一种快速检测疟原虫核酸的重组酶扩增方法的建立

iLABMED Pub Date : 2025-05-13 DOI:10.1002/ila2.70014
Xinxin Yang, Xiaoxue Lu, Junlian Yang, Wen Xu, Qian Li, Weiwei Chen
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引用次数: 0

摘要

疟疾仍然是一项全球卫生挑战,2022年报告了2.49亿例病例和60.8万人死亡。虽然中国实现了消除疟疾,但2023年输入病例激增194.4%,凸显了快速诊断的必要性。显微镜和快速诊断测试(RDTs)等传统方法在灵敏度和基础设施要求方面面临限制。本研究旨在建立并优化一种用于恶性疟原虫和间日疟原虫分子检测的“一锅法”酶重组酶扩增(ERA)方法,并通过方法学验证和临床表现来评价该方法的有效性。方法设计针对恶性疟原虫和间日疟原虫遗传物质保守区域的特异性ERA检测方法。我们使用合成质粒和基因组材料评估了该检测的敏感性和特异性。此外,我们通过将潜在干扰物质加入反应体系来测试反应的稳定性。最后,我们分析了ERA方法对实时荧光定量PCR和临床样品快速诊断检测的检测性能。结果在35°C ~ 40°C条件下,检测过程可在25 min内完成,结果可在紫外灯下或GeneScope仪器下进行解释。ERA法检测限为250拷贝/mL,灵敏度是荧光定量PCR的40倍。在对75份临床标本进行临床性能评价时,ERA法的检出率为94.54%,而荧光定量PCR法的检出率为89.09%。当血液稀释1024倍甚至4096倍时,ERA法和荧光定量PCR均能检测出阳性。相比之下,快速诊断试验的检测能力明显低于ERA试验。结论ERA方法对恶性疟原虫和间日疟原虫的检测效果良好,可作为疟疾筛查和临床诊断的辅助工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Development of an Enzymatic Recombinase Amplification Assay for the Rapid Detection of Plasmodium Nucleic Acids

Development of an Enzymatic Recombinase Amplification Assay for the Rapid Detection of Plasmodium Nucleic Acids

Background

Malaria remains a global health challenge, with 249 million cases and 608,000 deaths reported in 2022. While China achieved malaria elimination, imported cases surged by 194.4% in 2023, underscoring the need for rapid diagnostics. Traditional methods like microscopy and rapid diagnostic tests (RDTs) face limitations in sensitivity and infrastructure requirements. This study aimed to establish and optimize a “one-pot” enzymatic recombinase amplification (ERA) assay for the molecular detection of Plasmodium falciparum and Plasmodium vivax, and to evaluate the efficacy of this assay through methodological verification and clinical performance.

Methods

We designed a specific ERA assay targeting the conserved regions of P. falciparum and P. vivax genetic material. We evaluated the sensitivity and specificity of this assay using synthetic plasmids and genomic material. Additionally, we tested the stability of the reaction by incorporating potential interfering substances into the reaction system. Finally, we analyzed the detection performance of the ERA method against real-time fluorescent quantitative PCR and rapid diagnostic tests using clinical samples.

Results

The detection process could be completed within 25 min at 35°C–40°C, and the results could be interpreted either under UV light or using a GeneScope instrument. The detection limit of the ERA assay was 250 copies/mL, which was 40 times more sensitive than fluorescent quantitative PCR. When evaluating the clinical performance using 75 clinical specimens, the detection rate of the ERA method was 94.54% compared with 89.09% for fluorescent quantitative PCR. The ERA assay and fluorescent quantitative PCR can achieve positive detection when blood samples were diluted 1024 times or even 4096 times. Comparatively, the detection capabilities of rapid diagnostic tests were significantly lower than that of the ERA assay.

Conclusion

The ERA method shows good performance in the detection of P. falciparum and P. vivax, and can be used as a complementary tool for malaria screening and clinical diagnosis.

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