Validation of the purity of acarid mite cultures used for allergen extract preparation and identification of contaminants by ribosomal DNA sequencing via a PCR-cloning- and sequence homology-based approach*1

Abstract Rationale Contamination in mite cultures is not easily detected and purity of raw materials used for allergen extract preparation difficult to validate. Identification of mites can be difficult for non-specialists. Compounding this, the presence of closely related ( Dermatophagoides ) and morphologically indistinct genera ( Acarus and Tyrophagus ) can led to misidentification. Methods Using a test mixed culture of Acarus siro , Tyrophagus putrescentiae and Suidasia medanensis (three morphologically related species), we PCR amplified and sequenced the 18S ribosomal DNA genes and compare these with reference mites that have been correctly identified. Randomly selected 18S PCR products were then cloned to examine if the PCR amplicons would show mixed sequences. To rule out the possibility of experimental error, experiments were repeated four times by different individuals on separate occasions. Three commercial sources of mites were then evaluated. Results Fragments of the 18S rDNA from these cultures was amplified, gel purified and cloned. A total of 45 clones for each test were sequenced (774 bases) and aligned to sequences of reference mites. Direct sequencing of the 18S amplification showed overlapping chromatogram peaks in contaminated cultures. One out of the three raw material obtained commercially was found to be contaminated with other mite species. Conclusions We reiterate Colloff and Spieksma (1992) call for mite cultures to be examined prior to preparation of allergen extracts. We propose a PCR-cloning approach using the 18S rDNA as a marker to augment visual morphological screening. This method may also be extended to other raw materials e.g., pollen or fungi cultures.