Multiplex LAMP assay for detecting the prevalent species of dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus in the domestic environment.

Dermatophagoides farina (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus) are the prevalent kinds of house dust mites (HDMs). HDMs are common inhalant allergens that cause a range of allergic diseases, such as rhinitis, atopic dermatitis, and asthma. The epidemiology of these diseases is associated with exposure to mites. Therefore, in the present study, a method named multiplex loop-mediated isothermal amplification (LAMP) was developed to detect environmental dust mites. The multiplex LAMP assay allows amplification within a single tube and has an ITS plasmid detection limit as low as 40 fg/µL for both single dust mites and mixed dust mites (D. pteronyssinus and D. farinae), which is up to ten times more sensitive than classical PCR techniques. Furthermore, the multiplex LAMP method was applied to samples of single dust mites and clinical dust to confirm its validity. The multiplex LAMP assay exhibited higher sensitivity, simpler instrumentation, and visualization of test results, indicating that this method could be used as an alternative to traditional techniques for the detection of HDMs.

Asthmatic Eosinophils Alter the Gene Expression of Extracellular Matrix Proteins in Airway Smooth Muscle Cells and Pulmonary Fibroblasts.

The impaired production of extracellular matrix (ECM) proteins by airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) is a part of airway remodeling in asthma. This process might be influenced by eosinophils that migrate to the airway and abundantly secrete various cytokines, including TGF-ß. We aimed to investigate the effect of asthmatic eosinophils on the gene expression of ECM proteins in ASMC and PF. A total of 34 study subjects were recruited: 14 with allergic asthma (AA), 9 with severe non-allergic eosinophilic asthma (SNEA), and 11 healthy subjects (HS). All AA patients underwent bronchial allergen challenge with D. pteronyssinus. The peripheral blood eosinophils were isolated using high-density centrifugation and magnetic separation. The individual cell cultures were made using hTERT ASMC and MRC-5 cell lines and the subjects' eosinophils. The gene expression of ECM and the TGF-ß signaling pathway was analyzed using qRT-PCR. We found that asthmatic eosinophils significantly promoted collagen I, fibronectin, versican, tenascin C, decorin, vitronectin, periostin, vimentin, MMP-9, ADAM33, TIMP-1, and TIMP-2 gene expression in ASMC and collagen I, collagen III, fibronectin, elastin, decorin, MMP-2, and TIMP-2 gene expression in PF compared with the HS eosinophil effect. The asthmatic eosinophils significantly increased the gene expression of several canonical and non-canonical TGF-ß signaling pathway components in ASMC and PF compared with the HS eosinophil effect. The allergen-activated AA and SNEA eosinophils had a greater effect on these changes. In conclusion, asthmatic eosinophils, especially SNEA and allergen-activated eosinophils, imbalanced the gene expression of ECM proteins and their degradation-regulating proteins. These changes were associated with increased gene expression of TGF-ß signaling pathway molecules in ASMC and PF.

Characterisation of three novel ß-1,3 glucanases from the medically important house dust mite Dermatophagoides pteronyssinus (airmid).

The European house dust mite, Dermatophagoides pteronyssinus is a major source of airborne allergens worldwide and is found in half of European homes. Interactions between microbes and house dust mites (HDM) are considered important factors that allow them to persist in the home. Laboratory studies indicate the European HDM, D. pteronyssinus is a mycophagous mite, capable of utilising a variety of fungi for nutrients, however specific mycolytic digestive enzymes are unknown. Our previous work identified a number of putative glycosyl hydrolases present in the predicted proteome of D. pteronyssinus airmid and validated the expression of 42 of these. Of note, three GH16 proteins with predicted ß-1,3 glucanase activity were found to be consistently present in the mite body and excretome. Here, we performed an extensive bioinformatic, proteomic and biochemical study to characterize three-novel ß-1,3 glucanases from this medically important house dust mite. The genes encoding novel ß-1,3 glucanases designated Glu1, Glu2 and Glu3 were identified in D. pteronyssinus airmid, each exhibited more than 59% amino acid identity to one another. These enzymes are encoded by Glu genes present in a tri-gene cluster and protein homologs are found in other acari. The patchy phyletic distribution of Glu proteins means their evolutionary history remains elusive, however horizontal gene transfer cannot be completely excluded. Recombinant Glu1 and Glu2 exhibit hydrolytic activity toward laminarin, pachyman and barley glucan. Excreted ß-1,3 glucanase activity was increased in response to D. pteronyssinus airmid feeding on baker's yeast. Active ß-1,3 glucanases are expressed and excreted in the faeces of D. pteronyssinus airmid indicating they are digestive enzymes capable of breaking down ß-1,3 glucans of fungi present in house dust.

