To begin, switch on the laminar airflow cabinet, allowing it to stabilize for three minutes. Aseptically clean and sanitize the cabinet's internal surfaces using the following disinfection agents. After disinfecting any material that will enter the cabinet, place napkins and nitrile gloves into the cabinet.
Then switch off the cabinet and expose it to ultraviolet light for 15 minutes. After the irradiation, restart the cabinet. Place the primer design reagents in an ice-filled cooler.
After sanitizing it with 70%ethanol, place the container inside the cabinet. Prepare the loop mediated isothermal amplification, or LAMP mix, in a 0.6 milliliter microcentrifuge tube. Pipette the mixture to homogenize it.
Next, incubate the closed tubes in a heating block at 95 degrees Celsius for five minutes. Then cool the tubes in an ice-filled polystyrene cooler for five minutes. After cooling, place the tubes in the laminar flow cabinet.
To complete the LAMP mix, add four microliters of DNA polymerase, followed by one microliter of reverse transcriptase. To perform colorimetric detection, add the dihydroxynaphthol blue. After reagent addition, pipette each solution to properly mix the LAMP reagents.
Next, pipette 22 microliters of the solution into PCR tubes, being careful not to create bubbles. For the negative control, add three microliters of 0.1%diethyl pyrocarbonate water. Keep aside any remaining tubes for the addition of genetic material.
Next, remove all materials from the cabinet. After sanitizing the cabinet surfaces with 70%ethanol, shut it off. In a separate area, add three microliters of the sample to each PCR tube and pipette well with a 20 microliter micropipette to thoroughly homogenize.
Before initiating the colorimetric reaction, use a high quality camera to take pictures of the PCR tubes. Now, carry out the reaction using a thermal cycler and a water bath. For the thermal cycler, place the tubes into the reaction block and set up thermo profile on the equipment.
If using the water bath, securely place the tubes in circular containers, then place these containers in the water bath at 66.3 degrees Celsius for 60 minutes. After the reaction time, remove the tubes and store them at four degrees Celsius until use. If a colorimetric reaction was performed, capture images of the PCR with a high quality camera.
A temperature gradient demonstrated that the optimal melting temperature was 66.3 degrees Celsius. The concentration of BST 3.0 enzyme was optimized to 3.2 international units per microliter. To define the colorimetric strategy, phenol red in neutral red dyes were evaluated.
However, no color change was observed after amplification. Standardization of different concentrations of hydroxynaphthol blue was performed with the optimum change occurring with 125 micromoles of hydroxynaphthol blue. The amplified samples demonstrated a change in the color in accordance with their concentration.
Sample amplification was further confirmed by electrophoresis.
