基于环介导等温扩增技术的暗褐书虱可视化鉴定

Visual identification of booklouse Liposcelis brunnea based on loop-mediated isothermal amplification

为防控暗褐书虱Liposcelis brunnea入侵和扩散,以mtDNA COI基因为靶标设计环介导等温扩增(loop-mediated isothermal amplification,LAMP)特异性内外引物,测定引物的特异性和灵敏度,并对暗褐书虱DNA粗提液进行LAMP可视化鉴定检测。结果显示,所设计的暗褐书虱LAMP引物特异性良好,DNA浓度的检测灵敏度为1.0×10^(-2) ng/μL;在65℃下恒温反应45 min后,暗褐书虱LAMP反应体系呈黄色(阳性反应),而其他啮虫昆虫的LAMP反应体系均呈紫红色(阴性反应),以DNA粗提液和以试剂盒法提取的DNA的LAMP可视化鉴定检测结果相同,但鉴定时间缩短到了1 h内,表明所建立的LAMP可视化鉴定技术高效、灵敏、操作简单,可快速准确地鉴定暗褐书虱。

In order to prevent and control the invasion and spread of booklouse Liposcelis brunnea,the specific internal and external primers targeting mtDNA COI gene for loop-mediated isothermal amplification(LAMP)were designed.The specificity and sensitivity of the primers were tested,and the LAMP visual detection was carried out based on the DNA crude extract of L.brunnea.The results showed that the LAMP primers of L.brunnea had good specificity,and the detection sensitivity of DNA concentration was 1.0×10^(-2) ng/μL.After 45 minutes of constant temperature reaction at 65℃,the LAMP reaction system for L.brunnea was yellow(positive reaction),while the LAMP reaction systems for other psocids were purple-red(negative reaction).The LAMP visual detection results of the DNA crude extract and the DNA extracted by the kit method were the same,but the identification time was shortened to less than one hour,indicating that the LAMP visual identification technique established in this study is efficient,sensitive,and simple to operate,and can identify L.brunnea quickly and accurately.