A TaqMan probe-based real-time PCR to differentiate porcine epidemic diarrhea virus virulent strains from attenuated vaccine strains

Publication date: Available online 17 April 2019Source: Molecular and Cellular ProbesAuthor(s): Jing Liu, Li-min Li, Jiu-qaun Han, Tai-ran Sun, Xue Zhao, Rui-tao Xu, Qin-ye SongAbstractPorcine epidemic diarrhea virus (PEDV) is an important pathogen causing severe watery diarrhea, vomiting, dehydration, and death in sucking piglets. Attenuated vaccines have been used widely in sows in order to protect piglets through passive lactogenic immunity. Rapid and sensitive detection methods for differentiating attenuated vaccine strains from virulent ones are essential and practical in PEDV prevention and control. Based on the deletion mutation in ORF3 gene sequence, a TaqMan probe-based real-time quantitative PCR (TaqMan qPCR) was developed to distinguish PEDV virulent strains from attenuated vaccine ones in this study. The TaqMan qPCR could specifically detect PEDV virulent strain but not attenuated vaccine strain and other viruses. At least 37 DNA copies and PEDV of 0.995 TCID50 could be detected by TaqMan qPCR. The reproducibility was evaluated using various dilution of plasmids carrying PEDV ORF3 gene and virulent PEDV, and the inter-assay coefficient of variation (CV) was less than 0.44%. The TaqMan qPCR was further applied to detect 38 samples including intestines and their contents, fecal swabs, and mesenteric lymph nodes. Meanwhile, indirect immunofluorescence assay (IFA) was employed to detect PEDV-specific antigen. PEDV positive rates were 31.58% (12/38) and 26.32% (10/38) ...
Source: Molecular and Cellular Probes - Category: Molecular Biology Source Type: research