The Establishment And Preliminary Application Of Indirect ELISA For The Diagnosis Of Calf Coronavirus And Rotavirus

Bovine coronavirus?BCV?and bovine rotavirus?BRV?are the main viruses that cause infectious diarrhea in calves.Calf infection leads to decreased immune function,severe diarrhea and dehydration.Therefore,for the development of indirect ELISA for fast BCV,BRV antibody diagnosis is particularly important in large scale dairy farms.Objective:?1?Bovine rotavirus of dairy farms in parts of northern and southern parts of Xinjiang,Investigation and Analysis of coronavirus.?2?Establish an indirect ELISA diagnostic method for rapid diagnosis of antibodies against calf coronavirus and rotavirus.Methods:A total of 172 diarrhoea calves and intestinal contents were collected from the northern Xinjiang area of Xinjiang and some parts of Southern Xinjiang,and were detected by ELISA and PCR methods.At the same time,in order to understand the infection of calf coronavirus disease in some areas of northern Xinjiang,141 diarrhoea calves in 10 dairy farms were collected and detected by PCR and ELISA at the same time.?2?Design and Synthesize primers for the conservative region of the calf rotavirus lingerie shell protein VP6,design specific primers,construct a recombinant expression vector pET-32a-VP6,express the bovine rotavirus VP6 protein,use Western Blot to analyze the immunoreactivity of the protein,establish a BRV indirect ELISA diagnosis method,and according to GenBank logged in calves at the same time.The N gene sequence of coronavirus was designed,specific primers were designed,recombinant expression vector pET-32a-N was constructed,and then subcloned into BL21?DE₃?expression vector,IPTG was used to induce and SDS-PAGE identification of recombinant bacteria,and Western blot was used to analyze the expression products.An indirect ELISA method for detecting the antibody of calf coronavirus was established.Using the established bovine rotavirus,the indirect ELISA of the coronavirus and the imported Belgian reel detection kit,the serum of 26 sera and the healthy calves from 10scale cattle farms in the northern part of Xinjiang were compared with that of the healthy calves.Results:?1?In the 172 samples,the ELISA test results showed that 100 calves rotavirus positive were detected,the positive rate was 72%,the results of PCR detection were 127 and the positive rate was 73.83%.In the 141 samples,the ELISA test results showed that 99calves positive coronavirus were detected,the positive rate was 70.21%,and the PCR test results 8 7,the positive rate was 61.7%.?2?The target gene was successfully amplified by PCR,and the pET-32a-VP6 expression vector was constructed by sequencing and enzyme digestion.SDS-PAGE electrophoresis showed that the expression product expressed the recombinant protein in 60kD.After Western Blot detection,the protein has good immune response,the coincidence rate of the indirect ELISA diagnosis method and the import kit reached 92.3%,and the bovine coronavirus N gene was cloned successfully,the size of the fragment was 1400bp,the relative molecular mass of the recombinant protein BCV-32a-N-DE₃ was 70kD,and the Western blot analysis showed that the recombinant protein was reorganized.Protein BCV-32a-N-DE₃ can react specifically with bovine coronavirus positive serum.The established indirect ELISA diagnostic method for detection of antibodies against calf coronavirus was 88.46%,and the serum of healthy calves did not respond.Conclusions:?1?The PCR and ELISA methods were used to investigate the main pathogenic rotavirus and coronavirus of calf diarrhea in some parts of Northern Xinjiang and some parts of Southern Xinjiang,and to find a high infection rate and provide a basis for the active prevention and control of the large-scale cattle farm.?2?An indirect ELISA method for rapid diagnosis of calf rotavirus and coronavirus antibody was established to screen the infection of calf rotavirus and coronavirus in large-scale cattle farms.