Surface Loading Proximity Ligation-Induced PCR Technique for Fluorescent Detection of Intact Methicillin-Resistant Staphylococcus aureus.

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA)-induced pneumonia in nursing patients with chronic obstructive pulmonary disease (COPD) necessitates rapid detection, timely intervention, and meticulous clinical management. Thus, the development of sensitive and accurate techniques for MRSA detection holds significant clinical nursing of infectious diseases. In this study, we designed a surface loading proximity ligation assay (PLA) for the precise detection of MRSA in pulmonary infections by simultaneously targeting three characteristic proteins. This assay utilizes three customized probes: the first probe targets protein A on the MRSA surface, the second probe immobilizes on the biological lipid layer, and the third probe identifies PBP2a (a protein responsible for drug resistance of MRSA). The proposed strategy integrates proximity ligation of these three probes with polymerase chain reaction (PCR) to perform "AND" logic-based analysis of the three key MRSA components, enabling sensitive detection of intact MRSA. Taking advantage of the high signal amplification efficiency of PCR and elevated target recognition capability of PLA, the method exhibited a low limit of detection of 2.9 CFU/ml. As a result, the proposed method demonstrated significantly improved accuracy for MRSA detection. We believe this novel integrated strategy could diversify existing bacterial detection approaches and may inspire the development of promising drug candidates in COPD nursing.