多通道Taqman-探针荧光定量PCR鉴定MRSA方法的建立

目的 建立基于mec A/nuc/fem B三基因联合的Taqman-探针荧光定量PCR鉴定耐甲氧西林金黄色葡萄球菌(MRSA)的方法。方法 以常规检验标本中分离和采用VITEK 2 Compact微生物分析仪鉴定为凝固酶阳性的MRSA为研究对象,通过PrimerPremier5.0和Beacon Designer 7软件设计针对mec A/nuc/fem B特异性PCR引物及Taqman荧光探针,荧光探针5'端分别采用FAM,HEX及ROX标记,3'端采用BHQ1标记,在荧光定量PCR仪进行检测。结果 ①1 g/dl凝胶电泳结果显示mec A/nuc/fem B三个基因引物特异性较好,扩增出的条带分子量与预期分子量一致且未见非特异性扩增; ②在单管单通道及单管多通道的PCR检测中mec A/nuc/fem B均获得特异性扩增,且三个基因在单管多通道的PCR扩增效果与单管单通道的相类似。结论 成功建立了多通道Taqman-探针荧光定量PCR鉴定MRSA的方法,mec A/nuc/fem B三种基因联合检测可有效区分凝固酶阴性和阳性的MRSA,提高鉴定MRSA的准确率。

Establishment of Muti-channel Taqman-ProbeFluorescence Quantitative PCR Identification MRSA Method

Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR basing on mecA/nuc/fem B threegene combined detecting.Methods Taking the coagulase positive MRSA,which isolated from the clinical samples and confirmed by VITEK 2 compact microbial analyzer,as the research object,designed mecA/nuc/fem B specific PCR primers and Taqman fluorescent probe by bio-software PrimerPremier 5 and Designer Beacon 7,FAM,HEX and ROX markers were used to label the fluorescentprobe at 5',and the end of 3'was labeled with BHQ1,detected by fluorescencequantitative PCR instrment.Results ①1 g/dl gel electrophoresis results showed that the primer's specificity of mec A/nuc/fem B were good,and molecular weight of the amplification band consistent with the expectedmolecular weight and no non-specific amplification band.②Three genes were obtained specific amplification in a single tube single channel and single tube multiple channel detection in PCR, and the three gene amplification effect in a single tube single tube single channel and multichannel PCR similar.Conclusion Successfully established a method of multi channel Taqman-probe fluorescence quantitative PCR identification of MRSA,mec A/nuc/fem B combined detection can effectively differentiate coagulase negative and positive MRSA,improve the accuracy of identification.