Utilização da técnica de High Resolution Melting (HRM) para identificação de diferentes haplótipos de Varroa destructor

Abstract

The Varroa destructor mite has spread worldwide parasitizing bees of the species Apis mellifera, and is currently considered the major threat to beekeeping. It is responsible for the clinical symptoms of varrose. Studies on the genetic variability of V. destructor mite showed that the virulence of varrose is also related to different haplotypes of the ectoparasite. Based on the sequence of the cytochrome C oxidase gene (COI) present in the mitochondrial DNA, researchers divided Varroa jacobsoni into two species: V. jacobsoni and V. destructor. Even within the species V. destructor, there are variations in the sequence of the above-cited gene, which allows differentiation of the species Japanese haplotypes (J) and Korean (K) haplotypes. Such differentiation is more than a genetic trait. Both haplotypes differ in their virulence, as the K haplotype is more virulent than J haplotype. It is possible to identify the haplotypes with PCR reaction followed by cleavage reaction. Both COI gene amplicons have cleavage site for XhoI enzyme, but only the J haplotype has cleavage site for the SacI restriction enzyme site for the enzyme. However, the time required in this technique is high, and hampers high-throughput analysis. Therefore, we propose the use of High Resolution Melting technique (HRM), which aims to distinguish these polymorphisms observing the denaturation curve of double-stranded DNA, for fast and large-scale analysis. This study aims to establish the HRM technique in order to distinguish the K and J haplotypes, and compare the results with the standard method used. The DNA sequences of the V. destructor COI gene were selected based on haplotype of polymorphic regions J and K; the selected fragments were synthesized artificially (gBlocks, Integrated DNA Technologies). From these sequences, primers were designed for HRM and cleavage reactions. Artificial fragments were subjected to PCR for standardization of experimental conditions using primers for HRM and subsequent cleavage. After the PCR reaction, the amplified fragments were subjected to cleavage reaction with SacI and XhoI restriction enzymes. After standardization, cleavage reactions were conducted using the restriction enzymes SacI and XhoI for both amplification products from the synthetic DNA and environmental samples. The results obtained were observed by electrophoresis in agarose gel. After, the samples were subjected to HRM. The resulting amplicons presented the expected size and cleavage pattern. Was also identified the environmental sample haplotype, which has been identified as K haplotype. In HRM haplotype analysis, the two samples comprising the variant 2 (K) showed the same melting curve pattern, differing only in the intensity. We conclude that this method could potentially be used for V. destructor haplotypes J and K identification. This technique improves the current method for J and K haplotypes identification, rendering a more fast and precise method, which can be used for V. destructor monitoring and control programs.