Development of a Nested-PCR detection for Aspergillus flavus Based on Aflatoxins Gene aflP

Aspergillus flavus is a devastating pathogen for human health and economic development,for the aflatoxins produced by A.flavus has strong toxicity for humans and animals.Therefore,to establish a rapid,accurate and sensitivity detection system for A.flavus,we developed a nested polymerase chain reaction(PCR)based on the aflP(GenBank:FN398191)gene.In the present study,aflP was used as a molecular target,alignment of the aflPregion sequences with other sequences belonging to Aspergillus species closely related to A.flavus and other fungi was used to identify conserved and differing regions,and then two pairs of PCR primers(aflP-1-F/aflP-1-R and aflP-2-F/aflP-1-R)were developed.For specificity testing,DNA extracted from 7 A.flavus,5different Aspergillus spp.and 21 other fungi were used,and our results showed that a 211 bp of specific band can be amplified only in aflatoxins produced A.flavus strains.A nested PCR procedure using aflP-1-F/aflP-1-R as the first-round primers and the followed using aflP-2-F/aflP-1-R increased the detection sensitivity 100-fold to10 fg.Furthermore,the aflP-based PCR was used for detection analysis of cDNA from artificially and naturally aflatoxins-contaminated corn and peanut samples and our results suggest that the aflP-based PCR detection is a specific,sensitive and rapid diagnostic tool for A.flavus monitoring.