The RT-LAMP Based Test For Coronavirus

The coronavirus disease 2019 (COVID-19) pandemic has infected millions of individuals. The new coronavirus (SARS-CoV-2) epidemic offers a significant health danger to the general public. As a result, early and correct detection of the virus is critical for disease treatment and lifesaving. The reverse transcription-polymerase chain reaction (RT-PCR) technology is the current standard for coronavirus detection. Isothermal PCR, such as loop-mediated isothermal amplification (LAMP), is a viable alternative to RT-PCR for pandemic surveillance. Amplification at a fixed temperature, the absence of a thermal cycler, and a speedier test result are all advantages of the LAMP test.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP):

LAMP stands for loop-mediated isothermal amplification, and it’s a viral RNA detection technique. The COVID-19 virus was successfully detected using reverse-transcription LAMP (RT-LAMP). Loop-mediated isothermal amplification eliminates the need for heat-induced DNA denaturation. The LAMP method requires only one steady temperature (often 65 °C).

RT LAMP test working:

The RT-LAMP technology was based on the mechanism of auto cycling strand displacement DNA synthesis. A polymerase carries out the reaction with a high strand displacement activity. Also, two primer pairs are used: one pair of inner primers and one pair of outside primers. These primers are custom-made for the reaction.

Because of the target sequences, the RT-LAMP test can achieve great specificity. RT-LAMP test, unlike other technologies, detects the target sequence utilising six different sequences at first and four independent sequences later on.

The recognition of the target genome by a primer results in a strong colourimetric reaction, allowing detection without highly specialised or expensive instruments. To evaluate viral content, similar approaches rely on the turbidity of the sample, which increases as the number of genetic material increases. Agarose gel analysis can also be used for isothermal amplification and detection.

Because of its reliability and precision of use, RT-LAMP is an ideal technology to be used in the COVID-19 pandemic. This means that tests can be performed in non-traditional settings such as airports, rural hospitals, and medical centres.

COVID-19 detection in less than 30 minutes using RT-LAMP:

A COVID-19 quick screening LAMP test with less than 30 minutes of assay time has been developed. The initial stage in developing the RT-LAMP assay is to determine the virus’s target sequences and construct appropriate LAMP primers. Online databases such as GenBank were used to find consensus sequences for 23 distinct strains. Similar simulated samples were created for MERS viruses or other coronaviruses studies without isolating RNA from samples. The developed RT-LAMP test was validated using healthy human samples.

The method was first tuned to run for 30 minutes at 63 degrees Celsius. Multiple testing with other pathogens such as MERS, MHV, and BtCoV revealed that the RT-LAMP test had a high specificity for COVID-19, as no signal was recorded. The sensitivity of LAMP primers is further demonstrated by comparison to SARS and other coronavirus sequences. With all COVID-19 strains, the primer had a 0% mismatch, whereas other common coronaviruses had a 27–54 percent mismatch. The assay sensitivity was tested using simulated samples, with the detection limit as low as 1.02 FG/reaction. Finally, the technique was shown to have excellent clinic usefulness, as it successfully screened for COVID-19 regardless of the spiked sample type. The COVID-19 sequence was spiked in serum, urine, saliva, oropharyngeal swab, and nasopharyngeal swab samples, and they were all accurately identified. It demonstrates the possibility of quickly diagnosing COVID-19.

Benefits of RT-LAMP:

The advantages of RT-LAMP are critical for the technology’s widespread adoption. The most important of these is that genetic material is amplified swiftly under isothermal conditions, eliminating the need for a thermal cycler. Because there is no need to wait for temperature changes, and thus no time is lost, better amplification efficiency is possible. The RT-LAMP test takes less than an hour, compared to 3 to 4 hours for standard PCR. The great specificity and selectivity for the genetic material in question are also advantages. In certain studies, RT-LAMP was 10 times more sensitive than standard RT-PCR tests. The specificity of RT-LAMP is also higher, with no false-positive results observed.

Conclusion:

Rapid, low-cost, and user-friendly molecular diagnostic technologies are required to combat infectious disease outbreaks. Especially during the COVID-19 pandemic, there is an urgent need to increase global testing capacity by 100 times what is now possible using normal methods. LAMP is a cutting-edge gene amplification approach that holds a lot of potential as a detection tool, especially in the midst of the coronavirus epidemic. Another advantage is the overall ease of usage, as the isothermal amplification test allows for the use of simple, low-cost equipment. Because there is no time loss due to thermal cycling in conventional RT-PCR, the isothermal setting also aids the LAMP in high amplification efficiency.