Taishi K, Muraki C, Yamashita K, Kumagai S, Sato M, Zufan I, Otsuka N, Kagawa S, Matsuoka A
Clinical Laboratory, Hyogo College of Medicine, Nishinomiya.
Rinsho Byori. 1992 May;40(5):541-6.
Two commonly used diagnostic tests for Staphylococcus aureus (MRSA) are cultivation of bacteria from clinical samples on mannitol-salt agar plate or on MRSA-screen agar plate with oxacillin. However, the use of PCR and DNA probes is considered more rapid and sensitive, as staphylococcal cells have high resistance to beta-lactam due to the novel penicillin-binding protein, PBP2'. Therefore, three different pairs of DNA primers (P1F-P1R, P2F-P2R and P3F-P3R) complementary with unique regions of the MRSA PBP2' gene were synthesized for use in polymerase chain reactions with DNA of MRSA. Amplified target DNA of 466, 480 and 630 bp could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. When applied to pure cultures on the MRSA screen agar, the DNA probes detected MRSA in 180 of 196 (P1F-P1R), 72 of 83 (P2F-P2R) and 66 of 71 (P3F-P3R) culture-positive specimens (accuracy range, 86.7-93.0%), while 61.6-89.3% of MRSA were detectable in culture-positive specimens streaked on the mannitol-salt agar. When applied directly to clinical samples, this DNA probe assay detected MRSA in culture-positive specimens within an accuracy range of 50.0-79.3%. It was thus clear that comparison of cultivation and DNA hybridization results yields good correlation with respect to detection of MRSA. These results suggest that the DNA probe assay may be useful in complementing existing culture techniques.
两种常用的金黄色葡萄球菌（耐甲氧西林金黄色葡萄球菌，MRSA）诊断测试方法是在甘露醇盐琼脂平板或含苯唑西林的MRSA筛选琼脂平板上从临床样本中培养细菌。然而，由于葡萄球菌细胞因新型青霉素结合蛋白PBP2'对β-内酰胺具有高度抗性，因此使用聚合酶链反应（PCR）和DNA探针被认为更快速且灵敏。因此，合成了三对与MRSA的PBP2'基因独特区域互补的不同DNA引物（P1F-P1R、P2F-P2R和P3F-P3R），用于与MRSA的DNA进行聚合酶链反应。扩增得到的466、480和630 bp的目标DNA可在溴化乙锭染色的凝胶上分辨出来，并与偶联碱性磷酸酶的DNA探针杂交。当应用于MRSA筛选琼脂上的纯培养物时，DNA探针在196个培养阳性标本中的180个（P1F-P1R）、83个中的72个（P2F-P2R）和71个中的66个（P3F-P3R）中检测到MRSA（准确率范围为86.7 - 93.0%），而在甘露醇盐琼脂上划线的培养阳性标本中，61.6 - 89.3%的MRSA可被检测到。当直接应用于临床样本时，这种DNA探针检测法在培养阳性标本中检测MRSA的准确率范围为50.0 - 79.3%。因此很明显，在检测MRSA方面，培养结果与DNA杂交结果的比较具有良好的相关性。这些结果表明，DNA探针检测法可能有助于补充现有的培养技术。
