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

Abstract

The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter-and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques.

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

“…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. It should also be noted that the study by Krause et al (19) reported C T values in the range of 35.8 to 43.7, which is marginally higher than that from the SH gene assay in our study (C T range, 31.6 to 39.6); however, direct comparison is problematic in the absence of a side-by-side evaluation of the available real-time methodologies for MuV detection.…”

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).…”

Detection of RNA of Mumps Virus during an Outbreak in a Population with a High Level of Measles, Mumps, and Rubella Vaccine Coverage

“…Previous studies have shown that reverse transcription-PCR (RT-PCR) is at least as sensitive as virus isolation for detecting mumps virus (2,4,9,12,13). Numerous samples were submitted to CDC by multiple state laboratories during the mumps outbreak of 2006.…”

“…All of the samples that were positive for mumps isolation at CDC were also positive when tested by RT-PCR, while mumps virus was not isolated from PCR-negative samples from clinically compatible cases (CDC, unpublished data). Therefore, we attempted to detect mumps viral RNA in the samples from the Kansas study by using a real-time RT-PCR that can reliably detect as few as 10 copies of mumps virus SH gene RNA (2). For the mumps primers and probes, a threshold cycle (C T ) of Ͻ38 was considered positive, a C T of Ն38 and Յ40 was considered equivocal, and a C T of Ͼ40 was considered negative.…”