Extraction and random primer PCR detection of genomic DNA of parasitic mites Demodex folliculorum and Demodex brevis(Acari:Demodicidae)

[Objective] To explore the extraction methods of genomic DNA of parasitic mites Demodex folliculorum (D.f.) and Demodex brevis (D.b.). [Methods] Mites were disrupted by repeated freezing and thawing in liquid nitrogen. Then,the improved DNA extraction method of mini-insects,alkaline lysis and the commercial extraction kit were used respectively to extract genomic DNA of D.f. and D.b.,which had been kept freezing for ﹤5 or 8-10 months. [Results] The results of protein and nucleic acid radiometer showed the purity and quantity of genomic DNA extracted by the commercial kit were obviously better than those by other methods of mini-insects and alkaline lysis. Clear DNA fingerprints were found in the amplification results of random amplified polymorphic DNA (RAPD). There were obviously different banding patterns between D.f. and D.b. The freezing time affected the quantity of DNA extraction,but had only small effect on the purity and RAPD fingerprints of genomic DNA. The same species of mites had similar banding pattern even using different extraction methods. More and clear bands were detected by the commercial extraction kits and improved DNA extraction method of mini-insects. The bands detected by alkaline lysis method were less and obscure. [Conclusions] The application of repeated freezing-thawing in liquid nitrogen acts effectively to disrupt mites. The mites had better not be kept frozen more than 6 months. The commercial extraction kit is an effective approach to extract genomic DNA of mites. RAPD technique provides a simple method for detecting and classifying D.f. and D.b.