毛细管直接PCR方法快速鉴定NDM-1阳性细菌  

NDM-1(+) bacteria screening using capillary PCR and direct PCR amplification method

作　　者：周小棉[1] 刘大渔[1] 黄华霖[2] 

机构地区：[1]广州医学院附属广州市第一人民医院检验科,510180 [2]广州医学院检验系,2006级广州510182

出　　处：《国际检验医学杂志》2011年第13期1404-1406,共3页International Journal of Laboratory Medicine

基　　金：国家自然基金资助项目(30870753);科技部国际合作项目(2010DFB3380);广东省自然基金资助项目(9151008901000190);广东省医学科研基金资助项目(A2009518;A2010458);广州市医药卫生科技重点项目(2009-ZDi-10;2009-ZDi-21);广州市科信局重大科技专项资助项目(2010U1-E00681);科技支撑计划资助项目(2010J-E241-1)

摘　　要：目的建立一种无需核酸提取预处理的毛细管PCR方法,用于新德里金属酰胺酶(NDM-1)阳性细菌的快速鉴定。方法考察在直接加入细菌培养液条件下,两种改良型DNA聚合酶Terra和MightyAmp DNA聚合酶对NDM-1编码基因的扩增效果。选用其中一种DNA聚合酶进行毛细管PCR,确定其对于NDM-1阳性细菌的检测限。结果当分别向25 μLPCR反应体系加入1、3、5、7μL细菌培养液条件下,两种DNA聚合酶均能有效扩增NDM-1编码基因序列,Terra DNA聚合酶效果较好。采用两步法毛细管PCR扩增NDM-1编码基因,3 μL反应体积条件下,40个循环反应在20 min内完成。结论应用改良型DNA聚合酶可以免去细菌核酸纯化步骤直接进行PCR。免核酸提取扩增结合毛细管PCR方法可以显著缩短核酸分析时间,适合于NDM-1阳性细菌的快速鉴定。Objective To develop a DNA extraction-free in-capillary PCR method for the fast identification of bacteria carrying New Delthi Metallo-bata-Lactamase（NDM-1）.Methods Amplification yields using two DNA polymerases developed for DNA extraction-free PCR were investigated,respectively.The limit-of-detection of this method was also identified.Results The two DNA polymerases could amplify directly from culture medium containing bacteria.Coarse bacteria sample could be added up to 30% the reaction volume and showed no inhibitory effect.The NDM-1 encoding gene was analyzed using a two-step capillary PCR method.With 3 μL reaction volume,an 40-cycle DNA amplification was completed in 20 minutes.Conclusion The improved DNA polymerase allows to skip DNA extraction and purification,and go directly to PCR amplification with coarse bacteria sample.The analysis time was greatly shortened using the method combined capillary PCR and direct PCR Amplification.The short analysis time,simplified operation and low sample consumption render the micro-device a potential point-of-care platform for NDM-1（＋） bacteria screening.

关 键 词：聚合酶链反应 基因 诊断 新德里金属酰胺酶 

分 类 号：R440[医药卫生—诊断学]