10.22376/ijpbs.2019.10.1.p1-12 Volume 11 Issue 2 2020 (April-June) Canine distemper virus (CDV) is a critical etiological agent responsible for distemper disease in dogs and many other carnivores. Clinical diagnosis of CDV is challenging due to the wide range of symptoms that resemble other respiratory and enteric diseases of dogs. Moreover, detection of CDV is difficult, due to genetic variation from strain to strain. Thus, laboratory-specific confirmation is needed. The current study has developed a molecular-based detection platform for CDV using highly conserved L gene sequences. In this study, multiple sequence alignment was carried out using various reported CDV sequences retrieved from GenBank and a highly conserved region from the L gene sequence was chosen for designing the specific primers. Further, RNA was isolated from various suspected samples and converted to cDNA employing reverse transcriptase PCR (RT-PCR). The converted cDNA was further used as a target for PCR amplification using highly specific primers in standardized PCR conditions. The specificity and sensitivity of the developed assay was evaluated employing related viruses and other bacteria. The assay was highly specific and can detect 10 ng/µL of RNA. Further, the applicability of the developed platform in clinical samples was validated using blood samples, ocular and nasal swabs from suspected dogs and compared with commercially available kit and plausible results were obtained. Altogether, the assay will be a suitable molecular platform for detection of CDV for routine laboratory testing. Soniya H and Bhairab Mondal Canine, Canine distemper virus (CDV), Negative-sense RNA, L gene, RT-PCR 77-83