Rapid and quantitative detection of mumps virus RNA by one-step real-time RT-PCR.
We developed a new TaqMan-based one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for detection and quantification of mumps virus RNA. Oligos targeting the matrix protein gene of mumps virus were designed by using our oligo designing and analyzing software, Oligoware 1.0. Oligos's specificity was tested with 5 strains (4 laboratory isolated and 1 Jeryl Lynn strain) of mumps virus. The suggested TaqMan-based one-step real-time RT-PCR assay correctly detected the 4 laboratory-isolated strains and 1 Jeryl Lynn strain. To confirm the specificity of the TaqMan PCR assay, parainfluenza type 1, 2, 3 strains, sendai virus, and measles virus (vaccine strain) were tested, and no cross-reactivity was observed between mumps and tested strains. In addition, a BLAST (NCBI) search showed no genomic cross-reactivity with other viruses or cells. Testing of the assay's reproducibility was repeated several times, and the same results were achieved. The new assay was able to quantify the concentrations of mumps virus gene ranging from 10(1) to 10(8) copies per reaction sensitively with generated plasmid standards. In addition, it was shown that a significant correlation (R2 = 0.9564) between genome number as determined by one-step real-time RT-PCR and the corresponding number of plaque in paired samples was found with regression analysis. The results of one-step real-time RT-PCR assay also corresponded well to those of nested PCR. We conclude that our one-step real-time RT-PCR assay is a reliable, specific, and sensitive tool for the diagnosis of mumps virus. We consider that these results come from highly conserved primers and probe set that were designed with Oligoware 1.0.
Kubar
Ayhan
A
Division of Virology, Department of Microbiology and Clinical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey. akubar@hotmail.com
Yapar
Mehmet
M
Besirbellioglu
Bulent
B
Avci
I Yasar
IY
Guney
Cakir
C
D001483:Base Sequence; D021121:Branched DNA Signal Amplification Assay / Q000379:methods; D005069:Evaluation Studies as Topic; D006801:Humans; D008969:Molecular Sequence Data; D009107:Mumps / Q000175:diagnosis; D009109:Mumps virus / Q000302:isolation & purification*; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012494:Sampling Studies; D012680:Sensitivity and Specificity
D012367:RNA, Viral