WO2022013761A1 - System and method for screening patients infected with severe acute respiratory syndrome coronavirus 2 - Google Patents

Abstract

A method, a system, and a kit for detecting presence of SARS-CoV-2 in a sample are provided. The method includes a plurality of first primers where the primers hybridize to the genome of the SARS-CoV-2 or cDNA thereof. The method further includes a plurality of second primers where the primers hybridize to the human beta-actin mRNA or cDNA thereof. The system includes a WarmStart reverse transcriptase, a Bst 2.0 WarmStart DNA polymerase, a first plurality of tubes for assaying the SARS-CoV-2 mRNA or the cDNA thereof; and a second plurality of tubes for independent and parallel assaying of the human beta-actin gene or mRNA thereof. A kit for diagnosis of SARS-CoV-2 in a sample is also provided.

Description

SYSTEM AND METHOD FOR SCREENING PATIENTS INFECTED WITH SEVERE ACUTE RESPIRATORY SYNDROME CORONA VIRUS 2

This patent application claims priority from the Sri Lankan Application No. 21222 filed on July 14, 2020.

TECHNICAL FIELD

The present disclosure relates to a method and a system of infection detection, and more particularly, for screening and diagnosis of infection in a subject.

BACKGROUND

Coronavirus disease 2019 or Covid-19 is a highly contagious disease caused by a vims known as Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). The contagious nature of the disease has led to a worldwide spread resulting in a pandemic causing millions of deaths. One of the challenges that pandemics such as Covid-19 bring is that the disease can be transmitted to others by asymptomatic individuals. Thus, early and rapid diagnosis becomes critical to contain the disease.

Unfortunately, problems associated with the current assays include quality control, time- consuming processes, and costly equipment. Conversely, such a scenario increases the likelihood of getting the infection especially during treatment in a hospital environment.

Diagnosis of COVID-19 through clinical signs alone is ineffective due to the extreme variability in time taken for expression of clinical signs and the non- specificity of clinical signs that are most shared with that of common flu. Therefore, confirmatory identification of a suspected infected individual even before the expression of clinical signs (based on contact tracing) is extremely helpful as it provides the opportunity to isolate the infected individuals and control the spread of the virus. The available standard for confirmatory diagnosis is real-time RT-PCR (L. E. Lamb, Bartolone, Ward, & Chancellor, 2020); which requires a real-time PCR machine and many other proprietary laboratory consumables which are extremely expensive to purchase and difficult to operate.

However, the diagnosis is critical to the containment of COVID-19 and widespread diagnostic testing over the upcoming years as vaccination is not fully effective in preventing infections in immunized individuals as per the current publicly available data. Furthermore, surveillance testing directed towards identifying diseased individuals will help minimize the spread of the disease. However, at present, many nations have a limited number of PCR laboratories due to high resource requirements in establishing PCR laboratories. Further, due to the high global demand for PCR consumables, their availability is limited to cater to the needs of the less privileged countries. As a result, the number of tests performed remains at a sub-optimal level in many countries compared to the developed world. Therefore, rapid, reliable, and cost-effective testing methods with minimum intrastate requirements are essential to ease the testing burden on governments. Early identification and isolation of infected individuals is key to the success of COVID-19 control. Thus, there is a dire need for a simple, cost-effective, portable, scalable, and universally applicable method and system to deal with a pandemic such as Covid-19.

SUMMARY

In one aspect of the present disclosure, a method for identifying a subject infected with a SARS-CoV-2 virus is disclosed. The method includes extracting SARS-CoV-2 RNA and human beta-actin mRNA from a sample obtained from the subject. The method further includes reverse transcribing the SARS-CoV-2 RNA to synthesize cDNA and amplifying the SARS-CoV-2 cDNA by a set of one or more first primers that hybridize to genome of the SARS-CoV-2 or the cDNA thereof. The method further includes reverse transcribing the human beta-actin mRNA to synthesize corresponding cDNA and amplifying the human beta-actin cDNA. The set of one or more second primers are completely bound to the beta- actin cDNA and partially bound to a beta-actin gene with a hanging 3’ end.

