Department of Medicine, University of California, San Francisco, California, USA.
J Clin Microbiol. 2013 Aug;51(8):2564-70. doi: 10.1128/JCM.00904-13. Epub 2013 May 29.
Genetic polymorphisms in the malaria parasite Plasmodium falciparum mediate alterations in sensitivity to important antimalarial drugs. Surveillance for these polymorphisms is helpful in assessing the prevalence of drug resistance and designing strategies for malaria control. Multiple methods are available for the assessment of P. falciparum genetic polymorphisms, but they suffer from low throughput, technical limitations, and high cost. We have optimized and tested a multiplex ligase detection reaction-fluorescent microsphere (LDR-FM) assay for the identification of important P. falciparum genetic polymorphisms. For 84 clinical samples from Kampala, Uganda, a region where both transmission intensity and infection complexity are high, DNA was extracted from dried blood spots, genes of interest were amplified, amplicons were subjected to multiplex ligase detection reactions to add bead-specific oligonucleotides and biotin, fragments were hybridized to magnetic beads, and polymorphism prevalences were assessed fluorometrically in a multiplex format. A total of 19 alleles from the pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps genes were analyzed by LDR-FM and restriction fragment length polymorphism (RFLP) analyses. Considering samples with results from the two assays, concordance between the assays was good, with 78 to 100% of results identical at individual alleles, most nonconcordant results differing only between a mixed and pure genotype call, and full disagreement at individual alleles in only 0 to 3% of results. We estimate that the LDR-FM assay offers much higher throughput and lower cost than RFLP. Our results suggest that the LDR-FM system offers an accurate high-throughput means of classifying genetic polymorphisms in field samples of P. falciparum.
疟原虫寄生虫恶性疟原虫中的遗传多态性介导了对重要抗疟药物敏感性的改变。对这些多态性进行监测有助于评估耐药性的流行情况，并设计疟疾控制策略。有多种方法可用于评估恶性疟原虫遗传多态性，但这些方法存在通量低、技术限制和成本高的问题。我们已经优化并测试了一种多重连接酶检测反应-荧光微球（LDR-FM）检测法，用于鉴定重要的恶性疟原虫遗传多态性。对来自乌干达坎帕拉的 84 个临床样本进行了测试，该地区的传播强度和感染复杂性都很高，从干血斑中提取 DNA，扩增感兴趣的基因，将扩增子进行多重连接酶检测反应，添加珠特异性寡核苷酸和生物素，片段与磁珠杂交，并以多重格式进行荧光检测评估多态性流行率。总共分析了来自 pfcrt、pfmdr1、pfmrp1、pfdhfr 和 pfdhps 基因的 19 个等位基因，通过 LDR-FM 和限制性片段长度多态性（RFLP）分析。考虑到两种检测方法的结果，两种检测方法的一致性很好，在各个等位基因上有 78%至 100%的结果一致，大多数不一致的结果仅在混合和纯基因型之间存在差异，只有 0%至 3%的结果在各个等位基因上完全不一致。我们估计 LDR-FM 检测法的通量比 RFLP 高得多，成本也低得多。我们的研究结果表明，LDR-FM 系统提供了一种准确的高通量方法，可用于对恶性疟原虫现场样本中的遗传多态性进行分类。
