Development of a TaqMan qPCR assay for trypanosomatid multi‑species detection and quantification in insects
MetadataShow full item record
AuthorBarranco Gómez, Olga; Carreira de Paula, Jessica; Solano Parada, Jennifer; Gómez Moracho, Tamara; Vic Marfil, Ana; Zafra, María; Orantes Bermejo, Francisco José; Osuna Carrillo De Albornoz, Antonio; Pablos Torró, Luis Miguel de
Barranco-Gómez, O... [et al.]. Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects. Parasites Vectors 16, 69 (2023). [https://doi.org/10.1186/s13071-023-05687-3]
SponsorshipSpanish Programme for Knowledge Generation and Scientific and Technological Strengthening of the R+D+I; System: Generacion del Conocimiento 2018 PGC2018-098929-A-I00 PID2021-126938OB-I00
Background Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. Methods A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. Results The assay showed a detection limit of 1 parasite equivalent/μl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. Conclusions These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.