Evaluation of interference to conventional and real-time PCR for detection and quantification of fungi in dust

Advances in polymerase chain reaction (PCR) have permitted accurate, rapid and quantitative identification of microorganisms in pure cultures regardless of viability or culturability. In this study, a simple sample processing method was investigated for rapid identification and quantification of fungal spores from dust samples using both conventional and real-time PCR. The proposed method was evaluated for susceptibility to interference from environmental dust samples. Stachybotrys chartarum and Aspergillus fumigatus were used as test organisms. The sensitivity of detection in pure culture was 0.1 spore DNA equivalents per PCR reaction corresponding to 20 spores ml(-1) in the sample. However, 1 spore DNA equivalent per PCR reaction corresponding to 200 spores ml(-1) in the sample was the lowest amount of spores tested without interference in dust samples spiked with spores of either fungal species. The extent of inhibition was calculated using conventional and real-time PCR reactions containing fungal spores, specific primers, specific probes (for real-time PCR) and various amounts of dust. The results indicate that the extent of inhibition by dust on PCR varies with the type and amount of dust, and number of spores. No interference in the analysis of spiked samples was detected from 0.2 mg ml(-1) of four real-life dust samples at p-value >0.05 using 2 x 10(4) spores for conventional PCR and 2 x 10(5) spores for real-time PCR. However, samples containing >0.2 mg ml(-1) real-life dust compromised the PCR assay. These results suggest the potential usefulness of a simple sample processing method in conjunction with PCR for monitoring the fungal content of aerosols collected from indoor environments.     

Organochlorine pesticides in the dust fall around Lake Chaohu, the fifth largest lake in China

The residual levels of organochlorine pesticides (OCPs) in the dust fall around Lake Chaohu were measured using gas chromatography mass spectrometry from April 2010 to March 2011. The fluxes, components, temporal–spatial variations, and sources of OCPs were also analyzed. Twenty-one types of OCPs were detected in the dust fall samples around Lake Chaohu, with a total concentration of 51.54?±?36.31 ng/g and a total flux of 10.01?±?13.69 ng/(m² day). Aldrin (35.3 %), endosulfan (39.1 %), dichlorodiphenyltrichloroethanes (DDTs) (49.8 %), and isodrin (37.1 %) were the major OCPs in the spring, summer, autumn, and winter, respectively. Both the residual level and the flux were higher in the spring than in other seasons and higher at the outer lake sampling sites than inner lake sampling site. The potential source of the hexachlorcyclohexanes in the dust fall may be recent lindane usage. The DDTs mainly came from historical dicofol usage, and a significant input of DDT was found during April and June. The presence of endosulfan may be due to the present use of technical endosulfan. The aldrin in the dust might be due to its occasional usage, and isodrin may be a result of long-distance transport from other countries.     

Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing

Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected.     

中国北方干旱地区沙尘矿物特征研究

为了开展对我国沙暴传输规律的研究,了解我国北方干旱地区沙尘矿物特征有重要意义。本文对我国新疆塔克拉玛干沙漠区,内蒙腾格里沙漠区,贺兰山及银川等处的沙尘样品用X射线粉末衍射法进行矿物分析鉴定,并用原子吸收法作了特征元素的分析对比,根据矿物的X射线粉末衍射结果,结合元素分析,对沙漠源区及其以东地区沙尘矿物特征作了比较,判析了两个源区的矿物组成,讨论了它们的传输过程。     

上海市降尘自动监测系统研发与应用

大气降尘量是重要的环境指标之一,可以较客观地反映和评价城市局部地区扬尘污染水平,对评价城市区域环境空气质量、分析大气污染来源和变化具有重要意义。国内外大气降尘监测普遍采用传统的手工监测方法,手工法因时效性差、效率低、能耗大、质控难、误差大等原因已不适应现代环境管理的需求。随着科技与网络的发展,自动监测方法成为发展趋势。文章主要阐述了上海市降尘自动监测技术的研发过程及实际应用,介绍了自主研发的降尘自动监测仪的原理、关键技术和系统组成。通过粉尘舱内实验、降尘自动监测方法与手工重量法比对测试及为期一年的试点应用,分析了降尘自动监测方法的精密度、准确度、相对误差及适用性,并探讨了降尘自动监测的应用前景。     

