Direct and Conventional Multiplex PCR Assays to Detect the Zearalenone Producing Fusarium Species in White and Brown Rice

Introduction: Zearalenone (ZEA) is an estrogenic mycotoxin produced by some species of Fusarium and commonly found in rice throughout the world. 

Purpose: The aim of this study is to detect ZEA producing Fusarium species in white and brown rice by conventional and direct multiplex polymerase chain reaction (mPCR).

Methods: The presence of 4 genes (PKS4, PKS13, ZEB1 and ZEB2) involved in ZEA biosynthesis was tested for 15 referenced Fusarium strains to optimize conventional and direct mPCR assays (in terms of specificity and sensitivity). Chemical analysis of ZEA was carried out for Fusarium cultures and rice samples by high performance-liquid chromatography. To check the practical usefulness of two mPCR assays, 51 Fusarium strains were evaluated by conventional mPCR, and artificially inoculated (10¹-10⁶) and naturally contaminated rice (n=41) were evaluated by direct mPCR.  

Results: The conventional mPCR was highly specific in detecting ZEA producing species containing these genes and was sensitive, detecting up to 1.25 pg/ml of genomic DNA. Direct mPCR without the necessity of time-consuming DNA isolation was shown to detect the presence of ZEA producing species at the lowest level of 10¹ macroconidia/g of white rice and 10² macroconidia/g of brown rice. 

Significance: These results suggest that both mPCR methods are suitable for the specific detection of ZEA producing Fusarium species in white and brown rice, but direct mPCR gives faster results.