Development of MRSA and MRAB detection kit employing loop-mediated isothermal amplification and colorimetric indicator dye

摘要

Methicillin-resistant Staphylococcus aureus (MRSA) and Multidrug-resistant Acinetobacter baumannii (MRAB) are serious life-threating pathogens. The rapid detection of those pathogens is a challenge for the most clinical microbiology laboratories. A nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP) that enables to amplify target DNA sequences in a short time period, is an excellent technique to detect the pathogens with high efficiency. We developed LAMP detecting kits against both MRSA and MRAB that utilizing a newly developed colorimetric Mg2+ ion indicator dye D-649 and novel sets of primers. An aqueous soluble dye D-649 is stable over physiological pH range and displays clear color change upon binding to Mg2+ ion. The addition of 0.12 mM of D-649 to LAMP solution did not disturb the amplification process, and the positive reaction would be easily confirmed by the color change of reaction mixture from ruby to purple in naked eye under ambient light. Each primer set was designed to recognize mecA/fernB gene of MASA and blaOXA-23/blaOXA51 like gene of MRAB, and the detection lirnits for both DNAs were shown as 1 pg. These LAMP detection kits will be beneficial for the detection of MRSA and MRAB in clinical laboratories.