Comparative Clinical Evaluation of a Novel FluA/FluB/SARS-CoV-2 Multiplex LAMP and Commercial FluA/FluB/SARS-CoV-2/RSV RT-qPCR Assays.

Influenza and coronaviruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Public health measures implemented during the current coronavirus disease (COVID-19) pandemic have gradually reduced influenza circulation worldwide. As COVID-19 measures have relaxed, it is necessary to monitor and control seasonal influenza during this COVID-19 pandemic. In particular, the development of rapid and accurate diagnostic methods for influenza and COVID-19 is of paramount importance because both diseases have significant public health and economic impacts. To address this, we developed a multi-loop-mediated isothermal amplification (LAMP) kit capable of simultaneously detecting influenza A/B and SARS-CoV-2. The kit was optimized by testing various ratios of primer sets for influenza A/B (FluA/FluB) and SARS-CoV-2 and internal control (IC). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed 100% specificity for uninfected clinical samples and sensitivities of 90.6%, 86.89%, and 98.96% for LAMP kits against influenza A, influenza B, and SARS-CoV-2 clinical samples, respectively. Finally, the attribute agreement analysis for clinical tests indicated substantial agreement between the multiplex FluA/FluB/SARS-CoV-2/IC LAMP and commercial AllplexTM SARS-CoV-2/FluA/FluB/RSV assays.

An inexpensive and rapid diagnostic method for detection of SARS-CoV-2 RNA by loop-mediated isothermal amplification (LAMP).

SARS-CoV-2 is a public pandemic health concern globally. Nasopharyngeal and oropharyngeal swab samples are used for Covid-19 viral detection. Sample collection procedure was tedious and uncomfortable and unsuitable for biochemical and CBC analysis in swab samples. Biochemistry and CBC tests are key determinant in management of Covid-19 patients. We developed a LAMP test to detect viral RNA in blood samples. LAMP is required four specific primers targeting the internal transcribed S-region and loop primers for viral RNA amplification. RNA was extracted from blood samples by TRIzol method. LAMP reaction was performed at 60 °C for 1 hour and amplicons were visualized in HNB dye. No cross-reactivity was seen with HBV, HCV, and HIV infected sample. Out of 40 blood samples, 33 samples were positive for LAMP and Q-PCR analysis, one sample was positive for LAMP and negative for Q-PCR, two samples were negative for LAMP but positive for Q-PCR, and four blood samples were negative for LAMP and Q-PCR. LAMP method has an accuracy of 92.50%, with sensitivity and specificity of 94.28% and 80%, respectively. Thus, LAMP diagnostic test has proved reliable, fast, inexpensive and can be useful for detection where the limited resources available.•LAMP method is a potential tool for detection of SARS-CoV-2.•Blood samples are the key determinant for routine diagnostics as well as molecular diagnostics.•LAMP assay is an appropriate diagnostics method which offers greater simplicity, low cost, sensitivity, and specificity than other methods in molecular diagnostics.

An immobilization-based, loop-mediated isothermal amplification device for nucleic acid detection of SARS-CoV-2 N gene

Confronting the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), simple, fast and specific non-laboratory SARS-CoV-2 diagnostic tests are urgently required. However, the current nucleic acid assays generally rely on the diagnostic laboratory, trained staff and specialized equipment for execution and analysis, presenting clear limitations in the field detection. Here, we describe a portable and reliable immobilization-based loop-mediated isothermal amplification (LAMP) device which is mobile, without the requirement of any complicated instrument and appropriate for high-throughput testing. This device was constructed by utilizing the interaction between a carboxyl-tagged primer and an amino-tagged substrate, and capable of catching the target sequence in SARS-CoV-2 produced via the immobilization-based LAMP. In this study, the immobilization conditions and immobilized primer structure were explored and optimized. With this proposed device, the analysis result can be obtained rapidly in 30 min with excellent specificity, even if the template is extracted from a complex sample containing pharyngeal swab or human blood. In addition, the device can be applied to detect the nucleic acid of SARS-CoV-2 and various other pathogens, showing attractive potential for rapid and high-throughput detection at airports, railway stations, cold-chain transportations, community hospitals and so on. Therefore, we believe that the immobilization-based LAMP device is an advanced approach to developing a portable, specific, low-cost and high-throughput diagnostic platform.

