Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma
Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma
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DOI：
10.1007/s11274-016-2078-4
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发表时间：
2016-07-01
影响因子：
4.1
通讯作者：
Hegde, Vinayaka
中科院分区：
文献类型：
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作者：
Nair, Smita;Manimekalai, Ramaswamy;Hegde, Vinayaka
椰子根枯萎病（RWD）和槟榔黄叶病（YLD）是影响印度南部棕榈树的两种主要植原体相关疾病。这些疾病极大地削弱了棕榈树的健康，导致产量大幅下降和农民的经济损失。快速而强大的诊断技术对于有效的疾病管理至关重要。我们建立了针对椰子 RWD 和槟榔 YLD 的植原体 16S rDNA 靶向环介导等温扩增 (LAMP) 和实时 LAMP 诊断。 LAMP反应设定在65℃，并使用羟基萘酚蓝(HNB)和琼脂糖凝胶电泳进行终点检测。使用限制性内切酶HpyCH4 V对LAMP产物进行分子分型。使用LAMP外部引物进行常规PCR并对扩增子进行测序。实时 LAMP 在 Genei II 平台（Optigene Ltd.，英国）上进行。在孵育结束时编程退火曲线分析以检查扩增子的保真度。植原体阳性样品在琼脂糖凝胶上产生典型的梯状条带，在HNB下显示颜色从紫色到蓝色的变化，并在实时检测中在85+/-0.5℃处产生独特的退火峰。限制性消化产生了预测大小的片段。测序和BLASTN分析证实扩增对应于植原体16S rRNA基因。研究发现，本文设计的 LAMP 方法比传统的巢式 PCR 更稳健，因此在从有症状的棕榈样本中检测植原体和快速筛选健康幼苗方面具有潜在的应用。
The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 degrees C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 +/- 0.5 degrees C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.