Loop-mediated isothermal amplification assay detects multiple alleles of genes in and other Gram-negative bacteria despite primer-template mismatches.

The known intrinsic and polymorphic genes of were recently reported in other non- Gram-negative pathogens. Accurate detection of this potentially transferrable carbapenemase gene in the clinical setting is critical. This study developed a loop-mediated isothermal amplification (LAMP) assay targetting multiple alleles of genes. Specifically, an alignment-based primer design, primer screening, and assay confirmation were conducted. Both and results revealed the tolerance of the LAMP assay to up to five primer-template mismatches outside the 3'-end primer regions. Within 90 min, the LAMP assay also detected the gene targets in other Gram-negative bacteria with known and novel genes. Finally, it showed a superior limit of detection (as low as 10 CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor genes in the clinical setting and provided important insights into LAMP primer design and screening.