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

Abstract

Lower respiratory tract infection (LRITs) including community-acquired pneumonia (CAP) is the fifth leading cause of death worldwide over last ten years, which has posed a serious threat to global healthcare. Conventional laboratory assays for detecting pathogens are hindered by complicated procedures, 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, Streptococcus pneumoniae, Mycoplasma pneumoniae and Haemophilus influenzae. FEN1-assisted LAMP utilized a sequence-specific probe with flap structure to generate amplification signal, which demonstrated to have high specificity and sensitivity, with a low limit of detection (down to 100 copies/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 polymerase chain reaction (PCR) in clinical samples testing. This simple, specific and multiple detection method has potential in identification of CAP and can also be applied to detect other pathogen infections.