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.     

Quantitative measurement of streptomycetes using real-time PCR

A quantitative real-time PCR method was developed and used for determination of streptomycetes in indoor dust samples of five homes collected during three years. The specificity of the method was tested with 14 Streptomyces and ten non-streptomycetous species, revealing a high specificity for mesophilic streptomycetes. Thermophilic species and S. albus were not efficiently detected. The method gave reproducible results in replicate analyses of the same dust DNA as well as of duplicate DNA isolations. The amount of streptomycetes in house dust was lowest in winter, followed by summer, and highest in spring and fall. The greatest variation in Streptomyces-concentrations was observed in the spring and fall samples.     

Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA

The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4 gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.     

Detection and quantification of major toxigenic Microcystis genotypes in Moo-Tan reservoir and associated water treatment plant

Two molecular methods, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time polymerase chain reaction (qPCR) with the Universal ProbeLibrary (UPL) probe, were developed and used for the characterization and quantification of several microcystin producers in Moo-Tan Reservoir (MTR), Taiwan and its associated water treatment plant (Shih-Men Water Treatment Plant, SMWTP). Internal transcribed spacer (ITS) sequence, a highly diversified region between the 16S rRNA and 23S rRNA genes, was used to further identify the isolated strains from MTR and also used in DGGE for the detection of the specific DNA fragments and biomarkers for 11 strains observed in MTR. These ITS-DGGE biomarkers were successfully applied to monitor the community changes of potential toxigenic Microcystis sp. over a period of five years. Two highly specific primers were combined with UPL probes to measure microcystins synthesis gene (mcyB) and phycocyanin intergenic spacer region (cpcB) concentrations in water samples. The copy concentrations of UPL-mcyB and UPL-cpcB correlated well with MC-RR concentrations/water temperature and Microcystis sp. cell numbers in the water samples, respectively. For SMWTP, toxin concentrations were low, but the DGGE bands clearly demonstrated the presence of potential microcystin producers in both water treatment plants and finished water samples. It was demonstrated that toxigenic Microcystis sp. may penetrate through the treatment processes and pose a potential risk to human health in the drinking water systems.     

Determination of response of real-time SidePak AM510 monitor to secondhand smoke, other common indoor aerosols, and outdoor aerosol

The amount of light scattered by airborne particles inside an aerosol photometer will vary not only with the mass concentration, but also with particle properties such as size, shape, and composition. This study conducted controlled experiments to compare the measurements of a real-time photometer, the SidePak AM510 monitor (SidePak), with gravimetric mass. PM sources tested were outdoor aerosols, and four indoor combustion sources: cigarettes, incense, wood chips, and toasting bread. The calibration factor for rescaling the SidePak measurements to agree with gravimetric mass was similar for the cigarette and incense sources, but different for burning wood chips and toasting bread. The calibration factors for ambient urban aerosols differed substantially from day to day, due to variations in the sources and composition of outdoor PM. A field evaluation inside a casino with active smokers yielded calibration factors consistent with those obtained in the controlled experiments with cigarette smoke.     

Airborne Alternaria and Cladosporium species and relationship with meteorological conditions in Eskisehir City, Turkey

Alternaria and Cladosporium, known as the most allergenic spores were first collected by means of Durham gravimetric sampler from Eskisehir atmosphere from January 1, 2000 to December 31, 2001. The daily, monthly and annual variations in spores/cm(2) of Cladosporium and Alternaria were recorded. During this period, a total of 10.231 spores belonging to Cladosporium and Alternaria genera were recorded. Of these spores, 5,103 were identified in 2000 and 5,128 in 2001. While 63.09% of the total spores were those of Cladosporium, 36.91% were of Alternaria. Relationships between airborne fungal spore presence and meteorological conditions were statistically investigated. A Shapiro-Wilk test revealed that the airborne Cladosporium and Alternaria spores differed from a normal distribution. Thus, a Friedmann test was performed followed by a Pearson Correlation Analysis. The effects of rainfall, temperature and wind speed on Cladosporium and Alternaria numbers were non-significant according to the sites and months (p > 0.05), but the effects of relative humidity on Cladosporium and Alternaria numbers were significant (p < 0.01). Spore concentrations reached to their highest levels in May 2001.     

