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A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus

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Bartolomé, Carolina and Buendía, María and Benito, María and Rúa, Pilar de la and Ornosa Gallego, Concepción and Martín Hernández, Raquel and Higes, Mariano and Maside, Xulio (2018) A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus. Journal of Invertebrate Pathology, 154 . pp. 37-41. ISSN 0022-2011, ESSN: 1096-0805

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Official URL: https://www.sciencedirect.com/science/article/pii/S0022201118300193



Abstract

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.


Item Type:Article
Uncontrolled Keywords:Crithidia mellificae; Crithidia bombi; Lotmaria passim; Apis mellifera; Bombus terrestris; Multiplex PCR
Subjects:Medical sciences > Veterinary > Parasitology
Medical sciences > Biology > Insects
ID Code:47452
Deposited On:09 May 2018 08:10
Last Modified:10 Dec 2018 15:25

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