Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

Modak, Sayli S.; Barber, Cheryl; Geva, Eran; Abrams, William R.; Malamud, Daniel; Ongagna, Yhombi Serge Yvon

doi:10.4137/idrt.s32162

Cited by 37 publications

(42 citation statements)

References 29 publications

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“…5, 6, 9, 12, 13 Sensitive detections of malaria parasites in these subpopulations, which are considered as important reservoirs of transmission, are particularly important for malaria elimination. 2, 12 Among various molecular amplification assays, loop-mediated isothermal DNA amplification (LAMP) has emerged as a promising technology for field use due to its simplicity, rapidness, sensitivity and specificity.…”

Section: Introductionmentioning

confidence: 99%

A field-deployable mobile molecular diagnostic system for malaria at the point of need

et al. 2016

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In response to the urgent need of a field-deployable and highly sensitive malaria diagnosis, we developed a standalone, “sample-in-answer-out” molecular diagnostic system (AnyMDx) to enable quantitative molecular analysis of blood-born malaria in low resource areas. The system consists of a durable battery-powered analyzer and a disposable microfluidic compact disc loaded with reagents ready for use. A low power thermal module and a novel fluorescence-sensing module are integrated into the analyzer for real-time monitoring of loop-mediated isothermal nucleic acid amplification (LAMP) of target parasite DNA. With 10 μL of raw blood sample, the AnyMDx system automates the nucleic acid sample preparation and subsequent LAMP and real-time detection. Under the laboratory conditions with whole-blood samples spiked with cultured Plasmodium falciparum, we achieved a detection limit of ∼0.6 parasites/μL, much lower than those for the conventional microscopy and rapid diagnostic tests (∼50-100 parasites/μL). The turnaround time from sample to answer is less than 40 minutes. The AnyMDx is user-friendly requiring minimal technological training. The analyzer and the disposable reagent compact discs are cost-effective, making AnyMDx a potential tool for malaria molecular diagnosis under field settings for malaria elimination.

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Section: Introductionmentioning

confidence: 99%

A field-deployable mobile molecular diagnostic system for malaria at the point of need

et al. 2016

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“…In addition, LAMP analysis is faster than qPCR because amplification can be accomplished without DNA extraction or using a crude lysate (Kostic et al, 2015; Modak et al, 2016). Previous studies demonstrated LAMP assays for quantification of Dehalococcoides spp.…”

Section: Introductionmentioning

confidence: 99%

Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater

Stedtfeld

Samhan

et al. 2016

Journal of Microbiological Methods

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Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 102 Dehalobacter cells per 100 mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100 mg per L of humic acid per reaction compared to 1 mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60 min) and highly sensitive quantification of microorganisms in environmental water samples.

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“…LAMP assay can be completed within 1h, and its sensitivity is 10 to 100-fold higher than that of conventional Polymerase Chain Reaction (PCR) when purified DNA is used for the template [4]. As LAMP is performed using three pairs of specific primers for DNA amplification, its specificity is higher than that of PCR [7].…”

Section: Introductionmentioning

confidence: 99%

“…When sensitive differential diagnosis need to be achieved by LAMP assay, researchers normally use as template either genome DNA purified from blood or samples that have been boiled and roughly cleaned up by centrifugation [9,10]. Recently, Modak, et al reported that high-sensitive LAMP can be performed using blood samples that have been heated at 90˚C for 5 min to lyse blood cells and parasites, releasing target DNA as templates [4]. Because at least two (one to be set at 90˚C, another at 65˚C) heating blocks are necessary for high-throughput analysis by LAMP assay a considerable amount of electricity is required, which might be not abundant or easily available in rather deprived areas or basic health care environments where the test need to be performed.…”

Section: Introductionmentioning

confidence: 99%

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Sensitivity of Loop-Mediated Isothermal Amplification Increases 100-Fold after TritonTM X-100 Treatment of Plasmodium-Infected Erythrocytes

Hashimoto¹,

Sakamoto²,

Ido³

et al. 2017

SOJMID

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Highly sensitive and rapid diagnostic methods that should also be appropriate for Point-of-Care (POC) testing are required for Mass Screening and Treatment (MSAT) programs to be effective in blocking transmission of malaria, and eventually eradicating the disease. Loop-Mediated Isothermal Amplification (LAMP) is an accurate, sensitive, rapid, and easy-to-perform method for gene amplification. Preparation of Plasmodium-Infected Red Blood Cell (IRBC) samples for the LAMP assay normally includes boiling and/or centrifugation. To increase the chances of success for MSAT programs in endemic countries where resources might be limited, it should become possible to perform the preparation in a short time and without having to resort to expensive equipment. Herein, we report that treating IRBCs with Triton TM X-100 prior to LAMP assay results in 100-fold increase in the test sensitivity. Our findings may contribute to improve the use and efficacy of LAMP assay as a malaria diagnostic test within MSAT programs in endemic countries.

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Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification

Cited by 37 publications

References 29 publications

A field-deployable mobile molecular diagnostic system for malaria at the point of need

A field-deployable mobile molecular diagnostic system for malaria at the point of need

Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater

Sensitivity of Loop-Mediated Isothermal Amplification Increases 100-Fold after TritonTM X-100 Treatment of Plasmodium-Infected Erythrocytes

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