Establishment Of Multiplex PCR Assay For Detection Of Porcine Diarrhea Pathogens And Phylogenetic Analysis Of Major Viruses

Porcine sapelovirus(PSV),Porcine kobuvirus(PKV),Porcine astrovirus(PAstV),Porcine Deltacoronavirus(PDCoV),Porcine torovirus(PToV),Porcine teschvoiru(PTV)and Porcine sapovirus(PSaV)were a kind of viruses which can arose pig gastrointestinal disease and often mix infection animal.As the multiplex RT-PCR method has high sensitivity,specificity and could simultaneously detect a variety of pathogen,it is more suitable for rapid diagnosis of mixed infection.Therefore,in this study,seven pairs of specific primers were synthetized to develop the multiplex PCR method,which can detect the seven virus infection,and the method was used in the detection of clinical samples.The clinical test of seven kinds of virus in diarrhea stool analysis showed that PSV,PKV,PAstV positive rate is higher,in order to understand the evolutionary characteristics of these three kinds of virus,three strains of PSV and PKV and two strains of PAstV were tested for the whole genome sequence.1.Establishment and preliminary application of multiplex RT-PCR for detecting PSV,PKV,PAstV,PDCoV,PToV,PTV and PSaV infectionSeven pairs of specific primers were used to amplify PSV,PKV,PAstV,PDCoV,PToV,PTV and PSa V gene sequences.The size of the target fragments were 624 bp,280bp,249 bp,504bp,421 bp,176bp,340 bp.Optimizing the reaction conditions of multiple RT-PCR,the method was established for the simultaneous detection of these seven viruses.The specificity and reproducibility of the multiplex RT-PCR were very good,and the sensitivity could reach the level of pg.Then the method was used to detect 492 samples of pig diarrhea fecal samples from Shanghai,where the infection rate of PKV reached the highest level of 41.9%(206/492),and the infection rate of PAstV and PSV was the second,which was 31.1%(153/492),15.7(77/492).The detection results of statistics found that 492 samples 28.7%(141/492)had two or more virus infection,mainly PKV and PAstV;13%(64/492)of samples had three or more virus samples,mainly PKV,PAstV and PSV;3.2%(16/492)of samples had four or more virus samples,mainly PKV,PAstV,PSV and PDCoV;two(0.6%)samples had PKV,PAstV,PSV,PSaV and PDCoV mixed infection;one samples had PKV,PAstV,PSV,PSaV,PDCoV and PTV mixed infection.2.Amplification and genetic evolution analysis of PSV,PKV,PAstV genome sequencesIn view of PSV,PKV,PAstV have a higher infection rate,and in order to deeply understand the genome molecular characteristics of these three types of viruses.Three strains of PSV and PKV,2 strains of PAstV were amplified by whole genome sequence and carried on genetic evolution analysis.The sequencing results showed that three strains of PSV whole genome sequence close affinity with strain reported in domestic sequence,and with the United States,Germany,South Korea representative strains of genetic distance is far,the sequences of the VP1 gene showed that VP1 gene of the three strains isolated in Germany and Po5116 the lowest homology,and with the Chinese isolates YC2011 the highest similarity.Among the three strains of PKV whole genome homology is not high,only 88.1%-89.1%,that there is a certain variation between different regions of Shanghai epidemic PKV strains,but the phylogenetic tree of VP1 gene indicated that their affinity with the Japanese strains PKV recently,thus speculate that popular in China in recent years PKV and Japan has a certain similarity between strains PKV,probably comes from the Japanese strains;In addition,two strains of PAstV whole genome homology is only 73.8%,belong to different subsets,with different countries PAstV similarity is 40.5% ~ 77.1%,the homology is generally low,further ORF2 gene sequence analysis showed that there are some similarities between Chinese and American popular PAstV,probably comes from United States strains.