NDLI: A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR)

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A Rapid and Sensitive Detection of Aflatoxin-producing Fungus Using an Optimized Polymerase Chain Reaction (PCR)

Content Provider	PubMed Central
Author	Bintvihok, Anong Treebonmuang, Supitchaya Srisakwattana, Kitiya Nuanchun, Wisut Patthanachai, Koranis Usawang, Sungworn
Copyright Year	2016
Abstract	Aflatoxin B1 (AFB1) is produced by Aspergillus flavus growing in feedstuffs. Early detection of maize contamination by aflatoxigenic fungi is advantageous since aflatoxins exert adverse health effects. In this study, we report the development of an optimized conventional PCR for AFB1 detection and a rapid, sensitive and simple screening Real-time PCR (qPCR) with SYBR Green and two pairs of primers targeting the aflR genes which involved aflatoxin biosynthesis. AFB1 contaminated maize samples were divided into three groups by the toxin concentration. Genomic DNA was extracted from those samples. The target genes for A. flavus were tested by conventional PCR and the PCR products were analyzed by electrophoresis. A conventional PCR was carried out as nested PCR to verify the gene amplicon sizes. PCR-RFLP patterns, obtained with Hinc II and Pvu II enzyme analysis showed the differences to distinguish aflatoxin-producing fungi. However, they are not quantitative and need a separation of the products on gel and their visualization under UV light. On the other hand, qPCR facilitates the monitoring of the reaction as it progresses. It does not require post-PCR handling, which reduces the risk of cross-contamination and handling errors. It results in a much faster throughout. We found that the optimal primer annealing temperature was 65°C. The optimized template and primer concentration were 1.5 μL (50 ng/μL) and 3 μL (10 μM/μL) respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at 88.0°C, 87.5°C, 83.5°C, and 89.5°C respectively. Consequently, it was found that the four primers had elevated annealing temperatures, nevertheless it is desirable since it enhances the DNA binding specificity of the dye. New qPCR protocol could be employed for the determination of aflatoxin content in feedstuff samples.
Related Links	http://dx.doi.org/10.5487/tr.2016.32.1.081
Ending Page	87
Page Count	7
Starting Page	81
File Format	PDF
ISSN	19768257
e-ISSN	22342753
Journal	Toxicological Research
Issue Number	1
Volume Number	32
Language	English
Publisher	Korean Society of Toxicology
Publisher Date	2016-01-31
Access Restriction	Open
Rights Holder	Korean Society of Toxicology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Health, Toxicology and Mutagenesis Toxicology

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