This article describes the loop-mediated isothermal amplification (LAMP) technique for detecting specific bacterial genes. The method utilizes a strand-displacing DNA polymerase and real-time fluorescence monitoring to achieve exponential amplification without thermal cycling.
Take a master mix containing a strand-displacing DNA polymerase, nucleotides, and a double-stranded DNA-binding dye for loop-mediated isothermal amplification or LAMP.
Add primers that contain a reverse-complementary sequence specific to the target bacterial gene.
Distribute the master mix into the tubes.
Add samples containing bacterial DNA templates to the tubes.
Place the tubes inside a LAMP instrument.
Initial denaturation allows the primer to bind to the target DNA.
As the strand-displacing DNA polymerase starts synthesizing a new strand, it displaces the existing strand, eliminating the need for thermal cycling.
The amplified product forms a looped structure due to the reverse-complementary pairing.
This structure contains multiple start sites for the next cycle, facilitating exponential amplification of target DNA.
The dye binds to the amplified DNA and emits fluorescence.
Monitor the fluorescence in real-time to detect the presence of the target bacterial gene in the samples.
Physically separate the areas used for preparing the LAMP Master Mix and adding the DNA templates.
Clean the work surfaces with isopropanol and a DNA and DNase degrading solution. Then, clean the pipettes and tube strip holders. Thaw the isothermal Master Mix, 10X Primer Mix, molecular grade water, positive control DNA, and DNA templates at room temperature.
Turn on the LAMP instrument, and enter the relevant sample information. Prepare the LAMP Master Mix according to manuscript directions, vortex and centrifuge it briefly, and distribute 23 microliters of the Master Mix into each well of a tube strip. Vortex all DNA templates and centrifuge them briefly.
Then, add two microliters of DNA template to the appropriate well, and cap the well tightly. Remove the tube strip from the holder, and flick it to ensure all reagents have pooled at the bottom of the tube. Load the tube strip into the LAMP instrument, and start the LAMP run.
While the LAMP reaction is in progress, tap the Temperature, Amplification, and Anneal tabs to see the dynamic changes of various parameters during the LAMP run.
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