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Article

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

1
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale”, Via Campo Boario 1, 64100 Teramo, Italy
2
Medicine and Health Science Department “V. Tiberio”, University of Molise, Via de Santis, 86100 Campobasso, Italy
*
Author to whom correspondence should be addressed.
Insects 2018, 9(4), 165; https://doi.org/10.3390/insects9040165
Received: 20 September 2018 / Revised: 31 October 2018 / Accepted: 13 November 2018 / Published: 16 November 2018
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. View Full-Text
Keywords: Paenibacillus larvae; optimized qPCR; quantification; honey; hive debris Paenibacillus larvae; optimized qPCR; quantification; honey; hive debris
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MDPI and ACS Style

Rossi, F.; Amadoro, C.; Ruberto, A.; Ricchiuti, L. Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris. Insects 2018, 9, 165. https://doi.org/10.3390/insects9040165

AMA Style

Rossi F, Amadoro C, Ruberto A, Ricchiuti L. Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris. Insects. 2018; 9(4):165. https://doi.org/10.3390/insects9040165

Chicago/Turabian Style

Rossi, Franca, Carmela Amadoro, Addolorato Ruberto, and Luciano Ricchiuti. 2018. "Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris" Insects 9, no. 4: 165. https://doi.org/10.3390/insects9040165

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