| Porcine Astrovirus(PAstV)is a single-stranded,positive-stranded RNA virus,resulting in viral diarrhea in pigs.PAstVs are worldwidely prevalent in pig populations,of which five PAstV genotypes are reported to be circulating in Chinese swine population.Studies reported that porcine astrovirus type 3(PAstV 3)can not only cause diarrhea in pigs,but also is one of the organisms inducing porcine encephalomyelitis.Meanwhile,little is known about co-infection with PAstV.The clinical manifestations of the disease can range from asymptomatic to encephalitis depending on host and virus genotype.However,due to frequent recombination events between strains,PAstV has a high degree of genetic variability and can be transmitted between different host species.Although the pattern and process of this transmission is unclear,there is growing evidence that recombination events lead to transboundary transmission from original host to others.This study aimed to establish a diagnostic method of PAstV type 3 TaqMan fluorescence quantitative PCR and a diagnostic method of PAstV multiplex RT-PCR,and apply it to determine the current epidemiological status of porcine astrovirus in Guangxi Province.The detailed results are as follows:1.Establishment and preliminary application of TaqMan fluorescent quantitative PCR method for porcine astrovirus type 3To establish a specific,sensitive and rapid PAstV 3 detection method and apply it to clinical detection,specific primers and TaqMan probes were designed for the conserved region of ORF1 b according to the PAstV 3 reference sequence retrieved from Gen Bank.After repeated conditions and reaction system optimization,the sensitivity and specificity of the established real-time fluorescent quantitative RT-PCR method were determined and applied to the detection of clinical fecal samples.The results show that the standard curve of this method presents a good linear relationship in which the R~2 value can reach0.997.In addition,there is no cross-reactions with many common swine disease viruses,indicating the analytical specificity is good.Compared with conventional PCR,the analytical sensitivity of this detection method is 100 times higher than that of conventional PCR method,and the sensitivity is high.Thirty fecal samples suspected of PAstV 3 were tested by this method,and the test prevalence was 40%,which is higher than the conventional PCR method.2.Establishment of porcine astrovirus multiplex RT-PCR methodCurrently,the diagnostic method for porcine astrovirus is mainly based on the reverse transcription-polymerase chain reaction(RT-PCR).To identify five different genotypes of PAstVs,a multiplex RT-PCR method based on PAstV type 1 ORF 2 gene and PAstV type 2-5 ORF1 ab gene was established in this study.By optimizing the sensitivity and specificity of multiplex RT-PCR detection,a multiplex RT-PCR detection method was established.A total of 275 fecal samples were collected from various regions in Guangxi,China between April 2019 and November 2020,and were subsequently tested using the newly established multiplex RT-PCR.The results show that the test prevalence of PAstV 1-5 genotypes are 11.2%(31/275),17.8%(49/275),13.1%(36/275),14.9%(41/275)and 8%(22/275),respectively,and there are also double infections 0.72%(2/275)as well as even triple infections0.36%(1/275).The test results show that the method can detect clinical samples rapidly,sensitively and accurately,indicating that the multiplex PCR method established in this study has high specificity,sensitivity and good clinical application value.Using the established diagnostic methods to investigate the prevalence of porcine astrovirus in Guangxi,it provides epidemiological information for the prevention and control of related diseases in the future. |
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