Lou Binghai, Song Yaqin, RoyChowdhury Moytri, Deng Chongling, Niu Ying, Fan Qijun, Tang Yan, Zhou Changyong
First author: College of Plant Protection, Southwest University, Chongqing 400715, P.R. China; first, second, fourth, fifth, sixth, and seventh authors: Guangxi Key Laboratory of Citrus Biology, Guangxi Academy of Specialty Crops, Guilin, Guangxi 541004, P.R. China; third author: Department of Biological Sciences, Idaho State University, 650 Memorial Dr., Pocatello 83201; and eighth author: Citrus Research Institute, Southwest University, Chongqing 400715, P.R. China.
Phytopathology. 2018 Feb;108(2):292-298. doi: 10.1094/PHYTO-06-17-0210-R. Epub 2018 Jan 2.
Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of 'Candidatus Liberibacter asiaticus', a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of 'Ca. L. asiaticus' with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of 'Ca. L. asiaticus'.
黄龙病（HLB）是全球柑橘生产中最具破坏性的病害之一。早期检测HLB病原体有助于及时清除田间受感染的柑橘树。然而，HLB病原体在寄主植物中的滴度低且分布不均，使得可靠检测具有挑战性。因此，开发具有高灵敏度的有效检测方法势在必行。本研究报告了一种用于检测“亚洲韧皮杆菌”（一种广泛分布的与HLB相关的细菌）的新方法——基于串联重复序列的聚合酶链置换反应（TR-PCDR）的开发。使用一组独特设计的引物（TR2-PCDR-F/TR2-PCDR-1R）和具有链置换活性的热稳定Taq DNA聚合酶突变体进行TR-PCDR扩增。在常规热循环仪中进行，TR-PCDR在每个扩增循环后可产生两个以上的扩增子。所开发的TR-PCDR的灵敏度为10个拷贝的目标DNA片段。已证明其灵敏度比传统PCR高100倍，与实时PCR相似。使用上述三种方法对田间样品检测“亚洲韧皮杆菌”的数据也显示了类似的结果。在健康柑橘样品和水对照中未观察到TR-PCDR的假阳性扩增。因此，这些结果表明，所开发的TR-PCDR方法可应用于“亚洲韧皮杆菌”的可靠、高灵敏度和经济高效的检测。
