Su Yu, Zhu Xuedong, Jing Haozheng, Yu Haiying, Liu Huai
College of Plant Protection, Southwest University, Chongqing 400716, China.
Southeast Chongqing Academy of Agricultural Sciences, Chongqing 408000, China.
Plants (Basel). 2024 Sep 26;13(19):2701. doi: 10.3390/plants13192701.
Pine wilt disease (PWD), which poses a significant risk to pine plantations across the globe, is caused by the pathogenic agent , also referred to as the pine wood nematode (PWN). A droplet digital PCR (ddPCR) assay was developed for the quick identification of the PWN in order to improve detection sensitivity. The research findings indicate that the ddPCR assay demonstrated significantly higher analysis sensitivity and detection sensitivity in comparison to traditional quantitative PCR (qPCR). However, it had a more limited dynamic range. High specificity was shown by both the ddPCR and qPCR techniques in the diagnosis of the PWN. Assessments of reproducibility revealed that ddPCR had lower coefficients of variation at every template concentration. Inhibition tests showed that ddPCR was less susceptible to inhibitors. There was a strong linear association between standard template measurements obtained using ddPCR and qPCR (Pearson correlation = 0.9317; < 0.001). Likewise, there was strong agreement (Pearson correlation = 0.9348; < 0.001) between ddPCR and qPCR measurements in the evaluation of pine wood samples. Additionally, wood samples from symptomatic (100% versus 86.67%) and asymptomatic (31.43% versus 2.9%) pine trees were diagnosed with greater detection rates using ddPCR. This study's conclusions highlight the advantages of the ddPCR assay over qPCR for the quantitative detection of the PWN. This method has a lot of potential for ecological research on PWD and use in quarantines.
松材线虫病（PWD）对全球范围内的松树种植园构成重大风险，该病由致病因子引起，该致病因子也被称为松材线虫（PWN）。为了提高检测灵敏度，开发了一种液滴数字PCR（ddPCR）检测方法用于快速鉴定松材线虫。研究结果表明，与传统定量PCR（qPCR）相比，ddPCR检测方法显示出显著更高的分析灵敏度和检测灵敏度。然而，其动态范围更有限。ddPCR和qPCR技术在松材线虫诊断中均表现出高特异性。重复性评估显示，ddPCR在每个模板浓度下的变异系数较低。抑制试验表明，ddPCR对抑制剂不太敏感。使用ddPCR和qPCR获得的标准模板测量值之间存在很强的线性关联（Pearson相关性 = 0.9317；<0.001）。同样，在评估松树木材样本时，ddPCR和qPCR测量值之间也存在很强的一致性（Pearson相关性 = 0.9348；<0.001）。此外，使用ddPCR对有症状（100%对86.67%）和无症状（31.43%对2.9%）松树的木材样本进行诊断时，检测率更高。本研究的结论突出了ddPCR检测方法在定量检测松材线虫方面优于qPCR的优势。该方法在松材线虫病的生态学研究和检疫应用方面具有很大潜力。