An Engineered Hybrid Protein from Dermatophagoides pteronyssinus Allergens Shows Hypoallergenicity.

The house dust mite (HDM) Dermatophagoides pteronyssinus is an important risk factor for asthma and rhinitis. Allergen specific immunotherapy that is based on recombinant proteins has been proposed for the safer and more efficient treatment of allergic diseases. The aim of this study was to design and obtain a hybrid protein (DPx4) containing antigenic regions of allergens Der p 1, Der p 2, Der p 7, and Der p 10 from this mite. DPx4 was produced in Escherichia coli and its folding was determined by circular dichroism. Non-denaturing dot-blot, ELISA, basophil activation test, dot blot with monoclonal antibodies, ELISA inhibition, and cysteine protease activity assays were performed. Mice that were immunized with DPx4 were also analyzed. We found that DPx4 had no cysteine protease activity and it showed significantly lower IgE reactivity than Der p 1, Der p 2, and D. pteronyssinus extract. DPx4 induced lower basophil activation than Der p 2 and the allergen extract. Immunized mice produced IgG antibodies that inhibited the binding of allergic patient's IgE to the allergen extract and induced comparatively higher levels of IL-10 than the extract in peripheral blood mononuclear cells (PBMC) culture. These results suggest that DPx4 has immunological properties that are useful for the development of a mite allergy vaccine.

Proteome and allergenome of the European house dust mite Dermatophagoides pteronyssinus.

The European house dust mite Dermatophagoides pteronyssinus is of significant medical importance as it is a major elicitor of allergic illnesses. In this analysis we have undertaken comprehensive bioinformatic and proteomic examination of Dermatophagoides pteronyssinus airmid, identified 12,530 predicted proteins and validated the expression of 4,002 proteins. Examination of homology between predicted proteins and allergens from other species revealed as much as 2.6% of the D. pteronyssinus airmid proteins may cause an allergenic response. Many of the potential allergens have evidence for expression (n = 259) and excretion (n = 161) making them interesting targets for future allergen studies. Comparative proteomic analysis of mite body and spent growth medium facilitated qualitative assessment of mite group allergen localisation. Protein extracts from house dust contain a substantial number of uncharacterised D. pteronyssinus proteins in addition to known and putative allergens. Novel D. pteronyssinus proteins were identified to be highly abundant both in house dust and laboratory cultures and included numerous carbohydrate active enzymes that may be involved in cuticle remodelling, bacteriophagy or mycophagy. These data may have clinical applications in the development of allergen-specific immunotherapy that mimic natural exposure. Using a phylogenomic approach utilising a supermatrix and supertree methodologies we also show that D. pteronyssinus is more closely related to Euroglyphus maynei than Dermatophagoides farinae.

Cross-Reactivity between Major IgE Epitopes of Family 5 Allergens from Dermatophagoides pteronyssinus and Blomia tropicalis.

