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
Abstract Background 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). All the assays demonstrated in this study were developed...
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doaj-e00eaed828504d5f8de54a169a3859092020-11-25T03:12:00ZengBMCBMC Biotechnology1472-67502020-06-0120111510.1186/s12896-020-00629-8Loop-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 samplePhiaw Chong Foo0A. B. Nurul Najian1Nuramin A. Muhamad2Mariana Ahamad3Maizan Mohamed4Chan Yean Yean5Boon Huat Lim6Acarology Unit, Infectious Disease Research Centre, Institute for Medical Research, Ministry of Health Malaysia, National Institutes of Health ComplexDepartment of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health CampusInstitute for Research in Molecular Medicine, Universiti Sains Malaysia, Health CampusAcarology Unit, Infectious Disease Research Centre, Institute for Medical Research, Ministry of Health Malaysia, National Institutes of Health ComplexFaculty of Veterinary Medicine, Universiti Malaysia KelantanDepartment of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health CampusSchool of Health Sciences, Universiti Sains MalaysiaAbstract Background 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). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model. Results A 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. Conclusions The 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.http://link.springer.com/article/10.1186/s12896-020-00629-8Loop-mediated isothermal amplificationNested PCRReal-time PCRLateral flow dipstickCalcein-manganese visualizationLAMP analytical sensitivity |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Phiaw Chong Foo A. B. Nurul Najian Nuramin A. Muhamad Mariana Ahamad Maizan Mohamed Chan Yean Yean Boon Huat Lim |
spellingShingle |
Phiaw Chong Foo A. B. Nurul Najian Nuramin A. Muhamad Mariana Ahamad Maizan Mohamed Chan Yean Yean Boon Huat Lim 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 BMC Biotechnology Loop-mediated isothermal amplification Nested PCR Real-time PCR Lateral flow dipstick Calcein-manganese visualization LAMP analytical sensitivity |
author_facet |
Phiaw Chong Foo A. B. Nurul Najian Nuramin A. Muhamad Mariana Ahamad Maizan Mohamed Chan Yean Yean Boon Huat Lim |
author_sort |
Phiaw Chong Foo |
title |
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 |
title_short |
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 |
title_full |
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 |
title_fullStr |
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 |
title_full_unstemmed |
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 |
title_sort |
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 |
publisher |
BMC |
series |
BMC Biotechnology |
issn |
1472-6750 |
publishDate |
2020-06-01 |
description |
Abstract Background 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). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model. Results A 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. Conclusions The 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. |
topic |
Loop-mediated isothermal amplification Nested PCR Real-time PCR Lateral flow dipstick Calcein-manganese visualization LAMP analytical sensitivity |
url |
http://link.springer.com/article/10.1186/s12896-020-00629-8 |
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