Establishment of Multiplex RT-PCR for Differentiating between the CD1901 and Lederle Strains of Canine Distemper Virus

Abstract

license/by-nc/3.0/). Canine distemper virus (CDV) is a life-threatening pathogen in dogs. Clinical, pathological and molecular methods are required to diagnose CDV infection, and it is important to differentiate between the Korean CDV strain CD1901 and Lederle CDV vaccine strain. Therefore, in this study, we used multiplex reverse-transcription polymerase chain reaction (RT-PCR) to differentiate between the CD1901 and Lederle strains. A primer set was designed based on the CDV nucleoprotein gene and nucleotide sequence variation in the fusion (F) gene. First, 224-bp DNA bands were amplified from viral RNA of the CD1901 and Lederle strains. Then, 428- and 326-bp DNA bands were amplified in the CD1901 and Lederle strain, respectively. The multiplex RT-PCR detection limits were 2.53 and 0.8 median tissue culture infectious dose/reaction for the CD1901 and Lederle strains, respectively. No cross-reactions were detected in non-CDV reference viruses, including rabies virus, parvovirus, canine adenovirus types 1 and 2, and parainfluenza virus. The results indicate that our one-step multiplex RT-PCR is useful for differentiating between wildtype and vaccine CDV distemper. The CD1901 and Lederle strains were propagated in Vero/dSLAM cells expressing the dog SLAM gene in Dulbecco’s modified Eagle’s medium containing two antibiotics, an antifungal agent, and 10% heat-inactivated fetal bovine serum (Gibco BRL, Grand Island, NY, USA). The CD1901 and Lederle strains were used as positive controls for multiplex RT-PCR, and the analytical sensitivity and analytical specificity of the primers were determined. Four commercial distemper/adeno/parvo/parainfluenza (DAPP) vaccines containing CDV, canine adenovirus type 1 (CAV-1) or canine adenovirus type 2 (CAV-2), canine parvovirus (CPV), and canine parainfluenza virus (CPIV) manufactured by Korean biological companies were used for multiplex showed high sensitivity and specificity for differentiating between