Development of a real-time RT-PCR assay for detection and quantitation of parainfluenza virus 3

A TaqMan-based real-time RT-PCR assay was developed to detect and quantify human parainfluenza virus 3 (PIV3). Two sets of primer-probe pairs were designed based on the nucleotide (nt) sequence of the nucleocapsid (N) gene. The primer-probe pairs were derived from the 3′ end of the N gene (set 1) and the 5′ region of the gene (set 2), respectively. Using real-time RT-PCR, the sensitivity of set 1 was determined to be about 9 copies of PIV3 genome, while the sensitivity of set 2 was about 93 copies of PIV3 genome. Set 1 was chosen for subsequent experiments. This primer-probe pair detected PIV3, but not any of several other respiratory viruses, indicating that the assay is PIV3 specific. For clinical evaluation, the assay was employed to test 80 nasopharyngeal aspirates from children with respiratory symptoms. The results confirmed the presence of PIV3 in 12 specimens previously identified as positive by culture confirmation, and showed all of which contained more than 100 copies of PIV3 genome. In addition, the method also detected PIV3 genomes in specimens found negative by culture confirmation, indicating the value of this RT-PCR assay. These data thus demonstrate the application of the real-time RT-PCR assay for the detection and quantification of PIV3 in clinical specimens.