Sun Xi-Meng, Ji Yong-Sheng, Liu Xian-Yong, Xiang Mei, He Guang, Xie Li, Suo Jing-Xia, Suo Xun
National Animal Protozoa Laboratory & College of Veterinary Medicine, China Agricultural University, Beijing, China.
State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing, China.
PLoS One. 2017 Jan 5;12(1):e0169125. doi: 10.1371/journal.pone.0169125. eCollection 2017.
Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries.
环介导等温扩增技术（LAMP）是一种颇具吸引力的DNA扩增方法，作为快速检测弓形虫的重要工具而被研发出来。在本研究中，通过靶向AF146527序列设计了种特异性LAMP引物，该序列是弓形虫529 bp片段的保守序列，重复200至300倍。对LAMP反应体系进行了优化，使其能够检测极少量的DNA样本，如单个速殖子或10个重组质粒拷贝。以其他寄生虫的DNA为模板时未发现交叉反应。随后，采用LAMP和传统PCR两种诊断方法对200份人类血液样本进行了直接检测。200份样本中有14份（7%）通过LAMP（本研究开发的引物）检测为弓形虫阳性，而传统PCR仅检测出200份中的5份（2.5%）为阳性。LAMP检测程序非常简单，因为反应在64°C等温条件下于单个管中进行，1小时内即可读出结果（使用环引物最早35分钟即可读出）。因此，该方法具有扩增快速、操作简单、检测容易等优点，对急性弓形虫病的快速可靠临床诊断具有重要意义，尤其在发展中国家。
