应用LAMP技术对4种疟原虫进行属种鉴定的初步研究

中国媒介生物学及控制杂志 ›› 2011, Vol. 22 ›› Issue (4): 355-358.

应用LAMP技术对4种疟原虫进行属种鉴定的初步研究

易海华¹, 祝长青², 孙慧宇¹, 吴萍兰¹, 徐波¹, 房超¹, 宋阳威¹, 王云飞¹, 徐政¹, 赵金伟¹, 徐继承³

1 徐州出入境检验检疫局技术中心,江苏徐州 221006;
2 江苏出入境检验检疫局;
3 徐州医学院公共卫生学院

收稿日期:2011-01-14 出版日期:2011-08-20 发布日期:2011-08-20
通讯作者: 赵金伟,Email: zhaojw@jsciq.gov.cn
作者简介:易海华(1977-),男,主管医师,从事病原微生物诊断研究。Email: yihaihua88@126.com
基金资助:
国家质检总局科研专项基金(2009IK217)

Detection of four Plasmodium species by genus-specific loop-mediated isothermal amplification

YI Hai-hua¹, ZHU Chang-qing², SUN Hui-yu¹, WU Ping-lan¹, XU Bo¹, FANG Chao¹, SONG Yang-wei¹, WANG Yun-fei¹, XU Zheng¹, ZHAO Jin-wei¹, XU Ji-cheng³

1 Xuzhou Entry-Exit Inspection and Quarantine Bureau, Xuzhou 221006, Jiangsu Province, China;
2 Jiangsu Entry-Exit Inspection and Quarantine Bureau;
3 Public Health School of Xuzhou Medical College

Received:2011-01-14 Online:2011-08-20 Published:2011-08-20
Supported by:
Supported by the Project Funds for Scientific Research of General Administration of Quality Supervision,Inspection and Quarantine(No. 2009IK217)

摘要/Abstract

摘要：

目的建立对4种感染人的疟原虫虫属及定量的环介导等温扩增法(loop-mediated isothermal amplification,LAMP)基因检测方法。方法根据恶性疟原虫、三日疟原虫、卵形疟原虫、间日疟原虫的18S rRNA基因共有保守序列及LAMP技术原理,设计一套含有6条疟原虫虫属特异性引物的LAMP反应体系,产物经SYBR GreenⅠ进行显色反应及电泳分析,观察其特征条带的情况,利用LAMP技术检测上述4种疟原虫DNA及其它12种相关寄生虫DNA评价反应特异性,利用PCR技术为参照对LAMP技术检测疟原虫虫属试验的敏感性,利用疟原虫重组质粒探索建立疟原虫LAMP定量的可能性。结果 4种疟原虫LAMP产物经显色后呈绿色,经电泳后呈特征性梯状条带,阴性对照及其它相关寄生虫DNA无明显扩增,重组质粒浓度在1.42×10⁴～1.42×10⁹拷贝/μl范围内时,反应循环阈值与模板浓度有良好的线性关系(r＝0.9995)。与PCR方法相比较,建立的疟原虫虫属LAMP检测方法的敏感性是其10倍,检出限达到1.42×10¹拷贝/μl。结论建立的检测疟原虫属种、数量的LAMP方法具有较高的特异性和敏感性,适合疟疾防治检测的需要。

关键词: 环介导等温扩增技术, 属种, 疟原虫, 18S rRNA基因

Abstract:

Objective To develop a quantitative detection method using loop-mediated isothermal amplification (LAMP) to detect Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Methods Based on the conserved nucleotide of the four species and the mechanism LAMP, a LAMP reaction system containing six primers was designed for genus detection and quantitation. LAMP products were stained by SYBR Green and analyzed by electrophoresis. The specificity of LAMP was evaluated by detection of Plasmodium spp. and twelve species of related parasites. The sensitivity of LAMP was evaluated in comparison with the PCR sensitivity, and the feasibility of quantitative LAMP was evaluated by detection of recombinant plasmids. Results After staining, LAMP products with Plasmodium DNA turned green while the color of the negative control and other parasites remained unchanged. Electrophoresis showed that LAMP products had characteristic ladders, which was not present in the negative control and other parasites. At a template concentration of 1.42×10⁴-1.42×10⁹ copies per microlitre, the standard curve established by recombinant plasmids showed a fine linear relationship between threshold cycle (Ct) and template concentration, with a correlation coefficient of 0.9995. The LAMP assay was found to have a detectability of 1.42×10¹ copies/μl, a 10-fold increase in sensitivity compared with traditional PCR. Conclusion The detection system had high specificity and sensitivity, which could be used for the prevention and control of malaria．

Key words: Loop-mediated isothermal amplification, Genus, Plasmodium, 18S rRNA gene

中图分类号:

R382.3⁺1

易海华, 祝长青, 孙慧宇, 吴萍兰, 徐波, 房超, 宋阳威, 王云飞, 徐政, 赵金伟, 徐继承. 应用LAMP技术对4种疟原虫进行属种鉴定的初步研究[J]. 中国媒介生物学及控制杂志, 2011, 22(4): 355-358.

YI Hai-hua, ZHU Chang-qing, SUN Hui-yu, WU Ping-lan, XU Bo, FANG Chao, SONG Yang-wei, WANG Yun-fei, XU Zheng, ZHAO Jin-wei, XU Ji-cheng. Detection of four Plasmodium species by genus-specific loop-mediated isothermal amplification[J]. Chines Journal of Vector Biology and Control, 2011, 22(4): 355-358.

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