Using Real-time PCR for Identification of Paenibacillus larvae

Vladimíra Kňazovická, Martina Miluchová, Miroslava Kačániová, Martin Melich, Miroslav Kročko, Anna Trakovická

Abstract


The aim of the study was identification of Paenibacillus larvae that causes American foulbrood disease (AFB) in colony of bees (Apis mellifera). Bacterial isolates originated from honey samples, because presence of P. larvae in honey is treated as early diagnostic of AFB. Intense proteolytic activity and no catalase activity are typical for Gram positive rod-shaped bacteria P. larvae. We diluted honey (1:2), heated at 80 °C for 10 min and inoculated on semiselective medium MYPGP agar with nalidixic acid. Plates were cultivated at 37 °C for 48 – 72 h under the aerobic conditions. Selected colonies were transferred on MYT agar and cultivated 24 h. We analysed 30 honey samples and found 27 bacterial isolates. All isolates were Gram positive and mainly rod-shaped. No catalase activity was documented for 6 from 27 isolates. Identification was finished by real-time PCR to detect the 16S rRNA gene of Paenibacillus larvae with real-time cycler Rotor-Gene 6000. As DNA template we used genomic DNA isolated with commercial kit and DNA lysate obtaining by boiled cells. We used 2 strains of P. larvae from CCM (Czech Collection of Microorganisms) as positive control. The reliable method of detection P. larvae has important rule for beekeeping.


Keywords


American foulbrood; Apis mellifera; Honey; Real-time PCR

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Scientific Papers Animal Science and Biotechnologies publish open access articles under the terms of the Creative Commons Attribution 4.0 Licence (read full legal code).


LUCRARI STIINTIFICE ZOOTEHNIE SI BIOTEHNOLOGII (SCIENTIFIC PAPERS ANIMAL SCIENCE AND BIOTECHNOLOGIES)

ISSN print 1841 - 9364
ISSN online 2344 - 4576
ISSN-L  1841 - 9364
(former ISSN 1221-5287, E-ISSN 1841-9364)

PUBLISHER: AGROPRINT Timisoara, Romania
PAPER ACCESS: Full text articles available for free
FREQUENCY: Semiannual
PUBLICATION LANGUAGE: English
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Banat´s University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania" from Timisoara
Bioengineering Faculty of Animal Resources
300645, Timişoara, Calea Aradului 119, Romania

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