Jantorn Pavarish, Nualla-Ong Aekkaraj, Sotthibandhu Dennapa Saeloh
Division of Biological Science, Faculty of Science, Prince of Songkla University, Songkhla, 90110, Thailand.
Faculty of Medical Technology, Prince of Songkla University, Songkhla, 90110, Thailand.
Vet Res Commun. 2025 Jan 13;49(2):74. doi: 10.1007/s11259-024-10638-z.
Staphylococcus pseudintermedius is a global animal pathogen. Traditional identification methods are time-consuming necessitating a more efficient approach. This study validated and enhanced the loop-mediated isothermal amplification (LAMP) technique by integration it with a lateral flow dipstick (LFD) assay for the detection of S. pseudintermedius and methicillin-resistant S. pseudintermedius (MRSP) strains. Conventional identification methods were compared with LAMP and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were tested for MRSP detection using oxacillin and cefoxitin disk diffusion tests alongside the LAMP assay targeting the mecA gene, a marker for methicillin resistance. Results showed that LAMP combined with LFD effectively detected S. pseudintermedius and MRSP. This study identified 53 isolates as S. pseudintermedius by conventional and LAMP methods, with MALDI-TOF MS correctly identifying 39.62% (21/53). The mecA gene, crucial for methicillin resistance, was detected in all PCR-positive isolates (n = 33) by LAMP, while the disk diffusion method identified 69.70% (23/33) of mecA-positive strains. LAMP and conventional methods exhibited superior accuracy, sensitivity, and specificity (all 100%) compared to MALDI-TOF MS, which showed lower sensitivity (39.62%) for S. pseudintermedius identification. Similarly, the LAMP assay demonstrated higher accuracy, sensitivity, and specificity (all 100%) for MRSP detection compared to the disk diffusion method (83.33%, 69.70%, and 94.87%, respectively). The LAMP assay coupled with the LFD method proved suitable for routine bacterial identification in laboratories, offering adequate sensitivity and specificity with simple steps and short reaction time.
中间型假丝酵母葡萄球菌是一种全球性的动物病原体。传统的鉴定方法耗时较长，因此需要一种更高效的方法。本研究通过将环介导等温扩增（LAMP）技术与侧向流动试纸条（LFD）检测法相结合，验证并改进了该技术，用于检测中间型假丝酵母葡萄球菌和耐甲氧西林中间型假丝酵母葡萄球菌（MRSP）菌株。将传统鉴定方法与LAMP和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行了比较。使用苯唑西林和头孢西丁纸片扩散试验以及针对mecA基因（耐甲氧西林的标志物）的LAMP检测法对分离株进行MRSP检测。结果表明，LAMP与LFD相结合能够有效检测中间型假丝酵母葡萄球菌和MRSP。本研究通过传统方法和LAMP方法鉴定出53株分离株为中间型假丝酵母葡萄球菌，MALDI-TOF MS正确鉴定出39.62%（21/53）。LAMP在所有PCR阳性分离株（n = 33）中均检测到了对耐甲氧西林至关重要的mecA基因，而纸片扩散法鉴定出69.70%（23/33）的mecA阳性菌株。与MALDI-TOF MS相比，LAMP和传统方法在鉴定中间型假丝酵母葡萄球菌时表现出更高的准确性、敏感性和特异性（均为100%），MALDI-TOF MS对中间型假丝酵母葡萄球菌的敏感性较低（39.62%）。同样，与纸片扩散法（分别为83.33%、69.70%和94.87%）相比，LAMP检测法在检测MRSP时也表现出更高的准确性、敏感性和特异性（均为100%）。LAMP检测法与LFD方法相结合被证明适用于实验室常规细菌鉴定，具有足够的敏感性和特异性，步骤简单且反应时间短。