宁波市镇海区大气降尘矿物组成及来源解析

为探讨宁波市镇海区大气降尘矿物组成及来源,用X射线衍射法分析了镇海区6个土地功能区2019年12月至2020年11月4个季度的大气降尘样品的矿物组成和含量,并开展了矿物来源解析。结果显示,降尘中以石英、长石、碳酸盐类矿物和黏土矿物为主,年均质量分数分别为29.2%、33.3%、24.2%和7.7%,且不同功能区各季度降尘中矿物含量普遍有显著差异。来源解析表明,镇海区降尘中矿物来源主要以地面扬尘输入为主,同时周边源区输入及人类活动和工业密集区燃煤等输入对降尘矿物组成有较显著影响,除黏土矿物、碳酸盐类矿物和长石类矿物之外,其他矿物的含量还与不同季节的气温和风向有关。     

Passive airborne dust sampling to assess mite antigen exposure in farming environments

The aim of this study was to estimate mite antigen exposure in farming environments by passive sampling of airborne dust. Antigen concentrations were measured with enzyme immunoassays specific for three storage mites (SM): Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae and the house dust mite (HDM) Dermatophagoides pteronyssinus. Dust samples were collected with electrostatic dust fall collectors (EDCs) in three different areas of cattle farms. EDCs were placed in cow stables (working area), in changing rooms (transit area) and in different rooms of farmer dwellings (living area). Mite concentrations in the living area of farm homes were compared to those of urban homes. In dust samples from stables, antigens of all four mite species could be detected. The highest exposure level was to L. destructor (median 56.7 μg/m(2)), the lowest to A. siro (median 14.4 μg/m(2)). Mite concentrations of different species showed no correlation within the cow stables. In comparison to stables, the median mite concentrations in farm homes were significantly lower, ranging from below the detection limit to 1.5 μg/m(2). Antigens of SM were predominantly found in changing rooms and kitchens, and HDM antigens were mainly detected in bedrooms. Antigens of all mites were measured the least often in living rooms. T. putrescentiae was the most prevalent mite in all room types, and the exposure levels correlated strongly between different rooms. The number of SM positive samples in farm homes was considerably higher than in urban homes, while the percentage of HDM positive samples did not differ significantly.     

应用化学质量平衡模型解析西宁大气PM2.5的来源

为研究影响西宁市大气环境PM_(2.5)污染水平的主要来源,于2014年采暖季、风沙季和非采暖季依托西宁市大气地面观测网络在11个监测点采集大气PM_(2.5)样品,对其化学组分(元素、离子和碳)进行分析。研究同步采集了4类固定源、14类移动源和4类开放源的PM_(2.5)样品,并构建源排放成分谱。应用化学质量平衡受体模型(CMB)开展源解析研究。源解析结果表明,观测期间西宁市PM_(2.5)主要来源包括城市扬尘(分担率为26.4%)、燃煤尘(14.5%)、机动车尾气(12.8%)、二次硫酸盐(9.0%)、生物质燃烧(6.6%)、二次硝酸盐(5.7%)、钢铁尘(4.7%)、锌冶炼尘(3.4%)、建筑尘(4.4%)、土壤尘(4.4%)、餐饮排放(2.9%)和其他未识别的来源(5.2%)。大力开展城市扬尘为主的开放源污染控制,严格控制本地燃煤、机动车等污染源的PM_(2.5)排放,是改善西宁市空气质量的重要途径。     

