Identification of Glycycometus malaysiensis (for the first time in Brazil), Blomia tropicalis and Dermatophagoides pteronyssinus through multiplex PCR

Abstract

Blomia tropicalis and Dermatophagoides pteronyssinus play an important role in triggering allergy. Glycycometus malaysiensis causes IgE reaction in sensitive people, but is rarely reported in domestic dust, because it is morphologically similar to B. tropicalis making the identification of these species difficult. The identification of mites is mostly based on morphology, a time-consuming and ambiguous approach. Herein, we describe a multiplex polymerase chain reaction (mPCR) assay based on ribosomal DNA capable to identify mixed cultures of B. tropicalis, D. pteronyssinus and G. malaysiensis, and/or to identify these species from environmental dust. For this, the internal transcribed spacer 2 (ITS2) regions, flanked by partial sequences of the 5.8S and 28S genes, were PCR-amplified, cloned and sequenced. The sequences obtained were aligned with co-specific sequences available in the GenBank database for primer design and phylogenetic studies. Three pairs of primers were chosen to compose the mPCR assay, which was used to verify the frequency of different mites in house dust samples (n = 20) from homes of Salvador, Brazil. Blomia tropicalis was the most frequent, found in 95% of the samples, followed by G. malaysiensis (70%) and D. pteronyssinus (60%). Besides reporting for the first time the occurrence of G. malaysiensis in Brazil, our results confirm the good resolution of the ITS2 region for mite identification. Furthermore, the mPCR assay proved to be a fast and reliable tool for identifying these mites in mixed cultures and could be applied in future epidemiological studies, and for quality control of mite extract production for general use.

Keywords: Allergy; Blomia tropicalis; Dermatophagoides pteronyssinus; Glycycometus malaysiensis; Multiplex PCR; Species identification.

Fig. 1

Schematic overview of the position of the annealing sites of the primers at the rDNA and the corresponding PCR products used for identification of Dermatophagoides pteronyssinus (Dp), Blomia tropicalis (Bt) and Glycycometus malaysiensis (Gm). Mite-specific primers are shown, targeting the end of the 18S (Fnav) and 5.8S sequences (ITS2f) and the start of the 28S region (Rnav and ITS2r) and the designed primers targeting ITS1 (for Dp and Bt) and ITS2 sequences of rDNA. The numbers near the arrows indicate the nucleotide positions of the designed primers, and are based on the GenBank sequences KC215364.1, KC215344.1 and MW763258.1 as template, respectively. The dotted region at the end of the 5.8S and part of the ITS2 regions shows the region used for the phylogenetic analyzes

Fig. 2

Maximum likelihood phylogenetic tree considering 1000 replicates of Bootstrap and T92 + G model constructed using alignment of the sequences obtained in our study for Blomia tropicalis and Glycycometus malaysiensis (blue dots before the accession numbers) and representative sequences available in the GenBank database for Dermatophagoides pteronyssinus, B. tropicalis, Glycyphagus domesticus and Lepidoglyphus destructor, consisting of the ITS2 region flanked by partial sequences of 5.8S and 28S subunits. (Color figure online)

Fig. 3

Venn diagram showing the positivity of the 20 dust samples analyzed in the multiplex PCR reaction: 95% of the samples were positive for Blomia tropicalis (Bt), 70% for Glycycometus malaysiensis (Gm) and 60% for Dermatophagoides pteronyssinus (Dp). Twenty % of the samples were positive just for Bt, 5% only for Dp + Bt, and 15% for Bt + Gm; 55% were positive for all studied mites

Fig. 4

Barcode gap analysis for the species Blomia tropicalis, Dermatophagoides pteronyssinus, Glycycometus malaysiensis, Glycyphagus domesticus and Lepidoglyphus destructor based on the ITS2 region and the intra- and interspecific distances. The top and bottom box represent the first and third quartiles of the data, the horizontal line in between indicates the median. The whiskers indicate 24.65% intervals, whereas the dots are outliers (remaining 0.35% of data)

Fig. 5

Maximum likelihood phylogenetic tree considering 1000 replicates of Bootstrap and T92 + G model constructed using alignment containing all sequences obtained herein and representative sequences available in the GenBank database for Dermatophagoides pteronyssinus, Blomia tropicalis, Glycyphagus domesticus and Lepidoglyphus destructor, consisting of the ITS2 region flanked by partial sequences of 5.8S and 28S subunits. The dust clone sequences obtained using primer pairs 3 and 6, for B. tropicalis and D. pteronyssinus, respectively, were trimmed, containing the end of the 5.8S region and partial ITS2 (see Fig. 1). Blue dots before the accession numbers indicate the sequences obtained for the ITS2 rDNA region flanked by partial sequences of the 5.8S and 28S subunits, the red dots indicate the sequences of the dust clones. (Color figure online)