In various embodiments, the method includes 55-95% bases in 5’ end of the set of one or more second primers complementary to one exon region of the mRNA or the cDNA thereof. In various embodiments, the method further includes 5-45% bases in the 3’ end of the second primers complementary to an adjacent exon. In an embodiment of the present disclosure, the method includes a reverse transcriptase to generate the cDNA and a DNA polymerase enzyme as part of a reaction mixture to amplify the cDNA. In an exemplary embodiment, the method includes separately performing the amplification of the SARS-CoV-2 and the human beta-actin for detection. In some embodiments, the set of one or more first primers include SEQ. ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the set of one or more second primers include SEQ. ID No. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the SARS-CoV-2 vims infection identification in the sample is performed via Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT- LAMP). In some examples, the RT-LAMP is colorimetric. In some exemplary embodiments of the present method, the sample is a tissue or a body fluid including blood, saliva, nasal drops, nasal swab, throat swab, pharyngeal swab, alveolar lavage fluid, plasma, teardrops, urine, stool, semen, or a tissue sample.

In another aspect of the present disclosure, a system for detecting the presence of SARS- CoV-2 in a sample from a subject is disclosed. The system including one or more extraction tubes configured to receive the sample from the subject including an open top portion and a closed bottom portion, and sidewalls encompassing a sample void disposed with a straining apparatus inside each of the tubes. The system includes one or more of first set of tubes configured to receive the sample from the subject comprising an open top portion and a closed bottom portion, and sidewalls encompassing a sample void and a cover configured to couple with the top portion. The cover is connected to the first set of tube body by a flexible joint. The system includes a plurality of racks configured to accommodate the one or more of first set of tubes and a set of PCR tubes for RT-LAMP assay. The plurality of the racks include a first rack channel encompasses the first set of tubes comprising an inner volume and a second channel encompasses the PCR tubes having a closed end, an open end and an inner diameter and comprising an inner volume and a set of one or more first primers to hybridize to genome of the SARS-CoV-2 or the cDNA thereof present in the one or more PCR tubes and a set of one or more second primers to amplifying the human beta-actin cDNA present in the one or more first set of tubes; and a reverse transcriptase and a DNA polymerase as part of a reaction mixture present in the first set of tubes and the PCR tubes and an RNAse inhibiting preparation present in the extraction tubes. In some exemplary embodiments of the present system, the sample is a tissue or a body fluid including blood, saliva, nasal drops, nasal swab, throat swab, pharyngeal swab, alveolar lavage fluid, plasma, teardrops, urine, stool, semen, or a tissue sample.

In various embodiments, the present disclosure includes a kit for performing RT-LAMP of a target nucleic acid sequence for identifying a subject infected with the SARS-CoV-2 virus. The kit comprising the sets of first and second primers, a DNA polymerase, and a reverse transcriptase according to the above method. In some exemplary embodiments, the RT- LAMP assay has a specificity up to 100% and a sensitivity of about 95% (CT value < 32). Other features and aspects of this disclosure will be apparent from the following description and the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A shows the initial color of the reaction mixture medium inside both tubes with white and grey color lids before incubating at 65 °C according to one embodiment of the present disclosure;

FIG. IB shows the color in grey of the reaction mixture medium after incubating at 65 °C for 30 minutes with specific mRNA (in both SARS-CoV-2 or Human beta-actin assays) according to one embodiment of the present disclosure;

FIG. 2 shows beta-actin2 B3 primer of the human beta-actin RT-LAMP assay amplifying beta-actin mRNA while avoiding the amplification of the gene;

FIG. 3 illustrates six LAMP primers bind to the double-stranded cDNA as SARS-CoV-2 and beta-actin Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) primers bind and extend similarly;

FIG. 4A is a schematic diagram showing the top view of heating apparatus;

FIG. 4B is a schematic diagram showing a lateral view of the two heating channels for first set of sample collection tubes and PCR tubes;

FIG. 4C is a schematic diagram showing a lateral view of the two heating channels with perforations;

FIG. 5 shows a tube from the first set of tubes for sample collection with a straining apparatus, according to an embodiment of the present disclosure; and

FIG. 6 shows the RT-LAMP reaction endpoint showing fluorescence indicating the reaction result.

