Abstract:

 Objective   To optimize conditions and screen for primers for  inter-simple sequence repeat-anchored(ISSR-PCR) of Demodex， using genome DNA as the template, preparing for the genetic diversity research of Demodex with ISSR mark. Methods   After mites were rinsed and precipitated in double distilled water(DDW), the genome DNA of Demodex was extracted with a kit. Then experiments were carried out using DNA of Demodex as the template, and (CA)8RG as the primer. The orthogonal array was conducted, and the involved factors were as follows： concentrations of Mg2+, dNTPs, primers，template DNA and Taq DNA polymerase and return temperature. An optimal reaction system of ISSR-PCR of Demodex was established, through which an optimal primer was obtained.  Results   An optimal 25μL of ISSR-PCR amplification system contained 2.0mmol/L of Mg2+, 0.4mmol/L of dNTPs, 30-60ng of template DNA, 30pmol of primers, and 2U of Taq DNA polymerase. The optional return temperature for primer was 49℃, and the cycle index is 35. 10 primers were selected, with clear bands, good polymorphism and high repetition. Conclusion   The optimal conditions of ISSR-PCR and primers of genome DNA of Demodex have been screened， which are the basis of molecular biological research of Demodex mites.

Key words: Mites; Minisatellite repeats; Optimization of the reaction system； DNA primers