RAPID DETECTION OF THE MECA GENE IN METHICILLIN-RESISTANT STAPHYLOCOCCI BY ENZYMATIC DETECTION OF POLYMERASE CHAIN-REACTION PRODUCTS
RAPID DETECTION OF THE MECA GENE IN METHICILLIN-RESISTANT STAPHYLOCOCCI BY ENZYMATIC DETECTION OF POLYMERASE CHAIN-REACTION PRODUCTS
复制标题
DOI：
10.1128/jcm.30.7.1728-1733.1992
复制
发表时间：
1992-07-01
影响因子：
9.4
通讯作者：
KONNO, M
中科院分区：
文献类型：
--
作者：
UBUKATA, K;NAKAGAMI, S;KONNO, M
为简便、可靠地鉴定临床来源的耐甲氧西林葡萄球菌，应用聚合酶链反应（ED-PCR）进行酶法检测。ED-PCR是基于通过生物素-链霉亲和素亲和性捕获扩增产物，并用酶联抗体检测扩增产物中掺入的半抗原。为了鉴定所有种属的甲氧西林耐药葡萄球菌，针对mecA基因的150 bp片段进行ED-PCR。用一对生物素和二硝基苯酚5 ′-标记的引物进行PCR后，将反应混合物加到用链霉亲和素预包被的微量滴定孔中。此后，结合的PCR产物用碱性磷酸酶缀合的抗二硝基酚抗体进行比色检测。简化了从葡萄球菌细胞中提取DNA用于PCR的过程，使其可以在一个试管中进行。包括PCR在内的整个检测时间不到3 h。mecA基因检测的灵敏度范围为金黄色葡萄球菌>5 x 10（2）CFU/管至表皮葡萄球菌>5 x 10（3）CFU/管。对161株供试菌株进行ED-PCR基因分型，其中97株为沙门氏菌。金黄色葡萄球菌和64株凝固酶阴性葡萄球菌）与菌株对苯唑西林的表型敏感性进行比较。ED-PCR结果与苯唑西林的MIC值基本一致，只有一株S.金黄色葡萄球菌和2株凝固酶阴性葡萄球菌具有mecA基因，但与表型不符。还通过ED-PCR检测了55份血培养样本。对于33个培养物中的葡萄球菌分离株，苯唑西林MIC>4 μ g/ml; 33个葡萄球菌分离株中的31个通过ED-PCR确定为mecA基因阳性。这些结果表明，ED-PCR可以在临床微生物实验室中使用，具有合理的信心。
In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphatase-conjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from >5 x 10(2) CFU per tube for Staphylococcus aureus to >5 x 10(3) CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4-mu-g/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.