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首页> 外文期刊>Journal of Nematology, with Annual of Applied Nematology >Combining maxRatio analysis with real-time PCR and its potential application for the prediction of Meloidogyne incognita in field samples
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Combining maxRatio analysis with real-time PCR and its potential application for the prediction of Meloidogyne incognita in field samples

机译：将maxRatio分析与实时PCR结合起来在预测田间根结线虫病中的潜在应用

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摘要

Diagnosing and quantifying plant-parasitic nematodes is critical for efficient nematode management. Several studies have been performed intending to demonstrate nematode quantification via real-time quantitative PCR. However, most of the studies useddilution of DNA templates to make standard curves, while few studies used samples with different nematode numbers to make the standard curve, resulting in a high standard error. The objective of the present study was to develop a high quality standard curve using samples containing different numbers of the root-knot nematode Meloidogyne incognita and evaluate the results of real time qPCR with maxRatio analysis. The results showed that a high quality standard curve was obtained with different nematode numbers using specific primers and cycle threshold (Ct)-PCR (R2=0.9962, P<0.001, n=9). With the maxRatio analysis, the fractional cycle number (FCN)-PCR cycle curve and adjusted FCN (FCNadj)-PCR cycle curve had similar patterns as those of the Ct-PCR cycle curve. For quantification of nematodes in field soil samples, qPCR estimations with a FCNadj-PCR cycle standard curve was very close to microscope counting of second-stage juveniles (R~2=0.9064, P<0.001, n=10), qPCR estimations with a FCN-PCR cycle standard curve was comparably good (R~2=0.8509, P<0.001, n=10), and the biases with a Ct-PCR cycle standard curve were large (R~2=0.7154, P<0.001, n=10). Moreover, we found that the concentration of Triton X-100 had less of an effect on FCN as compared to Ct, with delta FCN 0.52, and delta Ct 3.94 at 0.8% Triton. The present study suggests, that combined with maxRatio methods, real time qPCR could be a practical approach for quantifying M. incognita in field samples.
机译：诊断和量化植物寄生线虫对于有效的线虫管理至关重要。已经进行了一些研究，旨在通过实时定量PCR证明线虫的定量。然而，大多数研究使用DNA模板的稀释来制作标准曲线，而很少有研究使用具有不同线虫编号的样品来制作标准曲线，导致很高的标准误差。本研究的目的是使用包含不同数量的根结线虫Meloidogyne incognita的样品开发高质量的标准曲线，并通过maxRatio分析评估实时qPCR的结果。结果表明，使用特异性引物和循环阈值（Ct）-PCR（R2 = 0.9962，P <0.001，n = 9），可以得到不同线虫数的高质量标准曲线。通过maxRatio分析，分数循环数（FCN）-PCR循环曲线和调整后的FCN（FCNadj）-PCR循环曲线具有与Ct-PCR循环曲线相似的模式。为了量化田间土壤样品中的线虫，使用FCNadj-PCR循环标准曲线进行qPCR估计非常接近于对第二阶段幼虫的显微镜计数（R〜2 = 0.9064，P <0.001，n = 10），qPCR估计使用a FCN-PCR循环标准曲线比较好（R〜2 = 0.8509，P <0.001，n = 10），Ct-PCR循环标准曲线的偏差较大（R〜2 = 0.7154，P <0.001，n = 10）。此外，我们发现，与Ct相比，Triton X-100的浓度对FCN的影响较小，其FCN增量为0.52，Trit的Ct增量为3.94，为0.8％。本研究表明，结合maxRatio方法，实时定量PCR可能是定量田间样品中隐孢子虫的一种实用方法。

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