3种褐黄血蜱基因组DNA提取方法的比较  

Comparison of three methods for extracting genomic DNA from Haemaphysalis flava

作　　者：焦雪丽 张鑫 杨惠[1] 苏婧[1] JIAO Xueli;ZHANG Xin;YANG Hui;SU Jing(Nantong Center for Disease Control and Prevention,Nantong,Jiangsu 226007,China)

机构地区：[1]南通市疾病预防控制中心,江苏南通226007

出　　处：《中华卫生杀虫药械》2024年第4期354-358,共5页Chinese Journal of Hygienic Insecticides and Equipments

基　　金：南通市卫生健康委员会科研课题专项(MS2023092)。

摘　　要：目的在保持褐黄血蜱Haemaphysalis flava寄生蜱虫体形态完整的前提下,综合比较95℃水浴法、改良碱裂解法、离心柱法3种提取方法对蜱组织DNA提取质量的差异,筛选适用于蜱鉴定分型的高效稳定基因组DNA提取方法。方法取蜱标本的一只足作为实验样本,通过95℃水浴法、改良碱裂解法和离心柱法分别提取样品中基因组DNA,检测提取物浓度以及A260/A280和A260/A230比值,并通过PCR技术检测蜱分型相关基因16S rDNA(16S ribosoma DNA,16S核糖DNA)、ITS2(internal transcribed spacer 2,内转录间隔区2)的片段扩增效率,综合分析3种提取方法的优劣。多组之间使用单因素方差分析(ANOVA检验),两组之间比较采用t检验分析。P<0.05为差异有统计学意义。结果3种提取方法均可获得足量的蜱基因组DNA,改良碱裂解法提取效率显著高于其他2种方法(P<0.05),A260/A280和A260/A230比值结果表明离心柱提取法获得的DNA纯度更高,进一步的PCR实验表明,在模板量一致的情况下,以离心柱法提取的DNA为模板扩增产生的16S rDNA、ITS2片段扩增效率更高,而以95℃水浴法提取的DNA为模板扩增产生的16S rDNA、ITS2片段效率较低。结论离心柱法是提取蜱基因组DNA高效且实用的方法,能够在保持虫体形态完整的情况下满足对寄生蜱进行后续基因分型实验的要求。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

关 键 词：褐黄血蜱 寄生蜱 基因组DNA 核酸提取 PCR 

分 类 号：R384[医药卫生—医学寄生虫学]