It should be appreciated by those skilled in the art that any diagram herein represents conceptual views of illustrative systems embodying the principles of the present disclosure. DETAILED DESCRIPTION

Reference will now be made in detail to specific embodiments or features, examples of which are illustrated in the accompanying drawings. Wherever possible, corresponding or similar reference numbers will be used throughout the drawings to refer to the same or corresponding parts. Moreover, references to various elements described herein, are made collectively or individually when there may be more than one element of the same type. However, such references are merely exemplary in nature. It may be noted that any reference to elements in the singular may also be construed to relate to the plural and vice-versa without limiting the scope of the disclosure to the exact number or type of such elements unless set forth explicitly in the appended claim.

The present disclosure relates to a method for identifying a subject infected with a SARS- CoV-2 vims. The method comprises extracting SARS-CoV-2 RNA and human beta-actin mRNA from a sample obtained from the subject. The method includes reverse transcribing the SARS-CoV-2 RNA to synthesize cDNA and amplifying the SARS-CoV-2 cDNA by a set of one or more first primers which hybridize to genome of the SARS-CoV-2 or the cDNA thereof. As a control to ensure sample integrity, the method further includes internal control assay based on reverse transcribing the human beta-actin mRNA to synthesize cDNA and amplifying the human beta-actin cDNA. The method includes a set of one or more second primers completely bound to the beta-actin cDNA and partially bound to a beta-actin gene with a hanging 3’ end. In some embodiments, the amplification of the SARS-CoV-2 RNA and human beta-actin mRNA is performed separately.

As used herein, a "primer" refers to an oligonucleotide that can be hybridized to a complementary nucleic acid sequence and is utilized in a polymerase chain reaction for amplification of a target nucleic acid molecule.

As used herein, a "complementary sequence" or a "complementary nucleic acid sequence" or a "C-strand" or a “cDNA” refers to the opposite strand of one of the strands of a double- stranded nucleic acid molecule. A complementary sequence may also be complementary to at least a part of another sequence.

As used herein, a "PCR” or “PCR reaction" or "molecular photocopying" refers to a technique used to amplify sequences of nucleic acids. As used herein, a "real-time PCR reaction" refers to a PCR in which the amplified nucleic acid is detected simultaneously as the PCR reaction proceeds, after a desired threshold is achieved.

As used herein, “RT-LAMP” or “Reverse-transcription Loop-mediated isothermal amplification” refers to an amplification method that combines Loop-mediated isothermal amplification (LAMP) in a single-tube technique with reverse transcription to multiply specific sequences of RNA.

As used herein, "hybridization" or "hybridize" refers to the association between two at least in part complementary nucleic acid strands, a nucleic acid, and a nucleic acid analog, or two nucleic acid analogs to form a double- stranded molecule.

As used herein, "target nucleic acid" or "target sequence" or "target" refers to a nucleic acid sequence to be analyzed or detected.

As used herein, “COVID” or “COVID 19” or “COVID-19” or “covid” or “SARS-CoV-2 infection” or “coronavirus disease” or “coronavirus infection” are used synonymously and refer to infections and diseases, symptomatic or asymptomatic, variants of concern (VoCs) or variants of interest (Vols), caused by SARS-CoV-2 virus or SARS-CoV-2 variants, such as, alpha, beta, gamma, delta, delta plus, epsilon, zeta, eta, theta, iota, kappa, lambda and other known or unknown mutated forms of the virus.

As used herein, “SARS-CoV-2” or severe acute respiratory syndrome coronavirus 2 or 2019 novel coronavirus or 2019-nCoV or human coronavirus 2019 or HCoV-19 or 19-nCoV or “Coronavirus” are used synonymously.

As used herein, the WarmStart reverse transcriptase, and the Bst 2.0 WarmStart DNA polymerase refer to the registered trademarks and indicate the use of the enzymes.

