Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan™) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.     

Analysis of the Fungal Flora in Environmental Dust Samples by PCR–SSCP Method

Conventional microbiological techniques yield only limited information on the composition of fungal communities in dust. The aim of this study was to establish and optimize PCR-single strand conformation polymorphism (PCR–SSCP) analysis for investigation of fungal diversity in rural dust samples. Three different DNA extraction protocols were tested on 38 fungal cultures. A total of six known universal fungal primer pairs were tested targeting the 18S rRNA gene, the 28S rRNA gene and the ITS region, respectively. Objective evaluation was performed with respect to the following parameters: efficiency to amplify all 38 strains; separation of seven species from different phylogenetic groups on the SSCP gel; additional bands in PCR–SSCP analysis; possibility to classify the amplified gene fragments to species level. Primer ITS1/ITS4 and PowerSoil? DNA isolation showed the best performance in most cases and were chosen for further analysis. The detection limit of the developed system was 200 CFU/g dust. Moreover, the reproducibility of the system could be demonstrated, leading to average profile similarities of 94.94 % [SD = 2.51] within gels, 93.03 % [SD = 4.69] between different days and 87.66 % [SD = 6.62] between different gels when testing shed and mattress dust samples. Sequencing allowed identification on species level, in detail: Alternaria alternata, Cladosporium sphaerospermum, Cladosporium cladosporioides as well as the yeasts Candida cabralensis and Candida catenulata. This demonstrates the adaptability of the method. In this study, a standardized system for fungal community analysis was developed that provides reproducible results applicable for epidemiological purposes.     

Detection of Coxiella burnetii from Dust in a Barn Housing Dairy Cattle

We attempted to detect Coxiella burnetii in dust samples collected from a barn housing dairy cattle by the polymerase chain reaction (PCR) method. Ten dust samples (five from ventilation fans and five from crossbeams) were collected from two areas in a barn on a farm near Sapporo, Hokkaido. C. burnetii was detected in 5 of the 10 dust samples. It was believed that aerial contamination by C. burnetii occurred in the barn.     

PCR-based quantification of Pneumocystis carinii in in vitro systems

In many laboratories, PCR has become a routine method for the sensitive diagnosis of Pneumocystis carinii in patient samples. In contrast, quantification of fungal numbers in in vitro setups still largely relies on more conventional procedures such as histological stainings. These are time consuming and their applications are limited when dealing with small fungal numbers contaminated with tissue and cellular debris. This study presents a sensitive and rapid method for P. carinii quantification based on PCR analysis that can be easily integrated into standard detection procedures without requiring any major additional steps. P. carinii-specific PCR performed with total DNA extracted from both standard samples with known fungal numbers and experimental samples was quantified relative to PCR products of a standard concentration from a control plasmid added prior to DNA extraction. This measure controlled for variations in DNA extraction and PCR efficiency among the samples to be compared. The correlation between analyzed P. carinii-specific DNA and the actual fungal numbers employed was highly significant.     

通用PCR法在痰标本真菌检测中的临床应用价值分析

目的:探讨通用PCR法在痰标本真菌检测中的临床应用价值。方法:选择2015年9月至2017年9月本院收治的免疫力低下患者178例作为研究资料,各收集痰标本2份,其中一份用于PCR扩增,另一份经培养后若观察到真菌或可疑菌落,则提取后在此进行PCR扩增测序。比较3种方法对真菌的检出情况、真菌阳性率,与组织病理学结果进行比较,分析3种方法的诊断效能。结果:178份痰标本经平板培养可观察到107份真菌生长,其余71份未见真菌生长。挑取真菌及可疑菌落行PCR结果显示阳性、阴性分别115、63份。对痰标本直接PCR结果显示,阳性、阴性分别124、54份。PCR扩增产物测序结果显示大多是白色念珠菌感染,仅3份是曲霉菌感染。内对照扩增验证结果显示有1份阴性标本存在扩增抑制现象。3种方法的真菌阳性率:直接PCR法痰培养后PCR法痰培养,但组间无显著差异(P0.05)。3种方法诊断效能总体趋势:直接PCR法痰培养后PCR法痰培养,其中直接PCR法与痰培养后PCR法的特异度、准确度均显著高于痰培养法(P0.05),直接PCR法的敏感度显著高于痰培养法(P0.05)。结论:通用PCR法在痰标本真菌检测中的临床应用价值较高,直接PCR具有操作简单、快速等优势。     

