[1]陈昌国,李艳君,郭建巍,等.多通道Taqman-探针荧光定量PCR 鉴定MRSA方法的建立[J].现代检验医学杂志,2016,31(03):22-25.[doi:10.3969/j.issn.1671-7414.2016.03.007]
　CHEN Chang-guo,LI Yan-jun,GUO Jian-wei,et al.Establishment of Muti-channel Taqman-Probe Fluorescence Quantitative PCR Identification MRSA Method[J].Journal of Modern Laboratory Medicine,2016,31(03):22-25.[doi:10.3969/j.issn.1671-7414.2016.03.007]
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多通道Taqman-探针荧光定量PCR 鉴定MRSA方法的建立()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第31卷
期数:
2016年03期
页码:
22-25
栏目:
论著
出版日期:
2016-06-15

文章信息/Info

Title:
Establishment of Muti-channel Taqman-Probe Fluorescence Quantitative PCR Identification MRSA Method
文章编号:
1671-7414(2016)03-022-04
作者:
陈昌国; 李艳君; 郭建巍; 陈秋圆; 刘 敏; 马志家; 郝秀红; 赵强元
中国人民解放军海军总医院检验科,北京 100048
Author(s):
CHEN Chang-guo; LI Yan-jun; GUO Jian-wei;  CHEN Qiu-yuan; LIU Min; MA Zhi-jia; HAO Xiu-hong; ZHAO Qiang-yuan
Department of Clinical Laboratory,Navy General Hospital,PLA.,Beijing 100048,China
关键词:
Taqman-探针;  荧光定量PCR;  耐甲氧西里金黄色葡萄球菌;  基因;  联合检测
分类号:
Q503
DOI:
10.3969/j.issn.1671-7414.2016.03.007
文献标志码:
A
摘要:
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。
Abstract:
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.

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备注/Memo

备注/Memo:
基金项目:国家自然基金项目(编号:81401311); 首都临床特色应用研究,立项编号:Z141107006614009; 院内创新基金,立项编号:CXPY201412。 作者简介:陈昌国(1979-),男,博士,主管技师,从事分子诊断工作,研究方向:肿瘤免疫及新型血清肿瘤标志物的研究,Tel:010-66951224,E-mail:1234_chen@sina.com。
更新日期/Last Update: 2016-06-25