Objective While maintaining the intact morphology of Haemaphysalis flava,comprehensive comparison was conducted among three DNA extraction methods,namely the 95℃water bath method,modified alkaline lysis method,and centrifuge column method,to evaluate the differences in DNA extraction quality from tick tissues.An efficient and stable genomic DNA extraction method suitable for tick identification and typing was screened.Methods A leg of a tick specimen were taken as experimental samples.Genomic DNA was extracted using the 95℃water bath method,modified alkaline lysis method,and centrifuge column method,respectively.The concentration of the extracted material,as well as the values of A260/A280 and A260/A230,were determined.Additionally,the amplification efficiency of tick typing-related genes,16S rDNA(16S ribosomal DNA)and ITS2(internal transcribed spacer 2),was assessed using PCR method.The advantages and disadvantages of the three extraction methods were comprehensively analyzed.Comparison between multiple groups was analyzed using one-way analysis of variance(ANOVA),while comparison between two groups was analyzed using t-test.P＜0.05 means there was a significant difference compared with the control group.Results All three extraction methods yielded sufficient amounts of tick genomic DNA.The modified alkaline lysis method exhibited significantly higher extraction efficiency compared to the other two methods(P＜0.05).The values of A260/A280 and A260/A230 indicated higher DNA purity with the centrifuge column extraction method.Further PCR experiments demonstrated that,under consistent template amounts,DNA extracted using the centrifuge column method resulted in higher amplification efficiency for the 16S rDNA and ITS2 gene segments.Conversely,DNA extracted using the 95℃water bath method showed lower efficiency in amplifying the 16S rDNA and ITS2 segments.In conclusion,the centrifuge column method proved to be an efficient and practical approach for extracting tick genomic DNA,meeting the requirements for subsequent genetic typing experiments while preserving the intact morphology of the specimens.Conclusion The centrifuge column method is an efficient and practical approach for extracting tick genomic DNA.It can meet the requirements for subsequent genetic typing experiments while preserving the intact morphology of the specimens.
维普
万方数据
