An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5

A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay’s sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID₅₀/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen’s kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90–1.03) or 1.00 (1.00–1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.