Establishment Of A Triple Fluorescence Quantitative PCR Detection Method For The Main Vertical Transmitted Bacterial Diseases In Chicken

Salmonella,Escherichia coli and Proteus mirabilis are all of the main pathogens of chicken vertical spread bacterial disease.In order to differential diagnosis of these three kinds of diseases,the study isolated and identified three species of pathogenic bacteria,and established single and triple fluorescence quantitative PCR method for detection,and assembled the kit,designed for Salmonella,Escherichia coli,proteus differential diagnosis,which provides a simple,sensitive and less time and specific detection kit.1.Salmonella were isolated by culture.The homologous property of 16S rRNA partial base sequence was 99.7,which was in the same branch as AB594754,AB594759,AB594763,EU118085 and other intestinal salmonella in GenBank,and the homology was up to 99.8.Based on the Salmonella InvA gene design primer,the SYBR Green I fluorescence quantitative PCR assay was established to detect salmonella,which was 100 times higher than that of the conventional PCR method,and the mutation coefficient of repeatability test was 3.74%and 0.10%.2.Escherichia coli were isolated by culture.Isolates of 16 S rRNA partial base sequence homology between 99.3%¹00%,with GenBank AY098487?chicken source Danish isolates?,NZ_CP015855?Human source Korea isolates?,The homology of NZ_CP015995?chicken source Chinese isolates?was higher.Based on the design primer of Escherichia coli FimH gene,the SYBR Green I fluorescence quantitative PCR assay was established to detect Escherichia coli,which was 100times higher than that of normal PCR,the sensitivity was 0.91%and 1.59%.3.Proteus mirabilis were isolated by culture.The sequence homology of 16S r RNA partial base sequence was 99.7%¹00%,and AB272366?avian Japanese isolates?were in the same branch,the homologous sex was up to 100.Based on the design primer of urer gene,the SYBR Green I fluorescence quantitative PCR method for detection of Proteus Mirabilis was established,which was 100 times higher than that of conventional PCR method for detecting Proteus 10¹ cfu/ml.The variation coefficients of the repeatability test were 2.23%and 1.68%.4.A triple fluorescence quantitative PCR method for detection of Escherichia coli,Salmonella and Proteus mirabilis was established,which can reach the minimum limit of 10¹cfu/ml in detection of Escherichia coli,Salmonella and Proteus mirabilis,and the variation coefficient of repeatability test is less than 5%.5.The kit was assembled by the triple fluorescence quantitative PCR method of Escherichia coli,Salmonella and Proteus mirabilis,which has a good specificity,sensitivity,repeatability and stability for 6 months for the detection of the clinically suspected samples and 100%for the storage of the reagent box in the-20?refrigerator.