Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, No. 5, Dongdansantiao, Dongcheng District, Beijing, 10005, China.
Yunnan Institute of Parasitic Diseases & Yunnan Provincial Centre of Malaria Research, Pu'er, China.
Infect Dis Poverty. 2024 Oct 31;13(1):80. doi: 10.1186/s40249-024-01255-8.
Resource-limited regions face a greater burden of infectious diseases due to limited access to molecular tests, complicating timely diagnosis and management. Current molecular point-of-care tests (POCTs) either come with high costs or lack adequate sensitivity and specificity. To facilitate better prevention and control of infectious diseases in underserved areas, we seek to address the need for molecular POCTs that better align with the World Health Organization (WHO)'s ASSURED criteria-Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end users.
A novel molecular POCT, Pasteur Pipette-assisted isothermal probe amplification (pp-IPA), was developed for malaria detection. Without any microfluidics, this method captures Plasmodium 18S rRNA in a modified Pasteur pipette using tailed genus-specific probes. After washing, the bound tailed probes are ligated to form a template for subsequent novel isothermal probe amplification using a pair of generic primers, bypassing nucleic acid extraction and reverse transcription. The method was assessed using cultured Plasmodium and compared with real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP) in clinical blood samples.
The entire assay is completed in 60-80 min with minimal hands-on time, using only a Pasteur pipette and a water bath. The pp-IPA's analytical sensitivity is 1.28 × 10 parasites/μl, with 100% specificity against various blood-borne pathogens causing malaria-like symptoms. Additionally, pp-IPA needs only liquid-transfer skill for operation and the cost is around USD 0.25 per test, making it at least 300 times lower than mainstream POCT platforms.
Designed to improve the accessibility of molecular detection in resource-limited settings, pp-IPA's simplicity, affordability, high sensitivity/specificity, and minimal equipment requirements make it a promising point-of-care pathogen identification tool in resource-constrained regions.
资源有限的地区由于分子检测的获取受限，面临更大的传染病负担，这使得及时诊断和管理变得复杂。目前的分子即时检测（POCT）要么成本高昂，要么缺乏足够的灵敏度和特异性。为了促进资源匮乏地区更好地预防和控制传染病，我们寻求开发更符合世界卫生组织（WHO）的 ASSURED 标准（经济实惠、敏感、特异、用户友好、快速、稳健、无设备、可交付给最终用户）的分子 POCT。
我们开发了一种新颖的分子 POCT，即巴氏吸管辅助等温探针扩增（pp-IPA），用于疟疾检测。该方法无需任何微流控技术，而是在改良的巴氏吸管中使用带有长尾属特异性探针捕获疟原虫 18S rRNA。洗涤后，结合的长尾探针通过一对通用引物连接形成模板，随后进行新颖的等温探针扩增，从而绕过核酸提取和逆转录。该方法使用培养的疟原虫进行评估，并与实时定量逆转录 PCR（RT-qPCR）或逆转录环介导等温扩增（RT-LAMP）在临床血液样本中进行比较。
整个检测过程在 60-80 分钟内完成，手动操作时间极短，仅需巴氏吸管和水浴。pp-IPA 的分析灵敏度为 1.28×10 个寄生虫/μl，对引起疟疾样症状的各种血源性病原体具有 100%的特异性。此外，pp-IPA 仅需要液体转移技能即可操作，且每个测试的成本约为 0.25 美元，比主流 POCT 平台至少低 300 倍。
pp-IPA 的设计旨在提高资源有限环境中分子检测的可及性，其简单性、经济性、高灵敏度/特异性以及对设备要求低，使其成为资源有限地区有前途的即时病原体识别工具。
