Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections
Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections
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DOI：
10.1016/j.exppara.2016.01.009
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发表时间：
2016-04-01
影响因子：
2.1
通讯作者：
Valkiunas, Gediminas
中科院分区：
文献类型：
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作者：
Bernotiene, Rasa;Palinauskas, Vaidas;Valkiunas, Gediminas
不同种类和遗传谱系的血孢子虫寄生虫（血孢子虫）的混合感染在野生动物中占主导地位，这种感染是特别致命的。然而，目前使用的基于聚合酶链反应（PCR）的检测方法往往不能读取混合感染。不同的PCR检测混合感染的灵敏度还没有得到充分的测试，但这方面的知识是必不可少的研究解决寄生虫在野生动物的多样性。在这里，我们采用了五种不同的PCR检测，这是广泛用于野生动物禽血孢子虫的研究，并比较其灵敏度在检测实验设计的混合感染的血变形杆菌和疟原虫寄生虫。这些PCR检测中的三种使用扩增细胞色素B基因（cyt B）片段、细胞色素氧化酶亚基I（COI）基因片段和一个靶顶质体基因组片段的引物组。我们收集了野生捕获的鸟类的血液，并使用显微镜和PCR为基础的方法平行应用，确定了10个血孢子虫物种的单一感染，具有类似的寄生虫血症。然后，我们准备了15种不同血孢子虫寄生虫的实验混合物，这些寄生虫通常同时存在于野生鸟类中。在制备混合物期间，在每个寄生虫谱系中使用相似浓度的总DNA。对阳性扩增产物进行测序，并通过在序列电泳图中观察双碱基识别来报告混合感染的存在。这项研究表明，使用每个单一的PCR检测显着低估了血孢子虫寄生虫的生物多样性。应用至少3种平行的PCR检测法可检测到混合感染中存在的大多数，但仍不是所有谱系。我们确定了不同的引物在混合感染的血孢子虫属于不同属的寄生虫检测的偏好。(C)2016 Elsevier Inc. All rights reserved.
Mixed infections of different species and genetic lineages of haemosporidian parasites (Haemosporida) predominate in wildlife, and such infections are particularly virulent. However, currently used polymerase chain reaction (PCR)-based detection methods often do not read mixed infections. Sensitivity of different PCR assays in detection of mixed infections has been insufficiently tested, but this knowledge is essential in studies addressing parasite diversity in wildlife. Here, we applied five different PCR assays, which are broadly used in wildlife avian haemosporidian research, and compared their sensitivity in detection of experimentally designed mixed infections of Haemoproteus and Plasmodium parasites. Three of these PCR assays use primer sets that amplify fragments of cytochrome b gene (cyt b), one of cytochrome oxidase subunit I (COI) gene, and one target apicoplast genome. We collected blood from wild-caught birds and, using microscopic and PCR-based methods applied in parallel, identified single infections of ten haemosporidian species with similar parasitemia. Then, we prepared 15 experimental mixes of different haemosporidian parasites, which often are present simultaneously in wild birds. Similar concentration of total DNA was used in each parasite lineage during preparation of mixes. Positive amplifications were sequenced, and the presence of mixed infections was reported by visualising double-base calling in sequence electropherograms. This study shows that the use of each single PCR assay markedly underestimates biodiversity of haemosporidian parasites. The application of at least 3 PCR assays in parallel detected the majority, but still not all lineages present in mixed infections. We determined preferences of different primers in detection of parasites belonging to different genera of haemosporidians during mixed infections. (C) 2016 Elsevier Inc. All rights reserved.