【作者】 李杰；

【导师】 罗予；

【作者基本信息】 郑州大学 ， 免疫学， 2011， 硕士

【摘要】 背景与目的金黄色萄萄球茵(Staphylococcus aureus,SA)是人类和动物化脓性感染中最常见的病原菌,可引起食物中毒、化脓性感染、骨髓炎、毒素休克综合征、肺炎和心内膜炎等一系列不同程度的感染。一个不太严重的伤口感染也可由于该菌的存在发展为恶性的败血症、骨髓炎、脓毒性关节炎、心内膜炎等。近年来,随着抗生素的大量使用,金黄色葡萄球菌耐药菌株越来越多,其中,耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRS A)已经在全世界范围内流行,据文献报道,MRSA分离率已高达40%。因此,发展一种快速特异地检测金黄色萄萄球茵的方法成为必要。目前,金黄色萄萄球茵的检测方法多种多样。细菌分离培养是最常规的方法,是微生物检测的“金标准”,该法虽然可靠,但耗时、费力不能满足快速检测的需要；免疫学方法主要有免疫荧光法(IFA)、放射免疫法(RIA)和酶联免疫法(ELISA)等。有文献指出,ELISA方法检测肠毒素B的灵敏度为0.078μg/L,这些方法虽然具有一定特异性和敏感性,但通常需要结合细菌分离才能进行,也难满足快速检测需要；常规PCR法快速、特异、灵敏度很高,少至10cfu/ml的金黄色葡萄球菌也能被检测出来,但由于死菌中的DNA降解较慢,不能区分死菌和活菌。然而,对于外伤性眼内炎等某些感染性疾病来说,由于其感染部位比较特殊,明确标本中的细菌死活可以为临床提供更有针对性的治疗,探讨一种新的简便、快速、灵敏、特异并能区分细菌死活的检测方法成为必要。目前,关于细菌性眼内炎的报道中,革兰阳性球菌占大多数,并呈现逐年上升趋势。近期,在本实验室接收的眼内炎标本中,同样是革兰氏阳性球菌居多数。因此,实验选用金黄色葡萄球菌为目的菌,针对其mRNA利用RT-PCR方法进行检测。目的在于探讨RT-PCR检测金黄色葡萄球菌区分死菌和活菌的有关问题,为临床合理用药提供依据,避免抗菌药的滥用,减少耐药菌株的出现。方法将所获细菌包括金黄色葡萄球菌标准株(ATCC25923),临床分离株以及非金葡菌均通过常规表型方法验证,增菌后确定为纯培养的细菌,接种于营养肉汤。1.平板活菌计数法计数单菌落增菌后的金黄色葡萄球菌标准株(ATCC25923)及单个细菌增菌后活菌计数。2.平板活菌计数法计数替考拉宁和万古霉素不同剂量下体外抗金黄色葡萄球菌杀菌曲线。3.药敏纸片法及普通PCR方法检测菌株是否为MRSA。4.普通PCR和RT-PCR比较筛选可用于RT-PCR的目的基因。5. RT-PCR检测金黄色葡萄球的检测限。6. RT-PCR检测金黄色葡萄球的特异性。7. RT-PCR检测金黄色葡萄球活菌的时效。结果1.耐药菌株鉴定20株金黄色葡萄球菌临床株中有12株细菌药敏纸片结果为MRSA,占60%,其中mecA基因阳性的有9株细菌。2.杀菌曲线从杀菌曲线可以看出,替考拉宁(100μg/ml)、万古霉素(100μg/ml)作用10h后可将细菌杀死；替考拉宁(10μg/ml)作用12h后、万古霉素(10μg/ml)作用14h后可将细菌杀死。3.引物确定在针对几种目的基因设计的引物中, spa基因特异性较好,被用于后续RT-PCR实验。4.灵敏度测定RT-PCR检测金黄色葡萄球的最小检出量为1.0×104cfu/ml;单个细菌增菌6h后可以用RT-PCR方法检测到目的条带。5.特异性检测所设计的引物序列对所选择的金黄色葡萄球菌具有特异性,而对所选用的大肠埃希菌,铜绿假单胞菌,表皮葡萄球菌和化脓性链球菌无特异性扩增。6.活菌检测时效对于金黄色葡萄球菌标准株(ATCC25923)和MRSA活菌,RT-PCR均可以检测出目的条带；而对于灭活后的细菌,ATCC25923在高压蒸汽灭活后2h不能用RT-PCR检测到,MRSA在高压蒸汽灭活后12h不能用RT-PCR检测到。小结1.本文所设计的引物可用于RT-PCR方法检测金黄色葡萄球菌活菌。2. RT-PCR方法在检测敏感和耐药的金黄色葡萄球菌活菌方面无差异,但对于高压蒸汽灭活后的细菌,两者检测的时效性不同。更多还原

