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,...

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Autores principales:	Darja Kušar, Bojan Papić, Urška Zajc, Irena Zdovc, Majda Golob, Lucija Žvokelj, Tanja Knific, Jana Avberšek, Matjaž Ocepek, Metka Pislak Ocepek
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	American foulbrood (AFB) <i>Paenibacillus larvae</i> real-time or quantitative PCR (qPCR) digital PCR (dPCR) plate counting spore germination rate Science Q
Acceso en línea:	https://doaj.org/article/e3b7221369b140cd98273e7284ef979c
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spelling	oai:doaj.org-article:e3b7221369b140cd98273e7284ef979c2021-11-25T17:59:58ZNovel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples10.3390/insects121110342075-4450https://doaj.org/article/e3b7221369b140cd98273e7284ef979c2021-11-01T00:00:00Zhttps://www.mdpi.com/2075-4450/12/11/1034https://doaj.org/toc/2075-4450<i>Paenibacillus larvae</i> is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. <i>P. larvae</i> 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 <i>P. larvae</i>. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of <i>P. larvae</i> 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 <i>P. larvae</i> in honey and hive debris samples but can also be extended to other sample types.Darja KušarBojan PapićUrška ZajcIrena ZdovcMajda GolobLucija ŽvokeljTanja KnificJana AvberšekMatjaž OcepekMetka Pislak OcepekMDPI AGarticleAmerican foulbrood (AFB)<i>Paenibacillus larvae</i>real-time or quantitative PCR (qPCR)digital PCR (dPCR)plate countingspore germination rateScienceQENInsects, Vol 12, Iss 1034, p 1034 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	American foulbrood (AFB) <i>Paenibacillus larvae</i> real-time or quantitative PCR (qPCR) digital PCR (dPCR) plate counting spore germination rate Science Q
spellingShingle	American foulbrood (AFB) <i>Paenibacillus larvae</i> real-time or quantitative PCR (qPCR) digital PCR (dPCR) plate counting spore germination rate Science Q Darja Kušar Bojan Papić Urška Zajc Irena Zdovc Majda Golob Lucija Žvokelj Tanja Knific Jana Avberšek Matjaž Ocepek Metka Pislak Ocepek Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples
description	<i>Paenibacillus larvae</i> is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. <i>P. larvae</i> 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 <i>P. larvae</i>. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of <i>P. larvae</i> 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 <i>P. larvae</i> in honey and hive debris samples but can also be extended to other sample types.
format	article
author	Darja Kušar Bojan Papić Urška Zajc Irena Zdovc Majda Golob Lucija Žvokelj Tanja Knific Jana Avberšek Matjaž Ocepek Metka Pislak Ocepek
author_facet	Darja Kušar Bojan Papić Urška Zajc Irena Zdovc Majda Golob Lucija Žvokelj Tanja Knific Jana Avberšek Matjaž Ocepek Metka Pislak Ocepek
author_sort	Darja Kušar
title	Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples
title_short	Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples
title_full	Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples
title_fullStr	Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples
title_full_unstemmed	Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples
title_sort	novel taqman pcr assay for the quantification of <i>paenibacillus larvae</i> spores in bee-related samples
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/e3b7221369b140cd98273e7284ef979c
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Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples

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