RT Journal Article
T1 A new multiplex PCR protocol to detect mixed trypanosomatid infections in species of Apis and Bombus
A1 Bartolomé, Carolina
A1 Buendía, María
A1 Benito, María
A1 Rúa, Pilar de la
A1 Ornosa Gallego, Concepción
A1 Martín Hernández, Raquel
A1 Higes, Mariano
A1 Maside, Xulio
AB 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.
PB Elsevier
SN 0022-2011, ESSN: 1096-0805
YR 2018
FD 2018-05
LK https://hdl.handle.net/20.500.14352/12087
UL https://hdl.handle.net/20.500.14352/12087
LA eng
NO Instituto Nacional de Investigación y Tecnología Agraria (INIA)
NO Fundación Séneca (Plan of Science and Technology of the Region of Murcia, España)
NO COST Action FA1307
DS Docta Complutense
RD 27 abr 2024