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

Abstract

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 sporedecoating 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.

A real-time PCR-based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood

“…(2008) (2 vs 24 copies of 16S rRNA sequence per reaction, respectively). Also, our assay detection limit (0·6 spores per reaction or 2 spores g −1 of honey), contrasted to 20 spores per reaction (D’Alessandro et al. 2007), 10 5 spores g −1 of honey (Ryba et al.…”

“…The main difficulties associated with AFB control are (i) the disease vector is P. larvae spores, (ii) the low concentration of P. larvae spores in apiary supplies and products (Lindström et al 2008), (iii) the low number of spores able to germinate, and (iv) the low selectivity of the P. larvae growth media. To circumvent these difficulties, PCR methods have been developed for P. larvae detection (Bakonyi et al 2003;Alippi et al 2004;D'Alessandro et al 2007). Most of them are based on conventional PCR, which is time consuming (5-6 h) and has a relatively low technique detection limit.…”

“…To overcome this limitation, conventional PCR has been applied to detect P. larvae spores in honey (Bakonyi et al. 2003; D’Alessandro et al. 2007).…”

“…The latter is the most significant limitation of culture-based methods because it has been shown that only 6% of total P. larvae spores germinate under culture conditions (Dingman and Stahly 1983). To overcome this limitation, conventional PCR has been applied to detect P. larvae spores in honey (Bakonyi et al 2003;D'Alessandro et al 2007). These procedures have several pitfalls: (i) methods used to extract DNA from spores are lengthy and laborious, (ii) conventional PCR has restricted detection limit and, (iii) assay detection limit is determined against a suboptimal spread plate method.…”

American Foulbrood in Uruguay: Twelve years from its first report

“…P. larvae specific confirmation was carried out by PCR, using the primers PL 5 (5 0 -CGAGCG-GACCTTGTGTTTCC-3 0 ) and PL 4 (5 0 -TCAGTTATAGGCCAGAAAGC-3), which amplify a fragment of the P. larvae 16S rRNA gene (Piccini et al, 2002;D'Alessandro et al, 2007).…”