Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR
Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR
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DOI：
10.1007/s10658-013-0252-1
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发表时间：
2013-08
影响因子：
1.8
通讯作者：
F. Mokrini;L. Waeyenberge;N. Viaene;Fouad Abbad Andaloussi;M. Moens
中科院分区：
文献类型：
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作者：
F. Mokrini;L. Waeyenberge;N. Viaene;Fouad Abbad Andaloussi;M. Moens
穿透短体线虫是最具经济破坏性的植物寄生线虫之一，广泛存在于多种农作物上。对该线虫的正确识别和定量对于为农民提供建议是必要的，但通过传统的显微镜观察方式不容易实现。我们开发了一种 qPCR 检测方法来检测和定量 P。 penetransin 是一种简短但准确的方法。根据β-1,4-内切葡聚糖酶基因的序列设计了qPCR引物组，包括两个引物和一个TaqMan探针。通过在 qPCR 测定中使用引物和 SYBR green I 染料，并将 qPCR 程序设置为 60 °C 至 64 °C 的不同退火温度，对该测定进行了优化。根据 Ct 值，我们保留了退火温度为 63 °C 的程序。使用探针进行的测定非常灵敏，因为它能够检测到单个 P 个体。 penetrans，即使与多达 80 个 P.peetrans 个体混合。托尔奈。反应的特异性通过 18 个其他短体线虫属物种的 28 个种群和其他 9 个属的植物寄生线虫的 DNA 没有扩增得到证实。来自 P. 21 种不同分离株的 DNA。外显反式被扩增。从 80 个人中提取 DNA 并通过 qPCR 进行定量，重复四次； Ct 值显示一致的结果（Ct = 24.4 ± 0.4）。 P DNA 的一系列稀释液。 penetrans 产生的标准曲线显示 Ct 值和稀释率之间具有高度显着的线性关系（R2= 0.99；斜率 = −3.23；E = 104 %）。测试显示线虫的实际数量与 qPCR 检测到的数量之间存在高度相关性。开发的 qPCR 检测为快速检测和可靠定量该害虫个体提供了一种灵敏的方法。该方法不需要线虫分类学和形态学方面的专业知识，可以用作研究以及诊断实验室和推广服务中的快速诊断工具，为农民提供害虫管理建议。
Pratylenchus penetransis one of the most economically damaging plant-parasitic nematodes and is found on a wide variety of crops. Correct identification and quantification of this nematode are necessary for providing advice to farmers, but are not easily obtained with the traditional way of microscopic observation. We developed a qPCR assay to detect and quantifyP. penetransin a short but accurate manner. A qPCR primer set, including two primers and a TaqMan probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimized by using the primers in a qPCR assay with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 60 °C to 64 °C. Based on the Ct-values, we retained the program with an annealing temperature of 63 °C. The assay with the probe was very sensitive as it was able to detect a single individual ofP. penetrans, even when mixed with up to 80 individuals ofP. thornei. The specificity of the reaction was confirmed by the lack of amplification of DNA from 28 populations of 18 otherPratylenchusspecies and from plant-parasitic nematodes from nine other genera. DNA from 21 different isolates fromP. penetranswas amplified. DNA extraction from 80 individuals and quantification by qPCR was repeated four times; Ct-values showed consistent results (Ct = 24.4 ± 0.4). A dilution series from DNA ofP. penetransresulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R2= 0.99; slope = −3.23; E = 104 %). The tests showed a high correlation between the real numbers of nematodes and the numbers detected by the qPCR. The developed qPCR assay provides a sensitive means for the rapid detection and reliable quantification of individuals of this pest. This method does not require expertise in nematode taxonomy and morphology, and can be used as a rapid diagnostic tool in research, as well as in diagnostic labs and extension services advising farmers for pest management.