Allergy-causing Mite Identification based on PCR Amplification of their Ribosomal DNA

Resumo

RATIONALE: The aim of this study was to set up a system for the species identification of allergy-causing mites (order Astigmata), based on their rDNA sequence.METHODS: DNA was extracted from different Astigmata species (purified bodies, complete cultures and fecal particles), and rDNA was amplified using Astigmata specific primers. PCR products were cloned and 10 clones from each species were randomly selected for sequencing in order to obtain data on intra- and inter-species variation. A Neighbour-Joining tree (1000 replicates) was obtained after the ClustalW alignment of all full-length sequences. PCR products were digested with RsaI and HpaII restriction enzymes to obtain Restriction Fragment Length Polymorphism (PCR-RFLP) profiles for each species.RESULTS: rDNA comprising partial 18S and 28S, and full-length ITS1, 5.8S and ITS2 regions was amplified from the main allergy-causing mite species. Analysis of the PCR products showed the presence of a main band in each species. Complete direct sequencing was not possible due to the presence of insertions/deletions due to intra and/or inter-individual variability. Cloning and sequencing of PCR products demonstrated that sequence data can be used for the unambiguous identification of all species. PCR-RFLP profiles were unique for each species and could be used for species identification with no need of sequencing.CONCLUSIONS: Once the correlation between morphological identification and rDNA sequence is established, molecular biology techniques can be routinely used for domestic mite species identification with no need of observation of adult stages.This system could be useful for quality control of mite extracts intended for clinical use.