Establishment And Application Of Rapid Diagnosis Method For RPA Of Porcine Kobuvirus

Porcine kobuvirus(PKV)is a common enterovirus that causes diarrhea in pigs.Infected herds focus on pigs,especially the piglets which were less than one week old.Generally,clinical symptoms such as apathy,watery diarrhea,and severe dehydration and even death were occurred in these infected swine.At present,the clinical diagnosis of PKV mainly depends on the RT-PCR method.However,the RT-PCR method was limited for further use owing to several cause,such as the low sensitivity,in addition,it can't be accurately detected in some samples with low levels of toxicity,as a result,it was often missing detected.Therefore,it is very important to establish a clinical diagnosis method with higher sensitivity,specificity,and can be rapid detected rapidly.In this study,based on the basic RPA and LF-RPA,and the combination of with agarose gel electrophoresis as well as and lateral flow immunoassay,a rapid clinical detection method for PKV was established,aiming at providing a new technique for the clinical diagnosis and rapid detection of PKV in the future.The specific test results are as follows:1.Establishment of a rapid diagnosis method for Basic RPA of Porcine KobuvirusSpecific primers were designed and synthesized according to the genomic sequence of Porcine Kobuvirus(PKV)published in GenBank,and positive plasmid standards were constructed.Positive recombinants were used as templates to establish rapid detection methods,namley,recombinase polymerase amplification(RPA),for detecting PKV.Subsequently,the sensitivity,specificity and reproducibility of this method were evaluated.Results showed that no specific amplification band was detected when porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine delta coronavirus(PDCoV),swine foot-and-mouth disease virus(FMDV),and porcine reproductive and respiratory syndrome virus(PRRSV)was used as template following this method,However,specific bands could be amplifiable when repeated experiments with different concentrations of plasmid standards were performed.The minimum detectable limit was 3.36×10~2copies/?L.The above results show that this study successfully established a rapid detection method,known as basic RPA with high specificity,reproducibility and sensitivity,for detecting PKV.2.Establishment of a rapid diagnostic method for LF-RPA of Porcine KobuvirusAccording to the published Porcine Kobuvirus(PKV)genomic sequence published in GenBank,LF-RPA specific primers and probes were designed and then synthesized After the positive plasmid standards were constructed,it was used as a template to establish a rapid LF-RPA detection method.Subsequently,the specificity,repeatability and sensitivity of this method were evaluated.The results showed that there were no detection bands on the test strips when TGEV,PEDV,FMDV,PRRSV,and PDCoV were used as templates,and the test results were negative,indicating that the method has good specificity.Repeated experiments with different concentrations of plasmid standards showed strips on the test strips and the test results were positive.The minimum detectable limit is 3.36×10~2copies/?L.The above results indicate that a rapid detection method for PKV LF-RPA with high specificity,reproducibility,and sensitivity was successfully established.3.Clinical application of Basic RPA and LF-RPA detection methods for Porcine kobuvirusUsing the rapid detection method established in this study,276 samples of swine fecal samples collected from Henan,Xinjiang,and Heilongjiang provinces during2016-2017 were detected,while the conventional RT-PCR methods were used for the control tests.The results showed that 140 positive samples were detected by conventional RT-PCR method,and the positive detection rate was 50.7%.162 positive samples were detected using the Basic RPA method which established in this study.The positive detection rate was 58.7%.LF-RPA 154 positive samples were detected by LF-RPA method,and the positive detection rate was 55.8%.Overall,the two detection methods established in this study are simple to operate,time-saved compared to conventional RT-PCR,making them be suitable for large-scale sample detection in clinical trails.