Loop region-specific oligonucleotide probes for loop-mediated isothermal amplification–enzyme-linked immunosorbent assay truly minimize the instrument needed for detection process

Abstract

Enteric fever represents a significant public health burden in less-developed countries. Therefore, there is a great need for developing an improved diagnostic tool adapted to the demands of poor-resource clinical laboratories in those countries. The current study has developed a reliable loop-mediated isothermal amplification (LAMP)–enzyme-linked immunosorbent assay (ELISA) for diagnosis of enteric fever with a minimal equipment dependency. The LAMP–ELISA assay involves direct incorporation of a labeled nucleotide into amplicons during the amplification of the SPA3440 gene, their hybridization to the unique tagged oligonucleotide probes during the LAMP reaction, and finally detection of labeled LAMP amplicons by immunoassay technology. Because the designed oligonucleotide probes target the single-stranded DNA segment within the LAMP amplicons, the probe hybridization stage is performed simultaneously with the amplification process. This novel probe design strategy allows both the amplification and hybridization stages to be performed simultaneously and isothermally in a water bath. Among the bacteria tested, positive results were observed only with enteric fever causative bacteria. The LAMP–ELISA assay was successfully applied to artificially contaminated blood samples with a detection limit of 10 colony-forming units (CFU)/ml, which was 100 times more sensitive than polymerase chain reaction (PCR) and turbidity assessment-based conventional LAMP methods. The new assay is considered to be an effective method for diagnosis of enteric fever.