【Abstract】 BackgroudStaphylococcus aureus(SA) is an important zoonotic pathogens, which can cause skin and soft tissue infections, food poisoning, traumatic eye and so on.. Bacteria culture is a conventional method of detection of S. aureus at present,and it is reliable, but,the method usually spends over 24h which can not meet rapid detection. Immunological methods include immunofluorescence, radioimmunoassay and enzyme-linked immunosorbent assay. Although these methods have certain specificity and sensitity, it usually requires combination of bacteria culture,and it is difficult to meet the need for clinically rapid detection. Conventional PCR is rapid,specific and highly sensitive. Because DNA degradation of dead bacteria is slow, this method can not distinguish between dead and viable bacteria.For traumatic endophthalmitis, however,it can provide many targeted clinical treatment,if we know whether the bacteria in the samples are live or dead. So,it becomes necessary to develop a new method, which is rapid, sensitive and specific. Especially,the method can distinguish live bacteria. Staphylococcal protein A(SPA),encoded by spa gene, is species-specific and genus-specific. SPA is a superficial protein of the cell wall. Presence of SPA is a characteristic of Staphylococcus aureus. Half-life of prokaryotic’s mRNA is very short.Detection of mRNA can distinguish between dead and viable cells.The experiment was done to detect Staphylococcus aureus by RT-PCR with a pair of primers of the spa gene.AimInvestigate some releted questions about viable detection of Staphylococcus aureus by RT-PCR method in order to provide theoretical basis for clinical therapy, to avoid the overuse of antibiotics and reduce the emergence of resistant strains.MethodsAll of bateria,including a standard Staphylococcus awreus(ATCC25923),some clinical strains of Staphylococcus aureus,and some clinical isolates of non-Staphylococcus aureus were verified by conventional phenotypic methods. All the bateria were inoculated in nutrient broth and identified as pure culture.1. One colony-forming unit(CFU) of Staphylococcus aureus (ATCC25923) was dissolved in 2ml nutrient broth and cultured at 35℃for 6-8h. Number of the above baterial broth was counted by plate medium. Meanwhile, the baterial broth was diluted into five concentrations and the dilutions were detected by RT-PCR assay.2. Staphylococcus. aureus(ATCC25923) was killed by different antibotics with different concentrations in vitro envioment. The baterial numbers after effecting certain time were counted by plate medium. The bactericidal concentration-time curves were descriped using the numbers.3. All the strains of Staphylococcus. aureus were detected by two methods to select MRSA.4. The target gene was selected to be used to RT-PCR by comparing RT-PCR with unival PCR using different genes.5. The sensitivity of that Staphylococcus aureus was detected by RT-PCR was measured.6. The specificity of that Staphylococcus aureus was detected by RT-PCR was evaluated.7. The strain of Staphylococcus. aureus(ATCC25923) and a strain of MRSA were distinguished between dead and viable cells by RT-PCR. Results1. Detection of MRS A 12 of 20 strains, accounting for 60%, of Staphylococcus. aureus were MRSA, and 9 of the 12 Staphylococcus. aureus strains’mecA gene were amplified.2. Selection of pairs of primers For the several pairs of primers designed, the pairs of primers of nuc and spa gene could be used to RT-PCR.Contrastly, the pair of primers of spa gene was for follow-up experiments.3. Measuring of sensitivity The lowest concentration of Staphylococcus. aureus detected by RT-PCR was 1.0×104cu/ml,and one single piece of bacterium could be detected by RT-PCR if it was enriched for over 6h.4. Evaluation of specificity All the strains of Staphylococcus. aureus were detected by RT-PCR using the pair of primers primers.There was no crossreaction with E. coli、Pseudomonas aeruginosa、Staphylococcus epidermidis、Pyogenic streptococcus.5. Distinguishment of viable and dead cells mRNA from S. aureus (ATCC25923) became undetectable when dead cells were stored at 4℃temperature for under 2h; for MRSA, mRNA couldn’t be detected when cells were inactivited for over 2h.Conclusion1. This pair of primers can be used to detect viable Staphylococcus aureus.2. The method can be used to detect viable MSSA and MRSA,but the mRNA of MRSA can be stored for longer time than MSSA after inactivation by High-pressure steam sterilization.更多还原