BACKGROUND: The aim of this work was to understand the molecular features that trigger the cross-reactivity observed between Der p 5 from Dermatophagoides pteronyssinus, Blo t 5 from Blomia tropicalis, and Der f 5 from D. farinae. METHODS: We collected serum from 60 house dust mite (HDM)-allergic patients residing in the Dellys area of Boumerdès province in northern Algeria. The presence of specific IgE to Der p 5, Der f 5, and Blo t 5 was analyzed. We performed in silico analysis of the structure of the different allergens in order to identify epitopes that can elicit the cross-reactivity of the sera. Synthetic peptides corresponding to the linear epitope sequence of Der p 5, Der f 5, and Blo t 5 were used to evaluate its implication in the cross-reactivity between the allergens. We also modified the sequence of the conformational epitope of Der p 5 by site-directed mutagenesis to mimic Blo t 5. RESULTS: Several sera of patients allergic to HDM contained specific IgE antibodies to Der p 5 and Blo t 5. We demonstrated that the linear epitope of Der p 5 and Blo t 5 is not involved in the cross-reactivity of the sera. Furthermore, mutations introduced in the sequence of Der p 5 to mimic Blo t 5 could not modulate the cross-reactivity between them. CONCLUSIONS: The major linear IgE epitopes of Der p 5 and Blo t 5 are involved in species-specific recognition. Our results may be useful for the development of a hypoallergenic vaccine against HDM group 5 allergens.

Characterization of a hybrid protein designed with segments of allergens from Blomia tropicalis and Dermatophagoides pteronyssinus.

BACKGROUND: Sensitization to allergens of the house dust mites Dermatophagoides pteronyssinnus and Blomia tropicalis is an important risk factor for asthma and allergic diseases. Allergen-specific immunotherapy is currently based on natural allergen extracts, however, in the last years recombinant allergens with different modifications have shown promising immunological properties that may be advantageously applied for developing novel allergy vaccines. METHODS: A hybrid molecule (MAVAC-BD-2) containing epitopes of B. tropicalis (Blo t 5, Blo t 8 and Blo t 10) and D. pteronyssinus (Der p 1, Der p 2, Der p 7 and Der p 8) allergens was constructed, expressed in Escherichia coli and purified by affinity chromatography. Its folding was analyzed by circular dichroism. Antibody reactivities were evaluated by ELISA and non-denaturing dot blot assays using a battery of sera from mite allergic patients and non-allergic subjects. ELISA inhibition and dot blot assays with monoclonal antibodies were used to detect B-cell epitopes. Human basophil activation and induction of IgG-blocking antibodies in mice immunized with the hybrid protein were also evaluated. RESULTS: MAVAC-BD-2, expressed as a 22.8 kDa protein, showed a lower frequency and strength of IgE reactivity compared to Blo t 5, Der p 1, Der p 2 and the extracts of B. tropicalis and D. pteronyssinus. MAVAC-BD-2 inhibited 26% of IgE reactivity to Der p 2 and Blo t 5, reacted with anti-Der p 1 and anti-Der p 2 monoclonal antibodies and did not induce relevant basophil activation. MAVAC-BD-2 immunized mice produced specific antibodies that reacted against mite extracts and the purified allergens, as well as IgG antibodies that blocked the human IgE reactivity to mite extracts. CONCLUSION: MAVAC-BD-2 has hypoallergenic characteristics and in mice induces IgG antibodies that block the human IgE reactivity to mite extracts.

Proteases of Dermatophagoides pteronyssinus.

Since the discovery that Der p 1 is a cysteine protease, the role of proteolytic activity in allergic sensitization has been explored. There are many allergens with proteolytic activity; however, exposure from dust mites is not limited to allergens. In this paper, genomic, transcriptomic and proteomic data on Dermatophagoides pteronyssinus (DP) was mined for information regarding the complete degradome of this house dust mite. D. pteronyssinus has more proteases than the closely related Acari, Dermatophagoides farinae (DF) and Sarcoptes scabiei (SS). The group of proteases in D. pteronyssinus is found to be more highly transcribed than the norm for this species. The distribution of protease types is dominated by the cysteine proteases like Der p 1 that account for about half of protease transcription by abundance, and Der p 1 in particular accounts for 22% of the total protease transcripts. In an analysis of protease stability, the group of allergens (Der p 1, Der p 3, Der p 6, and Der p 9) is found to be more stable than the mean. It is also statistically demonstrated that the protease allergens are simultaneously more highly expressed and more stable than the group of D. pteronyssinus proteases being examined, consistent with common assumptions about allergens in general. There are several significant non-allergen outliers from the normal group of proteases with high expression and high stability that should be examined for IgE binding. This paper compiles the first holistic picture of the D. pteronyssinus degradome to which humans may be exposed.

