Webster B L, Rollinson D, Stothard J R, Huyse T
Wolfson Wellcome Biomedical Laboratories, Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK.
J Helminthol. 2010 Mar;84(1):107-14. doi: 10.1017/S0022149X09990447. Epub 2009 Aug 3.
Schistosoma haematobium and S. bovis are widespread schistosome species causing human and cattle schistosomiasis, respectively, in Africa. The sympatric occurrence of these two species and their ability to infect the same Bulinus intermediate snail hosts necessitates precise methods of identification of the larval stages. A rapid diagnostic 'mulitplex' one-step polymerase chain reaction protocol (RD-PCR) was developed using cytochrome oxidase subunit 1 (COX1) mitochondrial DNA (mtDNA) to discriminate between S. haematobium and S. bovis. A single forward primer and two species-specific reverse primers were used to produce a polymerase chain reaction (PCR) fragment of 306 bp and 543 bp for S. bovis and S. haematobium, respectively. Serial dilutions were carried out on various lifecycle stages and species combinations to test the sensitivity and specificity of the primers. This RD-PCR proved highly sensitive, detecting a single larval stage and as little as 0.78 ng of genomic DNA (gDNA) from an adult schistosome, providing a cost-effective, rapid and robust molecular tool for high-throughput screening of S. haematobium and S. bovis populations. In areas where human and cattle schistosomiasis overlap and are transmitted in close proximity, this mitochondrial assay will be a valuable identification tool for epidemiological studies, especially when used in conjunction with other nuclear diagnostic markers.
埃及血吸虫和牛血吸虫是广泛分布的血吸虫物种，分别在非洲导致人类和牛的血吸虫病。这两个物种的同域分布以及它们感染相同的双脐螺中间宿主的能力，使得需要精确的幼虫阶段鉴定方法。利用细胞色素氧化酶亚基1（COX1）线粒体DNA（mtDNA）开发了一种快速诊断“多重”一步聚合酶链反应方案（RD-PCR），以区分埃及血吸虫和牛血吸虫。使用一个正向引物和两个物种特异性反向引物，分别为牛血吸虫和埃及血吸虫产生306 bp和543 bp的聚合酶链反应（PCR）片段。对各种生命周期阶段和物种组合进行系列稀释，以测试引物的敏感性和特异性。这种RD-PCR被证明具有高度敏感性，能够检测单个幼虫阶段，并且能检测到来自成年血吸虫的低至0.78 ng基因组DNA（gDNA），为高通量筛选埃及血吸虫和牛血吸虫种群提供了一种经济高效、快速且可靠的分子工具。在人类和牛血吸虫病重叠且传播区域相近的地区，这种线粒体检测方法将是流行病学研究中一种有价值的鉴定工具，特别是与其他核诊断标记物联合使用时。
