Development of Taqman-Based Real-Time RT-PCR Assay Based on N Gene for The Quantitative Detection of Feline Morbillivirus

Abstract

Background: Feline morbillivirus (FeMV) is a member of genus Morbillivirus which has been associated with the chronic kidney disease in cats even though a definite relationship is still unclear. Morbilliviruses are associated with severe diseases such as Peste des petits ruminants, canine distemper and measles. FeMV has been detected in many countries including Malaysia. This study aims to develop a Taqman real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.Results: A specific assay was developed since no amplification was observed in viral strains from the same Paramyxoviridae family, such as canine distemper virus (CDV), Newcastle disease virus (NDV) and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 x 104 copies/L with Cq value of 34.32 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34% - 0.53% and 1.38% - 2.03%, respectively. Besides that, the clinical sample evaluation using this assay showed a higher detection rate, with 26 (37%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.Conclusions: The Taqman-based real-time RT PCR assay targeting the N gene described here is more sensitive, specific, rapid and reproducible compared to the conventional RT-PCR assay targeting the N gene and it could be used to detect early infection in cats.