| Objective The infection of Demodex humanis may result in acne, folliculitis, seborrheic dermatitis, rosacea and bletharitis. The parasitism of demodex mites are the possible pathogen or one of the pathogens in these diseases. Peoples are susceptible to mites, and the domestic infection rates are from20%to90%. Mites are really small, only100μm-400μm. It is difficult to collect, clean, isolate and grind mites, so that the molecular biology research of demodex mites has been retadated and few relative paper was published. Genome refers to the total DNA in the haploid nucleus, or is the amount of DNA in the single copy chromosomes. Genome size is described with C value, the unit is pg or Mb. The determination of genome size is important for a species, which are the basis to build a high quality gene library and an important parameter for genome research. It is also the foundation for screening the functional genes and studying the pathogenicity of demodex. This study focus on exploring the optimum method of demodex DNA extraction, and determinating the genome size of demodex by flow cytometry (FCM), which could lay the foundation for the construction of gene bank and the study of genome. These information are objective fact for the further research on mite’s molecular biology, genetics and bioinformatics.Methods Demodex caprae extracted from Boer Goat skin nodules is used as experimental material because it is difficult to collect enough samples of demodex humanis which have to be supplied by volunteers and are difficultly preserved. The genetic distance between Demodex caprae and Demodex brevis is closest. The samples of Demodex caprae can be easily obtained. The pathogenicty of Demodex caprae had been accepted. Collect clean mites which were washed with cleanser essence and selected under microscope. After smashing the mites by combination of tissue grinder and ultrasonic wave, mites’ nuclei were extracted in citric acid solution. The blood cell nuclei of domestic chicken were extracted in citric acid solution after treatment by ultrasonic wave. The nuclei were suspended by PBS and checked by optics microscope for appraising the mucleus density and cell debris. According to the counting result, we dilute the sample concentration for measuring by FCM. The mucleus DNA extracted by animal genome DNA extraction kit were used to agarose gel electrophoresis, in order to initially survey the DNA purity and size. Chicken erythrocytes were served as an internal reference. The mixed sample of demodex and chicken erythrocytes was dyed by propidium iodide (PI). After that the peak values of fluorescence intensity of the sample were measured by FCM. Comparing the multiple relationships between the two peak values with WinMDI, mites’ genome size was calculated.Results Citric acid solution can completely separate the nuclei, which satisfies the requirement of FCM. Animal genome DNA extraction kit have successfully extracted mite’s DNA. The DNA content of demodex is about1.05pg/2C determinated by FCM. As lpg converts to978Mb, the mite’s genome size is the equal of512Mb.Conclusion This study successfully separated the nuclei and extracted the genome DNA of demodex. Flow cytometry can rapidly and accurately determinate the genome size of Demodex. This study acquired the data of genome size, which enrich the genetic data of demodex, and it is the foundation work for the research of mite’s genomics. |
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