Abstract
The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a ~450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.
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Acknowledgements
Project support was provided by MURST contract year 2004, Prot. 2004078701-001 to LR. We thank all the people who supported the Department of Animal Production, Epidemiology, and Ecology, University of Turin—Italy, and the RNM118 investigation group. Junta de Andalucía—Spain is thanked for supporting SA’s investigation stay in Italy. XQZ is supported by a grant from the Program for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT0723). The experiments comply with the current laws of the countries in which the experiments were performed.
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Alasaad, S., Rossi, L., Maione, S. et al. HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA. Parasitol Res 103, 1455–1457 (2008). https://doi.org/10.1007/s00436-008-1127-9
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DOI: https://doi.org/10.1007/s00436-008-1127-9