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

Abstract

The application of quantitative PCR (qPCR) as a routine method to detect and enumerate Paenibacillus larvae in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of Apis mellifera. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for P. larvae detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of P. larvae spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired P. larvae 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 P. larvae 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 P. larvae strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of P. larvae 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.

Melissococcus plutonius Can Be Effectively and Economically Detected Using Hive Debris and Conventional PCR

“…Hive debris may indicate the health status of honey bee colonies and can be easily collected by beekeepers [ 32 , 33 , 34 , 35 , 36 ]. It is common and an easy task to sample honey, suspected broods, or adult bees [ 20 ]; however, this is time consuming, dependent on weather conditions, requires opening colonies, and increases the risk of losing the queen during the sampling process and requires significant logistics for sending the samples to the laboratory.…”

“…This is time consuming, demanding on human resources, and increases the costs for the prevention of honey bee diseases. Hive debris has been successfully used for AFB spore detection using cultivation [ 32 ] and PCR techniques [ 33 , 34 , 35 ], and for small hive beetle ( Aethina tumida ) using PCR [ 36 , 37 ].…”

Quantitative PCR (qPCR) vs culture-dependent detection to assess honey contamination by Paenibacillus larvae

“…Extraction of DNA from single colonies was done as described by Rossi et al (2018). Crude DNA extracts were 1:100 diluted before use in qPCR.…”

“…The qPCR method described by Rossi et al (2018) was used both for direct analysis of honey and for colony identification of presumptive P. larvae isolates.…”

The Foster method: Rapid and non-invasive detection of clinically significant American Foulbrood disease levels using eDNA sampling and a dual-target qPCR assay, with its potential for other hive pathogens

“…However the variability in Cq difference between the two markers among samples indicated the 16S copy number likely varied among AFB isolates and thus could not be relied on for quantification (Dahllof et al, 2000). This variability in copy number for diagnostic targets has been noted in other diagnostic qPCR assays for AFB (Rossi et al, 2018;Dainat et al, 2018). Therefore the use of the single copy ftsZ gene was used for quantification purposes using a standard curve prepared with a quantified synthetic template diluted in AFB-negative bee DNA.…”