Evaluation of PCR primers and PCR conditions for specific detection of common airborne fungi

Wu, Zhihong; Wang, Xiaoru; Blomquist, Göran

doi:10.1039/b200490a

Cited by 59 publications

(32 citation statements)

References 31 publications

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“…Other fungi were grown on 2% malt extract agar for a week at room temperature (22°C). The genomic DNAs were isolated from pure culture of each fungal strain by using a procedure described previously by Wu et al (26). Mycelial samples of ca.…”

Section: Methodsmentioning

confidence: 99%

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Zeng

Westermark²,

Rasmuson-Lestander

et al. 2004

Appl Environ Microbiol

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Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 10 7 m ؊3 by real-time PCR and 10 6 m ؊3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment.Wallemia sebi is a deuteromycete fungus capable of growth over a wide range of water activity from 0.69 to 0.997 (15). It can potentially grow in various environments and on different substances and has been isolated from jam, cake, cereals, salted meat, fish, and dairy products (12,23). Up to now, only one species is described in the genus Wallemia. W. sebi grows slowly on commonly used culture media, such as malt extract agar, and is often obscured by the fast-growing fungi. Thus, its presence in different environments has often been overlooked, which in turn hindered the studies on its distribution and ecology. Recently, with the use of selective media for xerophilic fungi, W. sebi has been found to be very common in the agricultural environments of many parts of the world (4,6,9,16).The conidium of W. sebi has a shape of a rough-surfaced sphere of 2.5 to 3.5 m in diameter (18); thus, it can reach the respiratory bronchioles when inhaled. Airborne W. sebi has been suspected to be a causative agent of human allergies, particularly bronchial asthma (17). Elevated levels of immunoglobulin G (IgG) antibodies were observed among Finnish farmers exposed to W. sebi (9). In eastern France, W. sebi has also been identified as playing a role in farmer's lung disease (16). The fungus produces a toxic metabolite, walleminol A, with a bioinhibitory dose effect similar to those of other mycotoxins such as penicillic acid (23).Conventional methods for the detection and quantification of W. sebi rely on microscopic or culture techniques that are time consuming and laborious. Molecular techniques are promising approaches complementary to the conventional detection methods. PCR-based methods have the advantage of detecting the presence of microorganisms in a sample regardless of the...

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Section: Methodsmentioning

confidence: 99%

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Zeng

Westermark²,

Rasmuson-Lestander

et al. 2004

Appl Environ Microbiol

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“…All PCR reactions were prepared in a dedicated PCR laminar flow cabinet (Esco Technologies Inc., PA, USA). PCR reaction mixtures and thermal cycling conditions were as described in Wu and Blomquist [15]. Briefly, each PCR reaction consisted of: 1×buffer (Denville Scientific, Saint-Laurent, QC, Canada), 500 µM of each primer, 1.5 U of Hot Start Taq DNA polymerase (Denville Scientific, SaintLaurent, QC, Canada), 200 mM of each dNTP (Bioshop Canada Inc., Burlington, ON, Canada), 1-5 ng of template DNA extracted from the spoiled samples, and molecular grade water (Sigma-Aldrich, Oakville, ON, Canada) in a total volume of 50 µl.…”

Section: Pcr Amplification Of Fungal 18s Rdnamentioning

confidence: 99%

Analysis of Fungal Diversity in Ready-to-Eat Pizza and Effectiveness of Pulsed Ultraviolet-Light Treatment for Inactivation of Mold on Agar Surface

Ngadi¹

2015

J Bioprocess Biotech

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Fungal contamination is a significant issue in the food production industry; therefore, identification and characterization of food spoilage fungi would allow for early intervention to limit the amount of fungal contamination, particularly in cereal-based industries. In the present study, culture-dependent and culture-independent methods were applied to study the microbiota of ready-to-eat pizza. The study was pursued by evaluating the effectiveness of a broad-spectrum pulsed ultraviolet light for the decontamination of Penicillium roqueforti (a dominant spoilage mold in bakery products) on the surface of solid agar as a representative of a flat food surface. The average population of mesophilic aerobic bacteria (MAB), mesophilic anaerobic bacteria (MANB), lactic acid bacteria (LAB), molds and yeasts (M+Y) on naturally spoiled pre-cooked pizza were 6.7 ± 0.5, less than 2.3, 2.8 ± 0.6 and 5.4 ± 0.4 log10 CFU g -1 , respectively. Cloning and sequencing identified at least 5 genera and species of fungi (Penicillium spp., Saccharomyces spp., Rhodotorula mucilaginosa, Monascus fuliginosus, Galactomyces geotrichum) from 2 phyla (Ascomycota and Basidiomycota). Pulsed light process parameters evaluated were treatment time (1, 3, 5, 7 and 10 min) and voltage input (500, 750 and 1,000 V) at 5 cm distance from the pulsed UV-light. An increase of the input voltage of the lamps and of the duration of the treatment resulted in a higher inactivation of P. roqueforti. The population of P. roqueforti was reduced after 10 min of exposure to pulsed light by 3.74, 5.36 and 6.14 log10 CFU ml -1 at 500, 750 and 1,000 V, respectively. The inactivation kinetics was best described by the Weibull model (2 parameters) with the smallest root mean squared error (RMSE) and R 2 ≥ 0.92. The results of this work show that pulsed light is a promising technique for fungi elimination or decontamination in the bakery industry.

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“…Culture-based methods are by far the most widely utilized, but these methods frequently underestimate the amount of or fail to detect the target organism in the environment (Wu et al 2002, Oliver 2005, Macey et al 2008a,b, Yamamoto et al 2010. Culture-based methods rely on the isolation and maintenance of axenic cultures of the causative pathogen and often depend on further manipulations like the induction of zoosporogenesis for positive identification.…”

Section: Introductionmentioning

confidence: 99%

“…Culture-based methods rely on the isolation and maintenance of axenic cultures of the causative pathogen and often depend on further manipulations like the induction of zoosporogenesis for positive identification. These methods are time consuming, laborious and at risk of misidentifying species due to intra-and inter-specific variations in species morphological characteristics (Wu et al 2002). Consequently, the use of culture-based techniques alone for disease management could be problematic.…”

mentioning

confidence: 99%

“…Unlike animal and bacterial cells, fungal cell walls are extremely tough and are often hard to disrupt by commonly used cell lysis methods, such as chemical and enzymatic digestion (Zhou et al 2000). However, physical disruption methods using glass or zirconia beads, freeze/thaw and a freeze press have been shown to be rapid, effective and convenient methods for disrupting cells and releasing DNA from fungi (Zhou et al 2000, Wu et al 2002. Karakousis et al (2006) also demonstrated that pestle grinding was an efficient method for lysing fungal hyphae.…”

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Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues

Greeff¹,

Christison²,

Macey³

2012

Dis. Aquat. Org.

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Evaluation of PCR primers and PCR conditions for specific detection of common airborne fungi

Cited by 59 publications

References 31 publications

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Detection and Quantification ofWallemia sebiin Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

Analysis of Fungal Diversity in Ready-to-Eat Pizza and Effectiveness of Pulsed Ultraviolet-Light Treatment for Inactivation of Mold on Agar Surface

Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues

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