Study On On-site Integrated Rapid Detection Method Of Common Pathogenic Bacteria In Manure

With the development of intensive livestock and poultry breeding,the amount of livestock and poultry manure waste also increases.The unreasonable treatment and disposal of livestock and poultry manure will pollute water,air and soil to some extent,especially the pathogenic microorganisms in manure may cause the spread of various zoonosis.Endanger the health of human body and the healthy development of animal husbandry,cause public health accidents.Therefore,it is necessary to master the pollution status and law of typical pathogenic microorganisms in manure,and to establish a set of field rapid integrated detection methods with strong operability and convenient result determination.In this study,based on the characteristics of the site conditions of livestock and poultry farms,the field rapid detection system of common pathogenic bacteria in manure was established with the help of highly efficient enrichment materials and rapid detection by Loop-mediated isothermal amplification?LAMP?technology.At the same time,the enzyme substrate technique and PCR were used to conduct on-site investigation of indicator microorganisms?total coliforms,fecal coliforms,Escherichia coli?and pathogenic microorganisms?Salmonella?in the feces of 9 farms of 3 livestock and poultry species in Tianjin.The main results are as follows:?1?There was a significant difference in the number of indicated microorganisms among different kinds manure.The number of indicated microorganisms in pig manure,chicken manure and cow manure without harmless treatment ranged from10⁷?10⁹ MPN·g^-1?10⁶?10⁸ MPN·g^-1?10⁶?10⁷ MPN·g^-1.After harmless treatment of pig manure and cow manure,the number of indicated microorganisms was greatly reduced,and the removal rate was above 90%.However,the value of fecal coliform bacteria was far less than the national standard,indicating that fecal harmless treatment was not ideal.Salmonella was detected in chicken manure and cow manure,which did not conform to the national standard.?2?Taking the invA gene of Salmonella as the target gene,five sets of primers were designed.The final primer group was determined by the primer efficiency experiment and LAMP reaction system for Salmonella was established.When the ratio of internal and external primers was 6:1,the system reacted at 65?for 50min,with a detection sensitivity of 20.8 fg·?L^-1,and a good specificity for 45 strains in the experiment.?3?Taking the nuc gene of Staphylococcus aureus as the target gene,four sets of primers were designed.The final primer group was determined by the primer efficiency experiment and LAMP reaction system of Staphylococcus aureus was established.When the ratio of internal and external primers was5:1,the system reacted at 65?for 30 min,and the detection sensitivity reached 13.5 fg·?L^-1,and it had good specificity for 45 strains in the experiment.?4?The field detection system of Salmonella and Staphylococcus aureus in manure was established.The system could complete the detection of manure samples with detection limit up to 10⁰ CFU·g^-1 by5.5 h enrichment,0.5 h concentration,0.5 h DNA extraction,1 h LAMP amplification and result interpretation,a total of 7.5 h.The on-site integrated rapid detection method established in this study has the advantages of strong specificity,high sensitivity,short detection time and strong operability,which is suitable for the detection of pathogenic microorganisms in manure.