Double QPCR Method For InvA And AadA Genes Of Salmonella Bovine Establishment And Separation And Identification

Most cases of calf diarrhea are caused by pathogenic microorganisms such as bacteria,viruses,and parasites in the intestines.Among them,calves within 2 months of age are prone to calf diarrhea.In severe cases,70%of calves can become ill,with a mortality rate of up to 50%.In calf diarrhea,common bacterial pathogens include pathogenic Escherichia coli,Clostridium weischii,and Salmonella.Among them,calf diarrhea caused by salmonella is also known as calf paratyphoid.In order to quickly detect the presence of Salmonella in cattle farms and the resistance of aminoglycoside drugs,this study referred to the Salmonella virulence gene invA and aminoglycoside drug resistance gene aadA on Gen Bank to design specific primers and probes.Establish a dual fluorescence quantitative PCR method for detecting Salmonella and aminoglycoside drug resistance genes,and apply this method to isolate and identify Salmonella and drug susceptibility tests on the detected positive stools.The test results show that the standard curve correlation coefficient(R2)of the dual fluorescence quantitative PCR method for detecting Salmonella invA and aadA genes established in this study is R~2=1.000,and the amplification efficiency(E)is 100%and 97.1%;it can effectively distinguish Bovine infectious rhinotracheitis virus,Pasteurella,Escherichia coli,etc.;the lowest sample DNA concentration that can be detected is 10 copies/?L;the coefficient of variation of Ct values within and between groups is less than 2%,which has good specificity,Sensitivity and repeatability.Three Salmonella isolates were isolated in this experiment,and their 16S r RNA sequence was between 86.6%and 95.5%homologous to 19 Salmonella 16S r RNAs at home and abroad.The phylogenetic tree analysis showed that 3 Salmonella isolates(NM-Sal1,NM-Sal2,NM-Sal3)are closely related to each other and belong to the same evolutionary group.The drug susceptibility test showed that the three strains were resistant to at least 14 of the 18 drugs,indicating that the drug resistance is serious.This method can be used for epidemiological investigation of Salmonella and aminoglycoside drug resistance genes,and provides a rapid detection method for cattle farms to effectively prevent and control calf diarrhea caused by Salmonella.