A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology
A simple, high-throughput method to detect Plasmodium falciparum single nucleotide polymorphisms in the dihydrofolate reductase, dihydropteroate synthase, and P. falciparum chloroquine resistance transporter genes using polymerase chain reaction- and enzyme-linked immunosorbent assay-based technology
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DOI：
10.4269/ajtmh.2005.72.155
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发表时间：
2005-02-01
影响因子：
3.3
通讯作者：
Bygjerg, IBC
中科院分区：
文献类型：
--
作者：
Alifrangis, M;Enosse, S;Bygjerg, IBC
恶性疟原虫二氢叶酸还原酶(DHFR)、二氢翼酸合成酶(DHPs)和氯喹抗性转运蛋白(Pfcrt)基因的单核苷酸多态性(SNPs)是恶性疟原虫对磺胺多辛/乙胺嘧啶和氯喹抗性的分子标记。然而，要成为监测耐药性的实用工具，高通量单倍型的简单方法是有必要的。在这里，我们描述了一种快速而简单的技术，它使用基于聚合酶链式反应(PCR)和酶联免疫吸附试验(ELISA)的技术来检测DHFR、DHPS和Pfcrt SNP。将DHFR、DHPs或P的生物素化PCR产物捕获在链霉亲和素包被的微孔板上，并将序列特异性寡核苷酸探针(SSOP)与PCR产物杂交。严格的洗涤程序能够检测到剩余的结合SSOP，并以高度特异性区分DHFR DHPs和Pfcrt的SNP。SSOP-ELISA法与标准的聚合酶链式反应-限制性片段长度多态性方法进行了比较，在检测的恶性疟原虫玻片阳性样本中，有90%以上的样本获得了相同的阳性结果。对88个样本的所有dhfr、dhps或Pfcrt SNPs的SSOP-ELISA法可以在一天内进行，并提供快速和可重复的结果。该系统可能会被修改，以检测其他基因中的SNPs。
Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum dihydrofolate reductase (dhfr), and dihydropteroate synthetase (dhps), and chloroquine resistance transporter (Pfcrt) genes are used as molecular markers of P. falciparum resistance to sulfadoxine/pyrimethamine and chloroquine. However, to be a practical tool in the surveillance of drug resistance, simpler methods for high-throughput haplotyping are warranted. Here we describe a quick and simple technique that detects dhfr, dhps, and Pfcrt SNPs using polymerase chain reaction (PCR)- and enzyme-linked immunosorbent assay (ELISA)-based technology. Biotinylated PCR products of dhfr, dhps, or P, were captured on streptavidin-coated microtiter plates and sequence-specific oligonucleotide probes (SSOPs) were hybridized with the PCR products. A stringent washing procedure enabled detection of remaining bound SSOPs and distinguished between the SNPs of dhfr dhps, and Pfcrt with high specificity. The SSOP-ELISA compared well with a standard PCR-restriction fragment length polymorphism procedure, and gave identical positive results in more than 90% of the P.falciparum slide-positive samples tested. The SSOP-ELISA of all dhfr, dhps, or Pfcrt SNPs on 88 samples can be performed in a single day and provides quick and reproducible results. The system can potentially be modified to detect SNPs in other genes.