“十一五”期间新疆大气降尘量时空分布特征及影响因素分析

通过对"十一五"期间新疆134个大气降尘监测点位数据进行统计分析发现,"十一五"期间,全疆年均降尘量维持在22.2~24.6 t/km2.月,变化幅度小于10%。一年内降尘主要集中在3~6月份,占全年降尘量的51%。在空间分布上,新疆年均降尘量呈现明显的南疆高于北疆的特征,南疆五地、州的总降尘量占全疆总降尘量的70.2%,为新疆降尘的主要集中区。特殊的气象、生态环境是决定新疆大气降尘时空分布特征的主要因素。     

大气可吸入颗粒物(PM10)中矿物组分的X射线衍射研究

利用X射线衍射技术对北京2002春季和夏季的可吸入颗粒物进行了研究.结果表明,北京春季和夏季可吸入颗粒物的矿物组成明显不同,春季可吸入颗粒物中的矿物以硅铝酸盐为主,同时存在碳酸盐、硫酸盐、硫化物、铁的氧化物、粘土矿物以及难以鉴定的矿物;在夏季的样品中,矿物的种类有所减少,却有新的物种出现,如氯化氨、硫酸氨等.XRD定量分析显示,在沙尘天气时,可吸入颗粒物中石英和粘土矿物以及非晶质分别占到24.1%、28.5%和2 0%,斜长石和方解石分别占到10.4%和8.1%,其他矿物总共不到10%.矿物组分的确定对可吸入颗粒物来源的识别有一定的指导作用.     

On-filter determination of collected wood dust by diffuse reflectance infrared Fourier-transform spectroscopy (DRIFTS)

A new analytical technique based on DRIFTS spectroscopy has been developed for the specific and sensitive determination of size-fractionated wood dust from 37 mm glass fiber filter samples collected with the Respicon sampler. A translational diffuse reflectance apparatus was modified to accept filter samples by incorporating a special filter holder in the sample stage and a clockwork motor to drive the translational stage during infrared scanning, thus providing an average analysis across the filter face. Filter samples were pre-treated with ethyl acetate to uniformly redeposit dust onto the filter and extract potential chemical interferences. Two absorbance maxima (1251 and 1291 cm(-1)), corresponding to the cellulose content of the wood, were suitable for quantitation of wood dust. Analysis of seven species of wood at 1291 cm(-1) showed an equivalent quantitative response for all species except maple. The response at 1251 cm(-1) was more variable across species but more sensitive for the softwoods. There was a statistically significant effect of particle size on the analytical response, so that analytical standards should be matched to the samples in terms of particle size distribution. Analytical limit of detection was approximately 0.08 mg of wood dust per sample with overall precision of about 6%. Comparison of DRIFTS and gravimetric analyses of 51 pure wood dust samples ranging from about 0.2 to 2 mg yielded a slope of 1.08 and r2 equal to 0.9. Other particulate contaminants common in the industrial wood processing industry showed little or no interference with the determination of wood dust by this method.     

南通市降尘时空分布特征及影响因素

对2004年—2009年南通市降尘监测数据进行分析,结果表明,市区降尘污染较轻,年均值全部达标,在6年间呈波状变化态势;从各月分布来看,3—7月降尘污染较为严重;在各功能区中,受降尘污染程度从大到小依次为:居民区>工业区>商业交通区>文教区。提出,应继续推进城区企业工艺改造升级,加快燃煤锅炉淘汰;施工(运输)期间对易产生扬尘的建筑材料应采取密闭存储、设置围挡等有效措施并保证建筑垃圾及时清运、施工车辆及道路及时清扫冲洗;加大城市的绿化建设和道路的硬化建设,尽量避免道路开挖。     

Suspended particulates and bioaerosols emitted from an agricultural non-point source

