Department of Reproductive Medicine, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China.
Modern Industrial College of Biomedicine and Great Health, Youjiang Medical University for Nationalities, Baise, Guangxi, China.
Parasit Vectors. 2024 Nov 24;17(1):488. doi: 10.1186/s13071-024-06575-0.
Malaria remains a serious public health problem worldwide, particularly in Africa. Resistance to antimalarial drugs is an essential issue for malaria control and elimination. Currently, polymerase chain reaction (PCR) combined with Sanger sequencing is regarded as the gold standard for mutation detection. However, this method fails to meet the requirements of point-of-care testing (POCT) because of its time-consuming, expensive instruments and professional dependence. To support this strategy, we developed a novel diagnostic platform that combines recombinase polymerase amplification (RPA) with the Pyrococcus furiosus argonaute (PfAgo) protein and was designed to detect gene mutations related to antimalarial drug resistance. The Pfcrt haplotypes CVMNK and CVIET of chloroquine resistance (CQR) were used as examples and were assessed in this study.
By meticulously designing strategies, RPA primers, guide DNAs, and probes were screened, the reaction was optimized, and the resulting parameters were employed to ascertain the genotype of Pfcrt. The recombinant plasmids pUC57/Pfcrt-CVIET and pUC57/Pfcrt-CVMNK were constructed and diluted for sensitivity detection. The pUC57/Pfcrt-CVIET plasmid mixture was added to the pUC57/Pfcrt-CVMNK plasmid mixture in different additions to configure several specific proportions of mixed plasmid mixtures. The RPA-PfAgo platform was used, and the mixed plasmid was detected simultaneously via nest-PCR (nPCR) and Sanger sequencing. The platform was then evaluated on 85 clinical samples and compared with Sanger sequencing.
The entire process achieves the key mutation Pfcrt-CVMNK/CVIET genotype identification of CQR within 90 min. The platform achieved 1.8 × 10 copies/μL sensitivity and could detect as little as 3% CVIET in mixed plasmids, which is a higher sensitivity than that of Sanger sequencing (5%). Notably, the platform shows 100% concordance with the gold standard method when 85 clinical samples are tested. The sensitivity and specificity were 100% for the 85 clinical samples.
This study established an RPA-PfAgo platform for genotyping the key mutation Pfcrt-CVMNK/CVIET of CQR. This method can rapidly produce reliable results and avoid the disadvantages of nPCR with sequencing. This approach has the characteristics of a short operation time, low device dependence, and a good match to the POCT strategy, suggesting that the platform can be easily applied locally or on site.
疟疾仍然是全球严重的公共卫生问题，尤其是在非洲。抗疟药物的耐药性是疟疾控制和消除的一个重要问题。目前，聚合酶链反应（PCR）结合 Sanger 测序被认为是突变检测的金标准。然而，由于其耗时、昂贵的仪器和专业依赖性，该方法无法满足即时检测（POCT）的要求。为了支持这一策略，我们开发了一种新的诊断平台，该平台结合了重组酶聚合酶扩增（RPA）和 Pyrococcus furiosus argonaute（PfAgo）蛋白，旨在检测与抗疟药物耐药性相关的基因突变。本研究以氯喹耐药（CQR）的 Pfcrt 单倍型 CVMNK 和 CVIET 为例进行评估。
通过精心设计策略，筛选 RPA 引物、引导 DNA 和探针，优化反应，并利用所得参数确定 Pfcrt 的基因型。构建并稀释重组质粒 pUC57/Pfcrt-CVIET 和 pUC57/Pfcrt-CVMNK 以进行灵敏度检测。将 pUC57/Pfcrt-CVIET 质粒混合物添加到 pUC57/Pfcrt-CVMNK 质粒混合物中，以不同的添加量配置几种特定比例的混合质粒混合物。使用 RPA-PfAgo 平台，通过巢式 PCR（nPCR）和 Sanger 测序同时检测混合质粒。然后，该平台在 85 个临床样本上进行了评估，并与 Sanger 测序进行了比较。
该平台在 90 分钟内实现了整个关键突变 Pfcrt-CVMNK/CVIET CQR 基因型的鉴定。该平台的灵敏度达到 1.8×10 拷贝/μL，可检测到混合质粒中低至 3%的 CVIET，灵敏度高于 Sanger 测序（5%）。值得注意的是，当检测 85 个临床样本时，该平台与金标准方法的一致性达到 100%。该平台对 85 个临床样本的灵敏度和特异性均为 100%。
本研究建立了一种用于 CQR 关键突变 Pfcrt-CVMNK/CVIET 基因分型的 RPA-PfAgo 平台。该方法能够快速产生可靠的结果，避免 nPCR 与测序相结合的缺点。该方法具有操作时间短、设备依赖性低、与 POCT 策略匹配良好的特点，表明该平台可以方便地在当地或现场应用。
