Effect of internal primer-template mismatches on loop-mediated isothermal amplification

The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (5 or 6) primers targeting 6 (7 or 8) regions within a fairly small genome segment for amplification. This technique has a potential for greater specificity than two-primer methods, such as polymerase chain reaction. There are still no reports for primer-template mismatch of LAMP. In this study, a set of LAMP primers was designed, targeting the 16S-23S rRNA intergenic spacer region of Streptococcus dysgalactiae. The selectivity of the LAMP method was tested with 25 bacterial strains. There was a non-specific amplification when the genomic DNA of Streptococcus agalactiae was used as a template and it was indicated by a nucleotide basic local alignment search tool (BLAST) in GenBank. There were three false priming sites on backward inner primer and the internal primer-template mismatches extended the detection time from 21 min to 47 min. This study would be of great reference value for targeting sequence selection, primer design of LAMP and detection of antibiotic-resistant bacteria with LAMP.