三重荧光定量PCR检测水产动物源细菌磺胺类耐药基因

根据Gen Bank中细菌磺胺类耐药基因Sul1、Sul2和Sul3的保守序列设计3对特异性引物及相应Taq Man探针,以重组质粒标准品为模板对反应条件进行优化,建立一种可同时检测上述3种耐药基因的三重荧光定量PCR方法,并对该方法进行敏感性、特异性、重复性及临床菌株检测试验。结果表明:该方法定量范围为1×10~1~1×10~8拷贝/反应时,其标准曲线呈良好的线性关系;该方法灵敏度高(3个基因的最低质粒检测限均可达到10拷贝/反应)、特异性强(与其他病原菌及病毒无交叉反应)、重复性好(变异系数均小于2%);对临床菌株的检测结果与药敏试验结果的符合率达94.2%,且整个检测过程可在2 h内完成。所建立的可同时检测Sul1、Sul2和Sul3基因的三重荧光定量PCR方法可用于临床检测细菌的磺胺类耐药基因。

Detection sulfonamides-resistance genes of pathogenic bacteria derived from aquatic animals using triple real-time PCR

Triple TaqMan real–time PCR method was developed to simultaneously detect sulfonamides–resistance genes Sul1,Sul2 andSul3, the three pathogenic bacteria in aquatic animals in the study. Three pairs of specific primers and fluorogenic–labeled probes were designed and synthesized in accordance with the above target genes using software PrimerExpress3.0. The reaction system and procedure were optimized, as well as the detection sensitivity, specificity, repeatability and clinical application of the method. Results showed that the method had a wide quantitative range from 1×101 to 1×108 copies per reaction, which presented a good linear relationship in its standard curve. The triple real–time PCR method had a high specificity in detecting mixed DNA ofSul1,Sul2 andSul3, but not those DNA from other bacteria and viruses; it also had a high sensitivity to the detection limit low to 10 copies per reaction for the purified recombinant plasmids ofSul1,Sul2 andSul3. The variation coefficients of the established method were less than 2%. Sulfonamides–resistance genes of 72 clinical isolates were analyzed using triple real–time PCR method and they were compared to drug sensitive test, the coincidence rate could reach to 94.2%. The entire detection could be completed within 2 h. In conclusion, the tripleTaqMan real–time PCR method developed in the present study could be applied to the rapid detection and molecular epidemiology survey in sulfonamides–resistance genesSul1,Sul2 andSul3 of clinical pathogenic strains isolated from aquatic animals.