Establishment And Application Of Double RT-PCR Detection Method For Goose Astrovirus And Novel Duck Reovirus

In 2020,the slaughter volume of meat geese in China was about 500 million.As a major province of goose breeding industry,the slaughter volume of meat geese in Shandong Province was about 67.2 million.With the vigorous development of goose breeding industry,large-scale and intensive breeding led to more and more epidemic diseases.Since 2017,Goose astrovirus and Novel duck reovirus began to appear in some parts of China,and then began to spread and erupt throughout the country,Mixed infection often occurs in clinic,which seriously threatens the healthy development of goose breeding industry;In order to quickly and simply detect the single or mixed infection of Goose astrovirus and Novel duck reovirus,a dual RT-PCR detection method was established to provide effective technical support for rapid diagnosis and early warning of the disease.According to the ORF1b gene of goose astrovirus and the novel duck reovirus the conserved sequence ofσC gene was used to design specific primers,and the conditions such as annealing temperature,specificity,sensitivity and repeatability were optimized to establish double RT-PCR method for the detection of goose astrovirus and novel duck reovirus;The clinical samples of dead goslings and goose farms of different sizes collected in Binzhou,Jining,Heze,Liaocheng and Weifang in Shandong Province were detected,verified by single RT-PCR,and the detected positive samples were analyzed for genetic evolution.The results showed that the nucleic acid fragments of two viruses were amplified by the double RT-PCR method established in this study,which were 903 bp（Goose astrovirus）and226 bp（Goose derived noel duck reovirus）,respectively.The optimumannealing temperature of this method was determined to be 54℃;There was no specific amplification of viral nucleic acids of RNA or DNA such as Duck hepatitis-1,Duck hepatitis-3,Muscovy duck reovirus and Goose circovirus.The lowest sensitivity of this method was 1.0×10~3copies/μL,and the repeatability is stable and feasible.The 256 suspected samples collected were detected by the established double RT-PCR method.94 samples were single infected with Goose astrovirus,71 samples were single infected with Novel duck reovirus,and 63 samples were mixed infected with Goose astrovirus and Novel duck reovirus;The method was applied to detect 320 clinical samples randomly taken from goose farms of different sizes.It was found that the detection rate of single infection of Goose astrovirus was 21.56%（69/320）,the detection rate of single infection of Novel duck reovirus was 3.44%（11/320）,and the detection rate of mixed infection of Goose astrovirus and Novel duck reovirus was 2.81%（9/320）;After verified by single RT-PCR,the positive rates of the two detection methods were completely consistent with each other.The nucleotide sequences of 9 Goose astrovirus RT-PCR positive products obtained had nucleotide homology between 97.1%-98.2%and Goose SDPY strain,GD strain,GTF-07strain and HBXG strain.Genetic evolution analysis showed that the positive product was closely related to Goose derived astrovirus;Two novel reovirus isolated from goose the nucleotide homology between the positive product of RT-PCR ofσC full-length gene and the published virus strains such as NDRV 091 and HN 5d is 89.4%-96.2%.The detected virus has the closest genetic relationship with the Novel duck reovirus,but there is a certain distance from Muscovy duck reovirus and Avian reovirus in genetic evolution.In conclusion,this study successfully established a double RT-PCR method for the detection of Goose astrovirus and novel duck reovirus,which provides a basis for the rapid diagnosis of Goose astrovirus and Novel duck reovirus from goose.