Application of a low-cost, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Plasmodium falciparum imported from Africa

IF 1.4 4区医学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular and biochemical parasitology Pub Date : 2022-11-01 DOI:10.1016/j.molbiopara.2022.111529

Jiaqi Zhang , Xi Chen , Maohua Pan , Yucheng Qin , Hui Zhao , Qi Yang , Xinxin Li , Weilin Zeng , Zheng Xiang , Yanrui Wu , Mengxi Duan , Xiaosong Li , Xun Wang , Dominique Mazier , Yanmei Zhang , Wenya Zhu , Kemin Sun , Yiman Wu , Liwang Cui , Yaming Huang , Zhaoqing Yang

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引用次数: 2

Abstract

Background

Chinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa.

Methods

We developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection.

Results

The new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5–100%] and 99.1% (95% CI 96.7–99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 10² copies/μL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/μL of blood (equal to 0.04 parasites/μL of DNA) using cultured P. falciparum and 1–7 parasites/μL of blood (4–28 parasites/μL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays.

Conclusion

The novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

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应用低成本、特异、灵敏的环介导等温扩增(LAMP)检测非洲输入恶性疟原虫

出国旅游的中国公民将输入性疟疾带回中国。目前的疟疾诊断测试，包括显微镜和抗原检测快速测试，不能可靠地检测低密度感染。为了补充现有的诊断方法，我们旨在建立一种新的环介导等温扩增(LAMP)方法来检测和鉴定从非洲返回的中国旅行者的恶性疟原虫。方法建立小型LAMP法扩增恶性疟原虫肌动蛋白I基因。每个反应只消耗传统LAMP测定中所用试剂的25%和巢式PCR中所用DNA模板的相同数量。我们利用466例从非洲输入的疑似疟疾病例评估了LAMP检测的性能，并将其与显微镜和巢式PCR检测进行了比较。我们使用培养的恶性疟原虫、临床样本和质粒构建物评估了新的LAMP检测方法的敏感性，在定量检测限时实现了前所未有的精度。结果LAMP检测灵敏度高，比巢式PCR多检出2例疟疾病例。与巢式PCR相比，新型LAMP检测的灵敏度和特异性分别为100%[95%置信区间(CI) 98.5-100%]和99.1% (95% CI 96.7% - 99.9%)。通过对质粒结构进行连续稀释，新LAMP的检出限低至102拷贝/μL，比PCR低10倍。LAMP法在培养恶性疟原虫的血液中检测到0.01个寄生虫/μL(相当于0.04个寄生虫/μL DNA)，在临床样品中检测到1-7个寄生虫/μL(相当于4-28个寄生虫/μL DNA)，与以前报道的和商业许可的检测方法一样或更好。结论基于恶性疟原虫肌动蛋白I基因的LAMP检测方法特异性强，灵敏度高，成本低，仅为LAMP反应所需试剂的1/4。

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来源期刊

Molecular and biochemical parasitology 医学-寄生虫学

CiteScore

2.90

自引率

0.00%

发文量

审稿时长

63 days

期刊介绍： The journal provides a medium for rapid publication of investigations of the molecular biology and biochemistry of parasitic protozoa and helminths and their interactions with both the definitive and intermediate host. The main subject areas covered are: • the structure, biosynthesis, degradation, properties and function of DNA, RNA, proteins, lipids, carbohydrates and small molecular-weight substances • intermediary metabolism and bioenergetics • drug target characterization and the mode of action of antiparasitic drugs • molecular and biochemical aspects of membrane structure and function • host-parasite relationships that focus on the parasite, particularly as related to specific parasite molecules. • analysis of genes and genome structure, function and expression • analysis of variation in parasite populations relevant to genetic exchange, pathogenesis, drug and vaccine target characterization, and drug resistance. • parasite protein trafficking, organelle biogenesis, and cellular structure especially with reference to the roles of specific molecules • parasite programmed cell death, development, and cell division at the molecular level.