3.4.3. Molecular detection of Acarapis woodi in Apis mellifera
Because morphological detection is time -consuming and requires detailed and sustained attention by the screener, detection of A. woodi using a molecular technique is currently being developed by various laboratories for routine screening and quarantine checking. A real-time PCR assay for A. woodi was designed by Giles Budge at the UK Food and Environment Research Agency (Fera) which amplifies a section of the internal transcribed spacer region 2 (ITS2); however when tested, it was found to also amplify the ITS sequence from other Acarapis species. Evans et al. (2007) developed a nested PCR to amplify part of the cytochrome oxidase1 gene (CO1) of A. woodi. The PCR was designed to detect a low level of infestation of A. woodi from the entire thorax of a bee. At the time of development, the assay was not tested against other Acarapis species, but subsequent testing has shown that these primers amplify sequences from them also (Delmiglio et al., in prep.).
Kojima et al. (2011b) published a conventional PCR method shown to amplify A. woodi DNA and not A. externus, but this method has not been tested on A. dorsalis DNA. Although their test was able to detect A. woodi when four mites were present in a single bee, it was not able to detect the presence of a single mite unless a nested-PCR was performed. Furthermore, Kojima et al. (2011b) did not test the sensitivity of detection at different bulking rates for extractions and the need for post amplification handling (i.e. electrophoresis) increases the length of the detection procedure
Delmiglio and Ward (unpublished) obtained sequences from the CO1 region for A. woodi, A. externus and A. dorsalis and designed real-time PCR primers and a TaqMan probe for A. woodi within a single variable region of the CO1 gene. The assay was able to amplify A. woodi DNA from a single mite (specimens obtained from Canada and UK).
Tests showed that
the assay was able to detect A. woodi
down to 1% and 2% prevalence in bees, and 200 copies of the target DNA when
using plasmid standards. The real-time assay was not found to cross-react with
mites of other genera associated with bees; however a very low level of cross-reaction occurs with the other two Acarapis species when sequence from
these species are present at high concentrations in the form of plasmid DNA, or
when there are a very high number Acarapis
mites. To counter this, bees are externally washed before testing and a lower Ct
cut-off of 35 cycles is used.
Nucleic acid extraction:
Note: the extraction method is based on the semi-automated system Thermo Kingfisher & InviMag® DNA Mini Kit (Invitek GmbH; Germany):
- Using this semi-automated extraction method, 15 bees maximum can be sampled per extraction to allow reliable detection of A. woodi from a single infected bee.
- Prepare bees by removing the abdomens using a clean scalpel, and place bee heads and thoraces into a filter grinding bag (Bioreba; Switzerland) of suitable size.
- Add lysis Buffer P (Invitek GmbH; Germany) to the grinding bag at a rate of 0.5 ml buffer per bee (i.e. use 7.5ml for 15 bees), and using a Homex grinder (Bioreba, Switzerland) grind the bees to form a lysate.
- Decant 600 µl of the lysate into a clean micro-centrifuge tube, add 30 µl of proteinase K (Invitek GmbH; Germany) and incubate at 65oC for 30 min in a thermomixer (Eppendorf; Germany).
- Centrifuge the resulting lysate at 8000 g for 1 minute to pellet debris.
- Extract DNA from the cleared lysate using a KingFisher® ml Magnetic Particle Processor system (Thermo Fisher Scientific Inc.; USA) using the protocol and program recommended by the manufacturer, and using buffers and magnetic beads supplied in an InviMag® DNA Mini Kit (Invitek GmbH; Germany).
- The resulting DNA is eluted in 260 ml of elution buffer D (Invitek), transferred to a fresh 1.5 ml tube and stored at -80°C prior to use.