Our research aims to standardize LAMP as a prediagnostic test suitable for field or primary care settings, like in advanced laboratory infrastructure. We seek to promote the use of locally producible buffers and alternative dyes, reducing independence on imported commercial kits. Currently, CRISPR-Cas is an efficient system deployed in the development of fast, specific, and sensitive diagnostic methods for pathogens.
Now, this system has a broad range of Cas enzymes and supplements to detect RNA or DNA targets in different samples of interest. The current experimental challenges involve preventing non-target amplification resulting from amplicon contamination and primary cell amplification, especially with lengthy primers prone to dimer formation. Additionally, we aim to determine optimal reaction conditions to achieve habitually distinguishable color change in the dye.
Once standardized and fully validated, this method can be easily implemented and adapted to any laboratory or point of care within 60 minutes at low cost, and using inexpensive equipment to detect almost any pathogen. Thus, it can potentially be used in primary care centers to carry out timely epidemiological surveillance. Our multidisciplinary group is focused on the discovery of molecules from native Colombia plants, trying to identify the potential to inhibit the replication of viruses such as dengue, Zika, or SARS-CoV-2.
In addition, we are conducting studies to develop molecular diagnostic methods for these viruses. 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 the 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 dye, hydroxynapthol 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%diethylpyrocarbonate water.
Keep aside any remaining tubes for the addition of genetic material. Next, remove all materials from the cabinet. After sanitizing the cabinet's 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 the 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 4 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 and neutral red dyes were evaluated. However, no color change was observed after amplification. Standardization of different concentrations of hydroxynapthol blue was performed, with the optimum change occurring with 125 micromoles of hydroxynapthol blue.
The amplified samples demonstrated a change in the color in accordance with their concentration. Sample amplification was further confirmed by electrophoresis.
