The overall goal of this procedure is to demonstrate the use of a multiplexed point-of-care diagnostics platform that does not require extensive sample preparation and expensive instrumentation and it's used in a variety of biological samples. This method can help answer key questions in the field of point-of-care diagnostics such as detection of different targets in single reaction tube. By using loop-mediated isothermal amplification coupled with target-specific strand displacing probes, three different viruses can be efficiently detected.
The main advantage of this technique is that it uses isothermal amplification instead of PCR and it requires minimum sample preparation and can be applied to a range of clinical samples. Begin by cutting Q-paper sheets into small rectangles of three millimeters by four millimeters using a paper punch. Then use a micro pestle to crush Aedes aegypti female mosquitoes potentially infected with Zika onto each paper.
Pipette 20 microliters of one molar aqueous ammonia solution onto each piece of paper. After five minutes, wash each paper with 20 microliters of 50%ethanol, followed by 20 microliters of nuclease-free water. Air dry the paper in a BSL-2 biosafety cabinet for about one hour.
After drying, use tweezers to fully immerse each paper in 100 microliters of RT-LAMP reaction mixture and incubate at 65 to 68 degrees Celsius for 45 minutes. Read and record the fluorescence arising from Zika virus using a 3D printed observing box with an LED light source and an orange or yellow filter. Begin by adding 90 microliters of 1.1 fold rehydration buffer to each reaction tube containing lyophilized RT-LAMP reagents and dissolve by shaking.
Then briefly spin down at 1, 000 times gravity. Following centrifugation, add 10 microliters of urine sample spiked with the viral RNA to the reaction tubes and mix by pipetting. If testing mosquitoes, add a rectangle of Q-paper holding mosquito carcasses along with 10 microliters of purified water instead of urine.
Place the closed tubes in a heat block preset at 65 to 68 degrees Celsius and incubate for 30 to 45 minutes. After the incubation, keep the tubes closed to prevent contamination of future assays and place in the observing box. Turn on the switch on the back of the observing box.
Observe the sample through the orange filter and capture the image by a cellphone camera which is held at a distance where the image fills 80%of the field of view. After visualization is complete, turn off the switch. Store the sealed tubes in the dark preferably with refrigeration if later visualization is needed.
In both singleplex and multiplex cases, RT-LAMP products appear as a letter of concatemers when run on agarose gel. Negative control samples containing water as sample gave only the bands for primers themselves including the nonspecific target control where total nucleic acid extracted from a noninfected female mosquito was used as a template. Green fluorescence is observed for Zika using probes as five prime NT labeled with FAM.
Light green yellow fluorescence is assigned to Chikungunya where the probe is five prime NT labeled with HEX. An orange red fluorescence is used for Dengue-1 where its probe is five prime NT labeled with TAMRA. Two papers containing mosquitoes were directly submerged in the RT-LAMP mixture and after incubation at 65 degrees Celsius for 30 minutes, bright green fluorescence was observed in the presence of Zika virus.
Using nine volumes of rehydration buffer and one volume of urine sample, visible green fluorescence can be generated within 30 minutes and the image can be captured by any cellphone camera. After its development, this technique paved the way for researchers in the field of clinical diagnostics to explore point-of-care platforms to detect mosquito-borne viruses in urine, blood, saliva, or mosquito carcasses. After watching this video, you should have a good understanding of how to use an isothermal amplification system, in this case it's lamp, to detect Zika and other arboviruses in multiplex fashion in a variety of biological samples.
