A rapid identification method for common astigmatid species based on multiplex polymerase chain reaction

Abstract

Astigmatid mites are economically significant pests of stored products and sources of inhalant allergens causing allergic rhinitis and asthma worldwide. The morphological identification of astigmatid mites at the species level is often a difficult task due to their small size, phenotypic similarity and lack of diagnostic characters. We used multiplex polymerase chain reaction (PCR) to identify astigmatid mite species, which could complement the morphological data for the species-specific identification of mites. Internal ribosomal transcribed spacer (ITS) sequences (i.e., partial 18S, the full length of ITS1-5.8S-ITS2 and partial 28S) from eight astigmatid species (Acarus siro, Tyrophagus putrescentiae, Suidasia nesbitti, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Lepidoglyphus destructor, Chortoglyphus arcuatus and Gohieria fuscus) were obtained by DNA extraction and then sequenced after PCR amplification. Specific primers were designed in the ITS2 region manually. Results revealed that an identification method for eight common astigmatid species was established based on multiplex PCR, which should be effective for the identification of other species of mites by redesigning species-specific primers in future experiments.

Keywords: Astigmatid mite; Internal ribosomal transcribed spacer; Multiplex polymerase chain reaction; Species identification; Species-specific primers.

Fig. 1

Morphological images of eight common astigmatid mites. A) Acarus siro (female, scale 100 µm); B) Acarus siro (male, bar = 100 µm); C) Tyrophagus putrescentiae (female, bar = 100 µm); D) Tyrophagus putrescentiae (male, scale 100 µm); E) Suidasia nesbitti (female, bar = 100 µm); F) Suidasia nesbitti (male, bar = 100 µm); G) Dermatophagoides pteronyssinus (female, bar = 50.00 µm); H) Dermatophagoides pteronyssinus (male, bar = 50.00 µm); I) Dermatophagoides farinae (female, scale 50 µm); J) Dermatophagoides farinae (male, bar = 50.00 µm); K) Lepidoglyphus destructor (female, bar = 100 µm); L) Lepidoglyphus destructor (male, bar = 100 µm); M) Chortoglyphus arcuatus (female, bar = 50.00 µm); N) Chortoglyphus arcuatus (male, bar = 50.00 µm); O) Gohieria fuscus (female, bar = 50.00 µm); P) Gohieria fuscus (male, bar = 50.00 µm)

Fig. 2

Results of multiplex polymerase chain reaction with DNA extracted from 8 astigmatid mite species (Lanes 1-8). Lane 1: Acarus siro, 429 bp; Lane 2: Tyrophagus putrescentiae, 355 bp.; Lane 3: Suidasia nesbitti, 192 bp; Lane 4: Dermatophagoides pteronyssinus, 215 bp; Lane 5: Dermatophagoides farinae, 288 bp; Lane 6: Lepidoglyphus destructor, 306 bp; Lane 7: Chortoglyphus arcuatus, 409 bp; Lane 8: Gohieria fuscus, 382 bp; Lane 9: negative control (A universal forward primer and eight reverse primers were added without extracted gDNAs); Lane M: DNA ladder A Plus (50 bp DNA ladder marker)