The study with rapid detection for the toxins of Methicillin-resistant Staphylococcus aureus and with the action of the toxins

1.建立了葡萄球菌肠毒素和TSST-1基因的多聚酶链式反应检测方法。制备了5对寡核苷酸引物(ENT、A-D和TST)，并对临床分离的金黄色葡萄球菌进行了检测。用聚合酶链式反应和免疫学方法检测A、B、D和TST的结果基本一致。而对免疫学方法未检测到肠毒素C的金黄色葡萄球菌，用聚合酶链式反应检测到Ent C基因。结论：1.术后MRSA肠炎病原菌的毒力类型依次为A、C和TST1。然后，直接从临床标本(痰和胃液)中提取葡萄球菌DNA，进行mecA和TST的聚合酶链式反应。检测MRSA最小菌落数的灵敏度为MecA约800CFU，TST约200CFU。新西兰大白兔注射TSST-1。监测他们的动脉血压，并检查他们的白细胞计数。观察动脉血压下降和白细胞计数下降的情况。但用生物法检测不到血清中的肿瘤坏死因子和白介素1。我们检测了在不同条件(单核细胞、单核细胞、单核细胞)、单核细胞(单核细胞)、单核各培养上清液中的肿瘤坏死因子水平无明显差异。

1. We have established the method for detection of genes for staphylococcal enterotoxins and TSST-1 by the polymelase chain reaction (PCR). Five pairs (ent A - D and tst) of oligonucleotid primers were made and tested with clinical isolated strains of S.aureus. The results using PCR and immunological method are almost same within ent A, B, D and tst. But ent C genes were detected by PCR with the strain of S.aureus whose enterotoxin C were not detected by immunological method. The toxical type of most causative strains of post operative MRSA enteritis was ent A, C and tst.2. Next, PCR for mec A and tst was performed with directly extracted staphylococcal DNA from clinical samples (sputum and gastric juice). The sensibilitys for the detection of minimal CFU of MRSA were about 800 CFU for mec A and about 200 CFU for tst.3. New Zealand white Rabbits were injected with TSST-1. Their arterial blood pressure were monitored and their white blood cell count was examined. The decline of the arterial blood pressure and decrease of white blood cell count were observed. But we could not detect serum TNF and IL-1 with bioassay.4. We examined TNF production from human isolated monocyte (Mo) with stimulation of TSST-1 or LPS under various conditions (Mo, Mo + Tcell, Mo + IL-2, Mo + INF gamma, mono nuclear cells). There was no significant deferenciation of TNF level between each culture supernatant.