研究目的
To develop a simple, low-cost method for digital loop-mediated isothermal amplification (LAMP) and reverse-transcription LAMP (RT-LAMP) using a commercial membrane for absolute quantification of nucleic acids and viruses without complex equipment or chip fabrication.
研究成果
The membrane-based digital LAMP system provides a simple, low-cost, and effective method for absolute quantification of nucleic acids and viruses, with high specificity and minimal equipment requirements. It is suitable for point-of-care applications and can be further integrated with other technologies for enhanced functionality.
研究不足
The detection limit is relatively high (10 copies/μL) due to small droplet size, and there is a potential error of up to 3% from pore overlap. The method requires fluorescence microscopy for readout, which may not be universally accessible.
1：Experimental Design and Method Selection:
The study utilized a commercial track-etched polycarbonate (PCTE) membrane for sample partitioning, with a peel-off process to remove residual solution. Digital LAMP and RT-LAMP were performed isothermally at 65°C. Theoretical error analysis was conducted using Random Distribution Model and Multi-volume Theory.
2：Sample Selection and Data Sources:
Genomic DNA from Escherichia coli, Enterococcus faecalis, and Salmonella Typhi, and MS2 virus in wastewater samples were used. DNA and virus concentrations were quantified using standard methods like droplet digital PCR and plaque assays.
3：List of Experimental Equipment and Materials:
PCTE membranes from Sterlitech Corporation and GVS Filter Technology, PDMS films, LAMP reagents from New England Biolabs, primers from Integrated DNA Technologies, calcein and MnCl2 from Sigma-Aldrich, mineral oil, frame-seal from Bio-Rad, hotplate (MJ Research PTC-100), fluorescence microscope (Leica DMi8), and various chemicals and culture media.
4：Experimental Procedures and Operational Workflow:
LAMP mix was added to the membrane, sealed with PDMS films, peeled off to remove residue, covered with mineral oil and frame-seal, incubated at 65°C for 40 minutes, and imaged with a fluorescence microscope. Positive pores were counted using ImageJ software and analyzed with Poisson distribution.
5：Data Analysis Methods:
Data were analyzed using Poisson distribution for absolute quantification, with error analysis based on pore size distribution. Fluorescence images were processed with ImageJ, and statistical methods were applied for correlation and error calculations.
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PDMS film
Sylgard 184
Dow Corning
Used for sealing the membrane and facilitating the peel-off process to remove residual solution.
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Fluorescence microscope
DMi8
Leica
Used for imaging the membrane after amplification to count positive pores.
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PCTE membrane
25 μm pore size
Sterlitech Corporation
Used as a substrate for sample partitioning and digital LAMP reactions, with each pore acting as a nanoreactor.
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Hotplate
PTC-100
MJ Research
Used for isothermal incubation at 65°C during LAMP amplification.
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Droplet digital PCR system
QX200
Bio-Rad
Used for precise measurement of genomic DNA concentration as a reference method.
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Real-time PCR system
RealPlex2
Eppendorf
Used for qPCR and qLAMP experiments to compare with digital methods.
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Syringe filter
0.2 μm
GE Whatman
Used for purifying MS2 virus suspensions by removing bacterial cells and debris.
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Frame-seal
Bio-Rad
Used to cover the membrane to prevent evaporation during incubation.
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