Development of a duplex real-time RT-PCR for the simultaneous detection and differentiation of Theiler’s murine encephalomyelitis virus and rat theilovirus

Abstract

Theiler's murine encephalomyelitis virus (TMEV) and rat theilovirus (RTV), the member of the genus Cardiovirus, are widespread in laboratory mice and rats, and are potential contaminants of biological materials. Cardioviruses infection may cause serious complications in biomedical research. To improve the efficiency of routine screening for Cardioviruses infection, a duplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection and differentiation of TMEV and RTV. The duplex assay was specific for reference strains of TMEV and RTV, and no cross-reaction was found with seven other rodent viruses. The limits of detection of both TMEV and RTV were 4×10(1) copies RNA/reaction. Reproducibility was estimated using standard dilutions, with coefficients of variation <3.1%. 439 clinical samples were evaluated by both duplex real-time RT-PCR and conventional RT-PCR. For 439 clinical samples，95 samples were positive for TMEV and 72 samples were positive for RTV using duplex real-time RT-PCR approach, whereas only 77 samples were positive for TMEV and 66 samples were positive for RTV when conventional RT-PCR was applied. Mixed infections were found in 20 samples when analyzed by conventional RT-PCR whereas 30 samples were found to be mixed infection when duplex real-time RT-PCR was applied. This duplex assay provides a useful tool for routine health monitoring and screening of contaminated biological materials of these two viruses.

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

“…Forty eight frozen clinical samples (16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens) from previous experiment stored at − 80°C at our laboratory were subjected to the RT-RPA assay [36]. Homogenates of the tissues were produced with Dulbecco's Modified Eagle Medium (DMEM).…”

Establishment of a Real-Time Recombinase Polymerase Amplification Assay for the Detection of Avian Reovirus

“…The transcribed RNA concentration was determined using Nano2000 (GE, USA). The RNA copy numbers were calculated as the method previously reported (26).…”

Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

“…These limitations can be overcome by using multiplex TaqMan real-time PCR assay, which enables the simultaneous detection and identification of several viruses in a single reaction [14]. Multiplex TaqMan real-time PCR has been applied widely for pathogen detection in humans [14,[24][25][26][27] and animals [10,[28][29][30][31][32][33][34][35]. Recently, this method has also been used to detect viruses in sweet potatoes, pome fruit trees, stone fruit trees, and fig trees [36][37][38][39][40].…”