic acid.…”

“…We compared the RT-PCR assay described here to a previously published realtime assay performed by Uchida and colleagues that targeted the F gene of mumps virus in a two-step real-time RT-PCR (22). We chose the method of Uchida et al for comparative studies because, at the time we initiated this study, it was the only published real-time RT-PCR assay that had been tested with patient specimens.…”