Ultra-sensitive quantification of copper in food and water samples by electrochemical adsorptive stripping voltammetry

A new electrochemical adsorptive stripping voltammetry method was developed for the determination of trace amounts of copper in food and water samples. The study of electrochemical behavior of Cu ion indicated that Cu(II) and Schiff base formed a complex in H₃BO₄–NaOH buffer solution (pH?=?7.25). An accumulation potential of ?100 mV (vs Ag/AgCl) was applied while the solution was stirred for 60 s. The response curve was recorded by scanning the potential, and the peak current of ?0.31 V (vs Ag/AgCl) was recorded. The peak current and concentration of copper accorded with linear relationship in the range of 0.04–120 ng mL^?1. The relative standard deviation (for 12 ng mL^?1 of copper) was 1.73 %, and the detection limit was 0.007 ng mL^?1. The possible interference of some common ions was studied. The proposed method was applied to the determination of copper in water, rice, wheat, tea, milk, and tomato with satisfactory results.     

SEM-EDX analysis of various sizes aerosols in Delhi India

Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) was used to understand the differences in morphology, elemental composition and particle density of aerosols in different five size ranges to further investigate the potential sources as well as transport of pollutants from/at a much polluted and a very clean area of Delhi. Aerosol samples were obtained in five different size ranges viz. > or = 10.9, 10.9-5.4, 5.4-1.6, 1.6-0.7 and < or = 0.7 microm from a considerably very clean and a much polluted area of Delhi. It was observed that at polluted area most of the particles irrespective of size are of anthropogenic origin. At clean area, in coarse size fractions particles are of natural origin while in fine size range the presence of anthropogenic particles suggests the transport of particles from one area to the other.     

Visibility impairing aerosols in the urban atmosphere of Delhi

To study the visual air quality of Delhi, size fractionated aerosols – coarse and fine fractions of PM₁₀ – were collected and analysed for and EC at three sites with different background activities. The analysed species constitute a smaller portion of coarse fraction (39%) but a larger portion of fine fraction (69%). The sampling was performed from June 2003 to November 2003 which covers monsoon and post monsoon seasons.Aerosol data was used to describe the spatial variation of Visibility Range as a function of chemical composition of visibility impairing aerosols. During the study period, visibility was found to be poor varying between 4.7 and 13 km with an average value of 9.4 km. It is observed that visibility impairment was more due to carbonaceous aerosol followed by sulphate.     

Particle size distribution of aerosols and associated heavy metals in kitchen environments

Mass size distributions of total suspended particulate matter (TSPM) was measured from Sep 2002 to April 2003 in indoor kitchen environments of five locations in Jawaharlal Nehru University (JNU), New Delhi, with the help of a high volume cascade impactor. Particulate matters were separated in five different size ranges, i.e. >10.9 microm, 10.9-5.4 microm, 5.4-1.6 microm, 1.6-0.7 microm and <0.7 microm. The particle size distribution at various sites appears to follow uni-modal trend corresponding to fine particles i.e. size range <0.7 microm. The contributions of fine particles are estimated to be approximately 50% of TSPM and PM10.9, while PM10.9 comprises 80% of TSPM. Good correlations were observed between various size fractions. Regression results reveal that TSPM can adequately act as a surrogate for PM10.9 and fine particles, while PM10.9 can also act as surrogate for fine particles. The concentrations of heavy metals are found to be dominantly associated with fine particles. However, the concentration of some metals and their size distribution, to some extent is also site specific (fuel type used).     

Monitoring trace metals in urban aerosols from Buenos Aires city. Determination by plasma-based techniques

A study was undertaken, within the framework of a 3 years national project, to assess the content of 13 elements in airborne particulate matter collected in representative zones of the metropolitan area of Buenos Aires. The sampling strategy followed consisted in collecting simultaneously 67 samples of PM10 particulate matter in 9 sampling sites covering an area of about 30 km2 during one week. The collection was performed on ash-free fibre-glass filters using high volume samplers. A combination of aqua regia and perchloric acid was used for leaching metals from filters. Key elements, namely Al, Ca, Cu, Fe, Mn, Mo, Ni, Pb, S, Sb, Sn, Zn and Zr, were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS) at micro g g(-1) and ng g(-1) levels. Analyte concentration varied from 130 ng g(-1)(Mo) to over 30%(Ca). Multivariate statistical analysis was performed on the data set including the measured elemental compositions for the monitored period. The atmospheric concentration found for Pb confirms the decreasing levels of this element since the introduction of unleaded gasoline in 1995: 88 ng m(-3)(2001) < 220 ng m(-3)(1997) < 3900 ng m(-3)(1994). The average S concentration above 3 microg m(-3) is somehow unexpectedly high for Buenos Aires since the relatively low S content of liquid fuels and the massive usage of natural gas imply low emissions of this element from combustion activities. To the best of our knowledge, S concentrations are reported for the first time for this city.     

