Development and application of multiplex PCR assays for detection of virus-induced respiratory disease complex in dogs

PIEWBANG, Chutchai; RUNGSIPIPAT, Anudep; POOVORAWAN, Yong; TECHANGAMSUWAN, Somporn

The CIRDC is an important disease that impacts on dogs, especially puppies or immunosuppressed dogs, and is frequently associated with viral infections. It has gained attention recently, because many viruses have been discovered and co-infections with multiple pathogens are often fatal. Thus, the development of diagnostic tools for CIRDC-associated virus detection is necessary to enhance the diagnosis coverage. In this study, multiplex RT-PCR and multiplex PCR for the detection of CIRDC-associated RNA and DNA viruses, respectively, were developed and compared with conventional methods. Both developed multiplex PCRs could detect several viruses associated with CIRDC efficiently. The two multiplex PCRs gave similar results equivalent to that obtained from the conventional simplex PCRs that could only detect one pathogen per reaction and so required six separate reactions per sample. Nested amplification was performed for CRCoV detection in order to increase the sensitivity of detection (Poovorawan, personal communication). Although multiplex PCR has been developed previously to detect several pathogens of CIRDC, such as CIV, CDV and CRCoV [10], its application remained limited because of the narrow range of viruses covered, with other CIRDC-associated viruses being neither detected nor ruled out. Thus, our study might provide a novel platform for whole CIRDC-virus detection. The overall sensitivity of the multiplex RT-PCR and multiplex PCR was more than 90% and 87%, respectively, compared to their simplex counterparts. However, the detection of CRCoV was modified as a hemi-nested RT-PCR to increase its sensitivity. The false negative reactions when performing multiplex PCRs in this study might be resulted from the selection of the single optimized Ta for several primer pairs and the low amount of particular target genes [1] . These suggested for the decreased sensitivity of the developed multiplex PCRs. Moreover, there was 100% specificity in both modalities for clinical sample detection. Thus, these platforms could likely be used effectively in practice. Recently, some multiplex PCR assays were developed in order to detect the CIRDC pathogens [10]; however, the test might be immature, because only CIV, CDV and CRCoV could be detected but not for others. Thus, our study expanded the coverage of CIRDC virus detection. In an evaluation of the commercially available three-antigen rapid test kit (CAdV-2, CIV and CDV), we found only CIV detection showed an unexpected sensitivity and specificity. A previous study reported that the developed multiplex RT-PCR for H3N2 CIV, CDV and CRCoV detection had an almost 100% sensitivity and specificity compared with the conventional RT-PCR and rapid antigen test kit [10] . In contrast, our study showed that the CIV-positive samples by multiplex RT-PCR were negative when tested with the rapid antigen test kit. This is consistent with reports that many rapid test kits might have a low sensitivity to detect the influenza virus, but could still be suitable for rapid in-house clinical applications [11, 15] . This reflects that the type of kit, viral copy number, duration of storage, route of sample collection, and type or virus strain may all influence the test results [19] . Interestingly, in this study, about 70% (71/102) of samples from the clinical respiratory illness dogs were found to have multiple infections. This finding supports that symptomatically, the CIRDC is a complex disease, which is mostly caus