Method Description

The mumps virus laboratory-developed (LDT) real-time polymerase chain reaction (RT-PCR) assay is designed for the qualitative detect of mumps virus RNA from urine and buccal swabs of patients with suspected infection. Mumps virus RNA in clinical specimens is first extracted using the NucliSENS easyMag/EMAG (bioMerieux) instruments according to manufacturer instructions. As a component of extraction, a lysis buffer is first added to clinical specimens in a class II biosafety cabinet (BSC). At this step, any mumps virus that may be present in the sample is inactivated, rendering it non-infectious. Following the addition of lysis buffer, specimens are safe to remove from the BSC and placed onto an instrument for automated extraction. A sample input of 200 mcL will be extracted with an elution volume of 50 mcL.

This assay employs a reverse transcription reaction to convert RNA to complementary DNA (cDNA). Oligonucleotide forward and reverse primers specific to the matrix protein (M) gene region of the mumps virus amplify the target sequence. A TaqMan probe labeled with the fluorophore FAM and specific to the target region of mumps virus RNA bind to amplified mumps RNA virus product. Ribonuclease P (RNase P) is used as an internal control. Oligonucleotide forward and reverse primers specific to the p30 subunit of RNase P amplify the internal control target sequence. A TaqMan probe labeled with fluorophore Cy5 and specific to RNase P bind to the amplified RNase P product. The dye-labeled TaqMan probes allow for the detection of the target and internal control in the corresponding channel of the Roche LightCycler 480 II (LC480) instrument. Detection of the target M gene region indicates the presence of mumps virus RNA in the specimen. The clinical validity of RT-PCR for the detection of mumps virus RNA in urine and buccal swabs as well as the highly conserved nature of the mumps M gene target is documented in peer-reviewed literature.(Unpublished Mayo method)