Use of multiple displacement amplification to increase the detection and genotyping of Trypanosoma species samples immobilized on FTA filters
Use of multiple displacement amplification to increase the detection and genotyping of Trypanosoma species samples immobilized on FTA filters
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DOI：
10.4269/ajtmh.2007.76.1132
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发表时间：
2007-06-01
影响因子：
3.3
通讯作者：
MacLeod, Annette
中科院分区：
文献类型：
--
作者：
Morrison, Liam J.;McCormack, Gillian;MacLeod, Annette
全基因组扩增方法是最近开发的一种工具，用于从有限的模板中扩增 DNA。我们报告了其在锥虫感染中的应用，其特点是寄生虫血症低。多重置换扩增 (MDA) 通过简单的体外步骤扩增 DNA，并在 FTA 过滤卡上的小鼠血液样本上对已知数量的布氏锥虫寄生虫进行评估。数据显示，使用诊断多拷贝核糖体 ITS 区域或 177 bp 重复的引物，每个样品可能进行的 PCR 数量增加了 20 倍，并且与针对单拷贝微卫星的巢式 PCR 相比，灵敏度增加了 20 倍。使用 MDA 进行微卫星基因分型会导致低 DNA 浓度下的等位基因丢失，通过合并多个 MDA 反应可以克服这一问题。使用非洲锥虫病患者的样本证实了使用 MDA 的有效性。 MDA 的使用可以最大限度地利用有限的 DNA 样本，并且可能在需要多个反应的研究（例如群体遗传分析）中证明是一种有价值的工具。
Whole genome amplification methods are a recently developed tool for amplifying DNA from limited template. We report its application in trypanosome infections, characterized by low parasitemias. Multiple displacement amplification (MDA) amplifies DNA with a simple in vitro step and was evaluated on mouse blood samples on FTA filter cards with known numbers of Trypanosoma brucei parasites. The data showed a 20-fold increase in the number of PCRs possible per sample, using primers diagnostic for the multicopy ribosomal ITS region or 177-bp repeats, and a 20-fold increase in sensitivity over nested PCR against a single-copy microsatellite. Using MDA for microsatellite genotyping caused allele dropout at low DNA concentrations, which was overcome by pooling multiple MDA reactions. The validity of using MDA was established with samples from Human African Trypanosomiasis patients. The use of MDA allows maximal use of finite DNA samples and may prove a valuable tool in studies where multiple reactions are necessary, such as population genetic analyses.