Department of Clinical Microscopy, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
Malar J. 2021 Feb 28;20(1):120. doi: 10.1186/s12936-021-03659-5.
Copy number variations (CNVs) of the Plasmodium falciparum multidrug resistance 1 (pfmdr1), P. falciparum plasmepsin2 (pfplasmepsin2) and P. falciparum GTP cyclohydrolase 1 (pfgch1) genes are associated with anti-malarial drug resistance in P. falciparum malaria. Droplet digital PCR (ddPCR) assays have been developed for accurate assessment of CNVs in several human genes. The aim of the present study was to develop and validate ddPCR assays for detection of the CNVs of P. falciparum genes associated with resistance to anti-malarial drugs.
A multiplex ddPCR assay was developed to detect the CNVs in the pfmdr1 and pfplasmepsin2 genes, while a duplex ddPCR assay was developed to detect CNV in the pfgch1 gene. The gene copy number (GCN) quantification limit, as well as the accuracy and precision of the ddPCR assays were determined and compared to conventional quantitative PCR (qPCR). In order to reduce the cost of testing, a multiplex ddPCR assay of two target genes, pfmdr1 and pfplasmepsin2, was validated. In addition, the CNVs of genes of field samples collected from Thailand from 2015 to 2019 (n = 84) were assessed by ddPCR and results were compared to qPCR as the reference assay.
There were no significant differences between the GCN results obtained from uniplex and multiplex ddPCR assays for detection of CNVs in the pfmdr1 and pfplasmepsin2 genes (p = 0.363 and 0.330, respectively). Based on the obtained gene copy number quantification limit, the accuracy and percent relative standard deviation (%RSD) value of the multiplex ddPCR assay were 95% and 5%, respectively, for detection of the CNV of the pfmdr1 gene, and 91% and 5% for detection of the CNV of the pfplasmepsin2 gene. There was no significant difference in gene copy numbers assessed by uniplex or duplex ddPCR assays regarding CNV in the pfgch1 gene (p = 0.276). The accuracy and %RSD value of the duplex ddPCR assay were 95% and 4%, respectively, regarding pfgch1 GCN. In the P. falciparum field samples, pfmdr1 and pfplasmepsin2 GCNs were amplified in 15% and 27% of samples from Ubon Ratchathani, Thailand, while pfgch1 GCN was amplified in 50% of samples from Yala, Thailand. There was 100% agreement between the GCN results obtained from the ddPCR and qPCR assays (κ = 1.00). The results suggested that multiplex ddPCR assay is the optional assay for the accurate detection of gene copy number without requiring calibration standards, while the cost and required time are reduced. Based on the results of this study, criteria for GCN detection by ddPCR analysis were generated.
The developed ddPCR assays are simple, accurate, precise and cost-effective tools for detection of the CNVs in the pfmdr1, pfplasmepsin2 and pfgch1 genes of P. falciparum. The ddPCR assay is a useful additional tool for the surveillance of anti-malarial drug resistance.
疟原虫多药耐药 1 基因（pfmdr1）、疟原虫原浆体 2 基因（pfplasmepsin2）和疟原虫鸟苷三磷酸环化水解酶 1 基因（pfgch1）的拷贝数变异（CNVs）与恶性疟原虫疟疾中的抗疟药物耐药性有关。液滴数字 PCR（ddPCR）检测已被开发用于准确评估几个人类基因中的 CNVs。本研究的目的是开发和验证 ddPCR 检测方法，以检测与抗疟药物耐药性相关的疟原虫基因的 CNVs。
开发了一种多重 ddPCR 检测方法来检测 pfmdr1 和 pfplasmepsin2 基因中的 CNVs，同时开发了一种双 ddPCR 检测方法来检测 pfgch1 基因中的 CNV。确定了基因拷贝数（GCN）定量限以及 ddPCR 检测方法的准确性和精密度，并与常规定量 PCR（qPCR）进行了比较。为了降低检测成本，验证了一种检测两个靶基因 pfmdr1 和 pfplasmepsin2 的多重 ddPCR 检测方法。此外，还通过 ddPCR 检测了 2015 年至 2019 年从泰国采集的 84 个现场样本的基因 CNVs，并与 qPCR 作为参考检测方法进行了比较。
pfmdr1 和 pfplasmepsin2 基因的 CNV 检测中，单重和多重 ddPCR 检测的 GCN 结果无显著差异（p=0.363 和 0.330）。基于获得的基因拷贝数定量限，多重 ddPCR 检测 pfmdr1 基因 CNV 的准确性和%相对标准偏差（%RSD）值分别为 95%和 5%，pfplasmepsin2 基因 CNV 的准确性和%RSD 值分别为 91%和 5%。pfch1 基因 CNV 的单重或双重 ddPCR 检测的基因拷贝数无显著差异（p=0.276）。双 ddPCR 检测 pfgch1 GCN 的准确性和%RSD 值分别为 95%和 4%。在恶性疟原虫现场样本中，15%的乌汶府泰国样本和 27%的泰国亚拉样本扩增了 pfmdr1 和 pfplasmepsin2 GCN，而 50%的泰国亚拉样本扩增了 pfgch1 GCN。ddPCR 和 qPCR 检测的 GCN 结果之间有 100%的一致性（κ=1.00）。结果表明，多重 ddPCR 检测是一种简单、准确、精确且具有成本效益的工具，可用于检测 pfmdr1、pfplasmepsin2 和 pfgch1 基因中的 CNV，而无需校准标准，同时降低了成本和所需时间。基于本研究的结果，生成了 ddPCR 分析的 GCN 检测标准。
所开发的 ddPCR 检测方法是一种简单、准确、精确且具有成本效益的工具，可用于检测疟原虫多药耐药 1 基因（pfmdr1）、疟原虫原浆体 2 基因（pfplasmepsin2）和疟原虫鸟苷三磷酸环化水解酶 1 基因（pfgch1）中的 CNVs。ddPCR 检测是一种有用的抗疟药物耐药性监测的附加工具。