Electromechanical RT-LAMP device for portable SARS-CoV-2 detection.

Rapid point-of-care tests for infectious diseases are essential, especially in pandemic conditions. We have developed a point-of-care electromechanical device to detect SARS-CoV-2 viral RNA using the reverse-transcription loop-mediated isothermal amplification (RT-LAMP) principle. The developed device can detect SARS-CoV-2 viral RNA down to 103 copies/mL and from a low amount of sample volumes (2 µL) in less than an hour of standalone operation without the need for professional labor and equipment. Integrated Peltier elements in the device keep the sample at a constant temperature, and an integrated camera allows automated monitoring of LAMP reaction in a stirring sample by using colorimetric analysis of unfocused sample images in the hue/saturation/value color space. This palm-fitting, portable and low-cost device does not require a fully focused sample image for analysis, and the operation could be stopped automatically through image analysis when the positive test results are obtained. Hence, viral infections can be detected with the portable device produced without the need for long, expensive, and labor-intensive tests and equipment, which can make the viral tests disseminated at the point-of-care.

Digital detection and quantification of SARS-CoV-2 in a droplet microfluidic all-fiber device

Silica fibers and capillaries offer opportunities for compact integration of optics with microfluidics while adding advantages such as;flexibility within a high aspect ratio format, uniaxial arrangements, and measurement-at-a-distance. Here, we describe droplet microfluidics-based nucleic acid detection of SARS-CoV-2 in a lab-in-a-fiber platform. The fiber component integrates three modules with key functions: droplet generation, incubation, and fluorescence detection. Within the scope of this work, we developed the component specifically to target the quantification of SARS-CoV-2 viral RNA through reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The all-fiber component could successfully generate uniform droplets and differentiate pre-amplified positive LAMP reaction from negative sample. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

DESIGN OPTIMIZATION OF MICROFLUIDIC DIAGNOSTIC DEVICES FOR THE RAPID GENETIC DETECTION OF MULTIPLE INFECTIOUS VIRUSES

This paper presents a newly developed microfluidic flow control theory for autonomous sample dispensing into an array of reaction microchambers. The theoretical predictions for the possible dispensing number and maximum flow rate were validated by comparison to experimental results. Moreover, we successfully demonstrated the rapid genetic detection of multiple infectious viruses including SARS-CoV-2 in fabricated polydimethylsiloxane (PDMS)-based microfluidic devices based on the loop-mediated isothermal amplification (LAMP) method. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

PCR to CRISPR: Role of Nucleic Acid Tests (NAT) in detection of COVID-19.

COVID-19 infection has emerged as an unparalleled pandemic with morbidity and mortality tolls challenging diagnostic approaches and therapeutic interventions, and raising serious questions for healthcare policy-makers. From the diagnostic perspective, Reverse transcriptase polymerase chain reaction remains the gold standard. However, issues associated with gene primer variation in different countries, low analytical sensitivity, cross-reactivity with certain human coronaviruses have raised serious concerns within the scientific community. Alongside longer turnaround times, requirements of sophisticated equipment and trained technicians are the other challenges for conventional reverse transcriptase polymerase chain reaction testing. The recent biotechnological boom has now allowed newer nucleic acid testing options for diagnosing severe acute respiratory syndrome Coronovairus 2 (SARS-CoV2) with much better diagnostic efficiency, reduced turnaround times and possible benefit for use as a point-of-care test. Isothermal techniques with simple equipment requirements along with uniform temperature for analysis have emerged to be more sensitive and specific with turnaround times as low as 10-15 minutes. Similarly, Cluster Regularly Interspaced Short Palindromic Repeats have also been seen to play a very decisive role in COVID-19 diagnostics with much superior diagnostic efficiency and feasibility as a point-of-care test and its possible use for sequencing. The current narrative review was planned to consolidate data for all possible nucleic acid testing options under research/clinical use, and to provide a comparative assessment from the perspective of both the clinician and the laboratory.