一种快速高效获取丝状真菌PCR反应模板的方法

建立一种快速高效获取丝状真菌PCR反应模板的方法,提高丝状真菌PCR鉴定效率。通过单因素法对机械破壁联合微波法进行条件优化,利用优化后的方法获取13株不同种属丝状真菌的PCR反应模板,同时与Chelex-100法、机械破壁法作对比,以试剂盒抽提法作为阳性对照,进行ITS序列扩增,琼脂糖凝胶电泳检测扩增结果。机械破壁联合微波法获取丝状真菌PCR反应模板的最佳条件为40 Hz机械破壁1 min、微波700 W高温裂解3 min,采取该法与试剂盒抽提法获得的模板均成功扩增13株不同种属丝状真菌ITS序列,且PCR鉴定结果一致;Chelex-100法获得的模板成功扩增6株丝状真菌ITS序列;机械破壁法获得的模板虽成功扩增9株丝状真菌ITS序列,但扩增效果欠佳。机械破壁联合微波法能够有效获取丝状真菌PCR反应模板,与试剂盒抽提法相比具有操作简便、快速高效的优点,提高丝状真菌PCR鉴定效率。     

Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gram-negative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r = 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r = 0.88 +/- 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16- or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.     

Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification

The increasing incidence of infectious diseases caused by fungi in immunocompromised patients has encouraged researchers to develop rapid and accurate diagnosis methods. Identification of the causative fungal species is critical in deciding the appropriate treatment, but it is not easy to get satisfactory results due to the difficulty of fungal cultivation and morphological identification from clinical samples. In this study, we established a microarray system that can identify 42 species from 24 genera of clinically important fungal pathogens by using a chemical color reaction in the detection process. The array uses the internal transcribed spacer region of the rRNA gene for identification of fungal DNA at the species level. The specificity of this array was tested against a total of 355 target and nontarget fungal species. The fungal detection was succeeded directly from 10³ CFU/ml for whole blood samples, and 50 fg DNA per 1 ml of serum samples indicating that the array system we established is sensitive to identify infecting fungi from clinical sample. Furthermore, we conducted isothermal amplification in place of PCR amplification and labeling. The successful identification with PCR-amplified as well as isothermally amplified target genes demonstrated that our microarray system is an efficient and robust method for identifying a variety of fungal species in a sample.     

Air- and dustborne mycoflora in houses free of water damage and fungal growth

Typically, studies on indoor fungal growth in buildings focus on structures with known or suspected water damage, moisture, and/or indoor fungal growth problems. Reference information on types of culturable fungi and total fungal levels are generally not available for buildings without these problems. This study assessed 50 detached single-family homes in metropolitan Atlanta, Ga., to establish a baseline of "normal and typical" types and concentrations of airborne and dustborne fungi in urban homes which were predetermined not to have noteworthy moisture problems or indoor fungal growth. Each home was visually examined, and samples of indoor and outdoor air and of indoor settled dust were taken in winter and summer. The results showed that rankings by prevalence and abundance of the types of airborne and dustborne fungi did not differ from winter to summer, nor did these rankings differ when air samples taken indoors were compared with those taken outdoors. Water indicator fungi were essentially absent from both air and dust samples. The air and dust data sets were also examined specifically for the proportions of colonies from ecological groupings such as leaf surface fungi and soil fungi. In the analysis of dust for culturable fungal colonies, leaf surface fungi constituted a considerable portion (>20%) of the total colonies in at least 85% of the samples. Thus, replicate dust samples with less than 20% of colonies from leaf surface fungi are unlikely to be from buildings free of moisture or mold growth problems.     

