摘要
目的建立一种毛细管聚合酶链反应(PCR)芯片电泳方法,实现快速、准确地检测新德里金属-β-内酰胺酶(NDM-1)耐药菌。方法根据NDM-1基因设计1对特异引物,对临床微生物检测的80株耐药肠道杆菌和60株鲍曼不动杆菌的细菌培养液进行毛细管振荡流PCR扩增,芯片电泳快速检测PCR产物。将NDM-1阳性标本进行测序验证,同时对NDM-1阳性菌细菌悬液定量梯度稀释进行PCR,考察免核酸提取PCR的敏感度。结果毛细管PCR芯片电泳方法检测出2例阳性标本,其产物经测序验证,确定携带NDM-1基因,阳性率为100%。该方法在40min内实现产NDM-1耐药菌扩增产物的快速分离检测,细菌检测线为1.15×101 CFU/mL。结论毛细管PCR芯片电泳方法检测NDM-1基因准确性高、特异性强,具有快速、廉价等特点,适合NDM-1阳性菌的早期快速现场诊断。
Objective To establish a method based on microfluidic chip electrophoresis combined PCR for rapid detect bacteria carrying New Delthi Metallo-bata-Lactamase(NDM-1).Methods A pair of specific primers were designed according to the gene of NDM-1,the capillary oscillatory flow PCR was used to amplify80strains of drug-resistant Enterobacter and60strains of Acinetobacter baumannii cultures,and the PCR products were rapidly detected by chip electrophoresis.The NDM-1positive samples were sequenced and validated.At the same time,the quantitative gradient dilution of NDM-1positive bacterial suspension was carried out by PCR,and the sensitivity of DNA-free extraction of PCR was investigated.Results Two positive samples were detected by capillary polymerase chain reaction chip electrophoresis.The product was confirmed to carry NDM-1gene by sequencing.The positive rate was100%.The rapid isolation and detection of NDM-1-resistant bacterial amplification products were achieved within40min.The bacterial detection line was1.15×101CFU/mL.Conclusion Capillary PCR chip electrophoresis is a rapid and inexpensive method with high accuracy and specificity for NDM-1gene detection.It is suitable for early and rapid field diagnosis of NDM-1positive bacteria.
作者
王秋平
WANG Qiuping(Department of Laboratory Medicine,the First Affiliated Hospital of University ofSouth China,Hengyang,Hunan 421001,China)
出处
《检验医学与临床》
CAS
2018年第24期3674-3677,3681,共5页
Laboratory Medicine and Clinic
基金
湖南省教育厅科学研究资助项目(16C1390)