Dietary shifts have consequences for the repertoire of allergens produced by the European house dust mite.

Products manufactured from mass-cultured house dust mites, currently commercialized for the diagnosis and immunotherapy of allergy, are heterogeneous in terms of allergen composition and thus present concerns to regulatory authorities. The most abundant species, Dermatophagoides pteronyssinus (Trouessart) (Astigmata: Pyroglyphidae), produces 19 allergenic proteins. Many of these are putatively involved in mite digestive physiology and metabolism. This study aimed to evaluate the effects of mite-rearing media on allergen production. Mites were adapted to feed on culture media supplemented with proteins, lipids, carbohydrates or beard shavings, and collected to quantify major allergens (Der p 1 and 2) by immunodetection, transcription of allergen genes by real-time quantitative polymerase chain reaction, and allergen-related enzymatic activities. All culture media significantly affected the content of major allergens. Modification of macronutrients in the diet produced minor effects on the transcription of allergen genes, but significantly altered mite allergen-related activities. The most remarkable impacts were detected in mites feeding on beard shavings and were reflected in reductions in the content of major allergens, alterations in the transcription of nine allergen genes, and changes in eight allergen-related activities. These results demonstrate the importance of culture media to the quality and consistency of mite extracts used for pharmaceuticals, and highlight the need to further elucidate allergen production by mites in the laboratory and in domestic environments.

Effects of domestic chemical stressors on expression of allergen genes in the European house dust mite.

The expression of allergen genes in house dust mites is influenced by temperature and relative humidity, but little is known of the impacts of other environmental factors that may alter the repertoire of allergens released by mites in home microhabitats. Bioassays were conducted in concave microscope slides in combination with real-time quantitative polymerase chain reaction (RT-qPCR) to analyse gene expression of 17 allergens of Dermatophagoides pteronyssinus (Acariformes: Pyroglyphidae) exposed to three chemical stressors that can be present in domestic environments. Short-term exposure (5-12 days) to diesel exhaust particles (DEPs) (1 µg/cm2 ), bacterial lipopolysaccharide endotoxin (0.1 µg/cm2 ) and benzyl benzoate (3.2 µg/cm2 ), at concentrations exceeding those expected in homes, had no significant effect on allergen transcription. A significant increase in the transcription of allergens Der p 3, Der p 8 and Der p 21 was observed only after exposing mites to a higher concentration of DEPs (10 µg/cm2 ) over a whole generation. In combination, the present results suggest that the analysed factors have low impact on allergen production. The methodology described here offers a sound and rapid approach to the broad-spectrum study of factors affecting allergen-related mite physiology, and allows the simultaneous screening of different factors in a relatively short period with consideration of the full spectrum of allergen genes.

Allergen expression in the European house dust mite Dermatophagoides pteronyssinus throughout development and response to environmental conditions.

House dust mites are a major source of allergy worldwide. While diagnosis and treatment based on mite extracts have remarkably advanced, little information exists on the expression of allergens in mites. We have studied gene expression of eight Dermatophagoides pteronyssinus (Trouessart) (Acari: Pyroglyphidae) allergens (Der p 1, 2, 3, 4, 5, 7, 10 and 21). All allergens showed higher transcription in nymphs compared with larvae or adults, with the only exception of Der p 10. The transcription of Der p 4 and Der p 10, together with the transcription and protein ratios Der p 1 to Der p 2, were higher in males than in females. One-week exposure of mite cultures to 16 or 35 °C (versus 24 °C) or low RH (44% versus 76%) significantly influenced the allergen gene transcription profile. Our results demonstrate that allergen expression is quantitatively and/or qualitatively influenced by mite development and sex, as well as by the environment. We suggest that monitoring allergen gene expression may be a useful tool to assist the optimization of mite cultures in the production of standardized allergenic extracts for clinical use.