Validation of atmospheric aerosols parallel sampling in a multifold device

In this work, particulate matter was collected using an active sampling system consisting of a PM₁₀ (<10 μm) inlet coupled to a multifold device containing six channels, connected to a vacuum pump. Each channel was equipped with a filter holder fitted with adequately chosen filters. The system was fixed on a metallic structure, which was placed on the roof of the laboratory building, at the Faculty of Sciences, in Lisbon. Sampling took place under flow-controlled conditions. Aerosols were extracted from the filters with water, in defined conditions, and the water-soluble fraction was quantified by ion chromatography (IC) for the determination of inorganic anions (Cl^?, NO₃ ^? and SO₄ ^2?). Equivalent sampling through the various channels was validated. Validation was based on the metrological compatibility of the content results for the various filters. Ion masses are metrologically equivalent when their absolute difference is smaller than the respective expanded uncertainty. When this condition is verified, the studied multifold device produces equivalent samples.     

Identification and quantification of bisphenol A by gas chromatography and mass spectrometry in a lab-scale dual membrane system

Endocrine disruptor contamination is an emerging issue of concern in the field of water quality engineering. In this study, a lab-scale microfiltration (MF) and reverse osmosis (RO) based water reclamation system was set up to monitor and evaluate the removal of bisphenol A (BPA), which is a known oestrogenic compound. The identification and quantification of BPA were performed by using gas chromatography coupled with mass spectrometry. It was noted that the detection method used in this study was able to achieve an average recovery ranging from 88.2 to 94.1% of BPA with standard deviations of less than 10% in different spiked samples. The detection limit of the analytical protocol was determined at 20 ng L(-1). Based on the analytical protocol, it was noted that a low level of BPA (1.18-3.04 microg L(-1)) could be detected in feed water (effluent of an activated sludge treatment system) to the dual membrane water reclamation system. The results obtained suggested that BPA could be easily chlorinated by sodium hypochlorite with a dosage of 4 to 5 mg L(-1) and a contact time of 1 to 2 min. In this lab-scale study, a satisfactory removal of BPA was readily obtained by RO and BPA was abated to an undetectable level in the product water. It was noted that the RO rejection characteristic of BPA was not sensitive to the variations in raw feed water characteristics experienced in this study. In addition, it was noted that BPA concentration present in raw feed water did not exert any significant impact on RO performance in terms of BPA rejection. The results of this study demonstrated that membrane technology could be effectively used for BPA removal.     

Detection and extraction of endosulfan by metal nanoparticles

One of the most common pesticides in the developing world, endosulfan, can be detected in ppm levels using gold nanoparticles. Endosulfan adsorbs on the nanoparticle surface and upon interaction for a long time, the nanoparticles precipitate from the solution. Interaction with silver is weak, yet adsorption occurs leading to removal of endosulfan from the solution. A multilayer assembly of gold nanoparticles prepared on a glass substrate shows excellent spectrophotometric response suggesting potential applications.     

Attribution of aerosol light absorption to black carbon and volatile aerosols

We investigated the contribution of volatile aerosols in light-absorption measurement by three filter-based optical instruments [aethalometer, continuous light-absorption photometer (CLAP), and continuous soot monitoring system (COSMOS)] at Gosan Climate Observatory (GCO) from February to June 2012. The aerosol absorption coefficient (σ _abs) and the equivalent black carbon (BC) mass concentration (M _BC) measured by the aethalometer and CLAP showed good agreement with a difference of 9 %, which is likely due to the instrumental uncertainty. However, σ _abs and M _BC measured by the COSMOS with a heated inlet were found to be approximately 44 and 49 % lower than those measured by the aethalometer and CLAP under ambient conditions, respectively. This difference can be attributed to the light absorption by the volatile aerosols coexisting with the BC. Even considering inherent observational uncertainty, it suggests that approximately 35–40 % difference in the σ _abs and M _BC can be contributed by volatile aerosols. Increase in the difference of M _BC measured by the aethalometer and COSMOS with the increasing thermal organic carbon (OC) measured by Sunset OC/EC analyzer further suggests that the filter-based optical instruments without the use of a heater are likely to enhance the value of σ _abs and M _BC, because this sample air may contain both BC and volatile aerosols.     

Chemical mass balance model for source apportionment of aerosols in Bombay

Aerosol samples collected within an industrial region of Bombay were analyzed for elemental concentrations using inductively coupled plasma emission spectroscopy, ultraviolet/visible spectrophotometry and X-ray fluorescence spectroscopy. Nineteen elements were selected as tracers of identified sources of aerosol in the region. The U.S. EPA chemical mass balance model was employed for source apportionment. Seven major source types were identified and the performance of the model was evaluated at different sampling locations. Model results were unsatisfactory at highly polluted sites in the study regions. It was found that U.S. EPA source profiles are not suitable for such regions in India and site-specific source profiles should be used in the application of chemical mass balance for source apportionment.     

