摘要
目的构建mecA、nuc、内标基因3种基因联合检测的单管多通道Taqman探针荧光定量PCR法,用于鉴定耐甲氧西林金黄色葡萄球菌(MRSA),并与传统细菌培养和鉴定方法进行比较,评估其临床应用价值。方法以169例临床标本和经常规细菌培养和VITEK2 Compact全自动微生物分析仪鉴定的金黄色葡萄球菌(SA)156株[其中MRSA 106株、甲氧西林敏感的金黄色葡萄球菌(MSSA)50株]作为研究对象;用Primer premier 5.0和Beacon Designer 7.0软件进行PCR引物和Taqman探针设计,探针5′端分别标记FAM、Cy5和VIC,3′端标记MGB,用荧光定量PCR仪进行检测。169例临床标本同时也做了常规细菌培养和鉴定。结果169例临床标本中,常规方法鉴定出MRSA 48例,检出率为28.40%;多重荧光定量PCR检测鉴定出MRSA 53例,检出率31.36%。2种方法比较,差异无统计学意义(P>0.05)。156例SA的检测中,使用常规方法鉴定出MRSA 106例、MSSA 50例,MRSA检出率为67.95%;多重荧光定量PCR检测法鉴定出MRSA 108例、MSSA 48例,MRSA检出率为69.23%;两者比较差异无统计学意义(P>0.05)。结论多重荧光定量PCR检测法提高了MRSA的检出率,减少了漏检风险,检测时间较常规方法明显缩短,可用于临床MRSA的快速鉴定。
Objective To establish the method of identifying MRSA with Taqman-fluorescence quantitative PCR based on the detection for mecA,nuc,endogenous reference gene,which is a three-gene combined detection in a single tube multiple channel,and compare with the traditional bacterial culture and identification method to evaluate its clinical application value for the rapid detection of MRSA.Methods A total of 169 clinical samples and 156 SA strains were collected in the study.The SA strains were isolated from clinical samples and confirmed by VITEK2 Compact microbial analyzer.MecA,nuc,endogenous reference gene PCR primers and Taqman fluorescent probes were designed by using Primer premier 5.0 and Beacon Designer 7.0.FAM,Cy5 and VIC markers were used to label the fluorescent probe at 5′end,and 3′end was labeled with MGB,and the detection was carried out in the fluorescent quantitative PCR instrument,while the 169 cases of clinical samples were also cultured and tested by the traditional method.Results The traditional method confirmed 48 cases of MRSA,and the detection rate was 28.40%,while the multiple fluorescent quantitative PCR detected 53 cases of MRSA,and the detection rate was 31.36%.There was no significant difference between traditional method and Taqman-fluorescence quantitative PCR(P>0.05).In 156 SA strains,the traditional methord confirmed 106 strains of MRSA,and the detection rate was 67.95%,while the multiple fluorescence quantitative PCR detected 108 strains of MRSA,and the detection rate was 69.23%.There is also no significant difference between traditional method and Taqman-fluorescence quantitative PCR(P>0.05).Conclusion Taqman-fluorescence quantitative PCR improved positive detection rate and reduced the risk of missing detection of MRSA,it is much more time-saving than tradition method,which can be used in rapid clinical identification of MRSA.
作者
赵峻英
席家庄
谢铌奇
潘莉娟
王杉
董剑
ZHAO Junying;XI Jiazhuang;XIE Niqi;PAN Lijuan;WANG Shan;DONG Jian(Department of Clinical Laboratory,People′s Hospital of Dazu District,Chongqing 402360,China)
出处
《国际检验医学杂志》
CAS
2021年第4期402-404,411,共4页
International Journal of Laboratory Medicine
基金
重庆市科卫联合基金项目(2018MSXM059)。
作者简介
赵峻英,女,主管技师,主要从事临床微生物检验的相关研究;通信作者:董剑,E-mail:dongyjian163.com。