As used herein, the “CT value” is the cycle threshold value that emerges during PCR tests and is defined as the number of cycles required for the fluorescent signal to cross a threshold Ct levels are inversely proportional to the amount of target nucleic acid in the sample i.e., the lower the Ct level the greater the amount of target nucleic acid in the sample

As used herein, “SARS-CoV-2 variant” includes B.1.1.7, B.1.617.2, Variant of Concern 202012/01, VOC -202012/01, VUI-20d2012/01, 20I/501Y.V1, Variant of Concern 202012/02, VOC-202012/02, VUI-202012/02, lineage P.1, lineage P.3, VUI-21MAR-02, B.1.1.28.3, B.1.617.1, VUI-21APR-01, B.1.617, B.1.617.3, VOC-20DEC-01, VOC-20DEC-02, VOC-21JAN-02, VOC-21FEB-02, VOC-21APR-02, VOC-21FEB-03, 201/501 Y. VI, 20H/501Y.V2, 20J/501Y.V3, 20C/S:452R, 21A/S:478K, 21A/S:154K, and 20A/S:484K and any variant which can be characterized later.

As used herein, “Human Beta-Actin” or “ACTB” or BRWS1 or PS1TP5BP1 or Actin Beta, Actx or A-X actin-like protein or E430023M04Rik or PS 1TP5 -Binding Protein 1 or Beta Cytoskeletal Actin or B-Actin or b-Actin refer to actin genes or proteins in humans involved in cell motility and integrity.

As used herein, “patient” or “subject” refers to a mammal, such as a human.

As used herein, the term “about” refers to ±10% variation in reduction of infection from the nominal value unless otherwise indicated or inferred. For example, in certain catheters, the term “about” can refer to ±5% or ±2.5% or ±1% variation from the nominal value or a fixed variation from the nominal value.

As used herein, ‘a first set of tubes’ or ‘a set of collection tubes’ or ‘a first set of sample collection tubes’ or ‘the tubes’ are used synonymously.

The use of the terms “include,” “includes”, “including,” “have,” “has,” or “having” should be generally understood as open-ended and non-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise.

In an exemplary embodiment, the method includes 55, 60, 65, 70, 75, 80, 85, 90, 95% bases in 5’ end of the set of one or more second primers complementary to one exon region of the mRNA or the cDNA thereof. In various embodiments, the method further includes 5, 10, 15, 20, 25, 30, 35, 40, 45% bases in the 3’ end of the second primers complementary to an adjacent exon. In various examples, 30-95% of the bases allows a complete binding of the one set of one or more second primers to the beta actin mRNA or cDNA, while there is a partial binding to the beta actin gene with a hanging 3’ end. In various embodiments, 30- 95% of the bases complementary to the 2nd exon region over the 5’ end of the primer while a few bases complementary to the 1st exon region over the 3’ end of the one set of one or more second primer is restricted to the amplification of the cDNA. In various embodiments, the amplification of the SARS-CoV2 RNA and the human beta-actin mRNA is performed in parallel in a separate set of tubes based on an identical sample. In various embodiment, the method of the present disclosure maintains the beta-actin genomic sequences or associated genes unamplified. In various embodiments, the method includes verifying the extraction of mRNA of the human beta-actin in the sample and acts as a control confirming the desired extraction of the SARS-CoV2 RNA present in the same sample.

In some embodiments, the SARS-CoV-2 virus infection identification in the sample is performed via Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT- LAMP). In some examples, the RT-LAMP is a colorimetric RT-LAMP. In various embodiments, the method allows the identification of the SARS-CoV-2 virus infection via the RT-LAMP based on cDNA, and any binding of the set of one or more second primers to the human beta-actin gene is restricted. In some embodiments, the RT-LAMP assay is performed on the sample at a temperature between 50 to 70 °C. In an example, the temperature for the RT-LAMP assay is maintained at 65 °C. In various embodiments, the RNA into the cDNA by action of a reverse transcriptase and subsequent amplification of cDNA results in rapid accumulation of protons in the medium making the medium acidic. The change in the pH of the medium from basic to acidic changes the color of the medium from pink to yellow due to an indicator dye present in the medium (See FIG 1 A and FIG. IB). In an exemplary embodiment, the RT-LAMP comes with a separate internal control assay based on the human beta-actin gene so that each sample is subjected to two parallel assays in two separate tubes: As the first assay, the SARS-CoV-2 RNA is converted to cDNA amplified and the second assay in the tube amplifies the human beta-actin mRNA in a similar manner. The second assay confirms the integrity of the sample and quality of the RNA extraction process. In some examples, the cover of the tubes is given a specific color to distinguish the internal control from the SARS-CoV-2 assay. In an example, the color of the tube of the first assay amplifying the SARS-CoV-2 specific RNA is provided with a red lid and the tube of the second assay amplifying the human beta-actin mRNA with a green lid.

