Monti J R, Chilton N B, Qian B Z, Gasser R B
Department of Veterinary Science, University of Melbourne, Victoria, Australia.
Mol Cell Probes. 1998 Apr;12(2):71-8. doi: 10.1006/mcpr.1997.0151.
Necator americanus and Ancylostoma duodenale are the two most important species of human hookworm, and occur in sympatry over much of their distribution. The specific diagnosis of hookworm infections is central to control. Diagnosis currently relies on the detection of hookworm eggs in human faeces and/or the specific identification of larvae by 'copro-culture' combined with microscopic examination. However, the eggs of the two species are morphologically indistinguishable, and the procedure of copro-culture is tedious and time-consuming to carry out. To work toward overcoming these limitations, a molecular approach utilizing genetic markers in the first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was established. The ITS-1 sequences of both hookworm species were determined, and specific oligonucleotide primers designed to regions of major sequence difference between the species were evaluated in polymerase chain reaction (PCR). Using a range of control samples, the primers allowed the specific identification of as little as 10 pg DNA of A. duodenale or N. americanus. The findings indicate clearly the potential for specific PCR to confirm the identity of eggs from faeces and larvae from the environment or host tissues. This should have important implications for studying fundamental aspects relating to anthelmintic efficacy and the epidemiology of hookworms.
美洲板口线虫和十二指肠钩口线虫是两种最重要的人体钩虫，在其分布的大部分地区共存。钩虫感染的特异性诊断对于控制钩虫病至关重要。目前的诊断依赖于在人类粪便中检测钩虫卵和/或通过“粪便培养”结合显微镜检查对幼虫进行特异性鉴定。然而，这两种钩虫的卵在形态上无法区分，而且粪便培养过程繁琐且耗时。为了克服这些局限性，建立了一种利用核糖体DNA（rDNA）第一内部转录间隔区（ITS-1）中的遗传标记的分子方法。测定了两种钩虫的ITS-1序列，并在聚合酶链反应（PCR）中评估了针对两种钩虫主要序列差异区域设计的特异性寡核苷酸引物。使用一系列对照样品，这些引物能够特异性鉴定低至10 pg的十二指肠钩口线虫或美洲板口线虫的DNA。研究结果清楚地表明，特异性PCR有潜力确认粪便中的虫卵以及环境或宿主组织中的幼虫的身份。这对于研究与驱虫效果和钩虫流行病学相关的基本问题应该具有重要意义。