Suspended particulate and bioaerosol levels were measured at three sites downwind of an agricultural non-point source during the wheat harvesting season. Suspended particulates were detected at mean values ranging from 10000 to 2420 micrograms m-3 at distances of from 20 to 60 m downwind of the source, respectively. Airborne viable bacterial counts were recorded at mean values ranging between 10(4) and 10(6) colony forming units (cfu) m-3, whereas, Gram negative (Gram -ve) bacteria varied between 10(3) and 10(5) cfu m-3. Fungi levels were detected at mean values varying between 10(5) and 10(6) cfu m-3. However, streptomycetes were found at lower counts than those recorded for viable bacteria and fungi. Total viable bacteria, Gram -ve bacteria, fungi and streptomycetes associated hay fragments were determined at mean values of 1.5 x 10(6), 1.6 x 10(3), 2.2 x 10(4) and 6 x 10(3) cfu g-1 of hay, respectively. Cladosporium, white and red yeasts as well as Alternaria were the predominant airborne fungi, whereas, Alternaria was the dominant species associated with hay fragments. Pseudomonas, Acinetobacter and Enterobacteriaceae were the dominant Gram -ve bacteria. The most common fungal genera, such as Cladosporium and Fusarium (minor short axis), as well as Streptomyces species have an aerodynamic diameter (dae) of less than 5 microns, which can penetrate and deposit in the alveoli. Farmers and nearby residents are exposed to high levels of organic dust and bioaerosols during the wheat harvesting season. This may cause health problems in exposed persons based on toxic or allergic reactions.     

Real-time PCR detection of toxigenic Fusarium in airborne and settled grain dust and associations with trichothecene mycotoxins

Inhalation of immunomodulating mycotoxins produced by Fusarium spp. that are commonly found in grain dust may imply health risks for grain farmers. Airborne Fusarium and mycotoxin exposure levels are mainly unknown due to difficulties in identifying Fusarium and mycotoxins in personal aerosol samples. We used a novel real-time PCR method to quantify the fungal trichodiene synthase gene (tri5) and DNA specific to F. langsethiae and F. avenaceum in airborne and settled grain dust, determined the personal inhalant exposure level to toxigenic Fusarium during various activities, and evaluated whether quantitative measurements of Fusarium-DNA could predict trichothecene levels in grain dust. Airborne Fusarium-DNA was detected in personal samples even from short tasks (10-60 min). The median Fusarium-DNA level was significantly higher in settled than in airborne grain dust (p < 0.001), and only the F. langsethiae-DNA levels correlated significantly in settled and airborne dust (r(s) = 0.20, p = 0.003). Both F. langsethiae-DNA and tri5-DNA were associated with HT-2 and T-2 toxins (r(s) = 0.24-0.71, p < 0.05 to p < 00.01) in settled dust, and could thus be suitable as indicators for HT-2 and T-2. The median personal inhalant exposure to specific toxigenic Fusarium spp. was less than 1 genome m(-3), but the exposure ranged from 0-10(5) genomes m(-3). This study is the first to apply real-time PCR on personal samples of inhalable grain dust for the quantification of tri5 and species-specific Fusarium-DNA, which may have potential for risk assessments of inhaled trichothecenes.     

基于K-means聚类的沙尘天气快速识别技术研究

依据沙尘天气判定条件分析2018年春季（2—4月）陕西省西安市环境空气质量小时监测数据，共识别9 d受沙尘影响。研究基于K-means聚类沙尘天气分析方法，分析沙尘天气监测数据特征，并对沙尘识别聚类模型进行优化研究，识别的沙尘影响天气与传统方法一致。分析表明，聚类方法可用于沙尘天气监测数据的识别，与传统方法相比较，基于K-means聚类方法能够快速、准确识别沙尘影响天气。     

Water-soluble species and heavy metal contamination of the petroleum refinary area,Jordan

The levels of lead, cadmium, copper, zinc, aluminum, chromium, and iron in street dust, soil, and plants in the Jordanian petroleum refinery were determined using flame and graphite-furnace atomic absorption spectrophotometry. Major cations (Li+, Na+, NH4+, K+, Mg2+, and Ca2+) and anions (F-, Cl-, NO3-, SO4(2-), and Br-) were also determined using suppression mode ion chromatography. Generally, higher levels of the heavy metals studied were found in street dust samples than in soil samples. On the other hand, except Cl-, and Li+ ions, other anions and cations showed higher concentrations in soil than in street dust samples. For plant samples, unwashed samples showed higher levels of heavy metals than their washed counterparts, indicating that dust fall is a source of heavy metal contamination.     

