Detection of Single-Nucleotide Polymorphisms in Plasmodium falciparum by PCR Primer Extension and Lateral Flow Immunoassay
Detection of Single-Nucleotide Polymorphisms in Plasmodium falciparum by PCR Primer Extension and Lateral Flow Immunoassay
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PCR引物延伸和侧流免疫分析检测恶性疟原虫单核苷酸多态性
DOI：
10.1128/aac.03395-14
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发表时间：
2014
影响因子：
4.9
通讯作者：
A. Amerongen
中科院分区：
文献类型：
--
作者：
Antoine Moers;R. Hallett;R. Burrow;Henk D F H Schallig;C. Sutherland;A. Amerongen
摘要 恶性疟原虫对某些抗疟药物的耐药性与单核苷酸多态性 (SNP) 有关。目前，还没有足够简单、便宜且足够快的方法来识别耐药寄生虫，以便在护理点（即在开具药物的当地医院）进行。开发了引物延伸方法 (PEXT) 以鉴定恶性疟原虫中的 4 个 SNP，这些单核苷酸多态性位于恶性疟原虫多药耐药 1 基因 (pfmdr1) 的氨基酸 86、184 和 1246 以及氯喹耐药转运蛋白基因 (pfcrt) 的氨基酸 76 处。 PEXT 产品通过核酸侧流免疫分析 (NALFIA) 以碳纳米颗粒作为检测标记进行可视化。在对 17 个现场样本进行的初始开放标签评估中，PCR-PEXT-NALFIA 显示出与参考方法、定量 PCR (qPCR) 或直接扩增子序列分析的良好相关性。这些测试在一项盲法研究设计中对 150 名患者分离株进行了进一步评估。所有 4 种 PCR-PEXT 基因分型检测均具有 98% 至 100% 的高特异性。当考虑所有 PEXT 阳性测试时，敏感性范围为 75% 至 100%。通过参考方法成功地表征了许多寄生虫密度较低的样品，但未能在 PCR-PEXT-NALFIA 中产生结果，特别是那些显微镜检查阴性的亚隐性感染样品。这项原理验证研究验证了 PCR-PEXT-NALFIA 用于检测恶性疟原虫耐药性相关突变的用途，特别是对于镜检阳性感染。虽然它需要标准的热循环仪，但该过程便宜且快速，因此是发展中国家床旁检测的潜在有价值的工具。
ABSTRACT The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.