Establishment And Application Of Droplet Digital PCR For Detection Of Bovine Coronavirus

Bovine coronavirus(BCo V)is one of the most important pathogens which can cause winter dysentery,calf diarrhea and respiratory diseases.At present,the prevalence of BCo V has been reported in many countries and regions in the world.The diseased animals could be discharged continuously for a period of time after being cured.BCo V has a character of well adhesion and can persist in the environment for a long time,leading to repeated infections in cattle.However,the method of existing detection for BCo V have problems of poor specificity,time-consuming,complex operation,and difficult to detect samples with low viral load.Droplet Digital PCR(dd PCR)has many advantages,such as simple operation,more sensitivity and accuracy,which can be used for absolute quantification directly without depending on standard curve.Aiming at the above problems,the development of droplet digital PCR for detection of bovine coronavirus were conducted in this experiment.1.Bovine diarrhea samples were detected,and BCo V positive samples were used to virus isolation and culture.N gene of BCo V was amplified and cloned into p GEM-T Easy vector.After gene sequencing,the N gene was blast with BCo V reference strains in the Gen Bank database,and the homology of nucleotide and amino acid was analysed for construction of genetic evolution tree.The results showed that the nucleotide homology of N gene between BCo V-p GEM-T Easy-N recombinant plasmid and 28 BCo V reference strains was 97.8 ~ 99.7%,and the homology of amino acids was 98.0% ~ 99.6%.N gene in recombinant plasmid BCo V-p GEM-T Easy-N was in the same branch as the reference strain BCo V-China-SWUN-LN2-2018 in genetic evolution analysis.Compared with other 28 BCo V reference strains,N gene has 8 conserved amino acid residues FYYLGTGP in N2 terminal.2.The specific primers and probes were designed according to N gene in recombinant plasmid BCo V-p GEM-T Easy-N and the genome sequence of BCo V in Gen Bank database.BCo V-p GEM-T Easy-N was served as plasmid standard to optimize the primer and probe concentration of Taq Man q PCR and dd PCR method.Finally,two PCR methods for BCo V detection were constructed,and their sensitivity,specificity and reproducibility were compared.A total of 216 bovine diarrhea samples were detected in parallel by Reverse transcription PCR(RT-PCR),Taq Man q PCR and dd PCR method.The results showed that the optimal concentration of primers and probes of Taq Man q PCR detection method was 400 n M and450 n M,respectively,and standard curves were constructed using optimized reaction conditions with a minimum detection limit of 10 copies/?L,and only BCo V was specifically amplified.The coefficient of variation of reproducibility test within and between groups were all less than 2% in Taq Man q PCR detection method.The optimal concentration of primers and probes for dd PCR detection method were400 n M.The minimum detection limit was 1 copies/?L.Specific amplification was found only for BCo V.The coefficient of variation of reproducibility test within and between groups were all less than 2%.Compared with Taq Man q PCR method,dd PCR method has higher sensitivity,specificity and reproducibility,and could detect gene copy number in samples without establishing a standard curve.216 samples of bovine diarrhea collected from cattle farms in different regions of Xinjiang were tested.The results showed 67 positive samples detected by RT-PCR,99 positive samples detected by Taq Man q PCR and 108 positive samples detected by dd PCR,with a positive rate of 31.02%,45.83% and 50%,respectively.In conclusion,in our study the BCo V were isolated and cultured from positive samples,and the similarity and genetic evolution were analyzed based on sequencing analysis.The Taq Man q PCR and dd PCR detection methods for BCo V were established.dd PCR method was 10 times more sensitive than Taq Man q PCR method,with good specificity and reproducibility.Two PCR methods were used to detect clinical samples,and dd PCR method increased the positive detection rate by 4.17%.In this study,a more concise,rapid and sensitive molecular biological detection method for BCo V was established,which provided technical support for the differential diagnosis of bovine coronavirus disease.