| Bovine coronavirus(bcov)is the main pathogen causing diarrhea in calves.It also causes winter dysentery and respiratory diseases in adult cattle,which is a major problem in cattle industry.The most important thing for the prevention and control of bcov is to detect the pathogens in time and understand the infection of bcov.The purpose of this study is to establish a fluorescent RT-PCR method for detection of bcov,and apply this method to detect bcov in some areas of China,so as to further understand the infection situation of bcov in China and enrich the data of molecular characteristics and genome of bcov,The results obtained as follows:1.Successfully established a insulated isothermal RT-PCR method for detecting BCoVBovine coronavirus(bcov)is an important pathogen causing diarrhea and respiratory diseases in cattle.Although there are many PCR based methods to detect bcov,there is no report on the method of insulated isothermal RT-PCR(iirt PCR),A pair of specific primers and probes were designed in the conserved region,the optimal reaction system was optimized,and the iirt PCR method for detection of bcov was established.The specificity of this method is good,only bcov can be detected,other unrelated pathogens can not be detected;The coefficient of variation within and between batches was less than 5%;The detection limit is 11 copies·?L-1?The detection rate of bcov in calf diarrhea samples and bovine respiratory tract samples by this method was significantly higher than that by Taq Man fluorescence quantitative RT-PCR reported abroad.It can be used in combination with pockettm series handheld nucleic acid analyzer and petnad nucleic acid extraction kit.The operation is convenient and fast.It provides an effective new tool for bcov on-site detection.2.Molecular detection of bovine coronavirus in four provinces in ChinaFrom November 2019 to December 2020,177 samples of sick cattle(diarrhea feces and respiratory secretions)were collected from 15 large-scale cattle farms in 4provinces(Sichuan,Henan,Inner Mongolia and Ningxia Hui Autonomous Region),including 110 samples of diarrhea feces of calves under 3 months old(22 samples from 1 Farm in Sichuan Province,9 samples from 3 farms in Henan Province,26samples from 1 Farm in Inner Mongolia There are 8 farms in Ningxia(53);49samples of adult cattle with winter dysentery(2 farms in Henan Province);18 calf respiratory tract samples(from the same Inner Mongolia cattle farm).The results showed that the detection rate of bcov was 26.36%(29/110),and the field positive rate was 81.82%(9/13);The detection rate of bcov was 20.41%(10/49)in 49 adult cattle samples and 38.89%(7/18)in 18 calf respiratory samples,which indicated that bcov was widely prevalent in cattle farms.The complete S gene and he gene were successfully amplified from 16 positive samples(12 calf diarrhea,1 adult winter dysentery and 3 bovine respiratory tract)from 6 cattle farms in 4 provinces.The phylogenetic tree results showed that the cloned S gene had the closest genetic relationship with bcov strains in China,showing a common evolutionary trend,We found a S gene with four consecutive amino acid deletions in S1B region.Compared with all 195 complete bcov he genes in Gen Bank,14 of them were clustered into an independent branch with 29 Chinese strains(1 from yak and 28 from cow);One of them was clustered with four Chinese strains;Another one was clustered with two American strains.The results of recombination analysis software showed that 13 he genes(13/16)were recombined,and the recombined region was located between the he esterase and lectin domains,which may affect the receptor binding function of he.The 13 recombined he genes came from five cattle farms in four provinces and were distributed in three different clinical samples:calf diarrhea samples,adult bovine winter dysentery samples and bovine respiratory tract samples,These results indicate that the recombinant strain of he gene has no tissue specificity,which is of great significance for further understanding the molecular characteristics and genetic variation of bcov and providing important theoretical reference for the prevention and control of bcov.3.Genomic research of bovine coronavirusIn order to further study the genomic characteristics of bcov S gene amino acid deletion strain,the whole genome sequence of bcov SWUN/NMG-D10/2020 strain was successfully amplified by using 44 pairs of primers designed in our laboratory.The results showed that the complete genome of the strain was 31008 BP,and the content of G+C%was 37.05%.The SWUN/NMG-D10/2020 strain has the closest genetic relationship with three Chinese bovine bcov strains in Gen Bank,with nucleotide homology of 99.0%-99.2%.The orf1a and ORF1b genes of the strain were also closely related to the three Chinese bcov strains.There is a unique amino acid mutation(T121I)in the he gene of this strain and in the acetylesterase domain(E)of11 Chinese cow strains,which is different from others.The amino acid mutation in this region may affect the function of receptor destruction.Amino acid sequence analysis of 255 bcov complete s genes in Gen Bank showed that 12 nucleotides were continuously deleted at sites 541-544 of receptor binding domain in S1B region,resulting in 4 amino acid deletions(SKST).The deletion of this region may change the binding ability of the virus to its receptor,thus changing its pathogenicity and histotropism.This is the first time that an amino acid deletion event has been found in the S gene of bcov in China,which enriches the genetic evolution database of bcov. |
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