Stem loop-mediated isothermal amplification test: comparative analysis with classical LAMP and PCR in detection of Entamoeba histolytica in Kenya.
BMC Res Notes
; 10(1): 142, 2017 Mar 31.
Article
in En
| MEDLINE
| ID: mdl-28359328
ABSTRACT
BACKGROUND:
Entamoeba histolytica, the causative agent for amoebiasis is a considerable burden to population in the developing countries where it accounts for over 50 million infections. The tools for detection of amoebiasis are inadequate and diagnosis relies on microscopy which means a significant percent of cases remain undiagnosed. Moreover, tests formats that can be rapidly applied in rural endemic areas are not available.METHODS:
In this study, a loop-mediated isothermal test (LAMP) based on 18S small subunit ribosomal RNA gene was designed with extra reaction accelerating primers (stem primers) and compared with the published LAMP and PCR tests in detection of E. histolytica DNA in clinical samples.RESULTS:
The stem LAMP test indicated shorter time to results by an average 11 min and analytical sensitivity of 10-7 (~30 pg/ml) compared to the standard LAMP and PCR which showed sensitivities levels of 10-5 (~3 ng/ml) and 10-4 (~30 ng/ml) respectively using tenfold serial dilution of DNA. In the analysis of clinical specimens positive for Entamoeba spp. trophozoites and cysts using microscopy, the stem LAMP test detected E. histolytica DNA in 36/126, standard LAMP test 20/126 and PCR 17/126 cases respectively. There was 100% agreement in detection of the stem LAMP test product using fluorescence of SYTO-9 dye in real time machine, through addition of 1/10 dilution of SYBR® Green I and electrophoresis in 2% agarose gel stained with ethidium bromide.CONCLUSION:
The stem LAMP test developed in this study indicates potential towards detection of E. histolytica.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Polymerase Chain Reaction
/
Nucleic Acid Amplification Techniques
/
Entamoeba histolytica
/
Entamoebiasis
Type of study:
Diagnostic_studies
Limits:
Child
/
Humans
Country/Region as subject:
Africa
Language:
En
Journal:
BMC Res Notes
Year:
2017
Document type:
Article
Affiliation country:
Kenya
Publication country:
ENGLAND
/
ESCOCIA
/
GB
/
GREAT BRITAIN
/
INGLATERRA
/
REINO UNIDO
/
SCOTLAND
/
UK
/
UNITED KINGDOM