Simple and Rapid Colorimetric Detection of Canine Parainfluenza Virus 5 (Orthorubulavirus mammalis) Using a Reverse-Transcription Loop-Mediated Isothermal Amplification Assay

Despite its many advantages, a reverse-transcription loop-mediated isothermal amplifica-tion (RT-LAMP) assay has yet to be developed for canine parainfluenza virus 5 (CPIV5). In this study, a visual RT-LAMP (vRT-LAMP) assay was developed for the rapid detection of CPIV5 in clinical samples. At a constant reaction temperature of 62 C-?, the assay was completed within 40 min, and the results could be directly detected with the naked eye using a hydroxynaphthol blue (HNB) metal indicator without any additional detection apparatuses. The assay specifically amplified CPIV5 RNA with a limit of detection of 10 RNA copies/reaction, which was 10-fold more sensitive than the previ-ously reported conventional reverse-transcription polymerase chain reaction (cRT-PCR) assay and was comparable to the previously reported real-time RT-PCR (qRT-PCR) assay. In a clinical evaluation using 267 nasopharyngeal swab samples collected from hospitalized dogs with respiratory symptoms, the CPIV5 detection rate using the vRT-LAMP assay was 5.24% (14/267), which was higher than that of the cRT-PCR assay (4.49%, 12/267) and consistent with that of the qRT-PCR assay, demonstrating 100% concordance with a kappa coefficient value (95% confidence interval) of 1 (1.00-1.00). The discrepancies in the results of the assays were confirmed to be attributed to the low sensitivity of the cRT-PCR assay. Owing to the advantages of a high specificity, rapidity, and simplicity, the developed vRT-LAMP assay using an HNB metal indicator will be a valuable diagnostic tool for the detection of CPIV5 in canine clinical samples, even in resource-limited laboratories.

Automated summary

A visual RT-LAMP assay using a hydroxynaphthol blue (HNB) metal indicator was developed for the rapid detection of canine parainfluenza virus 5 (CPIV5) in clinical samples. The assay showed high specificity and sensitivity, and the results can be directly observed with the naked eye without any additional detection apparatuses. The detection rate of CPIV5 using this assay was higher than that of conventional reverse-transcription polymerase chain reaction (cRT-PCR) assay and comparable to real-time RT-PCR (qRT-PCR) assay.