2013 | OriginalPaper | Chapter
Detection of Toxic Aspergillus Species in Food by a Multiplex PCR
Authors : Nguyen Thi Hue, Nguyen Huu Nhien, Pham Minh Thong, Chu Nguyen Thanh, Pham Van An
Published in: 4th International Conference on Biomedical Engineering in Vietnam
Publisher: Springer Berlin Heidelberg
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Aspergillus
parasiticus
and
flavus
are known as the main producers carcinogen aflatoxins. The presence of these fungus and aflatoxins in food is a serious risk for human and animal health. The identification of these fungi is not straightforward due to its similarities with closely related species. In this study, a multiplex PCR assay have been developed which overcame the disadvantages in detection of
Aspergillus
parasiticus
and
flavus
by conventional morphological method. Twelve isolates of filamentous fungi, representing six
Aspergillus
and
Aspergillus
-related species were used. DNA was extracted from mycelium following SDS method modified from Plaza’s method. The extracted DNA was used for the PCR method to identify the presence of selected fungi using specific primer sets. The result of identification was analysed by agarose gel electrophoresis. The sensitivity and specificity of the detection were checked by result of a gradient PCR assays. Two specific set of primers were successful designed based on Aflatoxin biosynthesis gene cluster to detect A.
parasiticus
and A.
flavus
species, with the limitation of detection (LOD) for each set of primer is 0.005ng and 0.008ng. A multiplex PCR assay set up with two these sets of primers was also successful in detection of both targets of A.
parasiticus
and A.
flavus
at the annealing temperature of 65°C with high sensitive and specificity. The limitation of detection (LOD) is 0.5ng. A highly specific multiplex PCR assay have been developed to detect the presence of
Aspergillus
parasiticus
and
flavus
in food. This will permit prediction of the presence of aflatoxins type G and aflatoxins type B, the most potent natural carcinogens.