Der p 11 is a major allergen for house dust mite-allergic patients suffering from atopic dermatitis.

House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.

A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy.

Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), can be expressed in Human Embryonic Kidney 293 cells (HEK 293 T) and whether such a construct can be used as a DNA vaccine in asthma therapy. The eukaryotic expression vector pcDNA3.1 was used to express the R8 molecule in HEK 293 T cells and successful expression of R8 was confirmed using a fluorescence microscope and western blot analysis. The efficacy of R8 as DNA vaccine was also assessed in a mouse asthma model. The in vivo data showed that R8 rectified the TH1/TH2 imbalance typical of allergic inflammation and stimulated the proliferation of regulatory T (Treg) cells. Immunization with the R8 construct also decreased serum allergen-specific IgE production in this mouse asthma model. Our findings suggest that R8 may be a feasible potential DNA vaccine for specific immunotherapy (SIT) in the treatment of allergic asthma.

PCR detection of the 14.5 antibacterial NlpC/P60-like Dermatophagoides pteronyssinus protein in Dermatophagoides farinae (Acari: Pyroglyphidae).

House dust mites produce antibacterial proteins suppressing bacterial growth. The 14.5-kDa bacteriolytic protein (UniProtKB Q8MWR6) has been known in Dermatophagoides pteronyssinus Trouessart. We have applied polymerase chain reaction and reverse transcription-PCR to detect a homologous gene sequence coding for a Q8MWR6-related protein in Dermatophagoides farinae (Hughes) using genomic DNA and total RNA, respectively. The resulting PCR product of expected size, 243 bp, was obtained from both Dermatophagoides spp., while no amplification was achieved from stored product mite samples. Sequence of the gene fragment from D. farinae showed 83% similarity to the previously described one in D. pteronyssinus. Successful amplification of the expected product from cDNA generated with oligo-dT primer implies that the NlpC/P60-like protein in Dermatophagoides mites is of eukaryotic or mite origin.

Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

Production of a chimeric allergen derived from the major allergen group 1 of house dust mite species in Nicotiana benthamiana.

Plants are widely accepted as a general platform for the large-scale production of recombinant proteins, which has been demonstrated by the successful expression of various exogenous proteins. Using plants as a bioreactor for mass production of target proteins for vaccines is thought to show the most potential. This study explores whether a chimeric allergen R8, derived from the major allergen group 1 of house dust mites species (Dermatophagoides farinae and Dermatophagoides pteronyssinus), is expressed in tobacco. The highly efficient and useful Tobacco mosaic virus RNA-based overexpression (TRBO) vector was used to investigate expression of the R8 molecule in tobacco by agroinfection. Presence of R8 was detected using SDS-PAGE and Western blotting. Purified allergens were characterized using IgE-binding activity assay and allergen-specific immunotherapy (ASIT) in murine asthmatic models. The recombinant R8 was successfully expressed in tobacco leaves. The pro-peptide was observed in the herbaceous leaf extracts. This protein exhibits properties similar to the parental allergen ProDer f 1 expressed in Escherichia coli or tobacco with respect to IgE immunoreactivity. R8 also rectifies imbalance of TH1/TH2 cells. An herbaceous plant expression system model allows mass production of R8, which might be used in the future for diagnosis of asthma or production of a candidate vaccine for allergen-specific immunotherapy of asthma.

Dermatophagoides pteronyssinus major allergen 1 activates the innate immune response of the fruit fly Drosophila melanogaster.

Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein-LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein-LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1.

Multiplex PCR for identifying common dust mites species (Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis).

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.