• Toxicological Research
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• v.32 no.1
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• pp.81-87
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• 2016

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 $65^{\circ}C$. The optimized template and primer concentration were $1.5{\mu}L\;(50ng/{\mu}L)$ and $3{\mu}L\;(10{\mu}M/{\mu}L)$ respectively. SYBR Green qPCR of four genes demonstrated amplification curves and melting peaks for tub1, afIM, afIR, and afID genes are at $88.0^{\circ}C$, $87.5^{\circ}C$, $83.5^{\circ}C$, and $89.5^{\circ}C$ 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.

• Applied Biological Chemistry
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• v.13 no.1
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• pp.35-42
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• 1970

Five samples of Korean native Maeju(fermented soy-bean mash) loaves which were collected each from Kyunggi, Chungchung, Kangwon, Cholla and Kyungsang-Do were examined for their fermenting microorganisms. The results of taxonomic and ecological studies of fermentation microorganisms in these Maeju loaves were as the fellows. (1) The fungus flora grew only is the outer layer of Maeju loaves. Miscellaneous molds, 3 species of Mucor, 2 species of Pericallium., one species each of Scopulariopsis and Aspergillus, were isolated. None of them seemed exclusively predominant to be able to designate as the ecologically significant. (2) The bacterial flora which consisted of two species, Bacillus subtilis and Bacillus pumilus were distributed uniformly in th a entire Maeju loaves. The inner parts of Maeju loaves were especially inhabited solely by these bacterial flora. Probably the Korean native Maeju fermentation could be characterized by these bacterial flora. A Staphylococcus species was also isolated probably as a casual contaminant. (3) The yeasts, Rhodotorula flava and Torulopsis dattila, were isolated from Maeju loaves though their ecological significance was not clear. (4) The ecological aspects of fermentation microbes in the outer and inner parts of Maeju loaves were apparently different, consequently different fermentation processes might have occurred in these two parts and it brought quite different final outlooks in the final matured Maeju loaves. The outer part, rather rigid and dry, retained the light brown color of boiled soy-bean; whereas the inner part, soft and sticky, showed dark brown color indicating severe chemical changes. (5) The aflatoxin producing mold, Aspergillus oryzae was isolated from one sample among 5 of Maeju loaves. In addition to the low probability of isolability from Maeju loaves samples, since this mold grew only in the outer layer of Maeju loaves with such a low population density, about $10^4/g$, perhaps the aflatoxin problem in Korean native soysauce may not be critical.