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旨在建立嗜虫书虱的实时荧光定量PCR分析方法,为嗜虫书虱基因的定量分析提供技术支持。利用RT-PCR方法,从嗜虫书虱体内克隆获得了β-actin基因cDNA片段(GenBank登录号:FJ041117),该基因片段长度为822 bp,编码273个氨基酸残基(从第3个碱基开始编码)。根据此β-actin基因的序列设计引物,建立了基于SYBR Green I染料技术的实时荧光定量PCR方法。建立的嗜虫书虱β-actin实时荧光定量PCR法扩增效率高、检测范围广、检测周期短,为β-actin作为内参基因进行嗜虫书虱功能基因的定量分析奠定了基础。
The aim of this study is to establish a real-time PCR method for the identification of flies and ticks and provide technical support for the quantitative analysis of flies and lice genes. The cDNA fragment of β-actin gene (GenBank accession number: FJ041117) was cloned by RT-PCR and cloned in vivo from B. lugens. The fragment was 822 bp in length and encoded 273 amino acid residues (from the third base Start coding). According to the sequence of β-actin gene, primers were designed and real-time PCR method based on SYBR Green I dye technology was established. The established β-actin real-time PCR method has the advantages of high amplification efficiency, wide detection range and short detection period, and lays the foundation for quantitative analysis of the function genes of the tick lice by using β-actin as an internal reference gene.