Stability of Loop-Mediated Isothermal Amplification (LAMP) Reagents and its Amplification Efficiency on Crude Trypanosome DNA Templates
Stability of Loop-Mediated Isothermal Amplification (LAMP) Reagents and its Amplification Efficiency on Crude Trypanosome DNA Templates
复制标题
DOI：
10.1292/jvms.71.471
复制
发表时间：
2009-04-01
影响因子：
1.2
通讯作者：
Inoue, Noboru
中科院分区：
文献类型：
--
作者：
Thekisoe, Oriel M. M.;Bazie, Raoul S. B.;Inoue, Noboru
本研究评估了LAMP试剂在25℃和37℃下储存时的稳定性，并评估了其对不同DNA模板制剂的检测效率。因此，使用储存在25℃和37℃的试剂的LAMP从试剂储存的第1天到第15天扩增了体外培养的布鲁氏锥虫(GUTat 3.1)的DNA。在25℃、37℃和-20℃(推荐储存温度)下储存的试剂之间，LAMP的检测灵敏度没有显著差异(P>；0.05)。用上述温度保存的试剂进行LAMP扩增冈比亚结核杆菌(IL2343)连续稀释的DNA(苯酚-氯仿法提取的基因组DNA、FTA卡片和溶血)，灵敏度高。对存放1~30d的试剂进行反应。当新鲜血液作为DNA模板直接加入到反应液中时，LAMP检测灵敏度较低。这项研究的结果表明，LAMP具有在现场条件下诊断锥虫感染的潜力，而不受热带和亚热带国家环境温度的影响，这些国家是锥虫流行的地方。
This study evaluated the stability of LAMP reagents when stored at 25 degrees C and 37 degrees C, and also assessed its detection efficiency on different DNA template preparations. Accordingly, LAMP using reagents stored at 25 degrees C and 37 degrees C amplified DNA of in vitro cultured T. b. brucei (GUTat 3.1) from day 1 to day 15 of reagent storage. There were no significant differences (P>0.05) in detection sensitivity of LAMP among the reagents stored at 25 degrees C, 37 degrees C and -20 degrees C (recommended storage temperature). LAMP using the reagents stored at above-mentioned temperatures amplified serially diluted DNAs (genomic DNA extracted by phenol-chloroform method, FTA card and hemolysed blood) of T b. gambiense (IL2343) with high sensitivity. Reactions were conducted on the reagents stored from 1 day to 30 days. LAMP detection sensitivity was poor when fresh blood as DNA template was added directly into reactive solution. Results of this study demonstrated that LAMP has the potential to be used in field conditions for diagnosis of trypanosome infections without being affected by ambient temperatures of tropical and sub-tropical countries where trypanosomosis is endemic.