Malaria mitochondrial diagnosis: challenges and pitfalls

Gabriel Luíz Costa, D. A. Alvarenga, Gabriela Maíra Pereira de Assis, A. Aguiar, Jaime Louzada, D. Pereira, A. Pina-Costa, Z. Hirano, S. B. Moreira, A. Pissinatti, P. Brasil, C. Daniel-Ribeiro, T. Sousa, C. F. Alves de Brito
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引用次数: 0

Abstract

High-copy genomic sequences could be used as PCR targets for the detection of Plasmodium infections, providing increased sensitivity over single- or low-copy genes. Mitochondrial genomes of malaria parasites are present in multiple copies in a single mitochondrion, and each parasite has many mitochondria. Here, we describe the development of seven species-specific qPCR assays for the diagnosis of Plasmodium vivax and Plasmodium falciparum, targeting coding and non-coding mitochondrial genomic regions.The optimization of the qPCR protocols involved a gradient of annealing temperatures and concentrations of primers and probes, as well as the inclusion of PCR additives/enhancers [e.g., dimethyl sulfoxide (DMSO), glycerol, bovine serum albumin (BSA)] to improve the specificity of qPCR amplification.Non-specific amplification of other Plasmodium species and of human targets was observed in different levels for all assays. Regardless of the late Cq values for most non-specific amplifications, the application of a cutoff value did not completely exclude false-positive amplification, compromising the specificity and also the sensitivity of the assays.Therefore, although mitochondrial targets have higher sensitivity, they frequently lose specificity due to their high levels of sequence conservation. A screening to evaluate the cross-reaction between Plasmodium species and the non-specific amplification of human malaria-free samples must be performed for Plasmodium mitochondrial assays.
疟疾线粒体诊断:挑战和陷阱
高拷贝基因组序列可以用作检测疟原虫感染的PCR靶点,从而提高了对单拷贝或低拷贝基因的敏感性。疟原虫的线粒体基因组存在于单个线粒体中的多个拷贝中,每个寄生虫都有许多线粒体。在这里,我们描述了用于诊断间日疟原虫和恶性疟原虫的七种物种特异性qPCR检测的开发,靶向编码和非编码线粒体基因组区域。qPCR方案的优化涉及退火温度和引物和探针浓度的梯度,以及加入PCR添加剂/增强子[例如二甲基亚砜(DMSO)、甘油、牛血清白蛋白(BSA)]以提高qPCR扩增的特异性。对于所有测定,在不同水平上观察到其他疟原虫物种和人类靶标的非特异性扩增。不管大多数非特异性扩增的晚期Cq值如何,应用截止值并不能完全排除假阳性扩增,这损害了测定的特异性和敏感性。因此,尽管线粒体靶标具有更高的敏感性,但由于其高度的序列保守性,它们经常失去特异性。疟原虫线粒体测定必须进行筛选,以评估疟原虫物种之间的交叉反应和人类无疟疾样本的非特异性扩增。
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