细菌β-内酰胺酶和氨基糖苷磷酸转移酶耐药基因可视化LAMP检测方法的建立和初步应用

Establishment and Application of Visible Loop-mediated Isothermal Amplification Method for Detecting Antibiotics Resistance Genes bla and Aph in Bacteria

为了建立快速准确检测微生物携带氨苄青霉素和卡那霉素抗性基因的环介导等温扩增(LAMP)方法,根据抗性基因β-内酰胺酶(bla)和氨基糖苷磷酸转移酶(Aph)基因序列,利用Primer Explo-rer V4在线工具设计特异LAMP引物,成功建立了检测上述2个基因的可视化LAMP方法。该LAMP体系在65℃条件下反应1 h,以100μmol/L HNB为最佳浓度,可实现阳性结果的准确判读,bla基因的检测灵敏度达到1 pg/μL。应用该LAMP方法成功检测到污水环境的微生物携带bla抗性基因。建立的LAMP方法在快速、可视化检测环境中抗性基因方面具有潜在应用价值。

A loop-mediated isothermal amplification(LAMP)method was developed for rapid and precise detection of ampicilin and kanamycin resistance genes,of which specific primers were designed by using online software Primer Explorer V4 based on DNA sequences of bla and Aph.The approach was optimized to amplify target genes by incubating 65℃for 1 hour,adding HNB with 100μmol/L final concentration,and it was feasible to distinguish the positive result from the control.The sensitivity of this assay was 1 pg/μL for bla gene detection.In addition,bla gene was detected out from 3 different water samples by this approach.We concluded that this rapid and visible LAMP can be used for detection of antibiotics resistance genes in environment.