Plowe C V, Djimde A, Bouare M, Doumbo O, Wellems T E
Malaria Research and Training Center, National School of Medicine and Pharmacy, Bamaka, Mali.
Am J Trop Med Hyg. 1995 Jun;52(6):565-8. doi: 10.4269/ajtmh.1995.52.565.
As chloroquine resistance spreads across Africa, the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and proguanil are being used as alternative first-line drugs for the treatment and prevention of Plasmodium falciparum malaria. Resistance to these drugs is conferred by point mutations in parasite DHFR. These point mutations can be detected by polymerase chain reaction (PCR) assays, but better methods for sample collection, DNA extraction, and a diagnostic PCR are needed to make these assays useful in malaria-endemic areas. Here we report methods for collecting fingerstick blood onto filter paper strips that are air-dried, then stored and transported at room temperature. Cell lysis and DNA extraction are accomplished by boiling in Chelex-100. We also report a nested PCR technique that has improved sensitivity and specificity. These procedures readily detect mixed infections of parasites with both sensitive and resistant genotypes (confirmed by direct sequencing) and are reliable at parasite densities less than 250/mm3 in field surveys.
随着氯喹耐药性在非洲的蔓延，二氢叶酸还原酶（DHFR）抑制剂乙胺嘧啶和氯胍正被用作治疗和预防恶性疟原虫疟疾的一线替代药物。寄生虫DHFR中的点突变赋予了对这些药物的耐药性。这些点突变可通过聚合酶链反应（PCR）检测，但需要更好的样本采集、DNA提取方法以及诊断性PCR，以使这些检测方法在疟疾流行地区发挥作用。在此，我们报告了将指尖血采集到滤纸条上的方法，这些滤纸条经风干后，在室温下储存和运输。通过在Chelex-100中煮沸实现细胞裂解和DNA提取。我们还报告了一种具有更高灵敏度和特异性的巢式PCR技术。这些程序能够轻松检测出具有敏感和耐药基因型的寄生虫混合感染（通过直接测序确认），并且在现场调查中，当寄生虫密度低于250/mm³时也很可靠。
