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Loop-Mediated Isothermal Amplification Coupled With Nanoparticle-Based Biosensor: A Rapid and Sensitive Method to
Fei Xiao1, Juan Zhou1, Chunrong Sun1
1Experimental research center, Capital Institute of pediatrics, Beijing, China.
Abstract:
Mycoplasma pneumoniae (MP), the causative agent of MP pneumonia (MPP), has posed a substantial burden to public health owing to a lack of rapid and effective diagnostic methods. Here, we designed a loop-mediated isothermal amplification (LAMP)-based assay, termed LAMP, combined with a nanoparticle-based lateral flow biosensor (LAMP-LFB) for rapid and sensitive diagnosis of MP.-LAMP-LFB included a set of six primers targeting the community-acquired respiratory distress syndrome (CARDS) toxin gene and was performed optimally at 63°C for only 30 min. The resulting LAMP products could be visually indicated by LFB within 2 min, thus the whole process could be accomplished within an hour. MP-LAMP-LFB's sensitivity was 50 fg per reaction, which was in complete accordance with these results obtained from real-time turbidity and visual detection reagent (VDR). MP-LAMP-LFB had no cross-reactivity with other pathogens that had similar clinical presentations. Our assay was further validated using 100 nasopharyngeal swab samples collected from children suspected of MPP, and the result was compared with the real-time PCR method. With a positive rate of 50%, the data indicated that MP-LAMP-LFB is a sensitive test for MP detection in clinical settings. Collectively, the MP-LAMP-LFB assay targeting the CARDS toxin gene was a rapid, highly sensitive, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas.
Insights
A new loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow biosensor (LAMP-LFB) offers rapid and sensitive detection of Mycoplasma pneumoniae (MP). This Mycoplasma pneumoniae diagnostic test is highly accurate for clinical settings.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Biotechnology
Background:
- Mycoplasma pneumoniae (MP) causes MP pneumonia (MPP), a significant public health concern.
- Current diagnostic methods for MP lack speed and efficiency, hindering timely treatment.
- There is a critical need for rapid, sensitive, and specific diagnostic tools for MP detection.
Purpose of the Study:
- To develop a novel loop-mediated isothermal amplification (LAMP)-based assay coupled with a nanoparticle-based lateral flow biosensor (LAMP-LFB).
- To achieve rapid, sensitive, and specific diagnosis of Mycoplasma pneumoniae (MP) infections.
- To validate the assay's performance using clinical samples and compare it with existing methods.
Main Methods:
- A set of six primers targeting the community-acquired respiratory distress syndrome (CARDS) toxin gene of MP was designed.
- The LAMP assay was optimized to run at 63°C for 30 minutes.
- The LAMP products were detected using a lateral flow biosensor (LFB) within 2 minutes, enabling a total assay time of under one hour.
Main Results:
- The MP-LAMP-LFB assay demonstrated high sensitivity with a detection limit of 50 fg per reaction.
- The assay showed no cross-reactivity with other common respiratory pathogens.
- Validation with 100 nasopharyngeal swab samples from children suspected of MPP revealed a 50% positive rate, consistent with real-time PCR results.
Conclusions:
- The developed MP-LAMP-LFB assay is a rapid, highly sensitive, and specific diagnostic tool for Mycoplasma pneumoniae.
- This assay is suitable for point-of-care settings and basic medical facilities, especially in rural areas.
- The MP-LAMP-LFB assay offers a valuable alternative for timely MP diagnosis and management.
