Loop-Primer Endonuclease Cleavage-Loop-Mediated Isothermal Amplification Technology for Multiplex Pathogen Detection and Single-Nucleotide Polymorphism Identification

Abstract

Loop-mediated isothermal amplification (LAMP) provides effective diagnostic technology for infectious disease pathogen identification and is compatible with inexpensive instrumentation for use in disease-prevalent developing regions. However, simultaneous multiple-target detection and single-nucleotide polymorphism (SNP) identification, essential properties of nucleic acid diagnostics, are difficult to achieve using LAMP. This study introduces loop-primer endonuclease cleavage (LEC)-LAMP, a singleplex or multiplex LAMP technology with single-base specificity for variable SNP identification. We developed a singleplex LEC-LAMP Neisseria meningitidis assay that demonstrated complete analytical specificity and a limit of detection of 3.1 genome copies per reaction. Small-scale clinical testing of this assay demonstrated 100% diagnostic specificity and sensitivity when assessed with anonymized DNA extracts from confirmed cases of bacterial meningitis infection. The single-base specificity of this assay indicated effective SNP identification properties when challenged with DNA templates containing SNPs located within a specific six-base region. This assay was modified to generate an allele-specific LEC-LAMP N. meningitidis assay that successfully demonstrated single-tube differentiation of wild-type and mutant allele templates. The singleplex assay was further modified to generate a multiplex LEC-LAMP assay that successfully demonstrated simultaneous multiple-target detection of three bacterial targets, N. meningitidis, Streptococcus pneumonia, and Hemophilus influenzae. LEC-LAMP is the first report of single-tube, real-time, singleplex or multiplex LAMP technology with single-base specificity for variable SNP identification.