Ding Xiaoyan, Wang Hejia, Cui Mingquan, Cheng Min, Zhao Qi, Bai Yuhui, Zhang Chunping, Zhang Cunshuai, Xu Shixin, Li Ting
Key Laboratory of Animal Antimicrobial Resistance Surveillance, Ministry of Agriculture and Rural Affairs, National Reference Laboratory of Veterinary Drugs Residues, China Institute of Veterinary Drug Control, Beijing 100081, China.
Microorganisms. 2022 Nov 28;10(12):2351. doi: 10.3390/microorganisms10122351.
Methicillin-resistant staphylococcus aureus (MRSA) is a major pathogen responsible for human hospital and community-onset diseases and severe invasive livestock infections. Rapid detection of MRSA is essential to control the spread of MRSA. Conventional identification methods and antibacterial susceptibility tests of MRSA are time-consuming. The commonly used qPCR assay also has the disadvantages of being complicated and expensive, restricting its application in resource-limited clinical laboratories. Here, a real-time fluorescent recombinase-assisted amplification (RAA) assay targeting the most conserved regions within the mecA gene of MRSA was developed and evaluated to detect MRSA. The detection limit of this assay was determined to be 10 copies/reaction of positive plasmids. The established RAA assay showed high specificity for MRSA detection without cross-reactivities with other clinically relevant bacteria. The diagnostic performance of real-time RAA was evaluated using 67 clinical S. aureus isolates from dairy farms, which were detected in parallel using the TaqMan probe qPCR assay. The results showed that 56 and 54 samples tested positive for MRSA by RAA and qPCR, respectively. The overall agreement between both assays was 97.01% (65/67), with a kappa value of 0.9517 (p < 0.001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R2 = 0.9012, p < 0.0001), indicating that this RAA assay possesses similar detection performance to the qPCR assay. In conclusion, our newly established RAA assay is a time-saving and convenient diagnostic tool suitable for MRSA detection and screening.
耐甲氧西林金黄色葡萄球菌（MRSA）是导致人类医院感染和社区感染疾病以及严重侵袭性家畜感染的主要病原体。快速检测MRSA对于控制其传播至关重要。MRSA的传统鉴定方法和抗菌药敏试验耗时较长。常用的qPCR检测方法也存在操作复杂、成本高昂的缺点，限制了其在资源有限的临床实验室中的应用。在此，开发并评估了一种针对MRSA的mecA基因最保守区域的实时荧光重组酶介导的扩增（RAA）检测方法，用于检测MRSA。该检测方法的检测限确定为阳性质粒10拷贝/反应。所建立的RAA检测方法对MRSA检测具有高度特异性，与其他临床相关细菌无交叉反应。使用来自奶牛场的67株临床金黄色葡萄球菌分离株评估实时RAA的诊断性能，同时使用TaqMan探针qPCR检测方法进行平行检测。结果显示，RAA和qPCR分别检测出56份和54份样本为MRSA阳性。两种检测方法的总体一致性为97.01%（65/67），kappa值为0.9517（p<0.001）。进一步的线性回归分析表明，两种检测方法的检测结果显著相关（R2 = 0.9012，p<0.0001），表明该RAA检测方法具有与qPCR检测方法相似的检测性能。总之，我们新建立的RAA检测方法是一种省时便捷的诊断工具，适用于MRSA的检测和筛查。
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