**5. PCR based detection of aflatoxin producing** *Aspergillus* **spp**

## **5.1 PCR primers and amplification**

Primers *nor-1* FP (5′-ACCGCTACGCCGGCACTC TCGGCAC-3′) and *nor-1* RP (5′-GTTGGCCGCCAG CTTCGACACTCCG-3′) were set to amplify an amplicon of 400 bp of norsolorinic acid reductase; *omtA* FP (5′-GGCCCGGTTCCTTG GCTCCTAAGC3′) and *omtA* RP (5′-CGCCCCAGTGAGACCCTTCC TCG-3′) to amplify a 1024 bp fragment of sterigmatocystin O-methyltransferase; and *aflR* FP (5′-TATCT CCCCCCGGGCATCTCCCGG-3′) and *aflR* RP (5′-CCGTCAGACAGCCACTGGACACGG-3′) to amplify a 1032 bp fragment of regulatory protein (*aflR*), involved in aflatoxin biosynthesis. The nucleotide sequence of all these genes from *A. parasiticus* are available at NCBI, GenBank at aceession numbers L27801 (*nor-1*), SRRC 2043 (*aflR*) and SRRC 143 (*omt-1*). PCR was performed in 15 μL of reaction volume containing 7.5 μl master mix, 1 μl forward primer, 1 μl of reverse primer and 4.5 μl nuclease free water and 1 μl of extracted DNA as template (with a total concentration of 100 ng of genomic DNA per reaction). PCR condition for *nor 1* primer initial denaturation for 5 min at 94° C, followed by 35 cycles at 94° C for 30 s, at 67° C for 30 s and at 72° C for 30 s and then final elongation at 72° C for 10 min [71]. PCR condition for *omtA* and *aflR* primer initial denaturation for 10 min at 95° C, followed by 30 cycles at 94° C for 1 min, at 65° C for 2 min and at 72° C for 2 min and then final elongation at 72° C for 5 min [71]. PCR products were separated by electrophoresis on a 1 % agarose gel with 0.5 % ethidium bromide in 1× TBE buffer and visualized under a Gel documentation system (Dynamica, GelView Master). 1 kb plus DNA Ladder (BioLabs, New England) was used as molecular size marker for the analysis of fragment size.

*Aflatoxins and Fumonisins Contamination of Maize in Bangladesh: An Emerging Threat… DOI: http://dx.doi.org/10.5772/intechopen.101647*
