Jiangsu Province Blood Center, Nanjing, China.
Key Laboratory of National Health and Family Planning Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Jiangsu Institute of Parasitic Diseases, Wuxi, China.
Front Cell Infect Microbiol. 2022 Jun 24;12:922146. doi: 10.3389/fcimb.2022.922146. eCollection 2022.
Malaria is a global public health problem. China has had no case of indigenous malaria since 2016. However, imported cases of malaria remain an issue among travelers, overseas workers, and foreign traders. Although these cases are always asymptomatic, if they donate blood, there is a great risk of transfusion transmitted-malaria (TTM). Therefore, blood banks need a rapid screening tool to detect species.
We designed an assay using recombinase-aided amplification (RAA) and a lateral-flow dipstick (LFD) (RAA-LFD) to detect the 18S ribosomal RNA gene of species. Sensitivity was evaluated using a recombinant plasmid and Plasmodium genomic DNA. Specificity was evaluated using DNA extracted from the blood of patients with malaria or other infectious parasites. For clinical assessment, blood samples from patients with malaria and blood donors were evaluated.
The RAA-LFD assay was performed in an incubator block at 37°C for 15 min, and the amplicons were visible to the naked eye on the flow dipsticks within 3 min. The sensitivity was 1 copy/μL of recombinant plasmid. For genomic DNA from whole blood of malaria patients infected with , , , and , the sensitivity was 0.1 pg/μL, 10 pg/μL, 10-100 pg/μL, and 100pg/μL, respectively. The sensitivity of this assay was 100pg/μL. No cross-reaction with other transfusion-transmissible parasites was detected.
The results demonstrated that this RAA-LFD assay was suitable for reliable field detection of species in low-resource settings with limited laboratory capabilities.
疟疾是一个全球性的公共卫生问题。自 2016 年以来，中国已无本土疟疾病例。然而，旅行者、海外务工人员和外国商人仍存在输入性疟疾病例。尽管这些病例通常无症状，但如果他们献血，就存在很大的输血传播疟疾（TTM）风险。因此，血库需要一种快速的筛选工具来检测疟原虫。
我们设计了一种使用重组酶辅助扩增（RAA）和侧向流动纸条（LFD）（RAA-LFD）检测疟原虫 18S 核糖体 RNA 基因的检测方法。使用重组质粒和疟原虫基因组 DNA 评估了敏感性。使用来自疟疾病例或其他传染性寄生虫患者的血液提取的 DNA 评估了特异性。为了进行临床评估，评估了疟疾病例患者和献血者的血液样本。
RAA-LFD 检测在 37°C 的孵育盒中进行 15 分钟，在 3 分钟内可在流动纸条上肉眼观察到扩增子。重组质粒的灵敏度为 1 拷贝/μL。对于感染 、 、 和 的疟疾病例全血基因组 DNA，灵敏度分别为 0.1pg/μL、10pg/μL、10-100pg/μL 和 100pg/μL。该检测方法的灵敏度为 100pg/μL。未检测到与其他可经输血传播的寄生虫的交叉反应。
结果表明，该 RAA-LFD 检测方法适用于资源有限的现场环境中可靠地检测疟原虫，具有较低的实验室要求。
