PCR detection of Aspergillus niger producing ochratoxin A

Abstract:

[Objective] Rapid detection of ochratoxin A-producing Aspergillus niger. [Methods] A PCR procedure has been developed for the rapid detection of ochratoxin A-producing A. niger. Two specific primers were designed based on the nucleotide sequence of the acyl transferase (AT) domain of the polyketide synthase encoded by An15g07920 from A. niger CBS 513.88. [Results] Specificity was confirmed by testing primers towards purified DNA from 72 Aspergillus genus strains, including A. niger, A. carbonarius, A. ochraceus, A. petrakii, A. parasiticus and A. tubingensis. Two specific primers can amplify a unique band from OTA-producing A. niger, but not from other OTA-producing strains. However, the use of the primer pairs also allowed amplification of the DNA from three OTA-non-producing A. niger. The quantitative real-time PCR found that the part of AT homeotic gene of An15g07920 can be normally expressed under the OTA-producing condition in OTA-non-producing A. niger. Therefore, the reason of false positive was not the gene can not express. The detection limit of the developed PCR protocol was 25 pg for DNA templates and about 4.0×104–4.0×105 spores/g when it was evaluated directly on artificially inoculated food. [Conclusion] The developed PCR procedure could be used for rapid detection of OTA-producing A. niger and is a promising tool in the prediction of potential ochratoxigenic risk for A. niger in foods.