Guan Guiquan, Chauvin Alain, Luo Jianxun, Inoue Noboru, Moreau Emmanuelle, Liu Zhijie, Gao Jinliang, Thekisoe Oriel M M, Ma Miling, Liu Aihong, Dang Zhisheng, Liu Junlong, Ren Qiaoyun, Jin Yurong, Sugimoto Chihiro, Yin Hong
Key Laboratory of Veterinary Parasitology of Gansu Province, Department of Veterinary Parasitology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Science, Xujiaping 1, Lanzhou Gansu 730046, PR China.
Exp Parasitol. 2008 Sep;120(1):39-44. doi: 10.1016/j.exppara.2008.04.012. Epub 2008 Apr 18.
The loop-mediated isothermal amplification (LAMP) reaction is a method that amplifies with high sensitivity, efficiency, and rapidity, deoxyribonucleic acid (DNA) under isothermal condition in simple incubators. Two primer sets for the LAMP method were designed using the nucleotide sequences of 18S rRNA gene of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 isolated in China. The primers were used to detect parasite DNA extracted from infected blood and purified parasites by LAMP. The specific ladder bands were amplified from the autologous genomic DNA of two Babesia species, respectively, and did not cross-react with the genomic DNA of Theileria sp. China 1, Theileria sp. China 2, B. bovis, Theileria sp. (Japan) and sheep. The LAMP was sensitive enough to detect 0.02 pg and 0.2 pg genomic DNA of Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005, respectively, from 10-fold serially diluted samples corresponding to the amount of DNA present in 50 microl of 0.000002% and 0.00002% parasitemic erythrocytes. Furthermore, DNA extracted from blood of intact (non-splenectomized) sheep experimentally infected with Babesia sp. BQ1 (Lintan) and Babesia sp. Xinjiang-2005 was amplified by the LAMP from week 1 to 9 and week 2 and 3 post-infection, respectively, demonstrating the high sensitivity of these primers. Of 365 samples collected from Gansu province, 14.3% (52/365) were positively detected by the LAMP. Of 145 samples collected on filter papers (Whatman) from the grazing sheep in Xinjiang province, 3.5% (5/145) were positive. These results show that the LAMP could be an alternative diagnostic tool for the detection of babesial infection in sheep and goats.
环介导等温扩增（LAMP）反应是一种在简单培养箱的等温条件下，以高灵敏度、高效率和快速性扩增脱氧核糖核酸（DNA）的方法。利用在中国分离的巴贝斯虫属BQ1（临潭）和巴贝斯虫属新疆-2005的18S rRNA基因的核苷酸序列设计了两套用于LAMP方法的引物。这些引物用于通过LAMP检测从感染血液中提取的寄生虫DNA和纯化的寄生虫。分别从两种巴贝斯虫的自体基因组DNA中扩增出特异性梯状条带，且不与中华泰勒虫1、中华泰勒虫2、牛巴贝斯虫、（日本）泰勒虫和绵羊的基因组DNA发生交叉反应。LAMP灵敏度足以从10倍系列稀释样品中分别检测到0.02 pg和0.2 pg巴贝斯虫属BQ1（临潭）和巴贝斯虫属新疆-2005的基因组DNA，这些样品对应的DNA量分别存在于50微升0.000002%和0.00002%的虫血症红细胞中。此外，分别从实验感染巴贝斯虫属BQ1（临潭）和巴贝斯虫属新疆-2005的完整（未脾切除）绵羊血液中提取的DNA，在感染后第1至9周和第2至3周通过LAMP进行了扩增，证明了这些引物的高灵敏度。从甘肃省采集的365份样本中，14.3%（52/365）通过LAMP检测呈阳性。从新疆放牧绵羊的滤纸片（Whatman）上采集的145份样本中，3.5%（5/145）呈阳性。这些结果表明，LAMP可作为检测绵羊和山羊巴贝斯虫感染的一种替代诊断工具。
