Department of Parasitology, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia.
National Wildlife Forensic Laboratory, Ex-Situ Conservation Division Department of Wildlife and National Parks Peninsular Malaysia, Jalan Cheras, 56100 Kuala Lumpur, Malaysia.
Acta Trop. 2024 Jul;255:107249. doi: 10.1016/j.actatropica.2024.107249. Epub 2024 May 11.
Natural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique.
By using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal.
There was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 10 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 10 copies/µL.
This is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.
最近有报道称，人体感染疟原虫和疟原虫已被证实，并引起了东南亚国家的关注。非人类疟原虫通过受感染蚊子的叮咬从猕猴传播给人类，属于动物源性疟疾的传播。本研究的目的是建立实时荧光环介导等温扩增（LAMP）检测方法，通过将实时荧光技术与等温扩增技术相结合，检测动物源性疟原虫。
本研究以 18S rRNA 为靶基因，设计了疟原虫、疟原虫和疟原虫的新引物。建立了 4 种新型实时荧光 LAMP 检测方法，用于检测疟原虫、疟原虫、疟原虫和疟原虫。整个扩增过程在 60 分钟内完成，在 65°C 下进行。采用 SYTO-9 作为核酸嵌入染料，通过实时荧光信号进行监测。
不同种属的引物之间没有观察到交叉反应。实时荧光 LAMP 检测方法成功扩增了 70 份野外采集的猴样。疟原虫、疟原虫和疟原虫的检测限为 5×10 拷贝/μL。同时，疟原虫的检测限为 5×10 拷贝/μL。
这是首次报道用实时荧光 LAMP 方法检测四种动物源性疟原虫。它是一种有效、快速、易于使用的技术。该平台作为一种替代动物源性疟原虫检测方法具有很大的潜力。