天津市春季典型道路积尘负荷分布特征

道路积尘负荷表征路面清洁程度,是道路扬尘排放清单中一个十分重要的参数,也是各地环境管理的一个重要方面。根据AP-42道路扬尘排放因子模型,2015年春季用真空吸尘法采集了天津市11条道路的扬尘样品,得到了不同道路类型以及不同车道的积尘负荷,并分析了积尘负荷的变化规律。结果表明:天津市春季非机动车道和机动车道慢车道的积尘负荷分别为0.282 0~1.064 5 g/m~2和0.050 4~0.173 9 g/m~2;5种道路类型的非机动车道的积尘负荷中位值均大于机动车慢车道积尘负荷的中位值;对于不同道路类型积尘负荷而言,非机动车道从大到小顺序依次为次干道主干道环线支路快速路,机动车道慢车道从大到小顺序依次为次干道环线主干道支路快速路;东西走向和南北走向道路两侧积尘负荷差异均无统计学意义。     

Biological monitoring of wood dust exposure in nasal lavage by high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for biomonitoring of occupational wood dust exposure based on nasal lavage as a biomonitoring matrix was developed. Gallic acid (GA) was chosen as the indicator compound for oak dust exposure. From the chromatographic profile of ash dust, four peaks were chosen as indicator compounds. Phenolic indicator compounds were analysed by HPLC. Personal dust samples and corresponding nasal lavage samples were collected from 16 workers exposed to oak dust and six to ash dust. The dust concentrations in the workers' breathing zone varied between 0.7 and 13.8 mg m(-3). The indicators revealed the nature of the wood dust inhaled. For the workers who did not use respirators, the correlation between the dust and corresponding indicator compound in their nasal lavage was significant; r2 = 0.59 (n = 12) for oak dust and r2 = 0.58 (n = 6) for ash dust, respectively. Further, the correlation for oak dust workers who used respirators was r = 0.67 (n = 4). Nasal lavage sampling and HPLC analysis of polyphenol indicator compounds are promising tools for measuring wood dust exposure. Although further validation is necessary, determination of the individual dose may prove invaluable in prospective epidemiological studies.     

Real-time PCR for detection of the Aspergillus genus

Aspergillus is a genus of mold that has strong indoor sources, including several species capable of acting as opportunistic pathogens. Previous studies suggest that Aspergillus could serve as an indicator for abnormal mold growth or moisture, making it an important genus for environmental monitoring. Here, a quantitative polymerase chain reaction (qPCR, or real-time PCR) assay is presented for Aspergillus. The assay shows good specificity for the genus, detecting all Aspergillus species tested, although a few non-Aspergillus species are also amplified. Sensitivity testing demonstrates that DNA representing one conidium can be detected. A validation study compared qPCR results against direct microscopy counts using A. fumigatus conidia aerosolized into a laboratory chamber. The assay was then used to quantify Aspergillus in indoor air samples, demonstrating its utility for environmental monitoring. Analysis of a small number of clinical sputum samples showed complete agreement with culturing results.     

Comparison of EC and BC and evaluation of dust aerosol contribution to light absorption in Xi'an, China

The concentrations of EC, BC and dust aerosols were determined for atmospheric samples collected from an observation station in Xi'an, China. The results show that the averaged correlation coefficient between EC and BC was founded to be 0.72 with 0.81 (n = 49) in autumn, 0.70 (n = 112) in winter and 0.69 (n = 57) in spring, respectively. Absorption coefficients of dust aerosol were estimated to be 2.7 m² g⁻¹ in autumn and 4.4 m² g⁻¹ in winter. The comparison of absorption coefficients of dust aerosol with those of BC implies that BC is the principal light-absorbing aerosol over Xi'an atmosphere. By combining thermal analysis of elemental carbon and dust contents in the aerosol samples, however, the fraction of dust absorption to total light absorption is estimated to be 19% in autumn and 31% in winter, respectively.