Laboratory Medical Science Cluster, Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sungai Buloh, Malaysia.
Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kota Bharu, Malaysia.
J Microbiol Immunol Infect. 2014 Dec;47(6):484-90. doi: 10.1016/j.jmii.2013.06.004. Epub 2013 Aug 6.
BACKGROUND/PURPOSE: Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for significant numbers of nosocomial and community-acquired infections worldwide. Molecular diagnosis for MRSA nasal carriers is increasingly important for rapid detection and screening of MRSA colonization because the conventional methods are time consuming and labor intensive. However, conventional polymerase chain reaction (PCR) tests still require cold-chain storage as well as trained personnel, which makes them unsuitable for rapid high-throughput analysis. The aim of this study was to develop a thermostabilized PCR assay for MRSA in a ready-to-use form that requires no cold chain.
The thermostabilized PCR assay detects the following targets simultaneously: (1) 16S rRNA of the Staphylococcus genus; (2) femA gene specific for S. aureus; (3) mecA gene conferring methicillin resistance; and (4) lukS gene, which encodes the virulent toxin. The thermostabilized PCR incorporates an internal amplification control that helps to verify the presence of PCR inhibitors in samples. PCR reagents and specific primers were lyophilized into a pellet form with an enzyme stabilizer.
The PCR was validated with 235 nasal swabs specimens and was found to be 100% sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 6 months at 24 °C. The limit of detection of thermostabilized PCR assay was determined by probit regression (95% confidence interval) was 10(6) colony forming units at the bacterial cell level and 10 ng of DNA at the genomic DNA level, which is comparable with conventional PCR methods.
A rapid thermostabilized PCR assay that requires minimal pipetting steps and is cold chain-free was developed for detecting MRSA nasal carriers.
背景/目的：耐甲氧西林金黄色葡萄球菌（MRSA）是一种主要的病原体，在全球范围内导致了大量的医院内和社区获得性感染。对于 MRSA 鼻腔携带者的分子诊断对于快速检测和筛查 MRSA 定植越来越重要，因为传统方法既费时又费力。然而，常规聚合酶链反应（PCR）检测仍然需要冷链储存以及经过培训的人员，这使得它们不适合快速高通量分析。本研究的目的是开发一种即用型的热稳定 PCR 检测方法，用于 MRSA 的检测，无需冷链。
该热稳定 PCR 检测方法同时检测以下靶标：（1）葡萄球菌属 16S rRNA；（2）金黄色葡萄球菌特异性 femA 基因；（3）赋予甲氧西林耐药性的 mecA 基因；（4）编码毒性毒素的 lukS 基因。热稳定 PCR 检测方法包含一个内部扩增对照，有助于验证样品中是否存在 PCR 抑制剂。PCR 试剂和特异性引物被冻干成含有酶稳定剂的颗粒形式。
该 PCR 方法用 235 份鼻腔拭子标本进行了验证，发现其具有 100%的敏感性和特异性。使用 Q10 法评估了热稳定 PCR 的稳定性，发现其在 24°C 下约可稳定保存 6 个月。热稳定 PCR 检测方法的检测限通过概率单位回归（95%置信区间）确定，在细菌细胞水平为 10（6）菌落形成单位，在基因组 DNA 水平为 10ng DNA，与常规 PCR 方法相当。
开发了一种快速的、热稳定的、无需冷链的 PCR 检测方法，用于检测 MRSA 鼻腔携带者。
