Havryliuk Tatiana, Orjuela-Sánchez Pamela, Ferreira Marcelo U
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Exp Parasitol. 2008 Dec;120(4):330-6. doi: 10.1016/j.exppara.2008.08.012. Epub 2008 Aug 31.
We used mixtures of genomic DNA from two genetically distinct isolates from Brazil, 42M and 312M, to investigate how accurately 12-locus microsatellite typing describes the overall genetic diversity and characterizes multilocus haplotypes in multiple-clone Plasmodium vivax infections. We found varying PCR amplification efficiencies of microsatellite alleles; for example, from the same 1:1 mixture of 42M and 312M DNA we amplified predominantly 312M-type alleles at 10 loci and 42M-type alleles at 2 loci. All microsatellite alleles were accurately scored in 1:0.5 and 1:0.25 312M:42M DNA mixtures, even when minor peak heights did not meet previously suggested criteria for minor allele detection in multiple-clone infections. Relative proportions of major and minor alleles were unaffected by multiple displacement amplification of template DNA prior to PCR-based microsatellite typing. Although microsatellite typing may detect minor alleles in clone mixtures, amplification biases may lead to inaccurate assignment of predominant haplotypes in multiple-clone P. vivax infections.
我们使用来自巴西的两个基因不同的分离株42M和312M的基因组DNA混合物，来研究12位点微卫星分型在描述间日疟原虫多重克隆感染中的总体遗传多样性以及表征多位点单倍型方面的准确性。我们发现微卫星等位基因的PCR扩增效率各不相同；例如，从相同的42M和312M DNA 1:1混合物中，我们在10个位点主要扩增出312M型等位基因，在2个位点扩增出42M型等位基因。在1:0.5和1:0.25的312M:42M DNA混合物中，所有微卫星等位基因都能被准确计分，即使次要峰高未达到先前提出的多重克隆感染中次要等位基因检测标准。在基于PCR的微卫星分型之前，模板DNA的多重置换扩增不会影响主要和次要等位基因的相对比例。尽管微卫星分型可能会在克隆混合物中检测到次要等位基因，但扩增偏差可能会导致在间日疟原虫多重克隆感染中主要单倍型的错误分配。