In some embodiments, the RT-LAMP assay is performed on the sample within 30 minutes. In an example, the time for the RT-LAMP assay maintained at a constant temperature of 65 °C for between 10 to 50 minutes. The method according to various embodiments includes the RT-LAMP assay with a specificity up to 100% and a sensitivity up to 95% (CT value < 32) in detecting SARS-CoV-2 patients in parallel to qRT-PCR assays. In some embodiments, the specificity of the RT-LAMP is 70, 75, 80, 85, 90, 95, 98, 99, 99.9, or 100%. In various embodiments, the sensitivity of the assay is 70, 75, 80. 85, 90, or 95% compared to qRT- PCR. Thus, in an exemplary embodiment, the identification of the SARS- CoV-2 virus infection is performed via the RT-LAMP on the sample from the subject based on the cDNA amplified by the set of one or more second primers at 65 °C within 30 minutes, wherein the detection on human beta actin mRNA confirming the extraction of mRNA from the sample is parallelly performed within the same time. In some embodiments, the total time for the RT-LAMP is 20 minutes and the results are perceived in the change in the color of the composition including the sample and a reaction mixture. The color changes are visible to naked eye.

In an embodiment of the present disclosure, the method includes a reverse transcriptase to generate the cDNA and a DNA polymerase enzyme as part of the reaction mixture to amplify the cDNA. In an exemplary embodiment, the method includes the synthesis of the cDNA wherein the reverse transcriptase is a WarmStart reverse transcriptase, and the DNA polymerase is a Bst 2.0 WarmStart DNA polymerase. In such examples, the WarmStart enzymes show high throughput, consistency, and specificity for the amplification processes. In an exemplary embodiment, the method includes separately performing the amplification of the SARS-CoV-2 and the human beta-actin for detection. In some embodiments, the set of one or more first primers include SEQ. ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the set of one or more first primers target a segment of ORFlab gene responsible for production of nonstructural protein 2, also known as nsp2. The nsp2 protein varies among different Corona viruses and in SARS-CoV-2 genome, the nsp2 target is 100% conserved (Yip et al., 2020). The reaction endpoint (either positive or negative) can be visually observed (See FIG. 1) making the kit usable in laboratories with minimum resources. The whole process from the conversion of viral RNA (template) to the cDNA, the amplification of cDNA and the detection is completed in a single reaction tube. In some embodiments, the set of one or more second primers include SEQ. ID No. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In an exemplary embodiment, to verify the sample integrity and to ensure the proper functioning of the RT-FAMP reaction, the method includes a separate assay targeting the human beta-actin mRNA. The RT-FAMP assay is performed in separate tubes up to six one or more second primers targeting the human beta-actin mRNA developed in a way that the RT-FAMP reaction will avoid binding to the human beta-actin gene, but the reaction will yield a positive outcome only to the cDNA coming from conversion of mRNA to cDNA with the RT-FAMP reaction. This indirectly confirms the presence and integrity of mRNA in the sample and the successful conversion of mRNA into cDNA. The human beta-actin RT-FAMP assay shall be carried out parallel to the SARS-CoV-2 RT- LAMP using the same processed biological sample obtained from the subject and the reaction mixture of the human beta-actin RT-LAMP is kept identical to SARS-CoV-2 RT- LAMP except for the primers and how they function. In an exemplary embodiment, the one or more of first and the second primers are developed to target or bind to a region of the cDNA corresponding to the exon-exon junction of the beta-actin mRNA created during the process of splicing of mRNA after transcription allowing the complete binding of the set of one or more of the second primers to the beta-actin mRNA or the cDNA, while there is a partial binding to the beta-actin gene with the hanging 3’ end. In some exemplary embodiments of the present method, the sample is a body fluid including blood, saliva, nasal drops, nasal swab, throat swab, pharyngeal swab, alveolar lavage fluid, plasma, teardrops, urine, stool, semen, or a tissue sample.

