A rapid and visual loop-mediated isothermal amplification assay to detect Leifsonia xyli subsp. xyli targeting a transposase gene

作者：Ghai M^*; Singh V; Martin L A; McFarlane S A; van Antwerpen T; Rutherford R S

来源：Letters in Applied Microbiology, 2014, 59(6): 648-657.

DOI：10.1111/lam.12327

• diagnosis
• hydroxynaphthol blue
• ISLxx5
• Leifsonia xyli subsp
• loop-mediated isothermal amplification
• ratoon stunt
• Xyli

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 30min via an isothermal amplification method at 65 degrees 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.

• 出版日期2014-12