Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica  

作　　者：Windell L.Rivera Vanissa A.Ong 

机构地区：[1]Institute of Biology,College of Science,University of the Philippines,Diliman,Quezon City 1101,Philippines [2]Molecular Protozoology Laboratory,Natural Sciences Research Institute,University of the Philippines,Diliman,Quezon City 1101,Philippines

出　　处：《Asian Pacific Journal of Tropical Medicine》2013年第6期457-461,共5页亚太热带医药杂志（英文版）

基　　金：supported financially by a research grant from the Natural Sciences Research Institute,University of the Philippines ) BIO 1 l-l-05) to W.L.R.

摘　　要：Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.Objective:To develop a loop-mediated isothermal amplification(LAMP) assay for the detection of Entamoeba histolytica(E.histolytica),the causative agent of amebiasis.Methods:The LAMP primer set was designed from E.histolytica hemolysin gene HLY6.Genomic DNA of E.histolytica trophozoites strain HK9 was used to optimize the LAMP mixture and conditions.Amplification of DNA in the LAMP mixture was monitored through visual inspection for turbidity of the LAMP mix as well as addition of fluorescent dye.Results:Positive LAMP reactions turned turbid while negative ones remained clear.Upon addition of a fluorescent dye,all positive reactions turned green while the negative control remained orange under ambient light After elecrophoresis in 1.5% agarose gels,a ladder of multiple bands of different sizes can be oliserved in positive samples while no bands were detected in the negative control.The sensitivity of the assay was found to be S parasites per reaction which corresponds to approximately 1S.8 ng/μL DNA.The specificity of the assay was verified by the absence of amplified products when DNA from other gastrointestinal parasites such as the morphologically similar but non-pathogenic species,Entamoeba dispar. and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.Conclusions:The I.AMP assay we have developed enables the detection of E.histolytica with rapidity and ease,therefore rendering it is suitable for laboratory and field diagnosis of amebiasis.

关 键 词：AMEBIASIS Diagnosis DNA ENTAMOEBA HISTOLYTICA HEMOLYSIN gene Loop-mediated ISOTHERMAL AMPLIFICATION assay 

分 类 号：R531.1[医药卫生—内科学]