PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Somashekar, D.; Rati, E. R.; Chandrashekar, A.

doi:10.1016/j.ijfoodmicro.2003.10.011

Cited by 49 publications

(37 citation statements)

References 16 publications

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“…isolated from forage (98 strains) 71.40% were positive by PCR analysis and 51.40% of them were identified with A. flavus, however A. parasiticus was not isolated. The results obtained in this work are in agreement with SOMASHEKAR et al (2004), who showed that PCR-RFLP patterns obtained with HincII can be used to distinguish the two species. A. flavus cleaved into 3 fragments of 385, 250, 161bp whereas A. parasiticus, having one restriction site for the HincII, produced 2 fragments of 546 and 250bp.…”

Section: Resultssupporting

confidence: 91%

“…First, a new PCR was used to amplify two target fragments on A. flavus and A. parasiticus, and the aflR primer sequences were designed according to the described by SOMASHEKAR et al (2004) to amplify a fragment of 796pb (aflR1-Forward: 5´-AAC CGC ATC CAC AAT CTC AT-3´ and aflR2-Reverse: 5´-AGT GCA GTT CGC TCA GAA CA-3´). The reaction mixture consisted of 1.0µg of fungal template DNA, 50pmol of each primer, MgCl 2 -free reaction buffer, 2.0mM MgCl 2 , 0.5U of Taq polymerase and 0.2mM of each dNTP.…”

Section: Restriction Site Analysis Of Pcr Productsmentioning

confidence: 99%

“…The mycotoxigenic profile (regarding aflatoxins B and G and CPA) of these strains has been routinely used for identification (RODRIGUES et al, 2009). Molecular techniques have been used to differentiate these two species (SOMASHEKAR et al, 2004;RODRIGUES et al, 2009). However, it is to be emphasized that the PCR detection of A. flavus, A. parasiticus or A. nomius is no guarantee of aflatoxin production since gene other than those involved in the biosynthesis of aflatoxins are not target for amplification (LEVIN, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach

et al. 2015

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Section: Resultssupporting

confidence: 91%

Section: Restriction Site Analysis Of Pcr Productsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach

et al. 2015

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“…The use of two-dimensional thin-layer chromatography (2D-TLC) (44,45) and high performance thin-layer chromatography (HPTLC) for the determination of aflatoxins has been reported (46). An alternative complex media based method is reported to detect the natural fluorescence of aflatoxins released by the growing mycelium (47) or relies on multiplex polymerase chain reaction (PCR) and real time polymerase chain reaction (RT-PCR) detection of genes or their transcripts involved in the aflatoxin biosynthetic pathway (48,49). An enzyme-linked immunosorbent assay (ELISA) for AFB 1 , and levels of total aflatoxins were quantified and confirmed by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) (50).…”

Section: Introductionmentioning

confidence: 99%

Determination of Aflatoxins in Medicinal Plants by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Mujeeb

Ahmad

et al. 2013

J Pharm Pharm Sci

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-Purpose. The intention of the proposed work is to study the presence of the aflatoxins B 1 , B 2 , G 1 and G 2 in medicinal plants, namely Mucuna pruriens, Delphinium denudatum and Portulaca oleraceae. Methodology. The aflatoxins were extracted, purified by immunoaffinity column chromatography and analysed by high-performance liquid chromatography-tandem quadrupole mass spectrometry with electrospray ionisation (HPLC-MS/MS). Fungal count was carried out in PDA media. Results. A good linear relationship was found for AFB 1 , AFB 2 , AFG 1 and AFG 2 at 1-10 ppb (r>0.9995). The analyte accuracy under three different spiking levels was 86.7-108.1 %, with low per cent relative standard deviations in each case. The aflatoxins can be separated within 5 to7 min using an Agilent XDB C18-column. We found that AFB 1 and AFB 2 were in trace amounts below the detection limit in M. pruriens whilst they were not detected in D. denudatum. P. oleraceae was found to be contaminated with AFB 1 and AFB 2 . AFG 1 and AFG 2 were not detected in M. pruriens, P. oleraceae and were below the detection limit in D. denudatum. This was consistent with very low numbers of fungal colonies observed after 6 hr of incubation. Conclusion. The analytical method developed is simple, precise, accurate, economical and can be effectively used to determine the aflatoxins in medicinal plants and therefore to control the quality of products. The aflatoxin levels in the plant extracts examined were related to the minimal fungal load in the medicinal plants examined.

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“…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%

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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PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Cited by 49 publications

References 16 publications

Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach

Identification of toxigenic Aspergillus species from diet dairy goat using a polyphasic approach

Determination of Aflatoxins in Medicinal Plants by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Comparison of methods for isolating fungal DNA

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