FEN1-assisted LAMP for specific and multiplex detection of pathogens associated with community-acquired pneumonia

Abstract

Lower respiratory tract infections (LRITs), including community-acquired pneumonia (CAP), are the fifth leading cause of death worldwide over the last ten years, posing a serious threat to global healthcare. Conventional laboratory assays for detecting pathogens are hindered by complicated procedures, a long turnaround time and a lack of multiplex detection capabilities. In this study, a flap-endonuclease 1 (FEN1)-assisted loop-mediated isothermal amplification (LAMP) method was designed, and an assay based on this method was developed to identify three leading pathogens for CAP, namely, Streptococcus pneumoniae, Mycoplasma pneumoniae and Haemophilus influenzae. FEN1-assisted LAMP utilized a sequence-specific probe with a flap structure to generate an amplified signal, demonstrating high specificity and sensitivity with a low limit of detection (100 copies per μL). Based on the cleavage of flap probes by FEN1, our assay was able to detect three pathogens in a single reaction. This method is highly consistent with the polymerase chain reaction (PCR) in clinical sample testing. This simple, specific and multiple detection method has the potential to identify CAP and could be applied to detect other pathogen infections.