微滴式数字聚合酶链反应（ddPCR）检测疟原虫 knowlesi 和疟原虫 vivax。
Droplet digital polymerase chain reaction (ddPCR) for the detection of Plasmodium knowlesi and Plasmodium vivax.
机构信息
Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.
Department of Parasitology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.
出版信息
Malar J. 2020 Jul 10;19(1):241. doi: 10.1186/s12936-020-03314-5.
BACKGROUND
Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of P. knowlesi and P. vivax, and compare this method to nested PCR and qPCR.
METHODS
The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.
RESULTS
The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.
CONCLUSIONS
Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.
背景
在马来西亚，导致疟疾的主要疟原虫种为间日疟原虫（Plasmodium vivax）和诺氏疟原虫（Plasmodium knowlesi），它们在马来西亚无症状疟疾传播中发挥作用。现有的诊断疟疾的工具，如显微镜检查和快速诊断检测（RDT），在检测较低寄生虫密度时的灵敏度较低。 数字液滴聚合酶链反应（ddPCR）已被证明在诊断疟疾方面具有更高的灵敏度，它可以直接定量，而无需标准曲线。本研究旨在开发和使用用于检测间日疟原虫和诺氏疟原虫的双 ddPCR 检测方法，并将其与巢式 PCR 和 qPCR 进行比较。
方法
确定双 ddPCR 检测方法的一致性率、灵敏度和特异性，并与巢式 PCR 和双 qPCR 进行比较。
结果
与 qPCR 相比，双 ddPCR 检测方法具有更高的分析灵敏度（间日疟原虫 = 10 拷贝/μL，诺氏疟原虫 = 0.01 拷贝/μL）。此外，与巢式 PCR 相比，ddPCR 检测方法具有可接受的临床灵敏度（间日疟原虫 = 80%，诺氏疟原虫 = 90%）和临床特异性（间日疟原虫 = 87.84%，诺氏疟原虫 = 81.08%）。ddPCR 和 qPCR 都在样本中检测到更多的双重感染。
结论
总的来说，ddPCR 检测方法在检测间日疟原虫和诺氏疟原虫方面表现出可接受的效率，在检测混合感染方面比巢式 PCR 更灵敏。然而，双 ddPCR 检测方法仍需要优化，以提高检测方法的临床灵敏度和特异性。
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