title: Development of loop-mediated isothermal amplification for rapid detection of Entamoeba histolytica.
abstract: 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 electrophoresis in 1.5% agarose gels, a ladder of multiple bands of different sizes can be observed in positive samples while no bands were detected in the negative control. The sensitivity of the assay was found to be 5 parasites per reaction which corresponds to approximately 15.8 ng/μ L 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 39, and other diarrhea-causing organisms such as Blastocystis hominis and Escherichia coli were used.
  
  
  CONCLUSIONS
  The LAMP 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.
authors.affiliation: Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City 1101, Philippines. wlrivera@science.upd.edu.ph
authors.forename: Windell L
authors.initials: WL
authors.lastname: Rivera
authors.forename: Vanissa A
authors.initials: VA
authors.lastname: Ong
mesh_terms: D016054:DNA, Protozoan / Q000032:analysis; D004748:Entamoeba histolytica / Q000235:genetics / Q000302:isolation & purification*; D004749:Entamoebiasis / Q000175:diagnosis / Q000469:parasitology*; D005243:Feces / Q000469:parasitology; D017125:Genes, Protozoan; D006460:Hemolysin Proteins / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D016054:DNA, Protozoan; D006460:Hemolysin Proteins