Anthony T G, Trueman H E, Harbach R E, Vogler A P
Department of Biology, Imperial College at Silwood Park, Ascot, Berks, UK.
Parasitology. 2000 Aug;121 ( Pt 2):121-6. doi: 10.1017/s003118209900623x.
The oocysts of Plasmodiom falciparam that form on the midgut wall of anopheline vectors represent the meiotic products of the sexual cycle of the parasite and are useful for genetic analyses. Recognizing the limitations of current markers for the analysis of genetic variation, we describe a protocol for the DNA isolation and PCR amplification of microsatellite loci from individual oocysts. We present a simple method for the storage and isolation of individual Plasmodium oocysts from wild-caught mosquitoes and demonstrate that it is possible to extract sufficient oocyst DNA for approximately 50 amplifications. Genotyping was successful for 10 microsatellite loci and, using Genescan technology, we found extensive microsatellite polymorphism in an analysis of a dozen oocysts from each of 2 Anopheles gambiae female specimens collected within 6 months at the same location in northern Tanzania. All of the loci exhibited at least 2 alleles, with a maximum of 5. Homozygous and heterozygous oocysts could easily be discerned, but most of the oocysts were homozygous. The procedure has great potential for shedding light on genetic mechanisms operating during meiosis in mosquito vectors and the influence this may have on the genetic structure of natural populations of P. falciparum.
在按蚊媒介中肠壁上形成的恶性疟原虫卵囊代表了该寄生虫有性生殖周期的减数分裂产物，对遗传分析很有用。认识到当前用于分析遗传变异的标记的局限性，我们描述了一种从单个卵囊中分离DNA并进行微卫星位点PCR扩增的方案。我们提出了一种从野外捕获的蚊子中储存和分离单个疟原虫卵囊的简单方法，并证明有可能提取足够的卵囊DNA进行大约50次扩增。对10个微卫星位点进行基因分型获得成功，并且利用基因扫描技术，在对坦桑尼亚北部同一地点6个月内采集的2只冈比亚按蚊雌蚊标本中的每只所采集的十几个卵囊进行分析时，我们发现了广泛的微卫星多态性。所有位点均显示至少2个等位基因，最多5个。纯合和杂合卵囊很容易辨别，但大多数卵囊是纯合的。该方法对于阐明蚊媒减数分裂过程中起作用的遗传机制以及这可能对恶性疟原虫自然种群遗传结构产生的影响具有很大潜力。
