Establishment Of Fluorescence Quantitative RT-PCR Method For Detection Of Porcine Kobuvirus And Its Application In Clinical Detection

Porcine kobuvirus(PKV)is a member of the genus Kobuvirus and family Picornaviridae.The widespread porcine enterovirus known as PKV is prevalent on farms all over the world.PKV can be detected in healthy and diarrheal pigs.Porcine kobuvirus may be linked to pig diarrhea,according to studies that reveal the detection rate of PKV in diarrheal pigs is much greater than that in healthy pigs and that PKV is frequently combined with other porcine enteroviruses in diarrheal pigs.However,research on porcine kobuvirus is few,with the majority of studies concentrating on epidemiological studies.In this study,a fluorescent quantitative RT-PCR detection technique for porcine kobuvirus was established using primers and probes built in the 3D area of PKV.The samples of diarrhea piglets in some parts of China were collected to detect PKV and several other common porcine enteroviruses.The infection rate of PKV in diarrhea piglets and healthy piglets,the mixed infection of PKV and other porcine enteroviruses in diarrhea piglets,and the infection of PKV in different seasons and different regions were compared.Main research contents are as follows:1.Establishment of PKV fluorescence quantitative RT-PCR detection methodPrimers and probes were designed according to the 3D conserved region of PKV,and the specificity of the primers was preliminarily verified by BLAST tool.Optimize the primer probe reaction concentration and annealing temperature to determine the best reaction system.In the range of 3.19×10~7～3.19×10~1copies/μL,a standard curve was established.The linear relationship expression was y=-3.2388x+36.004,the correlation coefficient was 0.9992,E(Eff%)=103.7%,with a good linear relationship.The minimum detection limit can be detected to 3.19 copies/μL.Compared with conventional RT-PCR,the establishment of PKV fluorescence quantitative RT-PCR was 100 times more sensitive than conventional RT-PCR.The positive critical value of the established PKV fluorescence quantitative detection method was 37.79 determined by ROC curve.The fluorescence quantitative RT-PCR established in this study can provide a technical support for the monitoring of PKV in the future.2.Infection of PKV in diarrhea piglets and healthy pigletsIn this study,166 diarrhea piglet samples and 221 healthy piglet samples were collected from March 2022 to January 2023 to detect the infection of PKV.In addition to PKV,diarrhea piglet samples also detected PEDV,Po Sa V,PDCo V,TGEV and Po RV,five common porcine enteroviruses.The detection rate of PKV in diarrhea piglets(59.04%)was much higher than that in healthy piglets(32.08%).According to different seasons,the samples of diarrhea piglets collected in different seasons were tested for PKV.It was found that the detection rate of PKV in summer(69.70%)and winter(66.10%)was higher than that in spring(48.39%)and autumn(48.84%),and piglets could be infected with PKV throughout the year.Comparing the infection of PKV in Hubei Province,Henan Province,Zhejiang Province and Anhui Province,it was found that the positive rate of PKV was more than 50%.The highest infection rate of PKV was 75.00%(12/16)in Anhui Province,followed by 71.43%(10/14)in Zhejiang Province,63.79%(37/58)in Henan Province and 52.17%(24/46)in Hubei Province.PKV is widespread in these areas.Among the 166 diarrhea samples collected,only 28 samples were infected with PKV alone,while 75 samples were infected with two or more viruses,and 70 samples were PKV-related.The detection rates of PKV in PEDV,Po RV and Po Sa V positive samples were 66.7%,72.7%and 81.0%,respectively.It is indicated that PKV is often co-infected with other porcine enteroviruses in diarrhea piglet samples,while PKV infection alone is less.3.Phylogenetic analysis of porcine kobuvirus VP1 geneSix PKV positive samples were selected for VP1 amplification,and nucleotide homology comparison and phylogenetic tree analysis were performed on the VP1 gene.The results showed that the nucleotide homology between the six PKV strains sequenced in this study was between 71.5%and 88.9%,and the nucleotide homology with other PKV strains at home and abroad was between 71.1%and 88.1%.The VP1 gene evolution of PKV strains popular in the same region at different times is obvious,indicating that PKV is constantly evolving in the natural environment and among hosts.In summary,this study established a PKV fluorescence quantitative RT-PCR detection method.The detection rate of PKV in diarrhea piglets was higher than that in healthy piglets.PKV can occur throughout the year and is widespread in some parts of the country.PKV is often co-infected with other porcine enteroviruses in diarrheal piglets,and there is less infection alone.The variation of PKV VP1 gene is large,and the evolution of VP1 gene is obvious in different time epidemic strains in the same area.