Abstract
This study aimed to determine the role of polymerase chain reaction (PCR) and High-performance liquid chromatography (HPLC) technique in the discrimination between aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus. The isolates were identified based on macroscopical and microscopical characteristics, and extracted aflatoxin was detected by HPLC technique. Furthermore, DNA was extracted from the all isolates and carried out by PCR to amplify target genes encoding for toxin production (nor-1, ver-1 and aflR). The results showed that the genes (aflR, nor-1) were found in 11 (73%) of isolates, while the (ver-1) gene appeared in 10 (67%) of isolates. Both aflatoxigenic and non-aflatoxigenic isolates were also determined depending on the amplification of gene sites in the targeted DNA. HPLC technique has also used with high efficiency to ensure the aflatoxin-producing isolates and to evaluate the level of aflatoxin B1 production for 15 isolates of A. flavus. Ten isolates were able to produce aflatoxin with rates ranged from 0.78 to 45.03 ppm. PCR technique has proved high efficiency in the differentiation between aflatoxigenic and non-aflatoxigenic isolates of A. flavus. Moreover, aflatoxin production was directly associated with gene appearance and gene detection. Also, HPLC technique is a standard and superb technique in identifying and analyzing aflatoxin with high sensitivity and accuracy.
Highlights
Dr.mohamme dh24@yah oo.com Abs tract This study aimed to determine the role of polymerase chain reaction (PCR) and High-performance liquid chromatography (HPLC) technique in the discrimination between aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus
Conventional methods which used for detection of aflatoxins are microbiological identify, high-performance liquid chromatography (HPLC), thin layer chromatography (TLC) or enzyme-linked immunosorbent assay (ELISA)
The aims of our study are to )1( determinate the capability of A. flavus isolated from clinical and environmental sources for production of aflatoxin B1, (2) differentiate between aflatoxigenic and non-aflatoxigenic isolates of the above fungus depending on PCR in addition to molecular detection for aflatoxin encoding genes, and (3) investigate the role of HPLC in analyzing and identifying aflatoxin B1 (AFB1) level produced from A. favus obtained from well extracted samples
Summary
Dr.mohamme dh24@yah oo.com Abs tract This study aimed to determine the role of polymerase chain reaction (PCR) and High-performance liquid chromatography (HPLC) technique in the discrimination between aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus. DNA was extracted from the all isolates and carried out by PCR to amplify target genes encoding for toxin production (nor-1, ver-1 and aflR). Because of the spread contamination of food products with A.flavus and the difficulty of access to a cut-off diagnostic tool for differentiation of aflatoxin producing isolates in addition to molecular screening and detection for aflatoxin encoding genes (ver-1, nor-1, and aflR). The aims of our study are to )1( determinate the capability of A. flavus isolated from clinical and environmental sources for production of aflatoxin B1, (2) differentiate between aflatoxigenic and non-aflatoxigenic isolates of the above fungus depending on PCR in addition to molecular detection for aflatoxin encoding genes (ver-1, nor-1 and aflR), and (3) investigate the role of HPLC in analyzing and identifying aflatoxin B1 (AFB1) level produced from A. favus obtained from well extracted samples. All PCR reagents were provided and synthesized by (Bioneer-Korea), Primers were scientifically designed thematically and all PCR attempts were carried out in PCR Thermal Cycler (Applied Biosystems)
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