Loop-mediated isothermal amplification assay detects multiple alleles of blaOXA-51-like genes in Acinetobacter baumannii and other Gram-negative bacteria despite primer-template mismatches
Mark B. Carascal,
Lawrence S. Macalalad,
Joy Ann Petronio-Santos,
Raul V. Destura,
Windell L. Rivera
Affiliations
Mark B. Carascal
Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines; Clinical and Translational Research Institute, The Medical City, Ortigas Avenue, Pasig City 1605, Philippines
Lawrence S. Macalalad
Clinical and Translational Research Institute, The Medical City, Ortigas Avenue, Pasig City 1605, Philippines
Joy Ann Petronio-Santos
Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines; Biological Research and Services Laboratory, Natural Sciences Research Institute, University of the Philippines Diliman, Quezon City 1101, Philippines
Raul V. Destura
Clinical and Translational Research Institute, The Medical City, Ortigas Avenue, Pasig City 1605, Philippines; Institute of Molecular Biology and Biotechnology, National Institutes of Health, University of the Philippines Manila, City of Manila 1159, Philippines
Windell L. Rivera
Pathogen-Host-Environment Interactions Research Laboratory, Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City 1101, Philippines; Corresponding author. Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, 1101 Philippines.
The known intrinsic and polymorphic blaOXA-51-like genes of Acinetobacter baumannii were recently reported in other non-A. baumannii 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 blaOXA-51-like genes. Specifically, an alignment-based primer design, in silico primer screening, and in vitro assay confirmation were conducted. Both in silico and in vitro 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 blaOXA-51-like genes. Finally, it showed a superior limit of detection (as low as 101 CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor blaOXA-51-like genes in the clinical setting and provided important insights into LAMP primer design and screening.
