Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of Schistosoma haematobium.
Control
Diagnostics
Elimination
Isothermal
Molecular
Point-of-need (PON)
RPA
Schistosoma haematobium
Surveillance
Urogenital schistosomiasis
Journal
Parasites & vectors
ISSN: 1756-3305
Titre abrégé: Parasit Vectors
Pays: England
ID NLM: 101462774
Informations de publication
Date de publication:
04 Nov 2019
04 Nov 2019
Historique:
received:
02
06
2019
accepted:
19
10
2019
entrez:
6
11
2019
pubmed:
7
11
2019
medline:
1
1
2020
Statut:
epublish
Résumé
Accurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. While a number of diagnostic techniques are available there is a need for simple, rapid and highly sensitive point-of-need (PON) tests in areas where infection prevalence and intensity are low. Recombinase Polymerase Amplification (RPA) is a sensitive isothermal molecular diagnostic technology that is rapid, portable and has been used at the PON for several pathogens. A real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic repeat region was developed and was able to detect 1 fg of S. haematobium gDNA. Results were obtained within 10 minutes using a small portable battery powered tube scanner device that incubated reactions at 40 °C, whilst detecting DNA amplification and fluorescence over time. The assay's performance was evaluated using 20 urine samples, with varying S. haematobium egg counts, from school children from Pemba Island, Zanzibar Archipelago, Tanzania. Prior to RPA analysis, samples were prepared using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen, Manchester, UK). Positive assay results were obtained from urine samples with egg counts of 1-926 eggs/10 ml, except for two samples, which had inconclusive results. These two samples had egg counts of two and three eggs/10 ml of urine. The RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and was able to amplify and detect S. haematobium DNA in urine samples from infected patients. The assay's speed and portability, together with the use of crude sample preparation methods, could advance the rapid molecular diagnosis of urogenital schistosomiasis at the PON within endemic countries.
Sections du résumé
BACKGROUND
BACKGROUND
Accurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. While a number of diagnostic techniques are available there is a need for simple, rapid and highly sensitive point-of-need (PON) tests in areas where infection prevalence and intensity are low. Recombinase Polymerase Amplification (RPA) is a sensitive isothermal molecular diagnostic technology that is rapid, portable and has been used at the PON for several pathogens.
RESULTS
RESULTS
A real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic repeat region was developed and was able to detect 1 fg of S. haematobium gDNA. Results were obtained within 10 minutes using a small portable battery powered tube scanner device that incubated reactions at 40 °C, whilst detecting DNA amplification and fluorescence over time. The assay's performance was evaluated using 20 urine samples, with varying S. haematobium egg counts, from school children from Pemba Island, Zanzibar Archipelago, Tanzania. Prior to RPA analysis, samples were prepared using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen, Manchester, UK). Positive assay results were obtained from urine samples with egg counts of 1-926 eggs/10 ml, except for two samples, which had inconclusive results. These two samples had egg counts of two and three eggs/10 ml of urine.
CONCLUSIONS
CONCLUSIONS
The RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and was able to amplify and detect S. haematobium DNA in urine samples from infected patients. The assay's speed and portability, together with the use of crude sample preparation methods, could advance the rapid molecular diagnosis of urogenital schistosomiasis at the PON within endemic countries.
Identifiants
pubmed: 31685024
doi: 10.1186/s13071-019-3755-6
pii: 10.1186/s13071-019-3755-6
pmc: PMC6827214
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
514Subventions
Organisme : Royal Society
ID : RG160395
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