In various embodiments, the reaction mixture for RT-LAMP includes Tris-HCl, (NPDiSCL, KC1, MgSCL, Tween^®20, dNTPs, Bst 2.0 WarmStart DNA polymerase, Antarctic Thermolabile UDG, WarmStart reverse transcriptase, pH indicator or a fluorescent dye, glycerol, one or more of the first primers or one or more of the second primers and nuclease- free water, and target mRNA, cDNA, or DNA. In an exemplary embodiment, the total volume of the sample and the reaction mixture volume used in the method is up to 25 pi, wherein up to 5 mΐ is the sample volume. Thus, in various embodiments, the composition of the each of the 25 mΐ of the reaction for RT-LAMP includes IX WarmStart Colorimetric LAMP Master Mix (Cat. No M18000L; New England Biolabs, Ipswich, MA 01938-2723, USA) and the one or more of the first or the second primers at a concentrations of 1 - 6 mM FIP/BIP, 0 2 mM F3/B3, 0 4 mM FL/BL.

In another aspect of the present disclosure, a system for detecting presence of SARS-CoV-2 in a sample from a subject is included. The sample may include a body fluid such as blood, saliva, nasal drops, nasal swab, throat swab, pharyngeal swab, alveolar lavage fluid, plasma, teardrops, urine, stool, semen, or a tissue sample. The system including one or more extraction tubes configured to receive the sample from the subject including an open top portion and a closed bottom portion, and sidewalls encompassing a sample void disposed with a straining apparatus inside each of the tubes.

Referring to FIG 5, in some examples, the straining apparatus is a mesh network that allows RNA extraction from the sample. The sample obtained using a swab is soaked in the liquid inside the tube containing one or more of nuclease free water, a weak buffer such as 1 mM Tris-Cl pH 8.5, 0.1 mM EDTA and an RNase inhibitor and squeezed using the straining apparatus prior to the swab is being discarded. Then the liquid in the sample with the patient cells and other organisms is heated between 37-58°C for 1-15 minutes, followed by heating up to 95 °C between 1-5 minutes to extract RNA. The system further includes one or more of first set of tubes configured to receive the sample from the subject comprising an open top portion and a closed bottom portion, and sidewalls encompassing a sample void and a cover configured to couple with the top portion. The cover is connected to the first set of tube body by a flexible joint. In some embodiments, the flexible joint is made of a material that is flexible and inert. The system includes a plurality of racks configured to accommodate the one or more of first set of tubes and a set of PCR tubes for RT-LAMP assay. The plurality of the racks include a first rack channel encompasses the first set of tubes comprising an inner volume and a second channel encompasses the PCR tubes having a closed end, an open end and an inner diameter and comprising an inner volume and a set of one or more first primers to hybridize to genome of the SARS-CoV-2 or the cDNA thereof present in the one or more PCR tubes and a set of one or more second primers to amplifying the human beta-actin cDNA present in the one or more first set of tubes. The system of the present disclosure allows specific identification of SARS-CoV-2 any other organism while eliminating the need of a separate viral RNA extraction and cleaning up step by using commercial RNA extraction kits which is costly and time consuming. In an exemplary embodiment, the staining apparatus includes RNase inhibiting preparation including Tris(hydroxymethyl) aminomethane (TE) buffer or RNAse free water and RNAsecure™ (ThermoFisher Scientific) or RIBOPROTECT Hu RNase Inhibitor (BLIRT S.A.), and heated between 37-58°C for 1-15 minutes, followed by heating up to 95 °C between 1-5 minutes to extract RNA. The system also includes a reverse transcriptase and a DNA polymerase as part of a reaction mixture present in the first set of tubes and the PCR tubes for sample amplification. The first set of tubes are used for amplification of the beta-actin mRNA while as the PCR tubes are used for SAR CoV-2 mRNA.

