Vythilingam I, Boaz L, Wa N
Institute for Medical Research, Kuala Lumpur, Malaysia.
J Am Mosq Control Assoc. 1998 Sep;14(3):243-7.
Accurate identification of filarial parasites in mosquitoes poses a major problem for the coordination of filariasis control programs. Traditional methods are tedious, and some are not specific enough to give satisfactory results. Amplification of specific gene sequences by primer-directed polymerase chain reaction (PCR) has been increasingly utilized as a diagnostic tool. However, current protocols for the extraction of parasite DNA from mosquito samples are tedious and could lead to failure of PCR amplification. We demonstrate that the use of Chelex is an efficient method for DNA extraction from mosquitoes and the parasite and that PCR amplification with primers specific for Brugia malayi yields a band of the expected size. The PCR products were transferred to a nylon membrane with Southern blotting, and a B. malayi-specific digoxigenin-labeled probe confirmed the sequence similarity of the PCR-amplified fragment and increased the sensitivity of the PCR assay. Use of this probe enabled us to detect PCR-amplified product from B. malayi even when a product was not visible on an ethidium bromide-stained agarose gel. This increased sensitivity allowed us to detect the parasite in the heads of mosquitoes.
准确鉴定蚊子体内的丝虫寄生虫是丝虫病防治计划协调工作中的一个主要问题。传统方法繁琐，而且有些方法特异性不足，无法给出令人满意的结果。通过引物定向聚合酶链反应（PCR）扩增特定基因序列已越来越多地用作诊断工具。然而，目前从蚊子样本中提取寄生虫DNA的方案繁琐，可能导致PCR扩增失败。我们证明，使用螯合树脂是从蚊子和寄生虫中提取DNA的有效方法，并且用特异于马来布鲁线虫的引物进行PCR扩增可产生预期大小的条带。通过Southern印迹将PCR产物转移到尼龙膜上，并用马来布鲁线虫特异性地高辛标记探针证实了PCR扩增片段的序列相似性，并提高了PCR检测的灵敏度。使用该探针使我们能够检测到来自马来布鲁线虫的PCR扩增产物，即使在溴化乙锭染色的琼脂糖凝胶上看不到产物时也是如此。这种提高的灵敏度使我们能够在蚊子头部检测到寄生虫。
