Dilhari Ayomi, Sampath Asanga, Gunasekara Chinthika, Fernando Neluka, Weerasekara Deepaka, Sissons Chris, McBain Andrew, Weerasekera Manjula
Department of Microbiology, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka.
Department of Surgery, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka.
AMB Express. 2017 Sep 19;7(1):179. doi: 10.1186/s13568-017-0477-z.
Infected chronic wounds are polymicrobial in nature which include a diverse group of aerobic and anaerobic microorganisms. Majority of these communal microorganisms are difficult to grow in vitro. DNA fingerprinting methods such as polymerase chain reaction-denaturation gradient gel electrophoresis (PCR-DGGE) facilitate the microbial profiling of complex ecosystems including infected chronic wounds. Six different DNA extraction methods were compared for profiling of the microbial community associated with chronic wound infections using PCR-DGGE. Tissue debris obtained from chronic wound ulcers of ten patients were used for DNA extraction. Total nucleic acid was extracted from each specimen using six DNA extraction methods. The yield, purity and quality of DNA was measured and used for PCR amplification targeting V2-V3 region of eubacterial 16S rRNA gene. QIAGEN DNeasy Blood and Tissue Kit (K method) produced good quality genomic DNA compared to the other five DNA extraction methods and gave a broad diversity of bacterial communities in chronic wounds. Among the five conventional methods, bead beater/phenol-chloroform based DNA extraction method with STES buffer (BP1 method) gave a yield of DNA with a high purity and resulted in a higher DGGE band diversity. Although DNA extraction using heat and NaOH had the lowest purity, DGGE revealed a higher bacterial diversity. The findings suggest that the quality and the yield of genomic DNA are influenced by the DNA extraction protocol, thus a method should be carefully selected in profiling a complex microbial community.
感染性慢性伤口本质上是多微生物的,其中包括多种需氧和厌氧微生物。这些共生微生物中的大多数很难在体外培养。DNA指纹分析方法,如聚合酶链反应-变性梯度凝胶电泳(PCR-DGGE),有助于对包括感染性慢性伤口在内的复杂生态系统进行微生物分析。使用PCR-DGGE比较了六种不同的DNA提取方法,以分析与慢性伤口感染相关的微生物群落。从10名患者的慢性伤口溃疡中获取组织碎片用于DNA提取。使用六种DNA提取方法从每个标本中提取总核酸。测量DNA的产量、纯度和质量,并用于针对真细菌16S rRNA基因V2-V3区域的PCR扩增。与其他五种DNA提取方法相比,QIAGEN DNeasy血液和组织试剂盒(K法)产生了高质量的基因组DNA,并在慢性伤口中呈现出广泛的细菌群落多样性。在五种传统方法中,基于珠磨仪/酚-氯仿的STES缓冲液DNA提取方法(BP1法)产生的DNA产量高且纯度高,并导致更高的DGGE条带多样性。尽管使用加热和NaOH进行DNA提取的纯度最低,但DGGE显示出更高的细菌多样性。研究结果表明,基因组DNA的质量和产量受DNA提取方案的影响,因此在分析复杂微生物群落时应谨慎选择方法。