DataSheet_1_Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.docx

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

禽血孢子虫（avian haemosporidian parasites）凭借其全球分布范围与极高的生物多样性，成为研究寄生虫-宿主互作生态与演化的理想模式生物。该类寄生虫的检测技术已从显微镜检查发展至基于聚合酶链式反应（PCR）的方法，其中线粒体细胞色素b基因（mitochondrial cytochrome b gene）被用作条形码标记区域。然而，当前用于筛选与鉴定的标准PCR方案因扩增片段较短，存在难以检测混合感染、无法生成系统发育信息数据的缺陷。为解决上述问题，本研究开发了一种靶向禽血孢子虫的新型属特异性巢式PCR（nested PCR）方案。
该方案首先利用包含马达加斯加三种不同雀形目鸟类血液样本的大型数据集开展了严格测试；随后在另外两家实验室中，通过使用不同的聚合酶预混液（master mixes）与不同鸟类物种的小型数据集完成了验证。研究团队还将新型PCR方案的检测结果与当前广泛使用的标准巢式PCR方法的结果进行了对比分析。
该新型方案可特异性鉴定疟原虫属（Plasmodium）、血变虫属（Haemoproteus，Parahaemoproteus）以及住白细胞虫属（Leucocytozoon）的寄生虫。分析结果显示，其灵敏度与标准巢式PCR相当，但在检测混合感染方面实现了显著提升。此外，由于该方案可扩增更长的DNA片段，系统发育分辨率得到改善，有助于更深入地理解血孢子虫的生物多样性与演化历程。
总体而言，这款新型PCR方案是禽血孢子虫检测方法体系中的宝贵补充，可推动寄生虫生态、流行病学与演化领域的综合性研究。