Pratylenchus neglectus and P. thornei are economically important migratory plant endoparasitic nematodes and are widely found in a wide variety of crops. Based on 28 s rDNA D2D3 expansion region, a duplex real-time quantitative (qPCR) assay was developed to simultaneously detect and quantify these two nematodes. The qPCR system included two pairs of primers and two Taqman probes, that is the P. neglectus-specific primer pair 28sPnF5/28sPnR7 and probe pntaq3 and the P. thornei-specific primer pair 28sPtF1/28sPtR1 and probe pttaq3. The optimal conditions of the qPCR assay were 20 μL reaction volume comprising 10 μL THUNDERBIRD Probe qPCR Mix, 0.2 μM of primer 28sPnF5 and 28sPnR7, 0.25 μM of primer 28sPtF1and 28sPtR1, 0.1 μM of probe pntaq3 and 0.08 μM of probe pttaq3, 1 μL template DNA. And the amplification program were 95 °C for 2 min, 40 cycles of 95 °C for 10s, 65 °C for 25 s. The specificity of the qPCR assay was confirmed by the lack of fluorescence signal from non-target nematode populations including seven other Pratylenchus species and five other nematode species. The assay was very sensitive as it can detect a single target nematode and a single target nematode mixed with up to 100 individuals of non-target nematodes. A dilution series from P. neglectus and P. thornei DNAs resulted in two standard curves respectively showing a highly significant linearity between the Cycle threshold (Ct) values and the dilution rates. This study is the first to provide a molecular tool for simultaneous detection and quantification of P. neglectus and P. thornei.

Pratylenchus neglectus和P. thornei是重要的迁徙植物内寄生线虫，在各种农作物中广泛发现。基于28 s rDNA D2D3扩展区，开发了一种双工实时定量（qPCR）分析方法，可同时检测和定量这两个线虫。qPCR系统包括两对引物和两个Taqman探针，即P. neglectus特异性引物对28sPnF5 / 28sPnR7和探针pntaq3和P. thornei特异性引物对28sPtF1 / 28sPtR1和探针pttaq3。qPCR分析的最佳条件是20μL反应体积，包括10μLTHUNDERBIRD探针qPCR混合物，0.2μM引物28sPnF5和28sPnR7、0.25μM引物28sPtF1和28sPtR1、0.1μM探针pntaq3和0.08μM探针pttaq3、1μL模板脱氧核糖核酸。扩增程序为95°C 2分钟，95°C 10s 40循环，65°C 25 s。qPCR分析的特异性通过非目标线虫种群（包括其他7种Pratylenchus物种和5种其他线虫物种）缺乏荧光信号来证实。该测定法非常灵敏，因为它可以检测单个目标线虫和单个目标线虫，最多可混合100个非目标线虫。P. neglectus的稀释系列和P. thornei DNAs产生两条标准曲线，分别显示出循环阈值（Ct）和稀释率之间高度线性关系。这项研究是第一个提供同时检测和定量检测P. neglectus和P. thornei的分子工具的方法。