Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain

Sudharsan, S.; Britzi, Malka; Zakin, Varda; Kostyukovsky, M.; Trostanetsky, Anatoly; Quinn, Elazar; Sionov, Edward

doi:10.3390/toxins9100302

Cited by 91 publications

(64 citation statements)

References 69 publications

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“…To detect and differentiate aflatoxigenic and non-toxigenic Aspergilli, several methods have been developed including molecular marker-based methods and fungal culture methods [19,20]. Currently, in most cases, aflatoxigenic fungi are being identified by culture methods coupled with thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC).…”

Section: Introductionmentioning

confidence: 99%

A Liquid Chromatographic Method for Rapid and Sensitive Analysis of Aflatoxins in Laboratory Fungal Cultures

Alshannaq

2020

Toxins

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Culture methods supplemented with high-performance liquid chromatography (HPLC) technique provide a rapid and simple tool for detecting levels of aflatoxins (AFs) produced by fungi. This study presents a robust method for simultaneous quantification of aflatoxin (AF) B1, B2, G1, and G2 levels in several fungal cultivation states: submerged shake culture, liquid slant culture, and solid-state culture. The recovery of the method was evaluated by spiking a mixture of AFs at several concentrations to the test medium. The applicability of the method was evaluated by using aflatoxigenic and non-aflatoxigenic Aspergilli. A HPLC coupled with the diode array (DAD) and fluorescence (FLD) detectors was used to determine the presence and amounts of AFs. Both detectors showed high sensitivity in detecting spiked AFs or AFs produced in situ by toxigenic fungi. Our methods showed 76%–88% recovery from medium spiked with 2.5, 10, 50, 100, and 500 ng/mL AFs. The limit of quantification (LOQ) for AFs were 2.5 to 5.0 ng/mL with DAD and 0.025 to 2.5 ng/mL with FLD. In this work, we described in detail a protocol, which can be considered the foremost and only verified method, to extract, detect, and quantify AFs employing both aflatoxigenic and non-toxigenic Aspergilli.

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Section: Introductionmentioning

confidence: 99%

A Liquid Chromatographic Method for Rapid and Sensitive Analysis of Aflatoxins in Laboratory Fungal Cultures

Alshannaq

2020

Toxins

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“…Following the multiplex PCR assay with the primer pairs: nor-1, ver-1, omt-A, and aflR1 sequences, the results indicated that the DNA from Aspergillus flavus (4) and Aspergillus parasiticus(1) [AF4 and AP1] possessed all the four aflatoxin biosynthesis genes. A number of researchers have made similar reports on different agricultural and food products (Nwachukwu et al, 2019;Sadhasivam et al, 2017;Gherbawy et al, 2016;Degola et al, 2007). The DNA of AF2 and AF3 indicated positive reaction for the following genes: nor-1 gene, ver-1 gene and aflR gene.…”

Section: Discussionmentioning

confidence: 88%

Untitled

2020

IBSPR

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Contamination of feeds by aflatoxins has been of global concern as a result of their noxious effects on human and animal health as well as their importance in international trade. Timely assessment of fungal contaminants, characterization and identification of the mycotoxigenic species are very necessary, not just for assessing food quality, but also for the development of strategies to control and ensure food safety. In this study, fungal species isolated from selected grains and cashew nut samples from south east Nigeria were screened for aflatoxigenic potential by Multiplex PCR Technique. Primers were designed specifically for the amplification of genes responsible for aflatoxigenic biosynthesis pathways. The necessary experimental conditions were standardized to achieve optimum mPCR result. The DNA extracted from seven fungal isolates were subjected to multiplex PCR using nor-1 (F&R), ver-1 (F&R), omtA (F&R) and aflR (F&R) as the primer pairs. DNA samples from AF4 and AP1 possessed the four genes needed for aflatoxigenic biosynthesis pathways. AF1 and AP3 did not show any positive reaction to the four genes after amplification following PCR. However, AF2 and AF3 showed positive response to the two structural genes (nor-1and ver-1) and also for the regulatory gene (aflR) but were negative to the omtA structural gene. The DNA sample of AP3 was only reactive to the structural gene aflR but, did not have the 3 structural genes. Multiple PCR assay has proven to a more reliable, accurate and sensitive molecular tool for the detection and characterization of mycotoxinproducing fungi from agricultural commodities.

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“…A multiplex polymerase chain reaction (PCR) strategy was established for rapid identification of mycotoxigenic fungi (Sadhasivam et al, 2017). The RT-PCR results obtained with genomic DNA as a template indicated that all tested aflatoxigenic isolates expressed nor-1 gene with significant expression levels in chicken products.…”

Section: Expression Of Nor-1 Gene Of Aflatoxins and Ocra Gene Of Ochrmentioning

confidence: 99%

Determination of aflatoxins by HPLC and the identification of biosynthetic nor-1 gene of aflatoxins in poultry products by PCR assay

Awad¹,

Mahmoud²,

Rashed

2019

Benha Veterinary Medical Journal

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Aflatoxins residues in different poultry products were determined using HPLC technique and the expression of biosynthetic nor-1 gene of these aflatoxins by PCR assay. 90 different chicken products were analyzed for presence of aflatoxins (AFB1, AFB2, AFG1 and AFG2) using highly accurate, robust, selective, sensitive and precise HPLC-FLD assay. Incidence of aflatoxins in examined samples showed that the level of AFB1, AFB2 and AFG2 were 10 % for each and of AFG1 was 16.7%. The highest aflatoxins levels of analyzed samples were found in the liver. Expression of nor-1 gene of aflatoxins in polluted poultry products by PCR assay. A Real time PCR (RT-PCR) technique was applied to detect the gene expression of structural nor-1, which catalyzed the first step in the biosynthetic pathway of aflatoxins. The obtained results showed a very strong relationship between the presences of aflatoxins biosynthetic genes as analyzed by multiplex PCR and aflatoxins determination by HPLC/FLD.

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Rapid Detection and Identification of Mycotoxigenic Fungi and Mycotoxins in Stored Wheat Grain

Cited by 91 publications

References 69 publications

A Liquid Chromatographic Method for Rapid and Sensitive Analysis of Aflatoxins in Laboratory Fungal Cultures

A Liquid Chromatographic Method for Rapid and Sensitive Analysis of Aflatoxins in Laboratory Fungal Cultures

Untitled

Determination of aflatoxins by HPLC and the identification of biosynthetic nor-1 gene of aflatoxins in poultry products by PCR assay

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