The Establishment And Preliminary Application Of Multiple PCR Diagnostic Methods For Bovine Infectious Diarrhea Syndrome

Mainly with gastrointestinal symptoms is common infectious bovine coronavirus infection in cattle, bovine rotavirus infection, Campylobacter diarrhea, bovine enterotoxigenic E. coli disease, cattle diseases such as salmonella. This study is maintained by the laboratory cultured pathogens, established PCR after, in the hope for the diagnosis of these diseases provide a new way. Bovine coronavirus inoculated human rectal cancer cell line (HRT-18), bovine rotavirus inoculated rhesus kidney cell line (MA-104), to be collected when the virus CPE solution appears as a PCR template. Campylobacter jejuni inoculated on Columbia blood agar, the micro-aerobic conditions (5% O2,10% CO2 and 85% N2) were collected after 48h culture bacilli. Enterotoxigenic Escherichia coli and Salmonella typhimurium inoculated on LB plate, after overnight culture were picked from a single bacterial colony overnight shaking logarithmic phase, collection bacilli as a PCR template.According to published GenBank various pathogen-specific genes (bovine coronavirus N (Nucleocapsid protein) gene, bovine rotavirus VP6 (inner shell protein) gene, enterotoxigenic Escherichia coli k99 and the F41 pili genes, Campylobacter jejuni mapA genes and Salmonella typhimurium invA (invasion protein A) gene) with Oligo6 design software to design a pair of primers, respectively, and then designed primer pairs of bovine coronavirus and bovine rotavirus, bovine coronavirus, and Campylobacter jejuni bacteria; pairs of enterotoxigenic Escherichia coli K99, F41 fimbriae genes and Salmonella typhimurium to triple-PCR, and associated conditions for optimization, At the same time of its specificity and sensitivity analysis. At last, well-established PCR methods is testing clinical samples.With the establishment of bovine coronavirus and bovine rotavirus double RT-PCR to detect mixed samples obtained two purposes stripe:size was 244bp (bovine coronavirus) and 382bp (bovine rotavirus), and consistent with the experimental design and well-established two kinds of virus, RT-PCR results were consistent with a single. The sensitivity of the dual PCR bovine coronavirus:a TCID50/100μl, bovine rotavirus: 100 TCID50/100μl. Control enterotoxigenic E. coli, Campylobacter jejuni, bovine adenovirus-7, bovine infectious rhinotracheitis, bovine parainfluenza virus type 3 were not amplified by the purpose of bands. The PCR method using 57 clinical samples tested, the results of detection of bovine coronavirus infection in 7 cases were infected with bovine rotavirus cases in four copies, two copies of mixed infections.With the establishment of bovine coronavirus and bovine Campylobacter jejuni double PCR to detect mixed samples obtained two purposes stripe:size was 244bp (bovine coronavirus) and 510bp (Bovine Campylobacter jejuni), and consistent with the experimental design, and have been established a good two kinds of PCR results were consistent with a single pathogen. The sensitivity of the dual PCR bovine coronavirus:10 TCID50/100μl, Bovine Campylobacter jejuni:1.088×102 CFU/reaction. Control enterotoxigenic E. coli, Salmonella typhimurium, bovine coronavirus were not the purpose of amplification bands. The PCR method using 57 clinical samples tested, the results of detection of bovine coronavirus infection in 9 cases were infected with Campylobacter jejuni cases in one copy.With the establishment of bovine enterotoxigenic Escherichia coli and bovine Salmonella PCR detection of the triple hybrid samples obtained three purposes stripe:size were 232bp (cattle salmonella),314bp (Escherichia coli K99 pili) and 380bp (Escherichia coli F41 Fimbriae), and experimental designed, and consistent and well-established results are consistent with a single PCR. The sensitivity of PCR in the triple cattle Salmonella:2×101 CFU/reaction, E. coli:2×102 CFU/reaction. The control of Campylobacter jejuni, bovine parainfluenza virus type 3, bovine coronavirus, and bovine rotavirus were not the purpose of amplification bands. The PCR method using 57 clinical samples tested, the results of detection of cases of salmonella infection in three cattle were infected with bovine enterotoxigenic E. coli in 11 cases, mixed infections one copies.By testing clinical samples showed that PCR method established in this study can be fast, accurate and specific amplification of a pathogen can be used for clinical diagnosis.