三重荧光定量PCR检测水产动物源细菌磺胺类耐药基因.PDFVIP

() 2017, 43(1):64–70. DOI:10.13331/ki.jhau.2017.01.012 Journal of Hunan Agricultural University (Natural Sciences) 三重荧光定量 PCR 检测水产动物源细菌磺胺类耐药基因 * ( 530021) 摘 要GenBank Sul1Sul2 Sul3 3 TaqMan 3 PCR 1×1011×108 /(3 10 /) ()(2%) 94.2%2 h Sul1Sul2 Sul3 PCR 关 键 词PCR 中图分类号S917.1 文献标志码A 文章编号1007− 1032(2017)01−0064−07 Detection sulfonamides–resistance genes of pathogenic bacteria derived from aquatic animals using triple real–time PCR * HUANG Guoqiu, TONG Guixiang, WEI Xinxian, CHEN Jing, WU Mingyuan, LI Xiaozheng (Guangxi Academy of Fishery Science, Nanning 530021, China) Abstract: Triple TaqMan real–time PCR method was developed to simultaneously detect sulfonamides–resistance genes Sul1, Sul2 and Sul3, 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 Primer Express 3.0. 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 8 1×10 to 1×10 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 of Sul1, Sul2 and Sul3, 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 of Sul1, Sul2 and Sul3. The variation coefficients of the established method were less than 2%. Sulfon