摘要
为了实现快捷、准确检测抗生素抗性基因,提高环境监测与治理能力,建立了一种基于重组酶介导等温核酸扩增技术(recombinase-aided amplification,RAA)的四环素抗性基因tet(A)的快速检测方法.该检测方法以tet(A)基因序列保守区为靶序列,设计其RAA引物,筛选并优化RAA引物探针,建立荧光tet(A)-RAA快速检测方法.结果表明,该tet(A)-RAA法在39℃恒温条件下30 min内即可特异性实现对tet(A)基因片段的有效扩增,与无抗性细菌均无交叉反应.且采用该方法对18个环境样品进行检测,阳性率为100%,与qPCR方法检测结果一致.该四环素抗性基因tet(A)的检测方法灵敏度高、特异性强、反应时间短、操作简便,可用于对抗性基因tet(A)的快速检测,也为开发其他抗生素抗性基因快速检测方法提供了参考.
Antibiotic resistance genes(ARGs),as a new type of environmental pollutant,pose a serious threat to ecology and human health,and have attracted the attention of many disciplines.To achieve rapid and accurate detection of ARGs,and improve the ability of environmental monitoring and treatment,this study established a rapid detecting method of tetracycline resistance gene tet(A)based on recombinase-aided amplification(RAA)technology.The detecting method takes the conserved region of tet(A)gene sequence as the target sequence,designs its RAA primers,screens and optimizes RAA primer probes,and establishes a rapid detecting method for a fluorescencing tet(A)-RAA.Results show that the tet(A)-RAA method can specifically realize effective amplification of the tet(A)gene fragment within 30 minutes under the constant temperature of 39℃,and has no cross-reaction with non-resistant bacteria.18 environmental samples were detected by this method,and the positive rate was 100%,which was consistent with the detection result of qPCR method.The tetracycline resistance gene tet(A)detection method established in this study has high sensitivity,good specificity,short reaction time and simple operation,and can be used for the rapid detection of the resistance gene tet(A).It also provides a reference for the development of other rapid detection methods for antibiotic resistance genes.
作者
周志祥
赵子杰
李疆帅
薛姗
洪启浩
侯潇楠
任阵海
ZHOU Zhixiang;ZHAO Zijie;LI Jiangshuai;XUE Shan;HONG Qihao;HOU Xiaonan;REN Zhenhai(Faculty of Environment and Life,Beijing University of Technology,Beijing 100124,China;Chinese Research Acdemy of Environmental Sciences,Beijing 100085,China)
出处
《北京工业大学学报》
CAS
CSCD
北大核心
2023年第2期227-234,共8页
Journal of Beijing University of Technology
基金
国家自然科学基金资助项目(42077399)。
关键词
抗性基因tet(A)
四环素
重组酶
等温扩增
快速检测
环境检测
resistance gene tet(A)
tetracycline
recombinase
isothermal amplification
rapid detection
environmental detection