Referring to FIG 4A to FIG. 4C, in various embodiments, the system includes a plurality of cartridge heating sources, thermocouples, temperature controllers, timers, and a UV light- emitting source configured to emit UV light for visual detection of fluorescence coming from specific DNA binding fluorescent dye in the reaction mixture as the cDNA gets amplified in the RT-LAMP reaction tubes. In various examples, the desired UV light wavelength to visualize the fluorescence coming from DNA binding dyes such as QuantiFlour® dsDNA fluorescent DNA-binding dye (Promega Corporation, USA) detection is between 300 to 400 nm. In an exemplary embodiment, the UV light-emitting source is configured to emit UV light at 366 nm. Furthermore, in an embodiment, the 366 nm UV light in the system excites the DNA binding fluorescence dye in the PCR tubes. The results are visualized under the 366 nm wavelength UV source and an apple green fluorescence indicates a positive amplification of DNA/cDNA (See FIG. 6). In various embodiments, the system can hold up to 64 PCR tubes and the first set of tubes simultaneously allowing bulk assaying and the detection of SARS-CoV-2. In some embodiments, the heating source is placed in such a manner that the tubes present in the racks of the system receive an equal amount of heat. In various embodiments, an ideal temperature of heating is between 60 to 70 °C. In various embodiments, the RNA present in RT-LAMP reaction tubes is converted into the cDNA by the action of reverse transcriptase and the subsequent amplification of the cDNA results in rapid accumulation of protons in the medium making the medium acidic. The change in the pH of the medium from basic to acidic changes the color of the medium from pink to yellow due to an indicator dye present in the medium (See FIG 1 A and FIG. IB). The RT-LAMP reaction mixture can be incubated at a temperature between 60-70°C for a time duration of 10-50 min. In various embodiments, the system allows the identification of the SARS-CoV-2 virus infection via the RT-LAMP based on cDNA. In some embodiments, the RT-LAMP assay is performed on the sample within 30 minutes. In an example, the time for the RT-LAMP assay maintained at 65 °C is between 10 to 50 minutes. The system according to various embodiments includes the RT-LAMP assay with a specificity up to 100% and a sensitivity up to 95% (CT value < 32). In some embodiments, the specificity of the RT-LAMP is 70, 75, 80, 85, 90, 95, 98, 99, 99.9, or 100%. In various embodiments, the sensitivity of the assay is 70, 75, 80. 85, 90, or 95% compared to the PCR. Thus, in an exemplary embodiment, the identification of the SARS-CoV-2 virus infection is performed via the RT-LAMP on the sample from the subject based on the cDNA amplified by the set of one or more second primers at 65 °C within 30 minutes, wherein the detection on mRNA confirming the extraction of mRNA from the sample is performed in parallel within the same time. In some embodiments, the total time for the RT-LAMP is 10-50 minutes and the results are perceived in the change in the color of the reaction mixture. The color changes are visible to naked eye.

In an embodiment, the system includes a two-channel heating block comprising of four cartridge heaters with two for each channel wherein channel- 1 is provided for the first set of sample collection tubes and heated between 37 °C to 95 °C for the RNA extraction. The Channel-2 is provided for PCR tubes to be heated between 37 °C to 70 °C for the RT-LAMP. In some embodiments, the system includes at least two thermocouples and two digital temperature controllers with timers for temperature and time control and a UV light source which allows detection of fluorescence emitted by the sample due to binding of the DNA binding dye in the reaction mixture to the amplifying DNA in the PCR tubes through the continued slit on the opposite side in the block of the channel-2 so that a positive reaction can be detected visually.

