耐甲氧西林金黄色葡萄球菌的基因多态性分型研究

【摘要】  目的探索一种准确、快速、可靠的耐甲氧西林金黄色葡萄球菌(MRSA)的分子生物学分型方法，了解医院内MRSA的多态性，为临床合理、有效地使用抗生素和控制耐药基因在种群间的传播提供帮助。方法用MecA基因相关高变区PCR扩增(HVR-PCR)和随机扩增多态性DNA(RAPD)两种方法，对MRSA菌株进行多态性分析。HVR-PCR检测MecA基因相关高变区正相重复序列元件(DRUs)的长度，根据DRUs数目的不同对MRSA分型;RAPD根据电泳图谱中条带的数目及位置，经NTSYS统计软件进行聚类分析，得到遗传树状图，按MRSA菌株之间的相关性进行分型。结果应用HVR-PCR方法可将31株MRSA标本分为7型，分别含有7、8、9、10、11、12和16个DRUs，其中10DRUs型最多\[11/31(35.48%)\]，16DRUs型最少\[1/31(3.23%)\];应用RAPD分型，31株MRSA共分10型，其中以Ⅳ型为主\[10/31(32.26%)\]，其他型分布比较接近。在11株HVR-PCR分型的10DRUs型中RAPD Ⅳ型4株\[4/11(36.36%)\]，RAPD Ⅲ型和RAPD Ⅳ型各2株\[2/11(18.18%)\];10株RAPD Ⅳ型中9DRUs型、10DRUs型各4株\[4/10(40.00%)\]。结论RAPD方法和HVR-PCR方法对MRSA的分型率都比较高，而且这两种方法都比较简单、稳定、可靠、快速，实用性强，具有广阔的应用前景。

【关键词】  耐甲氧西林金黄色葡萄球菌;MecA基因;随机扩增多态性DNA;MecA基因相关高变区PCR扩增

　ABSTRACT: ObjectiveTo investigate the polymorphisms of methicillin-resistant Staphylococcus aureus (MRSA) in local hospitals in order to provide evidence for clinical doctors to use the antibiotics rationally and effectively.MethodsWe analyzed the polymorphisms of MRSA with themethods of PCR for mec-associated hypervariable region (HVR-PCR) and random amplifiedpolymorphic DNA (RAPD). MRSA strains typed by HVR-PCR according to the length polymorphisms which decided by the number of direct repeat unit elements (DRUs) of HVR-PCR products; Grouping of MRSA by RAPD was based on the dependability among different MRSA isolates.ResultsTotally 31 MRSA strains typed by HVR-PCR were divided into 7 types which contained 7, 8, 9, 10, 11, 12 and 16 DRUs. MRSA strains with 10 DRUs were most predominant, present in 11/31 cases (35.48%). Among the 31 MRSA strains typed by HVR-PCR, only 1 showed 16 DRUs. There were 10 types classed by RAPD of all MRSA strains.The dominant type was RAPD Ⅳ, present in 10/31 cases (32.26%). The others showed similar distribution.ConclusionRAPD and HVR-PCR methods not also possess a good ability to obtain interpretable results, but also are simple, stable, reliable and rapid. They both have perfect practicality and prospect of extensive application.

　　KEY WORDS: methicillin-resistant Staphylococcus aureus; mecA gene; random amplified polymorphic DNA; PCR for the mec-associated hypervariable region

　　葡萄球菌的分型方法很多，传统的分型方法包括血清学分型、抗生素分型等，但均无法满足精确辨别的要求。分子生物学技术的出现为这一领域带来了革命。目前，分子分型方法已成为研究细菌耐药性发生及传播的重要手段，常见的有：脉冲场凝胶电泳(pulsed-fiel gel electrophoresis, PFGE)，随机扩增DNA多态性(random amplified polymorphic DNA, RAPD)，mecA基因高变区长度多态性(PCR for the mec-associated hypervariable region, HVR-PCR)，荧光扩增片段长度多态性(fluorescent amplified fragment length polymorphrism, FAFLP)等。这些方法各有特点，应用于耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureu, MRSA)的分型在国内外均有报道，但这些方法尚未成熟，各地分型标准也不一样，且多采用单一的分型方法。RAPD 和HVR-PCR联合应用于MRSA多态性分型尚未见报道。本实验采用RAPD和HVR-PCR两种方法对MRSA菌株进行分型，了解其多态性分布状况、鉴别菌株之间的相关性，为合理、有效地使用抗生素和控制耐药基因在种群间的传播提供理论基础。

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