20.500.12556/RUL-133255 Novel TaqMan PCR assay for the quantification of Paenibacillus larvae spores in bee-related samples Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types. American foulbrood (AFB) Paenibacillus larvae real-time or quantitative PCR (qPCR) digital PCR (dPCR) plate counting spore germination rate honey hive debris Polymerase Chain Reaction true false true Angleški jezik Ni določen Članek v reviji 2021-11-18 13:59:39 2021-11-18 13:59:40 2022-09-12 04:15:30 0000-00-00 00:00:00 2021 0 0 18 str. iss. 11, art. 1034 Vol. 12 2021 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 577 2075-4450 10.3390/insects12111034 85522179 RAZ_Kusar_Darja_2021.pdf RAZ_Kusar_Darja_2021.pdf 1 95754D99DB639862BB3E2E11294AA673 508c5511d221ef8c9365f1f3f4fa0d0c805dc513d5fbda3b93b989f00210fe7f a58549fa-4870-11ec-abdb-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=151156 https://www.mdpi.com/2075-4450/12/11/1034 1 520f6561-486f-11ec-abdb-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=151155 Veterinarska fakulteta 0 0 0