Fu Hua-Ying, Sun Sheng-Ren, Wang Jin-Da, Ahmad Kashif, Wang Heng-Bo, Chen Ru-Kai, Gao San-Ji
National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
Biomed Res Int. 2016;2016:2681816. doi: 10.1155/2016/2681816. Epub 2016 Sep 28.
Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent subsp. (). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the gene of were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for , especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.
甘蔗宿根矮化病（RSD）是严重影响甘蔗作物生产力的最重要病害之一，由细菌病原体subsp.（）引起。本研究建立了一种基于TaqMan探针的实时定量聚合酶链反应（qPCR）检测方法，用于定量检测甘蔗茎汁中的。一对靶向基因的PCR引物（Pat1-QF/Pat1-QR）和一个荧光探针（Pat1-QP）用于qPCR检测。该检测方法对质粒DNA的检测限为100拷贝，对基因组DNA的检测限为100 fg，比传统PCR灵敏100倍。来自两个田间试验的174个茎汁样品中，有50个（28.7%）经qPCR检测呈阳性，而采用已发表的引物对CxxITSf#5/CxxITSr#5进行传统PCR检测时，只有12.1%（21/174）呈阳性，采用新设计的引物对Pat1-F2/Pat1-R2检测时，有15.5%（27/174）呈阳性。这种新的qPCR检测方法可作为目前诊断方法的替代方法，特别是在处理大量健康甘蔗幼苗的认证以及准确测定商业田地中的发病率时。