Kinetics of Isothermal Dumbbell Exponential Amplification: Effects of Mix Composition on LAMP and Its Derivatives.

Loop-mediated isothermal amplification (LAMP) is an exponential amplification method of DNA strands that is more and more used for its high performances. Thanks to its high sensitivity and selectivity, LAMP found numerous applications from the detection of pathogens or viruses through their genome amplification to its incorporation as an amplification strategy in protein or miRNA biomarker quantification. The LAMP method is composed of two stages: the first one consists in the transformation of the DNA strands into dumbbell structures formed of two stems and loops thanks to four primers; then, in the second stage, only two primers are required to amplify the dumbbells exponentially in numerous hairpins of increasing lengths. In this paper, we propose a theoretical framework to analyze the kinetics of the second stage of LAMP, the isothermal dumbbell exponential amplification (IDEA) as function of the physico-chemical parameters of the amplification reaction. Dedicated experiments validate the models. We believe these results may help the optimization of LAMP performances by reducing the number of experiments necessary to find the best parameters.

Elimination of Carryover Contamination in Real-Time Reverse Transcriptase Loop-Mediated Isothermal Amplification for Rapid Detection of the SARS-CoV-2 Virus in Point-of-Care Testing.

Loop-mediated isothermal amplification (LAMP) is being used as a robust rapid diagnostic tool to prevent the transmission of infectious diseases. However, carryover contamination of LAMP-amplified products originating from previous tests has been a problem in LAMP-based bio-analytical assays. In this study, we developed a Cod-uracil-DNA-glycosylase real-time reverse transcriptase LAMP assay (Cod-UNG-rRT-LAMP) for the elimination of carryover contamination and the rapid detection of SARS-CoV-2 in point-of-care (POC) testing. Using the Cod-UNG-rRT-LAMP assay, the SARS-CoV-2 virus could be detected as low as 2 copies/µl (8 copies/reaction) within 45 min of amplification and 2.63 ± 0.17 pg (equivalent to 2.296 × 109 copies) of contaminants per reaction could be eliminated. Analysis of clinical SARS-CoV-2 samples using the Cod-UNG-rRT-LAMP assay showed an excellent agreement with a relative accuracy of 98.2%, sensitivity of 97.1%, and specificity of 95.2% in comparison to rRT-PCR. The results obtained in this study clearly demonstrate the feasibility of the use of the Cod-UNG-rRT-LAMP assay for applications toward the POC diagnosis of SARS-CoV-2 and on-site testing of other pathogens.

Assessment of Cost-Efficient Thermocycler Prototype for Polymerase Chain Reaction and Loop-Mediated Isothermal Amplification

DNA amplification-based diagnostic is the most accurate method among others, especially during the COVID-19 pandemic. Thus, increasing the global demand for instrumentation andamplification reagents locally, hence increasing import. It is a worrying state in terms of logisticsand the future domestic market. An effort for domestic production is a must. Previously a costefficientthermocycler prototype using Raspberry Pi and Phyton coding is constructed.Thermocycler prototype flow measurement and heat distribution have previously been tested. Thisresearch aims to compare thermocycler prototypes and commercial for in two types of DNAamplification reactions, polymerase chain reaction (PCR) and loop-mediated isothermalamplification (LAMP). PCR is the most and more common method than LAMP, with the maindifference of PCR require thermal cycling and LAMP operate in isothermal conditions. LAMP has aquicker reaction time and operates at a lower temperature. DNA pol with high strand displacementactivity is used for LAMP, in this research Bsm pol is used for LAMP and Taq pol for PCR. Since theprototype thermocycler is designed to be as simple and inexpensive as possible for ease ofmanufacture and accessibility for every layer of society. Hence, its heat control and stability are notas good as a commercial thermocycler, with huge temperature fluctuation resonance from its setpoint.That causes prototype incapability of performing PCR, no DNA band at 250-500 bp range ingel electrophoresis. However, the prototype is capable of performing LAMP, existing <100 bp DNAgradient band in gel electrophoresis. The prototype is also capable of performing LAMP below itsprotocol temperature and time separately, 62°C and 40 minutes compared to the protocol of 66°Cand 60 minutes © 2021,International Journal of Technology. All Rights Reserved.

Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis.

Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 µL reaction at isothermal 58℃ within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.

Microfluidic technology and its application in the point-of-care testing field.

Since the outbreak of the coronavirus disease 2019 (COVID-19), countries around the world have suffered heavy losses of life and property. The global pandemic poses a challenge to the global public health system, and public health organizations around the world are actively looking for ways to quickly and efficiently screen for viruses. Point-of-care testing (POCT), as a fast, portable, and instant detection method, is of great significance in infectious disease detection, disease screening, pre-disease prevention, postoperative treatment, and other fields. Microfluidic technology is a comprehensive technology that involves various interdisciplinary disciplines. It is also known as a lab-on-a-chip (LOC), and can concentrate biological and chemical experiments in traditional laboratories on a chip of several square centimeters with high integration. Therefore, microfluidic devices have become the primary implementation platform of POCT technology. POCT devices based on microfluidic technology combine the advantages of both POCT and microfluids, and are expected to shine in the biomedical field. This review introduces microfluidic technology and its applications in combination with other technologies.

Loop-mediated isothermal amplification (LAMP): An effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2.

The novel coronavirus disease-2019 (Covid-19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability of advanced diagnostics and current need for cost-effective methodology. The advent of the loop-mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point-of-care test for the diagnosis of Covid-19 in endemic areas, especially for low- and middle-income countries.

A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020.

INTRODUCTION: The Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo. METHODS: We diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method. RESULTS: As a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative. CONCLUSION: The false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.

An internet of things-based point-of-care device for direct reverse-transcription-loop mediated isothermal amplification to identify SARS-CoV-2.

Rapid and accurate testing tools for SARS-CoV-2 detection are urgently needed to prevent the spreading of the virus and to take timely governmental actions. Internet of things (IoT)-based diagnostic devices would be an ideal platform for point-of-care (POC) screening of COVID-19 and ubiquitous healthcare monitoring for patients. Herein, we present an advanced IoT-based POC device for real-time direct reverse-transcription-loop mediated isothermal amplification assay to detect SARS-CoV-2. The diagnostic system is miniaturized (10 cm [height] × 9 cm [width] × 5.5 cm [length]) and lightweight (320 g), which can be operated with a portable battery and a smartphone. Once a liquid sample was loaded into an integrated microfluidic chip, a series of sample lysis, nucleic amplification, and real-time monitoring of the fluorescent signals of amplicons were automatically performed. Four reaction chambers were patterned on the chip, targeting As1e, N, E genes and a negative control, so multiple genes of SARS-CoV-2 could be simultaneously analyzed. The fluorescence intensities in each chamber were measured by a CMOS camera upon excitation with a 488 nm LED light source. The recorded data were processed by a microprocessor inside the IoT-based POC device and transferred and displayed on the wirelessly connected smartphone in real-time. The positive results could be obtained using three primer sets of SARS-CoV-2 with a limit of detection of 2 × 101 genome copies/µL, and the clinical sample of SARS-CoV-2 was successfully analyzed with high sensitivity and accuracy. Our platform could provide an advanced molecular diagnostic tool to test SARS-CoV-2 anytime and anywhere.

Capillary-based reverse transcriptase loop-mediated isothermal amplification for cost-effective and rapid point-of-care COVID-19 testing.

