Establishment Of PCR Combined With Nucleic Acid Dot Blot For Detection Of Pseudorabies Virus And Epidemiological Investigation Of Pseudorabies In Some Pig Farms In Shandong

Pseudorabies(PR),which is caused by Pseudorabies virus(PRV),the main symptoms are fever,itching and encephalomyelitis.The virus could be transmitted by the sick pig,inapparent infection pig and infected rats,which caused serious economic losses to pig industry.In recent years,the diagnostic methods and control techniques for porcine pseudorabies have been continuously innovated.the gE-deleted PRV vaccines has been shown the effective vaccine for controlling the current PR in China.Differential diagnosis between gE-deleted vaccine strains and wild virus infection is significant for the prevention and control of the PR.In this study,two pairs of primers PRV-F1/R1 and PRV-F2/R2 were designed and synthesized according to the sequence of PRV on NCBI.PRV-F1/R1 was used to prepare the probe,PRV-F2/R2 was used for conventional PCR detection and preparation of plasmid standard products.Firstly,we established a PCR detection method using PRV-F2/R2 and explored the optimal reaction conditions for the PCR method to obtain the optimal primer concentration and the optimal annealing temperature.The established PCR was used to amplify the fragment size of 1049 bp.The PCR product was cloned into the PMD18-T vector to produce the new construct a recombinant plasmid named PRV-gE.The PRV gE gene DIG-labeled nucleic acid probe was labeled with the PRV-F1/PRV-R1 using the plasmid PRV-gE as a template.First,the sample DNA was extracted,and the DNA was amplified using the PRV-F2/R2.Then,the PCR amplification product was spotted on a nylon membrane,and dot blot hybridization was performed using the synthesized probe.The common pig diseases were detected by PCR and PCR combined with nucleic acid dot blot hybridization and the specificity of the two methods was tested.The plasmid PRV-gE was quantitated andserially diluted in multiples.The diluted plasmid was used as a template for PCR and PCR combined with nucleic acid dot blot hybridization to detect the sensitivity of these two methods.The results showed that only the DNA of PRV wild-type amplified the target band.AS the PCR combined with the nucleic acid dot blot hybridization method,the probe only reacted positively with the PCR product of the SDTA 2016 strain.It is indicated that the PCR hybridization detection method established in this paper has good specificity,the sensitivity of PCR-binding nucleic acid dot hybridization method can reach 10 pg/μL,which is 100 times for the PCR(1 ng/μL),which shows that the method established in this study has good sensitivity.A total of 53 samples were collected from different pig farms in parts of Shandong province,the DNA was extracted from the disease samples.PCR and PCR combined with nucleic acid dot blot hybridization were used to detect the DNA.As a result,4 of the PCR assays were positive,with a positive rate of 7.55%.PCR combined with nucleic acid dot blot hybridization methods detected 5 positives,with a positive rate of 9.43%,which was higher than the detection rate of PCR.The results showed that the detection method of PCR combined with nucleic acid dot blot hybridization in this study was effective in the detection of clinical samples and was suitable for clinical detection.More importantly,it is more specific than electrophoretic detection.In addition,according to the sequences of PRV gE and TK on NCBI,primers were designed to amplify the PRV gE and TK genes and perform sequencing analysis.The sequencing results were compared with the published strains on NCBI.The results showed that the five strains of PRV gE gene identified in this study were more homologous to the Asian strain than the European strain and the amino acid sequence of the gE gene.We found five strains identified in this study and mutant strains at position 48,there is an insertion of aspartic acid at position 495.In addition,we used ELISA to detect PRV-gE antibodies in 478 serum samples from 22 farms in Shandong.A total of 254 positive samples were detected,with a positive rate of53.1%,it shows PRV wild strain infection in many pig farms Through the detection of antibodies,the prevalence and incidence of pseudorabies in pigs in shandong province havebeen further understood.