Triplex real-time PCR method for the qualitative detection of European and American foulbrood in honeybee

Dainat, Benjamin; Grossar, Daniela; Ecoffey, Brigitte; Haldemann, Christoph

doi:10.1016/j.mimet.2018.01.018

Cited by 15 publications

(17 citation statements)

References 8 publications

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“…To determine the presence of M. plutonius in the extract of adult workers, we used the triplex qPCR described by Dainat et al [42]. This triplex real-time PCR allows us to analyze the following target organisms: M. plutonius, P. larvae, and Apis mellifera.…”

Section: Qpcr From Adult Honey Beesmentioning

confidence: 99%

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

Biová

Charrière

Dostálková

et al. 2021

Insects

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European foulbrood (EFB) is an infectious disease of honey bees caused by the bacterium Melissococcus plutonius. A method for DNA isolation and conventional PCR diagnosis was developed using hive debris, which was non-invasively collected on paper sheets placed on the bottom boards of hives. Field trials utilized 23 honey bee colonies with clinically positive symptoms and 21 colonies without symptoms. Bayes statistics were applied to calculate the comparable parameters for EFB diagnostics when using honey, hive debris, or samples of adult bees. The reliability of the conventional PCR was 100% at 6.7 × 103 Colony Forming Unit of M. plutonius in 1 g of debris. The sensitivity of the method for the sampled honey, hive debris, and adult bees was 0.867, 0.714, and 1.000, respectively. The specificity for the tested matrices was 0.842, 0.800, and 0.833. The predictive values for the positive tests from selected populations with 52% prevalence were 0.813, 0.833, and 0.842, and the real accuracies were 0.853, 0.750, and 0.912, for the honey, hive debris, and adult bees, respectively. It was concluded that hive debris can effectively be utilized to non-invasively monitor EFB in honey bee colonies.

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Section: Qpcr From Adult Honey Beesmentioning

confidence: 99%

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

Biová

Charrière

Dostálková

et al. 2021

Insects

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“…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.…”

Section: Discussionmentioning

confidence: 95%

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

Mackay¹,

Re²,

Nt³

et al. 2021

Preprint

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Clinical signs of American Foulbrood can be difficult to diagnose and thus disease is missed and leads to further spread. Diagnosis is centred on the beekeeper's skill in recognising clinical symptoms – a highly subjective and time-consuming activity. Previous testing methods have relied on sampling that necessitates dismantling the hive and/or requires multiple visits to retrieve passive samples. The Foster method is a new environmental DNA sampling method using entrance swabs together with a dual-target qPCR for AFB. The quantification data generated can be used to detect hives with clinically relevant infections, even when no visual symptoms are apparent. Such a method will be applicable to other bee pathogens and incursion pests.

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“…The oligonucleotide pairs previously proposed for the detection of P. larvae by qPCR were re-assessed in silico for exclusivity. The primer pair designed by Dainat et al [18] was not included in the analysis since BLAST alignment showed that it is targeted on phage DNA present in all P. larvae genomes but in a highly variable number of copies that ranges between tens and hundreds. Therefore, the cell number cannot be determined because there is not a stable correspondence between the target region copy number and the number of cells, though the method is supposed to be the most sensitive among those available for presence/absence determinations.…”

Section: Resultsmentioning

confidence: 99%

“…N. NZ_ADFW00000000), were aligned by Clustal Omega (). The target gene region of primers PL-F and PL-R designed by Dainat et al [18] was defined by BLAST analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Quintana et al [17] designed a qPCR test able to detect as little as 28 P. larvae spores in larval scales. In addition, a P. larvae -specific qPCR assay was included in a triplex test aimed at the qualitative detection of the microorganism in brood samples [18]. Quantification of P. larvae by qPCR was not applied to honey and hive debris so far.…”

Section: Introductionmentioning

confidence: 99%

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

Rossi

Amadoro

Ruberto

et al. 2018

Insects

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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.

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Triplex real-time PCR method for the qualitative detection of European and American foulbrood in honeybee

Cited by 15 publications

References 8 publications

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

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

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

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

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