As the SARS-CoV-2 pandemic continues to spread, the necessity for rapid, easy diagnostic capabilities could never have been more crucial. With this aim in mind, we have developed a cost-effective and time-saving testing methodology/strategy that implements a sensitive reverse transcriptase loop-mediated amplification (RT-LAMP) assay within narrow, commercially available and cheap, glass capillaries for detection of the SARS-CoV-2 viral RNA. The methodology is compatible with widely used laboratory-based molecular testing protocols and currently available infrastructure. It employs a simple rapid extraction protocol that lyses the virus, releasing sufficient genetic material for amplification. This extracted viral RNA is then amplified using a SARS-CoV-2 RT-LAMP kit, at a constant temperature and the resulting amplified product produces a colour change which can be visually interpreted. This testing protocol, in conjunction with the RT-LAMP assay, has a sensitivity of ∼100 viral copies per reaction of a sample and provides results in a little over 30 min. As the assay is carried out in a water bath, commonly available within most testing laboratories, it eliminates the need for specialised instruments and associated skills. In addition, our testing pathway requires a significantly reduced quantity of reagents per test while providing comparable sensitivity and specificity to the RT-LAMP kit used in this study. While the conventional technique requires 25 µl of reagent, our test only utilises less than half the quantity (10 µl). Thus, with its minimalistic approach, this capillary-based assay could be a promising alternative to the conventional testing, owing to the fact that it can be performed in resource-limited settings, using readily available apparatus, and has the potential of increasing the overall testing capacity, while also reducing the burden on supply chains for mass testing.

Laboratory-Based Resources for COVID-19 Diagnostics: Traditional Tools and Novel Technologies. A Perspective of Personalized Medicine.

The coronavirus infection 2019 (COVID-19) pandemic, caused by the highly contagious SARS-CoV-2 virus, has provoked a global healthcare and economic crisis. The control over the spread of the disease requires an efficient and scalable laboratory-based strategy for testing the population based on multiple platforms to provide rapid and accurate diagnosis. With the onset of the pandemic, the reverse transcription polymerase chain reaction (RT-PCR) method has become a standard diagnostic tool, which has received wide clinical use. In large-scale and repeated examinations, these tests can identify infected patients with COVID-19, with their accuracy, however, dependent on many factors, while the entire process takes up to 6-8 h. Here we also describe a number of serological systems for detecting antibodies against SARS-CoV-2. These are used to assess the level of population immunity in various categories of people, as well as for retrospective diagnosis of asymptomatic and mild COVID-19 in patients. However, the widespread use of traditional diagnostic tools in the context of the rapid spread of COVID-19 is hampered by a number of limitations. Therefore, the sharp increase in the number of patients with COVID-19 necessitates creation of new rapid, inexpensive, sensitive, and specific tests. In this regard, we focus on new laboratory technologies such as loop mediated isothermal amplification (LAMP) and lateral flow immunoassay (LFIA), which have proven to work well in the COVID-19 diagnostics and can become a worthy alternative to traditional laboratory-based diagnostics resources. To cope with the COVID-19 pandemic, the healthcare system requires a combination of various types of laboratory diagnostic testing techniques, whodse sensitivity and specificity increases with the progress in the SARS-CoV-2 research. The testing strategy should be designed in such a way to provide, depending on the timing of examination and the severity of the infection in patients, large-scale and repeated examinations based on the principle: screening-monitoring-control. The search and development of new methods for rapid diagnostics of COVID-19 in laboratory, based on new analytical platforms, is still a highly important and urgent healthcare issue. In the final part of the review, special emphasis is made on the relevance of the concept of personalized medicine to combat the COVID-19 pandemic in the light of the recent studies carried out to identify the causes of variation in individual susceptibility to SARS-CoV-2 and increase the efficiency and cost-effectiveness of treatment.

Biosensing Detection of the SARS-CoV-2 D614G Mutation.

The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis.

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 102 CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field.

A case of COVID-19 pneumonia successfully treated with favipiravir (Avigan) in which serum SARS-CoV-2 RNA detected by LAMP method was clinically useful.

A 49-year-old Japanese male was managed by mechanical ventilation due to coronavirus disease 2019 (COVID-19) pneumonia. Favipiravir as an antiviral therapy, and anti-inflammatory treatment were administered. SARS-CoV-2 RNA was detected in serum by the loop-mediated isothermal amplification (LAMP) method on Day 9; favipiravir treatment was continued. On Day 13, negative serum RNA was confirmed, followed by mechanical ventilation was removed. On Day 23, LAMP negative was confirmed in nasopharynx, after that the patient discharged on Day 27. We could treat successfully for severe COVID-19 pneumonia based on the LAMP method. We consider this method will be useful in COVID-19 treatment.