背景:泌尿生殖道血吸虫病是由寄生吸虫血吸虫引起的。为了消除这种疾病,需要敏感和特定的即时诊断。重组酶聚合酶扩增(RPA)测定符合这些标准,并且已经开发了诊断血吸虫链球菌的测定法(Sh-RPA)。然而,可能会出现假阳性结果,和优化反应条件以减轻这些是需要的。还必须考虑DNA提取方法的易用性和兼容性。
方法:使用合成DNA,S、嗜血杆菌基因组DNA(gDNA),和临床病例的尿液样本,掺入不同甜菜碱浓度的Sh-RPA反应(0M,1米,2.5M,12.5M)和样品与水的比率进行了测试,以确定对测定特异性和灵敏度的影响。此外,使用五种适用于资源有限环境的商业DNA提取试剂盒从单个血吸虫卵中获得gDNA,并根据DNA质量进行评估,数量,以及与Sh-RPA测定的兼容性。还通过定量聚合酶链反应(qPCR)评估所有样品以确认DNA获得。
结果:在所有甜菜碱浓度下,Sh-RPA的分析灵敏度≥10拷贝的合成Dra1标准品和0.1μg的血吸虫gDNA。甜菜碱的加入提高了Sh-RPA测定在所有反应条件下的特异性,添加2.5M甜菜碱以及最大可能的12.7μl样品体积被证明是最佳反应条件。使用所有五种商业DNA提取试剂盒,成功地从单个嗜血链球菌卵中分离出DNA,但是这些套件的Sh-RPA性能各不相同,其中一个被证明与RPA反应不相容。
结论:向Sh-RPA反应中添加2.5M甜菜碱改善了反应特异性,同时对灵敏度没有不利影响。这增加了测定的鲁棒性,推进在资源有限的环境中使用Sh-RPA测定的可行性。商业提取试剂盒的测试证明,快速,简单的方法就足以从单个血吸虫卵中获得DNA,并且这些提取物在大多数情况下可以与Sh-RPA一起使用。然而,观察到的特定试剂盒与Sh-RPA的不相容性凸显了在实施前需要对分子诊断平台的每个阶段进行稳健测试的必要性.
BACKGROUND: Urogenital schistosomiasis is caused by the parasitic trematode Schistosoma haematobium. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose S. haematobium has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered.
METHODS: Using synthetic DNA, S. haematobium genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single S. haematobium eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition.
RESULTS: The analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic Dra1 standard and 0.1 pg of S. haematobium gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single S. haematobium egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions.
CONCLUSIONS: The addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single S. haematobium eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a
molecular diagnostic platform to be robustly tested prior to implementation.