Mekata T, Sudhakaran R, Kono T, Supamattaya K, Linh N T H, Sakai M, Itami T
Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi, Miyazaki, Japan.
Lett Appl Microbiol. 2009 Jan;48(1):25-32. doi: 10.1111/j.1472-765X.2008.02479.x. Epub 2008 Nov 19.
White spot syndrome virus (WSSV) continues to be the most pathogenic virus among the crustacean aquaculture causing mass mortality. In the present study, we established a one-step, single tube, real-time accelerated loop-mediated isothermal amplification (real-time LAMP) for quantitative detection of WSSV.
A set of six specially designed primers that recognize eight distinct sequences of the target. The whole process can be completed in 1 h under isothermal conditions at 63 degrees C. Detection and quantification can be achieved by real-time monitoring in an inexpensive turbidimeter based on threshold time required for turbidity in the LAMP reaction. A standard curve was constructed by plotting viral titre against the threshold time (T(t)) using plasmid standards with high correlation coefficient (R(2) = 0.988).
Sensitivity analysis using 10-fold dilutions (equivalent to 35 ng microl(-1) to 35 ag microl(-1)) of plasmid standards revealed this method is capable of detecting upto 100 copies of template DNA. Cross-reactivity analysis with DNA/cDNA of IHHNV, TSV, YHV-infected and healthy shrimp showed this method is highly specific for quantitative detection of WSSV.
WSSV real-time LAMP assay appears to be precise, accurate and a valuable tool for the detection and quantification of WSSV in large field samples and epidemiological studies.
白斑综合征病毒（WSSV）仍然是甲壳类水产养殖中致病性最强的病毒，可导致大规模死亡。在本研究中，我们建立了一种一步单管实时加速环介导等温扩增法（实时LAMP）用于定量检测WSSV。
设计了一组六个特殊引物，可识别靶标的八个不同序列。整个过程可在63℃等温条件下1小时内完成。通过基于LAMP反应中浊度所需的阈值时间在廉价的浊度仪中进行实时监测来实现检测和定量。使用具有高相关系数（R² = 0.988）的质粒标准品绘制病毒滴度与阈值时间（T(t)）的标准曲线。
使用10倍稀释（相当于35 ng μl⁻¹至35 ag μl⁻¹）的质粒标准品进行敏感性分析表明，该方法能够检测多达100个模板DNA拷贝。与感染IHHNV、TSV、YHV的虾和健康虾的DNA/cDNA进行交叉反应分析表明，该方法对WSSV的定量检测具有高度特异性。
WSSV实时LAMP检测法似乎精确、准确，是在大量野外样本和流行病学研究中检测和定量WSSV的有价值工具。
