Asian Journal of Microbiology, Biotechnology & Environmental Sciences Paper

Vol 16, Issue 2, 2014; Page No.(361-372)

DEVELOPMENT OF A MOLECULAR METHOD FOR DETECTION OF MYCOTOXIN CONTAMINATION IN FOOD GRAINS

RAVICHARAN AMEDAA, JANAKIPRIYA SANNASIB, PRATHIMA NAMBURIC, ANURAG CHATURVEDIC, SREEDHAR MULINTIC, SUNIL C. KULKARNID, KRISHNA M.R. BHAGAVATULAE AND MANORAMA KANURIC

Abstract

Mycotoxin producing gene fragments in fungal strains were amplified using polymerase chain reaction (PCR) and real time PCR (RT-PCR) along with a Bioanalyzer in an attempt to develop a rapid assay for mycotoxin detection. Forward and reverse primers were designed from original gene sequences (obtained from GENBANK) of the three genes, aflQ (ordA), Tri13-DON, and pks, which are key genes involved in the production of three mycotoxins, namely, aflatoxin (AF), deoxynivalenol (DON), and ochratoxin (OTA), using DNASTAR Lasergene 8.0 version software. Primers were selected to amplify a 166 base pair fragment of aflQ from DNA of Aspergillus parasiticus and Aspergillus flavus, a 200 bp fragment of ochratoxin producing polyketide synthase gene from Aspergillus ochraceus, a 250 bp fragment of deoxynivalenol (DON) producing Tri13 gene from DNA of Fusarium culmorum and a 300 bp fragment of fumonisn producing FUM1 gene from DNA of Fusarium verticilloides. The fragments obtained were resolved on a DNA 1000 LabchipR in the Agilent 2100 Bioanalyzer for visualizing the amplified fragments. Amplification of the gene fragments was also achieved using RT-PCR with TAQMAN probes as well as SYBRGREEN, with cT values ranging from 19 to 30 for the four genes. Duplex PCR was successfully attempted to simultaneously detect two sets of genes in one PCR reaction, namely, aflQ and Tri13-DON, and FUM1 and aflQ. Mycotoxigenic ability of the fungi contaminating selected food grains, namely, groundnut, maize and rice, was confirmed using both molecular detection method for gene amplification, as well as LCMS method for mycotoxin detection in the contaminated grains.

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