Use of molecular biology methods for diagnosing fungal infections

A total of 130 various clinical materials taken from 48 children with suspected systemic fungal infection were used for the study. Clinical samples were tested by use of classical mycological procedures well as by use of molecular technique (PCR assay). The fragments of 125-bp (EO3) and 317 bp (HSP) specific for C. albicans were used for amplification. Fifty seven samples (48%) were positive for Candida albicans and eighty four (68%) by use of PCR. It should be stressed that 4 blood samples, 21 urine samples and 5 other samples were positive by use of molecular technique, only. PCR is sensitive and rapid method for detection and identification of Candida albicans from clinical materials of children with fungal infection. This technique can be applied for monitoring presence of fungal DNA in tested samples during antifungal therapy.     

Air- and Dustborne Mycoflora in Houses Free of Water Damage and Fungal Growth

Typically, studies on indoor fungal growth in buildings focus on structures with known or suspected water damage, moisture, and/or indoor fungal growth problems. Reference information on types of culturable fungi and total fungal levels are generally not available for buildings without these problems. This study assessed 50 detached single-family homes in metropolitan Atlanta, Ga., to establish a baseline of “normal and typical” types and concentrations of airborne and dustborne fungi in urban homes which were predetermined not to have noteworthy moisture problems or indoor fungal growth. Each home was visually examined, and samples of indoor and outdoor air and of indoor settled dust were taken in winter and summer. The results showed that rankings by prevalence and abundance of the types of airborne and dustborne fungi did not differ from winter to summer, nor did these rankings differ when air samples taken indoors were compared with those taken outdoors. Water indicator fungi were essentially absent from both air and dust samples. The air and dust data sets were also examined specifically for the proportions of colonies from ecological groupings such as leaf surface fungi and soil fungi. In the analysis of dust for culturable fungal colonies, leaf surface fungi constituted a considerable portion (>20%) of the total colonies in at least 85% of the samples. Thus, replicate dust samples with less than 20% of colonies from leaf surface fungi are unlikely to be from buildings free of moisture or mold growth problems.     

Fungi Identify the Geographic Origin of Dust Samples

There is a long history of archaeologists and forensic scientists using pollen found in a dust sample to identify its geographic origin or history. Such palynological approaches have important limitations as they require time-consuming identification of pollen grains, a priori knowledge of plant species distributions, and a sufficient diversity of pollen types to permit spatial or temporal identification. We demonstrate an alternative approach based on DNA sequencing analyses of the fungal diversity found in dust samples. Using nearly 1,000 dust samples collected from across the continental U.S., our analyses identify up to 40,000 fungal taxa from these samples, many of which exhibit a high degree of geographic endemism. We develop a statistical learning algorithm via discriminant analysis that exploits this geographic endemicity in the fungal diversity to correctly identify samples to within a few hundred kilometers of their geographic origin with high probability. In addition, our statistical approach provides a measure of certainty for each prediction, in contrast with current palynology methods that are almost always based on expert opinion and devoid of statistical inference. Fungal taxa found in dust samples can therefore be used to identify the origin of that dust and, more importantly, we can quantify our degree of certainty that a sample originated in a particular place. This work opens up a new approach to forensic biology that could be used by scientists to identify the origin of dust or soil samples found on objects, clothing, or archaeological artifacts.     

Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR-TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR-TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2+GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR-TTGE. However, few fungal species were detected by PCR-TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.     

Characterization of the microbial community in indoor environments: a chemical-analytical approach

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of (13)C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.     

Paxillus involutus/Pinus sylvestris Mycorrhizae from Heavily Polluted Forest II. Ultrastructural and Cytochemical Observations

Paxillus involutus/Pinus sylvestris mycorrhizae, collected from experimental plots (situated in a Pino-Quercetum forest) treated with cadmium dust, were previously reported to accumulate metals like Cd, P, S, N, Al and Si in vacuolar bodies. The Gomori-Swift test was applied to the same material to reveal if cysteine-rich proteins might be involved in element detoxification. Vacuolar deposits of high and low phosphate level were both stained. Higher intensity of the Swift reaction was found in the fungal mycelium from mycorrhizae sampled from polluted plots than in mycorrhizae from unpolluted sites. In both cases a higher intensity of the test was revealed in the host cytoplasm close to the Hartig net than in internal parts of roots. Different intensities of the Gomori-Swift reaction were also observed when the Hartig net was compared to the fungal mantle. A PATAg test was additionally processed and revealed an accumulation of glycogen in rosettes and net-like structures when samples from cadmium dust treated plots were observed.     

