Department of Plant Pathology, North Dakota State University, Fargo, ND 58108, USA.
Int J Mol Sci. 2021 May 30;22(11):5872. doi: 10.3390/ijms22115872.
A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode ( ) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of . The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R value of 0.95 between quantification cycle number and log number of . To validate the assays to distinguish from other spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of and discriminated it from other spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of .
本研究开发了一种快速准确的基于 PCR 的方法，用于检测和鉴定最近在美国北达科他州大豆田中发现的一种新的根结线虫（ ）。设计了针对核糖体 DNA 内部转录间隔区的物种特异性引物，用于常规和定量实时 PCR 检测鉴定。通过计算机分析和实验室 PCR 实验评估了引物的特异性。结果表明，只有 的 DNA 可在常规和实时 PCR 检测中被特异性扩增，而其他对照物种的 DNA 均未被扩增。检测灵敏度分析显示，常规 PCR 能够检测到相当于单个线虫 DNA 的 1/8，而实时 PCR 则能够检测到相当于单个线虫 DNA 的 1/32。根据生成的标准曲线，实时 PCR 中引物的扩增效率为 94%，定量循环数和对数之间的 R 值为 0.95。为了验证这些检测方法能够区分在北达科他州大豆田中常见的其他 spp.，从七个县采集了 20 个土壤样本进行了测试。PCR 检测方法扩增了 的 DNA，并将其与北达科他州大豆田中存在的其他 spp.区分开来。这是首次报道一种适合在诊断和研究实验室中用于检测 的物种特异性和快速 PCR 检测方法。
