Ghai M, Singh V, Martin L A, McFarlane S A, van Antwerpen T, Rutherford R S
School of Life Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa.
Lett Appl Microbiol. 2014 Dec;59(6):648-57. doi: 10.1111/lam.12327. Epub 2014 Oct 20.
Leifsonia xyli subsp. xyli (Lxx), causal organism of ratoon stunt (RSD), does not produce any reliable internal or external symptoms on sugarcane. Its detection on a large scale is solely based on microscopic and serological methods. These methods require well-equipped laboratories, are time consuming and are not feasible for near-field detection of Lxx. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive detection of Lxx without the use of sophisticated equipment. To the best of our knowledge, this is the first report on the detection of Lxx in 30 min via an isothermal amplification method at 65°C. A transposase gene, ISLxx5, was used to design a set of six primers specifically targeting eight genomic sequences. The xylem sap was used as template, thus circumventing the need to isolate pure genomic DNA. The positive reactions were visually detected through a colour change of hydroxynaphthol blue (HNB) from violet to light blue, thus, eliminating the need for gel electrophoresis. The LAMP method was 10 times more sensitive than serological detection and as sensitive as immunofluorescence microscopy (IFM). The simplicity and sensitivity of the ISLxx5 LAMP assay makes it suitable for near-field diagnosis of RSD.
Detection of Leifsonia xyli subsp. xyli (Lxx) on a large scale is based on serological assays such as evaporative-binding enzyme-linked immunoassay (EB-EIA). These methods are time consuming and require well-equipped laboratories. This study presents the development of a loop-mediated isothermal amplification (LAMP) assay which allows detection of Lxx in 30 min at 65°C, using xylem sap as the template. The assay requires minimal laboratory equipment and could be used at near farm conditions, thus saving time and money required to transfer samples from remote areas to diagnostic laboratories. The LAMP method shows potential as an alternative detection method for RSD.
宿根矮化病（RSD）的病原木糖李氏杆菌（Lxx）在甘蔗上不会产生任何可靠的内部或外部症状。对其进行大规模检测仅基于显微镜和血清学方法。这些方法需要设备精良的实验室，耗时较长，且对于Lxx的近场检测不可行。在本研究中，我们开发了一种环介导等温扩增（LAMP）检测方法，用于在不使用复杂设备的情况下快速灵敏地检测Lxx。据我们所知，这是首次报道通过65°C等温扩增方法在30分钟内检测Lxx。利用转座酶基因ISLxx5设计了一组六个引物，特异性靶向八个基因组序列。以木质部汁液为模板，从而无需分离纯基因组DNA。通过羟基萘酚蓝（HNB）从紫色变为浅蓝色的颜色变化直观检测阳性反应，因此无需进行凝胶电泳。LAMP方法比血清学检测灵敏10倍，与免疫荧光显微镜（IFM）一样灵敏。ISLxx5 LAMP检测方法的简便性和灵敏性使其适用于RSD的近场诊断。
对木糖李氏杆菌（Lxx）的大规模检测基于血清学检测方法，如蒸发结合酶联免疫吸附测定（EB-EIA）。这些方法耗时且需要设备精良的实验室。本研究提出了一种环介导等温扩增（LAMP）检测方法的开发，该方法允许在65°C下以木质部汁液为模板在30分钟内检测Lxx。该检测方法所需实验室设备最少，可在农场附近条件下使用，从而节省了将样本从偏远地区转移到诊断实验室所需的时间和费用。LAMP方法显示出作为RSD替代检测方法的潜力。