Characterization of the Microbial Community in Indoor Environments: a Chemical-Analytical Approach

An integrated procedure is presented whereby gas chromatography-ion trap mass spectrometry is used to determine chemical markers of gram-negative bacterial lipopolysaccharide (3-hydroxy fatty acids with 10 to 18 carbon atoms), gram-positive bacteria (branched-chain fatty acids with 15 and 17 carbon atoms), bacterial peptidoglycan (muramic acid), and fungal biomass (ergosterol) in samples of settled house dust. A hydrolysate of ¹³C-labeled cyanobacterial cells is used as an internal standard for the first three markers. These analyses require two dust samples, one for 3-OH fatty acids, branched-chain fatty acids, and muramic acid and another for ergosterol. The method may be used to characterize microbial communities in environmental samples.     

Isolation of fungal DNA from plant tissues and removal of DNA amplification inhibitors

Specific primers and the polymerase chain reaction (PCR) are increasingly used for detection of fungi in plants. Detection depends on isolation of DNA that is free of compounds that inhibit amplification. We report on the significance of inhibition in soybean stems with a focus on studies of the vascular fungal pathogen Phialophora gregata. A simple DNA extraction procedure based on a FastDNA® kit is described that allows consistent detection of fungi in soybean stems using PCR. The addition of polyvinylpyrrolidone and supplemental DNA purification steps overcame inhibition in over 90% of samples. These methods should also facilitate studies with other plant and fungal species.     

Molecular approach to the characterisation of fungal communities: methods for DNA extraction, PCR amplification and DGGE analysis of painted art objects

A protocol for efficient extraction of fungal DNA from micromycetes colonising painted art objects was developed. Polymerase chain reaction (PCR) inhibitors were successfully removed by a combined application of a Chelex-100 adsorption resin and a Geneclean Kit for Ancient DNA. Universal fungal primers for PCR amplification of 28S rDNA (U1 and U2) were tested for their applicability in denaturing gradient gel electrophoresis (DGGE) analysis of fungal communities. Artificially produced mortar samples inoculated with fungal pure cultures isolated from mural paintings were used as model objects for DNA extractions and DGGE analysis. Good resolution in DGGE was achieved using 260-bp rDNA fragments amplified with U1/DGGE and U2 primers directly from model communities.     

Fungi and fungal products in some Canadian houses

Building related illness prompted a study in the winter of 1986 to identify and quantify and fungal products present in c. 50 Canadian homes. Of these, 70% had been reputedly associated with health problems. Building parameters, i.e. air change rate and the internal moisture levels, were measured, and the fungi present were characterized and quantified along with their metabolites. Air and dust samples were analyzed and the fungal biomass in the dust was measured by a procedure which involved determination of ergosterol by a gas chromatograph/mass spectrometer system. Some 42 fungal species were identified in air, samples of which were further analyzed for fungal volatiles. Penicillium was the most common genus in both air and dust, together with Cladosporium and Alternaria. The potentially hazardous fungus Aspergillus fumigatus was found in only two houses, and Strachybotry atra in only one. New criteria are suggested to define the acceptable standards for indoor fungal levels in air during winter.     

Fungal molecular diagnostics: a mini review

Conventional methods to identify fungi have often relied on identification of disease symptoms, isolation and culturing of environmental organisms, and laboratory identification by morphology and biochemical tests. Although these methods are still fundamental there is an increasing move towards molecular diagnostics of fungi in all fields. In this review, some of the molecular approaches to fungal diagnostics based on polymerase chain reaction (PCR) and DNA/RNA probe technology are discussed. This includes several technological advances in PCR-based methods for the detection, identification and quantification of fungi including real-time PCR which has been successfully used to provide rapid, quantitative data on fungal species from environmental samples. PCR and probe based methods have provided new tools for the enumeration of fungal species, but it is still necessary to combine the new technology with more conventional methods to gain a fuller understanding of interactions occurring in the environment. Since its introduction in the mid 1980's PCR has provided many molecular diagnostic tools, some of which are discussed within this review, and with the advances in micro-array technology and real-time PCR methods the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual fungal species but also on whole communities.