Williams R H, Ward E, McCartney H A
Plant Pathology Department, IACR-Rothamsted, Harpenden, Herts, AL5 2JQ, United Kingdom.
Appl Environ Microbiol. 2001 Jun;67(6):2453-9. doi: 10.1128/AEM.67.6.2453-2459.2001.
Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.
本文描述了以罗克福特青霉作为模式真菌，用于检测空气中真菌孢子的综合空气采样和基于聚合酶链式反应（PCR）的方法。使用微型旋风式空气采样器将罗克福特青霉孢子直接收集到艾本德管中。然后将它们悬浮在0.1%的聚山梨醇酯-40中，并通过显微镜计数。对孢子进行系列稀释。采用三种方法制备用于PCR检测的DNA：将未处理的孢子加入PCR反应体系、使用玻璃珠破坏孢子（使孢子壁破裂以释放内容物）以及破坏孢子后进行DNA纯化。测试了三种罗克福特青霉特异性检测方法：一步法PCR、巢式PCR以及PCR后进行Southern印迹和探针杂交。结果发现，破坏孢子对于实现检测方法的最大灵敏度至关重要。将未处理的孢子加入PCR确实能够检测到罗克福特青霉，但当加入到PCR反应体系中的孢子少于1000个时，从未实现过检测。通过破坏孢子，无论后续是否进行DNA纯化，都有可能检测到单个孢子的DNA。当将已知数量的罗克福特青霉孢子加入到含有高浓度未鉴定真菌孢子、花粉和灰尘的空气样本中时，检测灵敏度会降低。将未处理或已破坏的孢子悬液加入PCR反应体系中无法检测到罗克福特青霉DNA。然而，使用纯化的DNA，在存在4500个其他孢子的背景下能够检测到10个罗克福特青霉孢子。对于所有DNA提取方法，巢式PCR比一步法PCR或PCR后进行Southern印迹更灵敏。
