Naidoo N, Ghai M, Moodley K, Mkize L, Martin L, McFarlane S, Rutherford S
School of Life Sciences, University of KwaZulu-Natal (Westville campus), Durban, South Africa.
South African Sugarcane Research Institute, Mount Edgecombe, South Africa.
Lett Appl Microbiol. 2017 Dec;65(6):496-503. doi: 10.1111/lam.12799. Epub 2017 Oct 10.
Ratoon stunt (RS) caused by bacterium Leifsonia xyli subsp. xyli (Lxx) results in substantial yield losses in sugarcane (Saccharum sp. L. hybrid). Since RS does not produce reliable symptoms in the field, laboratory-based techniques are necessary for detection. Loop-mediated isothermal amplification (LAMP) assay overcomes the limitations of laboratory-based techniques which are costly, time consuming and cannot be used for near-field detection. A sensitive LAMP assay was developed to detect Lxx at 65°C in 30 min. However, carry-over contamination affected the reliability of the assay. In the present study, contaminants were successfully eliminated by incorporation of uracil nucleoside glycosylase (1 U μl ) into the LAMP assay and incubation for 10 min at 37°C. To avoid the use of colorimetric reagents, lateral flow devices were successfully used for the detection of LAMP products and were equally sensitive to detection by agarose gel electrophoresis. The use of exudate from leaf sheath discs as an alternate template for the LAMP assay was found to be less sensitive than xylem sap. The preprepared master mix could be stored for up to 4 months at -20°C without any reduction in performance. These changes make the assay suitable for near-field detection in laboratories with basic facilities.
This study presents a modified loop-mediated isothermal amplification (LAMP) assay for the detection of Leifsonia xyli subsp. xyli. Modifications include incorporation of uracil nucleoside glycosylase to eliminate carry-over contamination and substitution of colorimetric detection for the use of lateral flow devices. LAMP master mix was preprepared and was stably stored up to 4 months at -20°C. Sugarcane leaf sheaths worked well as a substitute to xylem sap as template, although the sensitivity was lower. These modifications allow the assay to be conducted without contamination concerns and reduction in set up time, making it ideal for near-field diagnosis.
由木糖雷夫松氏菌木糖亚种（Lxx）引起的宿根矮化病会导致甘蔗（甘蔗属杂交种）大幅减产。由于宿根矮化病在田间不会产生可靠的症状，因此需要基于实验室的技术进行检测。环介导等温扩增（LAMP）检测克服了基于实验室的技术成本高、耗时且不能用于近场检测的局限性。开发了一种灵敏的LAMP检测方法，可在65°C下30分钟内检测到Lxx。然而，污染残留影响了检测的可靠性。在本研究中，通过将尿嘧啶核苷糖基化酶（1 U/μl）加入LAMP检测并在37°C下孵育10分钟，成功消除了污染物。为避免使用比色试剂，侧向流动装置成功用于检测LAMP产物，并且对琼脂糖凝胶电泳检测同样敏感。发现使用叶鞘圆盘的渗出液作为LAMP检测的替代模板比木质部汁液的敏感性更低。预制备的反应预混液可在-20°C下保存长达4个月而性能无任何下降。这些改变使该检测适用于具有基本设施的实验室进行近场检测。
本研究提出了一种改良的环介导等温扩增（LAMP）检测方法，用于检测木糖雷夫松氏菌木糖亚种。改进包括加入尿嘧啶核苷糖基化酶以消除污染残留，以及用侧向流动装置替代比色检测。制备了LAMP反应预混液，并在-20°C下稳定保存长达4个月。甘蔗叶鞘作为木质部汁液的替代模板效果良好，尽管敏感性较低。这些改进使得该检测可以在无污染问题且减少设置时间的情况下进行，使其成为近场诊断的理想选择。