Elemental and individual particle analysis of atmospheric aerosols from high Himalayas

Atmospheric aerosols were collected during the scientific expedition to Mt. Qomolangma (Everest) in May–June, 2005. The elemental concentrations of the aerosols were determined by inductively coupled plasma mass spectrometry. This yielded data for the concentration of 14 elements: Na, Mg, Al, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, and Pb. The mean elemental concentrations were generally comparable with those from central Asia and the Arctic, while much higher than those from Antarctic. Size, morphology, and chemical composition of 900 individual aerosol particles were determined by scanning electron microscopy and energy-dispersive X-ray microanalysis. Based on morphology and elemental composition, the particles were clustered into eight groups: soot (8%), tar ball (3%), alumosilicates/silica (55%), calcium sulfate (16%), Ca/Mg carbonate (2%), Fe/Ti-rich particles (3%), Pb-rich particles (1%), and biological particles (12%). The sampling site, located at 6,520 m in the Himalayas, is particularly remote and located at high altitude. Nonetheless, high aerosol enrichment factors for copper, chromium, lead, nickel, vanadium, and zinc all suggest the influence of long-range transported pollution, while enrichment in calcium and the presence of alumino-silicates in individual particle analyses indicates a distinct mineral dust influence. The backward air mass trajectories showed that the northwestern part of India may contribute to the atmospheric aerosol in the central high Himalayas.     

Speciation and sources of atmospheric aerosols in a highly industrialised emerging mega-city in central China

Monitoring air quality in large urban agglomerations is the key to the prevention of air pollution-related problems in emerging mega-cities. The city of Wuhan is a highly industrialised city with >9 million inhabitants in Central China. Simultaneous PM10 sampling was performed during 1 year at one urban and one industrial site. Mean PM10 daily levels (156 microg m(-3) at the urban site and 197 microg m(-3) at the industrial hotspot) exceed the US-EPA or EU annual limit values by 3-4 times. A detailed study of daily speciation showed that the mean chemical composition of PM10 presents minimal differences between peak and low PM episodes. This implies that PM10 aerosols in the study area result from local emissions, and air quality management and abatement strategies in Wuhan should thus focus on local anthropogenic sources. The levels of some elements of environmental concern are relatively high (409-615 ngPb m(-3), 66-70 ngAs m(-3), 116-227 ngMn m(-3), 10-12 ngCd m(-3)) due to industrial, but also urban emissions. Principal component analysis identified a mineral source (probably cement and steel manufacture) and smelting as the main contributors to PM10 levels at the industrial site (34%), followed by a coal fired power plant (20%) and the anthropogenic regional background (16%). At the urban site the major PM10 source is a mixed coal combustion source (31%), followed by the anthropogenic regional background (28%) and traffic (16%).     

工业废水中甲胺磷农药的气相色谱的分离与测定

对测定工业废水中甲胺磷的色谱条件及其干扰进行了试验,并对ＨＰ－５弹性石英毛细管柱和电子捕获检测器,氮磷检测器,火焰光度检测器作了分析比较,结果表明,３０ｍ柱和这３种检测器能满足要求。     

Chemical characteristics of aerosols and trace gas distribution over North and Central India

A field campaign on aerosol chemical properties and trace gases measurements was carried out along the Delhi-Hyderabad-Delhi road corridor (spanning about 3,200 km) in India, during February 1-29, 2004. Aerosol particles were collected on quartz and cellulose filters using high volume (PM(10)) sampler at various locations along the route (i.e., urban, semi-urban, rural, and forest areas) and have been characterized for major cations (Na(+), Ca(2+), Mg(2+), K(+), and NH (4) (+)), anions (Cl(-), NO (3)(-), and SO (4)(2-)), and heavy metals (Cu, Cd, Fe, Zn, Mn, and Pb). Simultaneously, we measured NO(2) and SO(2) gases. These species show large spatial and temporal variations. The ambient PM(10) concentration has been observed to be the highest (55 ± 4 μg m(-3)) near semi-urban areas followed by forest areas (48 ± 2 μg m(-3)) and in rural areas (44 ± 22 μg m(-3)). The concentrations of NO( x ) (NO(2)+NO) and SO(2) ranged from 16 to 69 μg m(-3) and 4 to 11 μg m(-3), respectively. Among anions, NO(3)(-) and SO(4) (2-) are the major constituents of PM(10). The urban and semi-urban sites showed enhanced concentrations of Fe, Zn, Mn, Cd, and Pb. This study provide information about atmospheric concentrations of various species in the northern to central India, which may be important for policy makers to better understand the air quality of the region.