Establishment And Preliminary Application Of A Double Droplet Digital Polymerase Chain Reaction For Rabbit Haemorrhagic Disease Virus

Rabbit hemorrhagic disease virus(RHDV)can cause severe infectious diseases in rabbits.There are two pathogenic genotypes of RHDV,namely RHDV GI.1 and RHDV GI.2.It is difficult to isolate the RHDV virus and no cell line with stable proliferation in vitro has been found.Therefore,rapid screening of a large number of samples and distinguishing different genotypes are still difficult in the established detection methods.In this study,Droplet digital PCR(ddPCR)was used to establish a quantitative,sensitive and rapid detection method for RHDV,especially for samples containing RHDV GI.1 and GI.2molecules,especially for low concentrations of RHDV.In this study,according to the VP60 gene sequences of RHDV GI.1 and GI.2,specific primers were designed to amplify the conserved fragments of RHDV GI.1 and RHDV GI.2VP60 gene of clinical screening,respectively,and the recombinant plasmids were successfully constructed.They were named p UC57-GI.1 and p UC57-GI.2,respectively.The concentration was determined by nucleic acid protein analyzer,and the copy number was calculated by formula.Using the online design software,the specific primer and probe sets of RHDV GI.1 F/R/P and RHDV GI.2 F/R/P were designed for the conserved fragments of the two genotypes.The detection methods of RHDV GI.1 and GI.2 ddPCR were established respectively.After the reaction system and program of single method were optimized with plasmid standard as template,the detection method of RHDV double droplet digital PCR was established and the effect was tested,the details are as follows:The reaction conditions of RHDV dual dd PCR established in this study were annealing temperature 56.5℃,final primer and probe concentrations of 500 nM and 250 nM,respectively,and cooling rate of 1.5℃/s.The sensitivity of this method was high,and the sensitivity of this method was 100%for 2.86×10~0-2.86×10~4 copies/μL of two recombinant plasmid standards.The correlation coefficients of the established standard curves were 0.9993 and 0.9992,respectively.The inter-assay and intra-assay repeated tests showed that the coefficients of variation were 9.26%and 8.90%,respectively.The data dispersion was small,and the reproducibility was good.This method was used to detect European hare syndrome virus,Pasteurella multocida rabbit and Salmonella enteritidis.It was found that this method could specifically identify RHDV GI.1 and GI.2,and had no cross-reaction with other pathogens.Both this method and qRT-PCR can detect 10~1 copies/μL or more copies,but the reproducibility of qRT-PCR is not high for 10~1 copies/μl samples,while ddPCR can detect all 30 replicates.Subsequently,329 clinical samples were tested by this method,which could clearly distinguish the GI.2 positive samples from the immune rabbit blood samples with low copy concentration.Genetic sequencing analysis of 24 RHDV GI.2 positive samples showed that one strain had close homology with the strains isolated in Sichuan,China in 2020,and the other strain had homology with the strains isolated in Canada and the United States in 2020.The clinical symptoms of RHDV GI.1 and GI.2 genotypes were similar,The dual detection method established in this study can achieve efficient differential diagnosis of RHDV strains of GI.1 and GI.2 genotypes at the same time,save experimental cost,and have high specificity,sensitivity and accuracy.It can be used for accurate quantitative detection of low-concentration samples and suspected samples that cannot be determined by qPCR,and has a wide application prospect in practical use.