Detection of aflatoxigenic fungi in feeds using the PCR method

Zachová, Iveta; Vytřasová, Jarmila; Pejchalová, Marcela; Červenka, Libor; Tavčar-Kalcher, Gabrijela

doi:10.1007/bf02931519

Cited by 20 publications

(12 citation statements)

References 5 publications

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“…Three genes were selected for PCR: apa-2, ver-1 and aflR, the regulatory gene of aflatoxin B1 biosynthesis. For PCR involving apa-2 and ver-1 (which were amplified simultaneously), the method according to Shapira et al (1996) was used as modified by Zachová et al (2003). For the regulatory gene aflR, the method according to Sweeney et al (2000) was used.…”

Section: Determining Dna Concentrationmentioning

confidence: 99%

“…The most difficult steps in the isolation of Aspergillus DNA is to disrupt the cell wall without causing damage to genomic DNA (Bir et al 1995). Most methods use mechanical or mechanical-physical techniques for the disruption of the cell wall, such as disruption with glass beads (van Burik et al 1998;Haugland et al 2002;Loeffler et al 2002;Kabir et al 2003;Somashekar et al 2004), grinding in liquid nitrogen (Raeder & Broda, 1985;Shapira et al 1996;Färber et al 1997;Sweeney et al 2000;Mayer et al 2003;de Aguirre et al 2004;Manonmani et al 2005), mill grinding (Cenis 1992), alternating freezing and thawing (Griffin et al 2002), ultrasound in combination with lysis buffer containing sodium dodecyl sulphate (SDS) (Van Burik et al 1998;Zachová et al 2003), and microwave radiation (Tendulkar et al 2003). Enzymes can also be used for digesting the cell wall (Jin et al 2004).…”

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confidence: 99%

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Comparison of methods for isolating fungal DNA

Moťková¹,

Vytřasová²

2011

Czech J. Food Sci.

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Moťková P., Vytřasová J. (2011): Comparison of methods for isolating fungal DNA. Czech J. Food Sci., 29 (Special Issue): S76-S85.In this study methods of fungal DNA isolation were optimised and compared. The aim of the isolation processes was to obtain DNA of sufficient quality and quantity necessary for its amplification, as most detection techniques require DNA amplification before the proper DNA detection itself. For this purpose, classic methods of DNA extraction were compared and optimised while isolations using commercial kits were also done. The methods were evaluated from several perspectives, with focus especially laid on the isolated DNA not contain PCR inhibitors which would prevent DNA amplification, thus inhibiting the detection itself. For optimising the individual methods, collection strains of the genus Aspergillus were used. After the evaluation, two most suitable methods were selected and chosen for isolating potentially aflatoxigenic moulds taken from food samples. These methods were the commercially supplied kit for isolating DNA from plant leaves from Sigma and a classic method according Cenis in combination with the cell wall disruption by means of liquid nitrogen.

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Section: Determining Dna Concentrationmentioning

confidence: 99%

mentioning

confidence: 99%

Comparison of methods for isolating fungal DNA

Moťková¹,

Vytřasová²

2011

Czech J. Food Sci.

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“…However, this approach is very time-consuming, laborious, and requires facilities and mycological expertise (Edwards et al 2002). PCR-based methods that target DNA are considered a good alternative for rapid diagnosis due to their high specificity and sensitivity, and have been used for the detection of aflatoxigenic strains of A. flavus and A. parasiticus (Shapira et al 1996;Fa¨rber et al 1997;Sweeney et al 2000;Criseo et al 2001;Chen et al 2002;Mayer et al 2003;Zachova´et al 2003;Somashekar et al 2004;Gonza´lez-Salgado et al 2008). However, as yet, none of these methods is capable of reliably detecting and discriminating A. parasiticus alone from other species of Section Flavi by conventional PCR assays.…”

Section: Introductionmentioning

confidence: 99%

Specific detection ofAspergillus parasiticusin wheat flour using a highly sensitive PCR assay

Sardiñas

Vázquez

Gil-Serna

et al. 2010

Food Additives & Contaminants: Part A

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Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.

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“…New methods developed in recent years include DNA-based analytical methods (e.g. Polymerase Chain Reaction-PCR) for fungal detection [11], and enzyme-linked immunoassays for mycotoxin detection [12]. Morphologic information during A. flavus growth also can be used for the determination of aflatoxin production [13].…”

Section: Introductionmentioning

confidence: 99%

Differentiation of toxigenic fungi using hyperspectral imagery

Yao

Hruska

Kincaid

et al. 2008

Sens. & Instrumen. Food Qual.

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Some pathogenic fungi, Aspergillus flavus for example, produce mycotoxins that can contaminate grain products including wheat and corn. The contaminated grain poses a threat to the health of both humans and animals. Therefore, from the perspective of food safety and protection, it is important to detect and identify the different toxin-producing fungi encountered in food production. Earlier studies examined various spectral-based, nondestructive methods for the detection of fungi and toxins. The present report focused on the feasibility of using spectral image data for fungal species classification. A tabletop hyperspectral imaging system, VNIR-100E, was used for spectral and spatial data acquisition. A total of five fungal species were selected for a two-part experiment: Penicillium chrysogenum, Fusarium moniliforme (verticillioides), Aspergillus parasiticus, Trichoderma viride, and Aspergillus flavus. All fungal isolates were cultured on media under laboratory conditions and were imaged on day 5 of growth. The objective of the study was to use visible near-infrared hyperspectral imagery to differentiate fungal species. Results indicate that all five fungi are highly separable with classification accuracy of 97.7%. In addition, all five fungi could be classified by using only three narrow bands (bandwidth = 2.43 nm) centered at 743 nm, 458 nm, and 541 nm.

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Detection of aflatoxigenic fungi in feeds using the PCR method

Cited by 20 publications

References 5 publications

Comparison of methods for isolating fungal DNA

Comparison of methods for isolating fungal DNA

Specific detection ofAspergillus parasiticusin wheat flour using a highly sensitive PCR assay

Differentiation of toxigenic fungi using hyperspectral imagery

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