The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China
The development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of Babesia spp. infective to sheep and goats in China
复制标题
用于检测巴贝虫属的环介导等温扩增（LAMP）方法的开发和评估。
DOI：
10.1016/j.exppara.2008.04.012
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发表时间：
2008-09-01
影响因子：
2.1
通讯作者：
Yin, Hong
中科院分区：
文献类型：
--
作者：
Guan, Guiquan;Chauvin, Alain;Yin, Hong
环介导等温扩增（LAMP）反应是一种在简单的培养箱中在等温条件下以高灵敏度、高效性和快速性扩增脱氧核糖核酸（DNA）的方法。根据我国分离的新疆巴氏杆菌BQ 1（临潭）和新疆巴氏杆菌Xinjiang-2005的18 SrRNA基因序列，设计了两对LAMP引物。将所设计的引物用于从感染血和纯化血吸虫中提取的血吸虫DNA的LAMP检测。从两种巴氏泰勒虫的自体基因组DNA中分别扩增出特异性梯状条带，与中国泰勒虫1号、中国泰勒虫2号、B的基因组DNA无交叉反应。牛泰勒虫（Theileria sp.）（日本）和绵羊。LAMP的灵敏度足以从10倍系列稀释的样品中分别检测到0.02 pg和0.2 pg的巴氏巴氏杆菌属BQ 1（临潭）和新疆巴氏杆菌属2005的基因组DNA，对应于50 μ l 0.000002%和0.00002%寄生虫血症红细胞中存在的DNA量。此外，从实验感染Bababelussp.BQ1（临潭）和Bababelussp. Xinjiang-2005的完整（未切除脾脏）绵羊的血液中提取的DNA分别在感染后1 - 9周和2 - 3周进行LAMP扩增，证明这些引物具有高灵敏度。从甘肃省采集的365份样品中，LAMP检测阳性率为14.3%（52/365）。从新疆放牧绵羊采集145份滤纸样品，阳性率为3.5%（5/145）。这些结果表明，LAMP可能是一种替代的诊断工具，用于检测绵羊和山羊的巴贝斯虫感染。(C)2008年爱思唯尔公司All rights reserved.
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 mu l 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. (C) 2008 Elsevier Inc. All rights reserved.