耐甲氧西林金黄色葡萄球菌微量凝集试验方法的建立及初步应用

Establishment and preliminary application of the microagglutination assay for detecting methicillin-resistant Staphylococcus aureus

为建立一种快速检测动物源耐甲氧西林金黄色葡萄球菌(MRSA)的方法,本研究以MRSA耐药性决定蛋白PBP2a的抗血清为检测抗体,通过对抗原浓度及反应温度等进行优化,确定最佳的反应条件后,建立了快速检测MRSA的微量凝集试验方法,并进行了敏感性、特异性、重复性试验以及药敏纸片法比较试验。其中,微量凝集试验最低检出MRSA的浓度为40亿/mL,该方法对MRSA的凝集反应具有良好的特异性,试验结果具有较好的重复性。采用该检测方法对分离自鸡、猪、家兔及小鼠的75株金黄色葡萄球菌进行检测,检出MRSA株占57.3%,经过与药敏纸片法检测进行比较,符合率为92%。该方法快速、准确、简单易行,适于临床或基层应用。

The objective of the study is to establish a method for rapid detection of methicillin-resistant Staphylococcus aureus （MRSA） in animals. The microagglutination assay was developed with the antiserum prepared from rabbits immunized by the purified recombinant MRSA PBP2a protein expressed in E.coli. Under the optimized conditions of rabbit antiserum dilution, bacteria concentration and reaction temperature, the results showed that the assay was specific and reproductive, with a detection limit of 4xl09 cfu/mL of MRSA, but no cross-reactions with other related bacteria. In addition, Application of this method to 75 isolates of S.aureus isolated from chickens, pigs, rabbits and mice indicated that 57.3% of the isolates were resistant to methicillin, which was 92% accordant with the disk diffusion method. Therefore, the rapid detection method is suitable for clinical or on-site application.