Bulletin "Veterinary biotechnology"

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Veterynarna biotekhnolohiia – Veterinary biotechnology, 2023, 43, 133-146 [in Ukrainian]. https://doi.org/10.31073/vet_biotech43-13

MOLOZHANOVA A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., NYCHYK S., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., HUDZ N., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., TARASOV O., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS

 

APPLICATION OF THE LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) FOR THE SARS-COV-2 VIRUS DIAGNOSTICS DETECTION

Introduction. The SARS-CoV-2 virus is a single-stranded enveloped RNA virus belonging to the Sarbeccovirus subgenus of the Betacoronavirus genus.

Timely diagnosis is an important measure in the system of preventing the spread of the pathogen. In addition to the classical polymerase chain reaction (PCR) and sequencing, detection of specific fragments of the viral genome is also carried out using LAMP technology, which is considered an alternative to reverse transcription PCR and is based on the unique feature of DNA polymerase from the bacterium Bacillus stearothermophillus (Geobacillus), which, in addition to DNA polymerase, also has high reverse transcription activity. This circumstance greatly simplifies the procedure of “rewriting” information from RNA to DNA with its subsequent one-step amplification, which is possible at a constant temperature in the range from 55°C to 65°C.

Thus, the application of new approaches to the diagnosis of viral and bacterial diseases is important for the rapid detection of the pathogen and the timely application of preventive measures aimed at preventing its spread among the susceptible population.

The goal of the work. To investigate the possibility of using loop-mediated isothermal amplification (LAMP) for the rapid and effective diagnosis of SARS-CoV-2 virus.

Materials and methods. RNA was isolated from biological samples using the IndiSpin Pathogen kit (Indical Bioscience) according to the manufacturer’s instructions.

Detection of specific fragments of the SARS-CoV-2 virus genome was performed by RT-qPCR using a commercial test system Allplex SARS-CoV-2 Assay (Seegene, South Korea), Ref RV10248X, LOT RV9120H23 according to the manufacturer’s guidelines on a BioRad 1000 plate-type amplifier with SFX96 module using the built-in software.

The RT-qPCR test results were evaluated using the SARS-CoV-2 Viewer V1 program (Seegene).

The detection of specific viral RNA fragments was also performed using LAMP technology (RT-LAMP), which is considered an alternative to reverse transcription PCR.

For the study, primer sets for RT-LAMP were used for the following gene regions: nucleocapsid gene N-gene and envelope gene S-gene of SARS-CoV-2.

The optimal conditions for RT-LAMP amplification with colorimetric evaluation of the endpoint results were selected experimentally, taking into account that the mixture should be of the minimum composition necessary for the reaction, since it was necessary to ensure that the change in pH of the solution, and the color after the reaction, would change only under the influence of specific amplification products that accumulate in the reaction mixture.

Results of research and discussion. Despite the fact that a number of authors claim an exceptionally high specificity and sensitivity of RT-LAMP, we were able to obtain results that coincide with those obtained by the classical RT-qPCR method only under conditions of a significant content of viral genomes in the reaction (sample). In this case, the Ct value should be within the range of no more than 25. For samples in the range of Ct 26-30, the sensitivity and specificity of RT-LAMP is significantly reduced. For samples with a low number of target genomes, which is typical for Ct 35-45, it is inappropriate to use RT-LAMP due to low specificity and a high risk of false-positive and false-negative results.

When analyzing the test results of the RT-LAMP method for the primer set for the N gene fragment, we observed a sensitivity limitation of about Ct ≈ 30-32. A direct comparison of the Ct values for the S and N gene primer sets for all samples revealed a difference of approximately 1.2-2.3 Ct. This suggested that the N gene primers were more sensitive than the S gene primers for the detection of SARS-CoV-2 RNA using this test.

To determine the specificity and sensitivity of the RT-LAMP assay, we analyzed all available positive samples. In total, we analyzed 25 RNA samples, repeating the assay at least 3 times. Visualization of the RT-LAMP analysis results 30 min after the start of incubation at 64°C showed the expected results, indicating that the RT-LAMP analysis had sufficient reproducibility. The consistency of the results during the assay confirmed the threshold of ΔOD >+0.3 as a reliable indicator for identifying samples that were positive for SARS-CoV-2 RNA.

Conclusions and prospects for further research: 1. The RT-LAMP method can be used to detect SARS-CoV-2 virus in samples with a significant number of pathogen genomes – 600 or more. For the RT-qPCR Ct values < 25, the method corresponds to the sensitivity and specificity of the standard RT-qPCR test. For colorimetric evaluation of the endpoint reaction, it is not recommended to use a reaction time of more than 30 minutes, as nonspecific late amplification may cause false-positive results.

2. The overall specificity of the RT-LAMP assay for the N gene was 99.4%, and the sensitivity for samples with a Ct score < 30 on RT-qPCR was 98.5%. For the S gene, the overall specificity was 92.8%, and the sensitivity for samples with a Ct < 30 was 96.8%. The overall specificity of RT-LAMP with colorimetric evaluation of the test results for the N gene was 85.7%, and the sensitivity was 98.5%. The overall specificity of RT-LAMP with colorimetric evaluation of the test results for the S gene was 75%, and the sensitivity was 98.5%.

Further studies are planned to investigate the sensitivity and specificity of the primer set for the detection of new mutant strains of SARS-CoV-2 virus.

Keywords: COVID-19, SARS-CoV-2, RT-qPCR, LAMP, diagnostcs.

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