Rosser A, Rollinson D, Forrest M, Webster B L
Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK.
Department of Life Sciences, Natural History Museum, London, UK.
Parasit Vectors. 2015 Sep 4;8:446. doi: 10.1186/s13071-015-1055-3.
Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources.
Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms.
The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.
准确诊断泌尿生殖系统血吸虫病对监测/控制项目至关重要。通过聚合酶链反应（PCR）扩增尿液中的血吸虫DNA具有敏感性和特异性，但需要基础设施、资金和技术人员，而这些在流行地区往往难以获得。重组酶聚合酶扩增（RPA）是一种等温DNA扩增/检测技术，操作简单、快速、便携，且所需资源较少。
在此开发并改进了一种埃及血吸虫RPA检测方法，以便能够使用寡色层析侧向流动（LF）试纸条检测DNA扩增子。该检测方法在30-45°C下10分钟内成功扩增了埃及血吸虫DNA，对低至100 fg的DNA敏感。在加入粗尿液（占总反应体积的5%）的情况下，该检测方法也取得了成功。其他血吸虫物种会出现交叉扩增，但其他常见尿液微生物则不会。
此处开发的LF-RPA检测方法能够扩增并检测低水平的埃及血吸虫DNA。反应快速，所需温度低，阳性反应通过侧向流动试纸条判读，减少了对基础设施和资源的需求。再加上能够耐受尿液中的抑制剂，RPA作为泌尿生殖系统血吸虫病分子诊断工具具有进一步开发的潜力。
