A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination

Malaria Journal

Research

Qin Cheng¹  Colin J. Sutherland²  Sumudu Britton³  James S. McCarthy³ 

[1] Australian Army Malaria Institute, Brisbane, Australia;London School of Hygiene and Tropical Medicine, London, UK;University of Queensland, Brisbane, Australia;QIMR Berghofer Medical Research Institute, Brisbane, Australia;

关键词: Loop mediated isothermal amplification;    LAMP;    Elimination;    High throughput;    Diagnosis;    Malaria;   

DOI  :  10.1186/s12936-015-0848-3

 received in 2015-07-07, accepted in 2015-08-11,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundTo detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput.MethodsA high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia.ResultsThe HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % CI 95–99) and specificity 83 % (n = 15/18, 95 % CI 59–96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % CI 92–99) and specificity of 96 % (n = 151/157, 95 % CI 92–99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % CI 80–100) and specificity of 95 % (n = 123/128, 95 % CI 90–98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % CI 69–97).ConclusionThis colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination.

【 授权许可】

CC BY   
© Britton et al. 2015