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FEN1-assisted LAMP for specific and multiplex detection of pathogens associated with community-acquired pneumonia
Guopeng Teng1, Gongde Lin2, Pengfan Wei1
1Department of Laboratory Medicine, Key Laboratory of Clinical Laboratory Technology for Precision Medicine, School of Medical Technology and Engineering, Fujian Medical University, Fuzhou 350005, China. cqq036@fjmu.edu.cn.
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.
Insights
A new flap endonuclease 1 (FEN1)-assisted loop-mediated isothermal amplification (LAMP) method offers rapid, multiplex detection of key community-acquired pneumonia (CAP) pathogens. This sensitive assay shows high consistency with PCR for clinical samples.
Area of Science:
- Molecular Biology
- Infectious Diseases
- Biotechnology
Background:
- Lower respiratory tract infections (LRITs), including community-acquired pneumonia (CAP), are a major global health concern.
- Conventional diagnostic methods for LRIT pathogens are often slow, complex, and lack multiplexing capabilities.
Purpose of the Study:
- To develop a novel, rapid, and multiplex diagnostic assay for common CAP pathogens.
- To utilize flap endonuclease 1 (FEN1)-assisted loop-mediated isothermal amplification (LAMP) for enhanced detection.
Main Methods:
- Designed a FEN1-assisted LAMP assay incorporating sequence-specific probes with flap structures.
- Evaluated the assay's specificity, sensitivity, and limit of detection (100 copies/μL).
- Validated the assay's performance against polymerase chain reaction (PCR) using clinical samples.
Main Results:
- The FEN1-assisted LAMP assay demonstrated high specificity and sensitivity.
- The assay successfully detected three leading CAP pathogens (Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae) in a single reaction.
- Results showed high consistency with conventional PCR methods in clinical sample testing.
Conclusions:
- FEN1-assisted LAMP provides a simple, specific, and multiplex method for detecting CAP pathogens.
- This assay has significant potential for diagnosing CAP and could be adapted for other infectious agents.
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