Summary
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.
References
Alippi AM, López AC, Aguilar OM (2002) Differentiation of Paenibacillus larvae subsp. larvae, the cause of American foulbrood of honeybees, by using PCR and restriction fragment analysis of genes encoding 16S rRNA. Appl Environ Microbiol 68: 3655–3660
Bickley J, Owen R (1995) Preparation of bacterial genomic DNA. In: Howard J, Whitcombe DM (eds) Diagnostic bacteriology protocols. Humana Press, Totowa, New Jersey, pp 141–147 (ISBN 0896032973)
Govan VA, Allsopp MH, Davison S (1999) A PCR detection method for rapid identification of Paenibacillus larvae. Appl Environ Microbiol 65:2243–2245
Hornitzky MAZ, Nicholls PJ (1993) J medium is superior to sheep blood agar and brain infusion agar for the isolation of Bacillus larvae from honey samples. J Apic Res 32:51–52
Lauro FM, Favaretto M, Covolo L, Rassu M, Bertoloni G (2003) Rapid detection of Paenibacillus larvae from honey and hive samples with a novel nested PCR protocol. Int J Food Microbiol 81:195–201
Levi K, Higham JL, Coates D, Hamlyn PF (2003) Molecular detection of anthrax spores on animal fibres. Lett Appl Microbiol 36:418–422
Paidhungat M, Setlow P (2000) Role of Ger proteins in nutrient and nonnutrient triggering of spore germination in Bacillus subtilis. J Bacteriol 182:2513–2519
Piccini C, D’Alessandro B, Antúnez K, Zunino P (2002) Detection of Paenibacillus larvae subspecies larvae spores in naturally infected bee larvae and artificially contaminated honey by PCR. World J Microbiol Biotechnol 18:761–765
Riesenman PJ, Nicholson WL (2000) Role of the spore coat layers in Bacillus subtilis spore resistance to hydrogen peroxide, artificial UV-C, UV-B, and solar UV radiation. Appl Environ Microbiol 66:620–626
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sargent MG (1980) A procedure for isolating high quality DNA from spores of Bacillus subtilis 168. J Gen Microbiol 116:511–514
Setlow B, Setlow P (1998) Heat killing of Bacillus subtilis spores in water is not due to oxidative damage. Appl Environ Microbiol 64:4109–4112
Vary JC (1973) Germination of Bacillus megaterium spores after various extraction procedures. J Bacteriol 116:797–802
Wilson IG (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63:3741–3751
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D’Alessandro, B., Antúnez, K., Piccini, C. et al. DNA extraction and PCR detection of Paenibacillus larvae spores from naturally contaminated honey and bees using spore-decoating and freeze-thawing techniques. World J Microbiol Biotechnol 23, 593–597 (2007). https://doi.org/10.1007/s11274-006-9261-y
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DOI: https://doi.org/10.1007/s11274-006-9261-y