Computational protocol: Molecular Prevalence of Acarapis Mite Infestations in Honey Bees in Korea

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Protocol publication

[…] The head and thorax of 20 adult bees randomly chosen from each hive were used for DNA extraction using DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Total DNA was eluted in 30 µl of elution buffer and used for PCR with LeGene Hot Start PCR Mix w/Sky Blue Dye (LeGene Biosciences, San Diego, California, USA). Two primer sets, including Acarapis (A) primer and Kojima (K) primer set, were used to identify Acarapis mite () which were capable of amplifying mitochondrial cytochrome c oxidase subunit I (COI) DNA fragments of Acarapis mites [,,]. These primers do not amplify honey bee COI DNA fragments; thus, total DNA isolated from bees was used as a template for PCR. As a positive control for PCR to verify the quality of DNA extraction, a honey bee genomic DNA fragment encoding a part of a honey bee Hymenoptera-specific transient receptor potential A channel (AmHsTRPA) was amplified by AmHsTRPA (T) primer set []. The thermal cycling conditions were as follows: 1 cycle of initial denaturation at 94˚C for 2 min, 35 cycles of denaturation at 94˚C for 30 sec, annealing at 55˚C for 30 sec, and extension at 72˚C for 30 sec. The PCR product was analyzed by 2% agarose gel electrophoresis. A negative control lacking template DNA was performed for each PCR reaction. Since A and K primer sets equally amplify COI genes of 3 species of Acarapis mites (A. woodi, A. dorsalis, and A. externus), the amplified products with T and A primer sets or T and K primer sets were sequenced and aligned with the sequences deposited in GenBank to determine the parasitized mite species. The nucleotide sequences were identified by the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI). Multiple nucleotide alignment was carried out using the published SBV sequences as references by BioEdit version 7.0.9.0 (). The phylogenetic tree was constructed by Mega 6.06.1 software [] using the neighbor-joining (NJ) method []. […]

Pipeline specifications

Software tools BLASTN, BioEdit
Application Phylogenetics
Organisms Apis mellifera, Meleagris gallopavo, Varroa destructor