Evaluation of Quantitative PCR (qPCR) Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris.

The application of quantitative PCR (qPCR) as a routine method to detect and enumerate in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of . However, none of the qPCR tests described so far has been officially proposed as a standard procedure for detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of a previously described method by shortening the forward primer, designing a new reverse primer and using more stringent amplification conditions. The new test allowed the detection of spores in honey and hive debris down to 1 CFU/g. The qPCR test optimized in this study proved suitable for quantification and also for identification of field strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.