Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

Phiaw Chong Foo,Nuramin A Muhamad,A B Nurul Najian,Chan Yean Yean,Mariana Ahamad,Boon Huat Lim,Maizan Mohamed

doi:10.1186/s12896-020-00629-8

Abstract

BackgroundThis study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.ResultsA set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.ConclusionsThe analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.

Highlights

This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional polymerase chain reaction (PCR), nested PCR and real-time PCR
The analytical sensitivity comparison among the conventional PCR, nested PCR (nPCR), Real-time polymerase chain reaction (qPCR) and LAMP reveals that the LAMP outperformed the others in terms of limit of detection (LoD) and amplification time
DNA-based detection method has been widely used for diagnosis of infectious diseases due to the presence of specific DNA sequences in pathogens that can served as reliable detection biomarkers [1, 2]

Summary

Introduction

This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). DNA-based detection method has been widely used for diagnosis of infectious diseases due to the presence of specific DNA sequences in pathogens that can served as reliable detection biomarkers [1, 2] This detection method is usually accompanied with amplification technology such as polymerase chain reaction (PCR), one of the most important scientific advances in molecular biology. Despite its popularity in disease diagnostic, PCR amplification possesses several inherent drawbacks such as primer mismatch due to high DNA similarity among species and low in copy number of specific gene for pathogen identification. These drawbacks have later paved the way for the emergences of several innovated PCR such as nested PCR (nPCR) and realtime PCR (qPCR). NPCR was not commonly used for disease diagnostics due to its long turnaround time, and its two-step-procedure made it susceptible to amplicon contamination [6]

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