Novel TaqMan PCR assay for the quantification of Paenibacillus larvae spores in bee-related samples

Kušar, Darja (author), Papić, Bojan (author), Zajc, Urška (author), Zdovc, Irena (author), Golob, Majda (author), Žvokelj, Lucija (author), Knific, Tanja (author), Avberšek, Jana (author), Ocepek, Matjaž (author), Pislak, Metka (author)

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.

• Language: English
• Keywords: 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
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: VF - Veterinary Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2021
• Number of pages: 18 str.
• Numbering: Vol. 12, iss. 11, art. 1034
• PID: 20.500.12556/RUL-133255
• UDC: 577
• ISSN on article: 2075-4450
• DOI: 10.3390/insects12111034
• COBISS.SI-ID: 85522179
• Publication date in RUL: 18.11.2021

Record is a part of a journal

• Title: Insects
• Shortened title: Insects
• Publisher: MDPI
• ISSN: 2075-4450
• COBISS.SI-ID: 519122457

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.
• Licensing start date: 17.11.2021