Simultaneous Detection of Canine Respiratory Disease Associated Viruses by a Multiplex Reverse Transcription-Polymerase Chain Reaction Assay

Abstract

ABSTRACT. A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for the simultaneous detection of canine distemper virus (CDV), canine respiratory coronavirus (CRCoV) and canine influenza virus (CIV). These viral pathogens are all causative agents of canine infectious respiratory disease (CIRD). The sensitivity and specificity of the mRT-PCR were determined by comparing it to a rapid antigen test (RAT) or immuno-chromatography test kit and reverse transcription-polymerase chain reaction (RT-PCR) in the detection of CDV, CRCoV and CIV antigens present in 100 clinical samples (nasal swabs and whole blood samples) from 50 dogs with respiratory disease symptoms. This study revealed that mRT-PCR had almost exactly the same performance or results were almost 100% in agreement with that of RT-PCR and RAT both in terms of the assay sensitivity and specificity which was more highly evident in detecting CIV, CDV and CRCoV antigens present in canine nasal swab samples. Therefore, this assay could be a better alternative for the definitive and simultaneous ante-mortem detection of the three viral pathogens that cause CIRD by using nasal swabs.

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

“…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.…”

“…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.…”

“…Various diagnostic tests are available for these infections. However, many are not practical due to their time-consuming process, poor specificity or sensitivity, and costly diagnostic tools [10]. Thus, a rapid molecular technique is an appropriate method of choice for CIRDC virus detection.…”

“…Thus, a rapid molecular technique is an appropriate method of choice for CIRDC virus detection. Because multiple viruses cause CIRDC, including co-infections, a multiplex polymerase chain reaction (PCR) was developed and has become commercially available as a test for respiratory tract infections [1, 10, 13, 18]. Recently, multiplex real-time PCR (qPCR) has largely replaced the conventional counterpart in order to increase the sensitivity.…”

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction

“…CDV immunoassays for detection of CDV antigen, such as immunofluorescence staining and ELISA [28][29][30][31][32], in general are not sensitive enough to detect CDV in asymptomatic dogs or during acute CD infection [31]. Various conventional, nested, and real-time reverse transcription polymerase chain reaction (RT-PCR) assays, which offer high degrees of detection sensitivity and specificity, have also been developed for CDV [33][34][35][36][37][38][39][40][41][42]. However, requiring an experienced technician and a relatively expensive thermocycler, these assays have been applied to CDV diagnosis mostly in professional laboratories.…”