Real-time PCR (qPCR) is the principal technique for the quantification of pathogen biomass in host tissue, yet no generic methods exist for the determination of the limit of quantification (LOQ) and the limit of detection (LOD) in qPCR. We suggest using the Youden index in the context of the receiver operating characteristic (ROC) curve analysis for this purpose. The LOQ was defined as the amount of target DNA that maximizes the sum of sensitivity and specificity. The LOD was defined as the lowest amount of target DNA that was amplified with a false-negative rate below a given threshold. We applied this concept to qPCR assays for Fusarium verticillioides and Fusarium proliferatum DNA in maize kernels. Spiked matrix and field samples characterized by melting curve analysis of PCR products were used as the source of true positives and true negatives. On the basis of the analysis of sensitivity and specificity of the assays, we estimated the LOQ values as 0.11 pg of DNA for spiked matrix and 0.62 pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03 pg for spiked matrix and 0.24 pg for field samples. The mean LOQ values correspond to approximately eight genomes for F. verticillioides and three genomes for F. proliferatum. We demonstrated that the ROC analysis concept, developed for qualitative diagnostics, can be used for the determination of performance parameters of quantitative PCR.

Eighty maize grain samples collected in Nigeria were investigated for fumonisin B 1 (FB 1 ) content and Fusarium verticillioides colonization. F. verticillioides DNA was quantified by species-specific real-time PCR and living propagules of the fungus were counted by agar-plating method. FB 1 was detected in 55 (68.7%) of the total samples (mean: 98.5 µg/kg, range: 10 to 714 µg/kg) at 10 µg/kg detection limit. The mean amount of F. verticillioides DNA determined by real-time PCR was 49.7 µg/kg (range: 10-126.7 µg/kg), while agar plate method showed the presence of F. verticillioides in 45 samples (mean incidence: 21.0%, range: 6.7-60.0%). There was correlation ties between F. verticillioides DNA by real time PCR and fungal colonization by agar plate method (R = 0.71, p = 00001 at 95% confidence level), and means of FB 1 and F. verticillioides DNA in the yellow and white maize were significantly different. Despite the high consumption of maize in Nigeria, the amount of FB 1 ingested by consumers appears to be low. The estimated daily intake of fumonisins was 0.21 µg/kg body weight per day.

No abstract