Quantitative reverse transcription-PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus
Quantitative reverse transcription-PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus
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DOI：
10.1111/j.1365-2672.2009.04476.x
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发表时间：
2010-03-01
影响因子：
4
通讯作者：
Nomoto, K.
中科院分区：
文献类型：
--
作者：
Wada, M.;Lkhagvadorj, E.;Nomoto, K.
目的：评估一种用于快速检测耐甲氧西林金黄色葡萄球菌（MRSA）的新型定量逆转录 - 聚合酶链反应（qRT - PCR）检测方法。
方法与结果：新设计了针对16S rRNA基因、spa基因和mecA基因的葡萄球菌特异性区域的引物。从肉汤培养菌株中提取的RNA通过针对每种引物的qRT - PCR进行检测，所获得的细菌计数与平板计数法所得计数相关性良好，检测限分别为100、10¹和10²菌落形成单位（CFU）。针对16S rRNA的qRT - PCR检测比qPCR检测灵敏6430倍或更多。所有受试的金黄色葡萄球菌菌株均被检测到，其他受试的葡萄球菌属和种的菌株与针对spa基因的检测均无交叉反应。所有受试的MRSA均被针对mecA基因的检测方法检测到。我们用该检测方法对临床样本、粪便样本和支气管冲洗液进行检测，在6小时内以高灵敏度检测到了MRSA。
结论：我们针对靶基因的三种新引物的qRT - PCR检测方法是一种直接从临床样本中检测MRSA的快速且灵敏的工具。
研究的意义与影响：由于其灵敏性和快速性，我们的qRT - PCR检测方法被认为是临床管理的一种有价值的工具。
Aim:To evaluate a new quantitative reverse transcription-PCR (qRT-PCR) assay for the rapid detection of methicillin-resistant Staphylococcus aureus (MRSA).Methods and Results:Primers for Staphylococcus-specific regions of 16S rRNA gene, spa gene and mecA gene were newly designed. RNAs extracted from broth-cultured strains were tested by qRT-PCR targeting each primer, and the bacterial counts obtained correlated well with those counted by the plating method with detection limits of 100, 101 and 102 CFU. The qRT-PCR assay targeting the 16S rRNA was 6430-fold or more sensitive than qPCR assay. All Staph. aureus strains tested were detected and none of the other Staphylococcus species and genus strains tested cross-reacted with the assay targeting the spa gene. All MRSAs tested were detected by the assay targeting the mecA gene. Clinical samples, faecal material and bronchial washout solutions were tested by our assay, and MRSAs were detected with a high sensitivity within 6 h.Conclusion:Our qRT-PCR assay targeting three new primers to the target genes is a rapid and sensitive tool for the detection of MRSA directly from clinical samples.Significance and Impact of the Study:Because of its sensitivity and rapidity, our qRT-PCR assay is considered to be a valuable tool for clinical management.