Grupo de Medicina Xenómica, CIMUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain; Grupo de Xenómica Comparada de Parásitos Humanos, IDIS, Santiago de Compostela, Galicia, Spain.
Laboratorio de Patología Apícola, Centro de Investigación Apícola y Agroambiental, Instituto Regional de Investigación y Desarrollo Agroalimentario y Forestal (CIAPA-IRIAF), JCCM, 19180 Marchamalo, Guadalajara, Spain.
J Invertebr Pathol. 2018 May;154:37-41. doi: 10.1016/j.jip.2018.03.015. Epub 2018 Mar 30.
Trypanosomatids are highly prevalent pathogens of Hymenoptera; however, most molecular methods used to detect them in Apis and Bombus spp. do not allow the identification of the infecting species, which then becomes expensive and time consuming. To overcome this drawback, we developed a multiplex PCR protocol to readily identify in a single reaction the main trypanosomatids present in these hymenopterans (Lotmaria passim, Crithidia mellificae and Crithidia bombi), which will facilitate the study of their epidemiology and transmission dynamics. A battery of primers, designed to simultaneously amplify fragments of the RNA polymerase II large subunit (RPB1) of L. passim, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of C. mellificae and the DNA topoisomerase II (TOPII) of C. bombi, was tested for target specificity under single and mixed template conditions using DNA extracted from cell cultures (L. passim ATCC PRA403; C. mellificae ATCC 30254) and from a bumblebee specimen infected with C. bombi only (14_349). Once validated, the performance of the method was assessed using DNA extractions from seven Apis mellifera (Linnaeus, 1758) and five Bombus terrestris (Linnaeus, 1758) field samples infected with trypanosomatids whose identity had been previously determined by PCR-cloning and sequencing (P-C-S). The new method confirmed the results obtained by P-C-S: two of the honeybee samples were parasitized by L. passim, C. mellificae and C. bombi at the same time, whereas the other five were infected with L. passim only. The method confirmed the simultaneous presence of L. passim and C. mellificae in two B. terrestris, where these parasites had not previously been reported.
锥虫是膜翅目昆虫的高度流行病原体；然而，大多数用于检测 Apis 和 Bombus 物种中锥虫的分子方法无法鉴定感染物种，这使得鉴定过程变得昂贵且耗时。为了克服这一缺点，我们开发了一种多重 PCR 协议，可以在单个反应中快速识别这些膜翅目昆虫中存在的主要锥虫（Lotmaria passim、Crithidia mellificae 和 Crithidia bombi），这将有助于研究它们的流行病学和传播动态。一组引物被设计用于同时扩增 L. passim 的 RNA 聚合酶 II 大亚基（RPB1）、C. mellificae 的甘油醛-3-磷酸脱氢酶（GAPDH）和 C. bombi 的 DNA 拓扑异构酶 II（TOPII）的片段，在单模板和混合模板条件下使用从细胞培养物（L. passim ATCC PRA403；C. mellificae ATCC 30254）和仅感染 C. bombi 的一只大黄蜂标本（14_349）中提取的 DNA 对其进行了靶特异性测试。方法验证后，使用先前通过 PCR-克隆和测序（P-C-S）确定身份的七种 Apis mellifera（Linnaeus，1758）和五种 Bombus terrestris（Linnaeus，1758）田间样本的 DNA 提取物评估了该方法的性能。新方法证实了 P-C-S 获得的结果：两个蜜蜂样本同时被 L. passim、C. mellificae 和 C. bombi 寄生，而其他五个样本仅被 L. passim 感染。该方法证实了两种 B. terrestris 同时存在 L. passim 和 C. mellificae，而这两种寄生虫以前并未在这些物种中报道过。
