Establishment And Application Of Fluorescence Quantitative RT-PCR Double Detection Method For Classic Strains And Variant Strains Of PEDV

Porcine epidemic diarrhea(Procine epidemic diaherra,PED)is a common disease caused by porcine epidemic diaherra virus(PEDV)in farms.Its main characteristics include acuteness,high contact,high mortality,and it is a high-incidence viral intestinal infectious disease.The commonly used diagnostic methods at this stage include the following:immunohistochemistry,immunofluorescence,ELISA,RT-PCR,etc.These methods have certain advantages,but the disadvantages are also obvious.For example,the detection is relatively time-consuming,not sensitive,and difficult.Achieve accurate diagnosis in the early stage of onset.The application of fluorescent quantitative RT-PCR diagnostic method can carry out high-throughput quantitative detection,and can better make up for the above mentioned defects.At present,most of the epidemic strains of PEDV in China are mutant strains,which may be one of the reasons for the disadvantages of commercial vaccines in the prevention and control of PED in recent years.Distinguishing between classic strains and variant strains of PEDV is of great significance for clinical prevention and vaccination of the disease.However,due to various factors,the existing PEDV detection technology cannot meet the distinguishing requirements in this respect.Based on the sequence of the S gene,this study set the corresponding primers and probes to optimize the amplification conditions,so as to study a fluorescent quantitative RT-PCR technology that distinguishes the classic strains and variants of PEDV,and verify the method’s effectiveness.Reliability,and verify the specificity,sensitivity,and repeatability of the method.The results show that the standard curve equation of the classic strain is Ct=-3.192×log copies+38.53,the correlation coefficient R2 is-0.999,the standard curve equation of the variant strain is Ct=-3.286×log copies+39.10,and the correlation coefficient R2 is-1,000.The method has strong specificity and can distinguish PEDV from other viral nucleic acids.The minimum detectable concentration of PEDV can reach 1×10~1 copies/μL;and the method has stable repeatability.Using this method to detect 119 suspected PEDV samples,71 of them were found to be positive,with a positive rate of 59.67%.Among them,9 were classic strains,with a positive rate of 7.56%for classic strains;62 were mutants,with a positive rate of52.10%for mutants.After sequencing the amplified products of the detected 1 classic strain PED-sampleC1 and 2 variant strains PED-sampleV1/PED-sampleV2,the sequence comparison found that the classic strain PED-sampleC1 has the highest homology with the classic strain JS2008,which is 99.6%,and the homology with the mutant strain is less than95%;the homology between the mutant strain PED-sampleV1 and the mutant strain YC2014is the highest at 99.4%,and the homology between PED-sampleV2 and the mutant strain AH2012 is the highest at 99.1%,The sequence homology between the variant strain PED-sampleV1/PED-sampleV2 and the classic strain(JS2008,CV777 and LZC)is less than95%.It can be judged that the method established in this paper can distinguish between classic and variant PEDV strains.This study provides a new method for clinical rapid diagnosis of PEDV infection.