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

Anong Bintvihok; Supitchaya Treebonmuang; Kitiya Srisakwattana; Wisut Nuanchun; Koranis Patthanachai; Sungworn Usawang

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저자정보: Anong Bintvihok (Chulalongkorn University) Supitchaya Treebonmuang (Chulalongkorn University) Kitiya Srisakwattana (Chulalongkorn University) Wisut Nuanchun (Chulalongkorn University) Koranis Patthanachai (Chulalongkorn University) Sungworn Usawang (Chulalongkorn University)

저널정보: 한국독성학회 Toxicological Research Toxicological Research Vol.32 No.1

발행연도: 2016.1

수록면: 81 - 87 (7page)

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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 aflatoxinproducing 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 65oC. 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 88.0oC, 87.5oC, 83.5oC, and 89.5oC 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.

#Polymerase chain reaction #Aflatoxin-producing fungus #Nested PCR #PCR-RFLP #SYBR Green real-time PCR

참고문헌 (25)

참고문헌 신청

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Bhat, R.V. (1991) Aflatoxin: successes and failures of three decades of research in Fungi and Mycotoxins in Stored Products (Champ, B.R., Highley, E., Hocking, A.D. and Pitt, J.I. edition). ACIAR Proc., 36, 80-85.

Norred, W.P. (1986) Occurrence and clinical manifestations of aflatoxicosis in Diagnosis of Mycotoxicosis (Richard, J.L. and Thurston, J.R. edition). Martinus Nijhoff, Dordrecht, pp. 11-

4. Pitt, J.L. and Hocking, A.D. (1985) Fungi and food spoilage, Academic Press, Australia, pp. 259-311.

Bintvihok, A., Kiatipattanasakul, W. and Doi, K. (1997) Acute toxicity of aflatoxin B1 in three species of domestic fowls. J. Toxicol. Pathol., 10, 149-152.

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