| With frequently using of sulfonamide drugs, most pathogenic bacteria developed drug resistance. Numerous studies showed that bacteria (especially gram-negative bacteria) developed resistance to sulphonamide drugs mainly by obtaining plasmid encoding resistance genes. At present, three kinds of sulphonamide resistance genes have been discovered, which named sull, sul2 and sul3 respectively. We developed a loop-mediated isothermal amplification (LAMP) assay, which can directly detect the sull, sul2 and sul3 gene effectively. Through system optimization and merging three LAMP systems, we successfully established a rapid, sensitive and specific m-LAMP assay, which can simultaneously amplify three kinds of sulphonamide resistance genes of avian Salmonella spp. and Escherichia coli. This assay can potentially serve as an important technical support to guide clinical treatment and control the prevalence of resistant strains.Section one:Identification of avian Salmonella spp. and Escherichia coli and detection of their resistance to sulphafurazoleWe obtained 468 strains of Salmonella spp. and 171 strains of Escherichia coli by identifying pathogens from clinical disease samples, which collected from large-scale poultry farms and departments of animal disease diagnostic. And we further detected the sensitivity of above isolates to sulphafurazole susceptibility paper. According to the results of drug sensitive test, there were 532 strains of sulphafurazole resistance bacteria, which accounted for 83.26%. Among them, there were 389 strains of Salmonella spp., of which resistance rate was 83.12% (389/468). And there were 143 strains of E.coli, of which resistance rate was 83.63%(143/171). In sulphafurazole resistance strains of Salmonella spp., the resistance rate of Salmonella Pullorum, Salmonella Enteritidis and Salmonella Indiana were higher than average level, the resistance rate of them reached to 83.61%(255/305),83.91%(73/87) and 84.48%(49/58). Salmonella Typhimurium, Salmonella Albany, Salmonella Heidelberg and Salmonella Saintpaul resistance rate were 71.43%,66.67%,66.67% and 71.43% respectively.Section two:development of m-LAMP method for detection of sulphonamide resistance genes (sull, sul2, sul3)According to reference sequences of sulphonamide resistance genes released on GenBank, we designed three groups of primers for LAMP assay based on the conserved region of sull, sul2 and sul3 gene with professional online software. Then we explored and optimized the concentration and conditions of LAMP reaction for detecting three kinds of sulphonamide resistance genes, and the sensitivity and specificity of the reaction system were verified.On this basis, we explored and optimized the concentration and conditions of m-LAMP reaction, such as the best proportion of outer primers and inner primers, concentration of Mg2+, dosages of Bst DNA polymerase, concentrations of dNTPs and reaction conditions. Then specificity and sensitivity test was also earned out, and amplification products were verified by enzyme digestion with Hinfl. Besides, the results of LAMP assay can be visualized directly with adding calcein reagent by observation of the color change. The results show that this method has good specificity and reproducibility. The detection limit of m-LAMP is 0.5 pg/tube, which is more sensitive than 5 pg/tube in conventional PCR. It’s quick, convenient and suitable for on-site rapid detection.Section three:detection of sulphonamide resistance genes of clinical isolates of Salmonella spp. and Escherichia coliAll strains of Salmonella spp. and Escherichia coli were detected of sulphonamide resistance genes and typed with LAMP assay we established.522 strains of them were tested positive for sulphonamide resistance genes, the detection rate was 81.69%(522/639). Compared with resistance phenotype,497 strains of LAMP detection results was consistent with the results of sulphafurazole sensitive test, the coincidence rate was 93.42%(497/532), which had a high correlation.In sulphonamide resistance strains, there were 383 strains of Salmonella spp., which accounted for 81.84% of the total isolates of Salmonella spp.. Among them,194 strains contained sull gene, the number of strains containing sul2 and sul3 was 242 and 23. In strains of Salmonella spp., the positive rate of sul2 gene was higher than sull, and the positive rate of sul3 was the lowest. There were 139 strains of Escherichia coli, which accounted for 81.29% of the total isolates of Escherichia coli. Among them,101 strains contained sull gene, the number of strains containing sul2 and sul3 was 121 and 17. According to the distribution of resistance genes, sul2 existed in Escherichia coli and seven serotypes of Salmonella spp.. In addition to Salmonella Saintpaul, other serotypes of Salmonella spp. detected sull positively. Each of three main popular serotypes(Salmonella Pullorum, Salmonella Enteritidis and Salmonella Indiana) contains three kinds of resistance genes, while Salmonella Saintpaul only contains sul2 gene. According to the positive rate of sul genes, the rate of sul2 (363/522,69.54%) of Salmonella spp. and Escherichia coli was higher than that of sull (295/522,56.51%), and positive rate of sul3 (40/522,7.66%) was also small. There were 172 strains of bacteria containing one kind of sul gene, which accounting for 32.95% of the total number of positive strains. And the number of posiive strains containing two kinds of sul genes were 322, accounting for 61.69%. Besides, the number of positive strains containing three kinds of sul genes were 28, accounting for 5.36%. Obviously, at present sulphonamide resistance strains are mainly mediated by sull and sul2.In summary, we established an m-LAMP assay for detection of sulphonamide resistance genes based on the rapid, simple, specific and sensitive LAMP technology in this study, to solve the shortcomings of traditional methods. This method can be used for detection and genotyping of resistance genes in clinical isolates and amplification products can be verified by enzyme digestion reaction with Hinfl. Besides, the results of LAMP assay can be visualized directly with adding calcein reagent by observation of the color change. The LAMP assay is not only applicable to detect clinical isolates of bacteria carrying sulphonamide resistance genes rapidly, but also beneficial to guide the use of drug reasonably and scientifically. And it can potentially serve as an important method to explore the relationship between resistance phenotype and resistance genes, which is of great significance for further studying on the mechanism of Sulphafurazole resistance of pathogenic bacteria and its evolution. |
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