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目的了解广州地区儿童分离株青霉素耐药肺炎链球菌(PNSP)的分子流行病学,为肺炎链球菌感染性疾病防控提供实验依据。方法设计特异性引物,PCR扩增PNSP青霉素结合蛋白基因PBP1A、PBP1B、PBP2A、PBP2B、PBP2X、PBP3,进行测序分析。并使用Hinf I对PCR产物进行酶切,分析其限制性片段性长度多态性(RFLP)。结果成功提取PNSP的DNA、PCR结果显示:50株耐青霉素肺炎链球菌中:含PBP1A、PBP1B、PBP2A、PBP2B、PBP2X及PBP3的菌株阳性率分别为48.9%、64.4%、71.1%、31.1%、40.0%、31.1%。测序显示,它们与GenBank已知序列的同源性分别为99%、98%、100%、97%、95%、100%。使用Hinf I的RFLP分析显示:PBP1A、PBP1B、PBP2A、PBP3仅有一种基因型;PBP2B、PBP2X有两种基因型的,阳性率分别为71.4%、28.6%、66.7%、33.3%。结论广州肺炎链球菌儿童分离株耐青霉素基因的分布以PBP2A、PBP1B、PBP1A为主。使用Hinf I酶切,PBP2B、PBP2X具有两种基因型,其中优势型均大于65%。
Objective To understand the molecular epidemiology of penicillin-resistant Streptococcus pneumoniae (PNSP) isolated from children in Guangzhou and to provide experimental evidence for the prevention and control of pneumococcal infectious diseases. Methods Specific primers were designed to amplify the penicillin-binding protein PBP1A, PBP1B, PBP2A, PBP2B, PBP2X and PBP3 of PNSP by PCR. The PCR products were digested with Hinf I and analyzed for restriction fragment length polymorphism (RFLP). Results The DNA of PNSP was successfully extracted. The results of PCR showed that the positive rates of the strains containing PBP1A, PBP1B, PBP2A, PBP2B, PBP2X and PBP3 were 48.9%, 64.4%, 71.1% and 31.1% respectively in the 50 strains of penicillin - resistant Streptococcus pneumoniae. 40.0%, 31.1%. Sequencing showed that they shared 99%, 98%, 100%, 97%, 95% and 100% homology with the known sequences in GenBank, respectively. RFLP analysis using Hinf I showed that there was only one genotype of PBP1A, PBP1B, PBP2A and PBP3, and two genotypes of PBP2B and PBP2X, with positive rates of 71.4%, 28.6%, 66.7% and 33.3%, respectively. Conclusion The distribution of penicillin-resistant gene of Streptococcus pneumoniae isolates in Guangzhou is mainly PBP2A, PBP1B and PBP1A. Using Hinf I digestion, PBP2B and PBP2X have two genotypes, of which dominance is> 65%.