Hynes S S, Chaudhry O, Providenti M A, Smith M L
Institute of Biology, Carleton University, Ottowa, ON, Canada.
Can J Microbiol. 2006 May;52(5):451-61. doi: 10.1139/w05-140.
The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (approximately 14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.
在复杂环境样本中快速鉴定和定量微生物菌株的能力在生态学、流行病学和工业领域有着广泛应用。在本研究中，我们描述了一种快速方法，可获得功能特异性遗传标记，这些标记可与标准或实时聚合酶链反应（PCR）结合使用，以确定选定环境样本中目标真菌菌株的丰度。该方法包括对来自目标生物体的随机克隆的扩增片段长度多态性（AFLP）产物进行测序，并设计AFLP片段内部的PCR引物。在为期4个月的土壤微观实验中，使用菌株特异性标记来确定三种与工业相关的真菌，即黑曲霉、米曲霉和球毛壳菌的命运。通过对直接从土壤中提取的DNA进行PCR分析，确定了分别接种到完整土壤微观环境中的三种真菌菌株各自的持久性。基于标准PCR的存在与否数据以及通过实时PCR对目标DNA的定量显示，接种后所有三种菌株均有所下降（黑曲霉、米曲霉和球毛壳菌分别下降约14倍、32倍和4倍），但在实验结束时仍可检测到，这表明如果释放到自然环境中，这些菌株将长期存活。
