Haugland Richard A, Varma Manju, Wymer Larry J, Vesper Stephen J
National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, Ohio 45268, USA.
Syst Appl Microbiol. 2004 Mar;27(2):198-210. doi: 10.1078/072320204322881826.
A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.
共开发并测试了65种定量聚合酶链反应（QPCR）检测方法，这些方法采用荧光5'核酸酶（TaqMan）化学技术，针对核糖体RNA操纵子、内部转录间隔区（ITS1或ITS2），用于检测特定的曲霉属、青霉属和拟青霉属物种。根据不同生物体中核苷酸序列变异的程度，这些检测方法的特异性有所不同，从物种或亚种到密切相关的物种组。还开发并测试了一种针对曲霉属、青霉属和拟青霉属所有目标物种的通用检测方法。使用先前报道的DNA提取方法，不同检测方法对目标物种分生孢子的估计检测限为每个样品少于1个至数百个。在几乎所有情况下，非目标物种的分生孢子检测限至少高1000倍。这些检测方法用于分析来自美国各地不同来源的10个暖通空调灰尘样本。通过通用检测方法以及特定检测方法的总和确定的样本中曲霉属、青霉属和拟青霉属分生孢子的总量总体一致，这表明特定检测方法涵盖了样本中所有数量上占主导地位的物种。对这些样本加入选定目标生物体后进行的QPCR分析表明，95%的时间内计数结果在预期值的大约半个对数范围内。有证据表明，将曲霉属和青霉属作为一个单一组或仅按属进行计数的常用做法，在理解这些生物体的室内种群及其潜在健康风险方面可能会产生误导。
