RESUMO
INTRODUCTION: Dust mites are known to be an important source of inhalant allergens causing allergic rhinitis and asthma worldwide. The sizes of dust mite populations in patients' houses are useful to monitor the risk of allergen exposure. However, mite identification using the conventional microscopic technique requires specific expertise and is time consuming; therefore a molecular technique has been developed in order to solve these drawbacks. OBJECTIVE: To develop a multiplex PCR assay for identifying the three common dust mite species in Thailand, namely Dermatophagoides pteronyssinus (Dp), D. farinae (Df) and Blomia tropicalis (Bt), and to evaluate the efficacy of the technique. METHODS: Pairs of primers were designed and tested in either singleplex PCR or multiplex PCR. The multiplex PCR technique was also optimized in order to obtain specific products. The reaction mixture contained 5 pmole of individual primers, 10 mM dNTP, 5 units Taq DNA polymerase and genomic DNA (gDNA). The reaction was run for 25 cycles at 94 degrees C for 20 seconds, 58 degrees C for 20 seconds and 72 degrees C for 30 seconds. The PCR products were analyzed by 1.5% agarose gel electrophoresis with GelRed fluorescence dye. The optimized multiplex technique was also tested with 30 house dust samples and dust samples spiked with DNA from other insect and mite species. RESULTS: Three PCR products were obtained with the relevant gDNA templates as expected; 143 bp for DF, 221 bp for DP and 318 bp for BT, respectively. The detection limit of the tests was found to be as low 1 ng of gDNA, whereas mixed gDNA species confirmed the 100% specificity of this assay. The total duration from the preparation of the PCR reaction mixture until the analysis by agarose gel electrophoresis was approximately 2 hours. No amplified product was obtained from mites and insects of other species. CONCLUSION: The multiplex PCR was successfully developed for identifying 3 common dust mite species. This technique can be helpful, not only for non-acarologist personnel for dust mite identification, but also for patients who are allergic to dust mites.
