19 research outputs found
Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: application to qPCR assays for Fusarium verticillioides and F. proliferatum
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 0.11Â pg of DNA for spiked matrix and 0.62Â pg of DNA for field samples for F. verticillioides. The LOQ values for F. proliferatum were 0.03Â pg for spiked matrix and 0.24Â 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
Dispersal of conidia of Fusicladium eriobotryae and spatial patterns of scab in loquat orchards in Spain.
Dispersal of conidia of Fusicladium
eriobotryae, the causal agent of loquat scab, was investigated
in two loquat orchards in Spain from 2010 to
2012. A volumetric spore sampler, horizontally and
vertically orientated microscope slides, and rain collectors
were placed in loquat fields to trap conidia of
F. eriobotryae. No conidia were collected in the volumetric
sampler, and highly variable numbers of conidia
were collected in the rain gatherers. Large numbers of
conidia were collected by microscope slides, particularly
by those held in a horizontal orientation compared
with those held in a vertical orientation. Approximately
90%of the F. eriobotryae conidia were collected during
rainy periods. Based on ROC and Bayesian analysis,
using 65 0.2 mm rainfall as a cut-off value resulted in a
high probability of correctly predicting actual conidial
dispersal, and had a low probability of failing to predict
actual conidial dispersal. Based on the index of
dispersion and the binary power law, the incidence of
loquat scab on fruit was highly aggregated in space
between and within trees, and aggregation was influenced
by disease incidence. Our results demonstrate, for
the first time, that F. eriobotryae is dispersed mainly in
rain splash. The results will be integrated into a mechanistic,
weather-driven, disease prediction model that
should help growers to minimize fungicide application
for the management of loquat scab