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    Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR

    Piccini, Claudia, D'Alessandro, Bruno, Antúnez, Karina, Zunino, Pablo
    World Journal of Microbiology and Biotechnology, 2002, Vol.18(8), pp.761-765 [Peer Reviewed Journal]
    Springer Science & Business Media B.V.
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    Title: Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR
    Author: Piccini, Claudia; D'Alessandro, Bruno; Antúnez, Karina; Zunino, Pablo
    Subject: American foulbrood ; American foulbrood-contaminated honey ; dead bee larvae ; subspecies ; polymerase chain reaction ; 16S rRNA ; spore detection
    Description: American foulbrood (AFB), a severe bacterial disease of honeybee brood, has recently been found in Uruguayan apiaries. Detection of the causative agent, Paenibacillus larvae subspecies larvae , is a very important concern in order to prevent disease dissemination and decrease of honey production. Since spores are the infective forms of this pathogen, in the present work we report the use of polymerase chain reaction (PCR) to detect P. l. subsp. larvae spores from in vitro cultures, larvae with clinical symptoms and experimentally contaminated honey. The set of primers was designed based on the published P. l. subsp. larvae 16S rRNA gene. Using this approach we could amplify the pathogen DNA and obtain a great sensitivity and a notable specificity. Detection limit for spore suspension was a 10 −2 dilution of template DNA obtained from 32 spores, as determined by plate count. For artificially contaminated honey, we could detect the PCR product at a 10 −3 dilution of template DNA obtained from 170 spores. In addition, when PCR conditions were set to improve specificity, we were able to amplify P. l . subsp. larvae DNA selectively and no cross-reactions were observed with a variety of related bacterial species, including P. l. subsp. pulvifaciens . Since spore detection is very important to confirm the presence of the disease, this method provides a reliable diagnosis of AFB from infected larvae and contaminated honey in a few hours.
    Is part of: World Journal of Microbiology and Biotechnology, 2002, Vol.18(8), pp.761-765
    Identifier: 0959-3993 (ISSN); 1573-0972 (E-ISSN); 10.1023/A (DOI)

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    DNA extraction and PCR detection of Paenibacillus larvae spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques

    D’Alessandro, Bruno, Antúnez, Karina, Piccini, Claudia, Zunino, Pablo
    World Journal of Microbiology and Biotechnology, 2007, Vol.23(4), pp.593-597 [Peer Reviewed Journal]
    Springer Science & Business Media B.V.
    Available
    More…
    Title: DNA extraction and PCR detection of Paenibacillus larvae spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques
    Author: D’Alessandro, Bruno; Antúnez, Karina; Piccini, Claudia; Zunino, Pablo
    Subject: American foulbrood ; Paenibacillus larvae ; Spores ; Spore-decoating ; Freeze-thawing ; Polymerase chain reaction
    Description: Paenibacillus larvae causes American foulbrood (AFB), a severe disease that affects the brood of honey bee Apis mellifera. AFB is worldwide distributed and causes great economic losses to beekeepers, but in many cases early diagnosis could help in its prevention and control. The aim of the present work was to design a reliable protocol for DNA extraction of P. larvae spores from naturally contaminated honey and adult bees. A novel method that includes a step of spore-decoating followed by an enzymatic spore disruption and DNA purification was developed. Also a freeze-thaw cycle protocol was tested and the results were compared. The DNA extracted was used as template for specific bacterial detection by amplification of a 16S rDNA fragment. Both methods allowed the direct detection by polymerase chain reaction (PCR) of P. larvae spores present in naturally contaminated material. The spore-decoating strategy was the most successful method for DNA extraction from spores, allowing specific and remarkably sensitive PCR detection of spores in all honey and bees tested samples. On the other hand freeze-thawing was only effective for detection of spores recovered from bees, and extensive damage to DNA affected detection by PCR. This work provides new strategies for spore DNA extraction and detection by PCR with high sensitivity, and brings an alternative tool for P. larvae detection in natural samples.
    Is part of: World Journal of Microbiology and Biotechnology, 2007, Vol.23(4), pp.593-597
    Identifier: 0959-3993 (ISSN); 1573-0972 (E-ISSN); 10.1007/s11274-006-9261-y (DOI)