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Tytuł pozycji:

A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR.

Tytuł :
A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution melt qPCR.
Autorzy :
Garrod G; Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Adams ER; Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Lingley JK; Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Saldanha I; Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Torr SJ; Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Cunningham LJ; Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
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Źródło :
PLoS neglected tropical diseases [PLoS Negl Trop Dis] 2020 Nov 25; Vol. 14 (11), pp. e0008308. Date of Electronic Publication: 2020 Nov 25 (Print Publication: 2020).
Typ publikacji :
Journal Article; Research Support, Non-U.S. Gov't
Język :
English
Imprint Name(s) :
Original Publication: San Francisco, CA : Public Library of Science
MeSH Terms :
DNA, Protozoan/*analysis
Trypanosoma brucei gambiense/*genetics
Trypanosoma brucei rhodesiense/*genetics
Trypanosomiasis, African/*diagnosis
Tsetse Flies/*parasitology
Animals ; DNA Primers/genetics ; DNA, Protozoan/genetics ; Humans ; Limit of Detection ; Mass Screening/methods ; Nucleic Acid Denaturation/genetics ; Proof of Concept Study ; Real-Time Polymerase Chain Reaction ; Trypanosoma brucei gambiense/isolation & purification ; Trypanosoma brucei rhodesiense/isolation & purification
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Grant Information :
MR/N013514/1 United Kingdom MRC_ Medical Research Council; BB/L019035/1 United Kingdom BB_ Biotechnology and Biological Sciences Research Council
Substance Nomenclature :
0 (DNA Primers)
0 (DNA, Protozoan)
Entry Date(s) :
Date Created: 20201125 Date Completed: 20210125 Latest Revision: 20210125
Update Code :
20210210
PubMed Central ID :
PMC7725321
DOI :
10.1371/journal.pntd.0008308
PMID :
33237917
Czasopismo naukowe
Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces results in less than 2 hours, avoiding gel electrophoresis and subjective analysis. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.
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