Development and validation of triplex quantitative real-time PCR assay for detection of human rhinovirus, human metapneumovirus and human bocavirus

OBJECTIVE  To develop a triplex quantitative real-time polymerase chain reaction （PCR） assay for the detection of human rhinovirus （HRV）, human metapneumovirus （hMPV） and human bocavirus （BoV）, and to preliminarily test nasopharyngeal swab specimens from patients with acute respiratory infections with this assay. The specificity, sensitivity and repeatability of the method were validated by comparison with sequencing results.

METHODS Specific primers and fluorescent probes were designed based on the sequences of HRV, hMPV and BoV. PCR reaction conditions were developed and optimized, and evaluations were conducted on its sensitivity, specificity, repeatability and clinical applicability.

RESULTS The optimal primer concentrations for HRV, hMPV and BoV were 400, 400 and 600 nM, respectively. The optimal probe concentrations were 200, 300 and 300 nM, respectively. This assay achieved optimal amplification under the following conditions: reverse transcription at 45 ℃ for 10 min, initial denaturation at 95 ℃ for 5 min, and 45 cycles of 95 ℃ for 15 s and 60 ℃ for 45 s. The detection sensitivity reached 10³ copies/ml, with a linear range of 10³–10⁹ copies/ml. The repeatability results showed a coefficient of variation （CV） ≤ 5%, with no cross-reactivity with other respiratory viruses, indicating good specificity. A total of 233 nasopharyngeal swab specimens from patients with acute respiratory infections at the clinical laboratory of the Sixth Affiliated Hospital of South China University of Technology were tested with this assay. These specimens were also subjected to sequencing, and the concordance rate between the two methods was 97.85%.

CONCLUSION The triplex quantitative real-time PCR assay for HRV, hMPV and BoV developed in this study exhibits high sensitivity and good specificity.