State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu 730046, PR China.
Exp Parasitol. 2012 May;131(1):125-9. doi: 10.1016/j.exppara.2012.02.012. Epub 2012 Feb 19.
We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/μl of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China.
我们开发了两种针对环形介导等温扩增（LAMP）检测的方法，用于检测环形泰勒虫，这是中国一种经济上重要的牛病，发生在亚热带和热带地区。这些方法针对核糖体 RNA（18S rRNA）和 ITS LAMP 序列。每个基因靶标的引物组由四个引物组成，每个组识别靶基因上的六个不同区域，以允许高度特异性地检测环形泰勒虫。从四个中国实验室保存的标准环形泰勒虫株的自身基因组 DNA 中扩增出了特异性梯带条带，与正常牛血液和其他原生动物物种的基因组 DNA 没有交叉反应。LAMP 检测法的灵敏度足以检测到 0.1pg/μl 的基因组 DNA。此外，从用环形泰勒虫人工感染的牛采集的血液中提取的 DNA 得到了扩增，表明这些引物具有很高的灵敏度。从中国采集的 351 个现场样本中，有 24.5%被两种 LAMP 引物阳性检测到，18.2%通过 PCR 发现感染了环形泰勒虫。这些结果表明，LAMP 检测法可能成为中国环形泰勒虫感染的流行病学研究的一种潜在诊断工具。
