实时荧光PCR直接检测样本中耐甲氧西林金黄色葡萄球菌应用研究  

Application of Real-time Fluorescence PCR Technique to Detect Samples of Methicillin-resistant Staphylococcus Aureus

作　　者：黄其建 张进[1] 李静[1] 丁燕[1] 曾韵[1] 王雅杰 喻华[2] 肖华亮[1] 吕云霞[1] 

机构地区：[1]四川省什邡市人民医院检验科,四川什邡618400 [2]四川省人民医院检验科,四川成都610072 [3]北京天坛医院临床医学实验室,北京

出　　处：《标记免疫分析与临床》2016年第2期194-196,222,共4页Labeled Immunoassays and Clinical Medicine

基　　金：德阳市科技局立项项目(2013SZ002)

摘　　要：目的探讨实时荧光PCR直接检测病人样本中耐甲氧西林金黄色葡萄球菌的应用价值。方法对80例经VITEK2COMPACT药敏鉴定系统鉴定为有金黄色葡萄球菌生长(其中MRSA40例,MSSA40例)及20例未检出金黄色葡萄球菌的患者样本进行实时荧光PCR扩增,检测样本中金黄色葡萄球菌特异性核酸片段(耐热核酸酶基因nuc)及耐药基因(mec A)并与药敏鉴定比较,判断实时荧光PCR法检测MRSA的敏感性及特异性;对于两种方法检测结果不一致的18例样本重新接种培养,收集金黄色葡萄球菌菌株进行实时荧光PCR扩增,检测nuc基因及mec A基因,以期发现两种方法检测结果差异原因。结果 100例临床样本两种方法检测结果一致共82例,其中MRSA一致40例,MSSA一致22例,非金葡20例;两种方法检测结果不一致18例,均为PCR基因检测为MRSA而药敏鉴定为MSSA。MRSA检测药敏鉴定阳性40例,检出率为40%,PCR基因检测阳性58例检出率为58%;PCR基因检出率明显高于药敏鉴定,二者结果差异有显著性(P<0.05);以药敏鉴定为标准,PCR基因检测MRSA的敏感性为100%,特异性为70%;18例金黄色葡萄球菌菌株实时荧光PCR检测MRSA结果与药敏鉴定一致。结论实时荧光PCR直接检测患者样本中MSSA具有高度敏感性及特异性,但检测MRSA会受到样本中其他含有mec A基因菌的干扰,对于临床结果解释需慎重。即便如此,该方法对于MRSA检测在院感防控方面仍有重要意义。Objective To explore the value of real-time fluorescent PCR in the detection of methicillin-resistant staphylococcus aureus in patient’ s samples. Methods 80 cases of samples by using VITEK2-COMPACT automatic bacteria identification and drug susceptibility analysis system identified staphylococcus aureus（ 40 cases of MRSA,40 cases of MSSA）,and 20 cases of samples who were not detected staphylococcus aureus were carried out the real-time fluorescent PCR amplification. The staphylococcus aureus DNA fragments（ heat resistant nucleic acid enzyme gene-nuc） and the drug resistance gene（ mec A） in samples were detected,and the specificity and susceptibility of real-time fluorescent PCR for MRSA were analyzed. Results 82 cases of samples detection results with two methods were consistent in total 100 cases of clinical samples,including 40 cases of MRSA,22 cases of MSSA and 20 cases of non-bacteria staphylococcus aureus. As to MRSA detection,40 cases of samples were tested as positive by drug susceptibility method with 40% detection rate,and 58 cases of samples were tested as positive by PCR technique with 58% detection rate. The detection rate with PCR technique was significantly higher than that of drug susceptibility method（ P 〈 0. 05）. On the basis of drug sensitivity evaluation,the sensitivity of the PCR technique for MRSA detection was 100%,and the specificity was 70%. Conclusion The real-time fluorescent PCR has high sensitivity and specificity in the direct detection of MSSA in patient samples,but detection of MRSA could be affected by other interference with mec A gene bacteria in the samples. The method for detection of MRSA in hospital still has important significance in prevention and control of MRSA.

关 键 词：耐甲氧西林 金黄色葡萄球菌 MEC A基因 nuc基因 实时荧光PCR 

分 类 号：R440[医药卫生—诊断学]