Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick
Shrimp Taura syndrome virus detection by reverse transcription loop-mediated isothermal amplification combined with a lateral flow dipstick
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DOI：
10.1016/j.jviromet.2008.06.025
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发表时间：
2008-11-01
影响因子：
3.1
通讯作者：
Flegel, T. W.
中科院分区：
文献类型：
--
作者：
Kiatpathomchai, Wansika;Jaroenram, Wansadaj;Flegel, T. W.
环介导的等温扩增技术(LAMP)允许在等温条件下使用四组特殊设计的引物识别六个不同的靶序列来快速扩增核酸，具有高度的特异性和敏感性。采用生物素标记的RT-LAMP(RT-LAMP)扩增Taura综合征病毒(TSV)基因的方法，与层析侧流试纸(LFD)相结合，可快速、简便地检测TSV特异性扩增产物。LFD过程包括RT-LAMP后5分钟的步骤，将cDNA与FITC标记的DNA探针进行特异性杂交，证实存在特定的生物素标记的TSV扩增片段。由此产生的DNA双链可以在样品暴露的5分钟内被捕获在LFD试条测试线上。使用RT-LAMP和LFD相结合的系统，总的检测间隔约为70min，不包括RNA提取时间。检测灵敏度与其他常用的套式RT-PCR检测TSV的方法相当。与电泳法相比，除了缩短分析时间外，RT-LAMP和LFD的结合还通过杂交确认了扩增子的特性，并消除了处理致癌溴化乙锭的需要。(C)2008爱思唯尔B.V.保留所有权利。
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acid under isothermal conditions using four sets of specially designed primers that recognize six distinct target sequences with high specificity and sensitivity. In this report, a 60-min reverse transcription LAMP (RT-LAMP) method for amplification of Taura syndrome virus (TSV) cDNA using biotin-labeled primer was combined with a chromatographic lateral flow dipstick (LFD) for rapid and simple visual detection of TSV-specific amplicons. The LFD process involved a 5-min post RT-LAMP step for specific hybridization of cDNA with an FITC-labeled DNA probe that confirmed the presence of specific, biotin-labeled TSV amplicons. The resulting DNA duplexes could be visualized trapped at the LFD strip test line within 5 min of sample exposure. Using the combined RT-LAMP and LFD system, the total assay interval was approximately 70 min, excluding RNA extraction time. Detection sensitivity was comparable to other commonly used methods for nested RT-PCR detection of TSV. In addition to reduced assay time when compared to electrophoresis, combination of RT-LAMP with LFD confirms amplicon identity by hybridization and eliminates the need to handle carcinogenic ethidium bromide. (C) 2008 Elsevier B.V. All rights reserved.