Establishment And Application Of Avian Hepatitis E Virus RT-PCR Combined With Nucleic Acid Dot Blot For Detection

Avian Hepatitis E virus(aHEV)is the main cause of chicken liver and spleen disease,Clinical symptoms include enlarged liver,enlarged spleen,decreased egg production performance,and peritoneal effusion.It is currently widespread and present in flocks worldwide.Since 2016,chicken liver and spleen disease,which was mainly manifested by liver spleen enlargement and rupture,have occurred in many chicken flocks in China,and new genotypes of aHEV have been identified in related chicken flocks.At present,the lack of detection methods for aHEV is still one of the difficulties in effectively monitoring and preventing and controlling them.In the current unstable in vitro culture system of aHEV,the establishment of sensitive,specific and stable nucleic acid detection methods is essential for conducting epidemiological investigations and implementing detection elimination.In this study,the RT-PCR combined with nucleic acid dot blot for detection method for aHEV was established,and a molecular epidemiological investigation was carried out in a number of suspected cases in China.In this study,primers F-2357 and R-2357 were designed to amplify the ORF2 gene based on the published ORF2 gene nucleic acid sequence of aHEV,The HEV Hebei isolate VaHEV-HB nucleic acid was used as a template to amplify its ORF2 gene,which was ligated to the pMD18-T vector to successfully construct the aHEV-ORF2 plasmid and further verified by sequencing.Two pairs of primers were designed based on the ORF2 gene sequence of VaHEV-HB and other reference strains,the primers F-P-HEV and R-P-HEV were used to prepare digoxin labeled nucleic acid probes,the primers F-W-HEV and R-W-HEV were used for preliminary RT-PCR amplification of sample nucleic acids,and the nucleic acid regions amplified by primers F-P-HEV and R-P-HEV were located within the nucleic acid regions amplified by primers F-W-HEV and R-W-HEV.Used the aHEV-ORF2 plasmid as a template,primers F-P-HEV and R-P-HEV were synthesized according to the instructions of the PCR DIG Probe Synthesis Kit,When detecting samples,first use primers F-W-HEV and R-W-HEV to perform RT-PCR amplification on sample nucleic acids,RT-RCR products are not detected by nucleic acid electrophoresis,but directly spotted on nylon membranes for nucleic acid dot hybridization detection using synthetic aHEV digoxin labeled nucleic acid probes.The results showed that the established method could reach 10 pg/?L sensitivity and only recognized the nucleic acids of aHEV.It did not develop color for ALV-A,ALV-J,REV,CIAV,FAdV and other virus nucleic acids,and had high specificity.Comparison of the sensitivity and detection rate of the established method with conventional RT-PCR and nested RT-PCR showed that the sensitivity and detection rate of this method were much higher than conventional RT-PCR,and it was also better than nested RT-PCR.It could detect more aHEV-positive samples,and provided a sensitive and specific detection method for conducting HEV molecular epidemiological investigation and clinical diagnosis.A total of 197 suspected cases of 15 chicken farms in 14 regions including Hebei,Shandong,Beijing,Liaoning,and Guangxi were tested for aHEV using the established RT-PCR combined with nucleic acid dot blot for detection method.The results showed that 78 of the 197 samples were positive for aHEV,with a positive rate of 39.6%,HEV positive rate of different farms ranged between 22.2%~69.2%.Cloned and sequenced the ORF2 gene amplification products of all positive samples and analyzed their homology and genetic evolution,The results showed that the homology of the obtained 78 sequences was between74.4% and 100%,which could be roughly divided into four groups according to the level of homology,And the homology of each group of sequences was similar,but there were some differences in homology between the four groups of sequences.Phylogenetic tree analysis revealed that the positive samples were aHEV genotype 3,genotype 5,and other unknown genotypes,among which the majority were genotype 5,and the genotype did not show obvious regional rules.In this study,the RT-PCR combined with nucleic acid dot blot for detection method for aHEV was established,which had high sensitivity and specificity,Used this method,amolecular epidemiological investigation was carried out on multiple suspected cases in China and a genetic evolution analysis was performed on its ORF2 gene,It provides new reference data for understanding the epidemic dynamics and variation of aHEV in China.