In some embodiment, the system includes the reverse transcriptase to generate the cDNA and the DNA polymerase enzyme as part of the reaction mixture to amplify the cDNA. In an exemplary embodiment, the method includes the synthesis of the cDNA wherein the reverse transcriptase is a WarmStart reverse transcriptase, and the DNA polymerase is a Bst 2.0 WarmStart DNA polymerase. In such examples, the Warmstart enzymes show high throughput, consistency, and specificity for the amplification processes. In an exemplary embodiment, the method includes separately performing the amplification of the SARS- CoV-2 and the human beta-actin for detection. In some embodiments, the set of one or more first primers include SEQ. ID No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the set of one or more first primers target a segment of ORFlab gene responsible for production of nonstructural protein 2, also known as nsp2. The nsp2 protein varies among different Corona vims strains and in SARS-CoV-2 genome which has a high mutation rate, the nsp2 target is 100% conserved. The reaction endpoint (either positive or negative) can be visually observed (FIG. 1) making the system usable in laboratories with minimum resources. The whole process from the conversion of viral RNA used as a template to cDNA, amplification of cDNA and detection is completed in a singular tube In some embodiments, the set of one or more second primers include SEQ. ID No. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In an exemplary embodiment, to verify the sample integrity and to ensure the proper functioning of the RT-FAMP reaction, the system includes a separate assay targeting the human beta-actin mRNA. The RT-FAMP assay is performed in separate tubes with the set of one or more second primers targeting human beta-actin mRNA provide a desired result by binding only to the mRNA. -In various embodiments, the human beta-actin RT-FAMP assay is carried out parallel to the SARS-CoV-2 RT-FAMP using the same sample obtained from the same subject. Additionally, the reaction mixture of the human beta-actin RT- FAMP is identical to SARS-CoV-2 RT-FAMP except for the sets of first and second primers. In an exemplary embodiment, the one or more of first and the second primers are developed to target or bind to a region of the cDNA corresponding to the exon-exon junction of the beta actin mRNA created during the process of splicing of mRNA after transcription allowing the complete binding of the set of one or more of the second primers to the beta- actin mRNA or the cDNA, while there is a partial binding to the beta-actin gene with the hanging 3’ end. In some embodiments, the SARS-CoV-2 virus infection identification in the sample is performed via RT-LAMP. In some examples, the RT-LAMP is a colorimetric RT-LAMP wherein the reaction mixture present in the tubes contains a pH indicator providing visualization of color changes visible to naked eye upon amplification of cDNA. In some exemplary embodiments of the present method, the sample is a body fluid including blood, saliva, nasal drops, nasal swab, throat swab, pharyngeal swab, alveolar lavage fluid, plasma, teardrops, urine, stool, semen, or a tissue sample.

In various embodiments, the present disclosure includes a kit for performing loop-mediated isothermal amplification (LAMP) of a target nucleic acid sequence for identifying a subject infected with the SARS-CoV-2 virus, the kit comprising the sets of first and second primers, a DNA polymerase, and a reverse transcriptase and a sample collection tube containing RNAse inhibitors according to the disclosed methods.

In various embodiments, the present disclosure includes a kit for performing loop-mediated isothermal amplification (LAMP) of a target nucleic acid sequence for identifying a subject infected with the SARS-CoV-2 virus, the kit comprising the sets of first and second primers, a DNA polymerase, and a reverse transcriptase according to the disclosed methods. The kit of the present disclosure should be stored at or below -20°C to prevent degradation of the ingredients in the LAMP reaction mixture. In various embodiments, shelf-life of the colorimetric LAMP master mix and DNA oligonucleotides used in the product are one year and two years, respectively.

In various embodiments, the reaction mixture for RT-LAMP in the kit includes Tris-HCl, (NH₄)₂S0₄, KCl, MgS0₄, Tween^®20, dNTPs, Bst 2.0 WarmStart DNA polymerase, Antarctic Thermolabile UDG, WarmStart reverse transcriptase, pH indicator or a fluorescent dye, glycerol, one or more of the first primers or one or more of the the second primers and nuclease-free water, and target mRNA, cDNA or DNA. In an exemplary embodiment, the total volume of the sample and the reaction mixture volume used in the kit for screening is up to 25 pi, wherein up to 5 mΐ is the sample volume. Thus, in various embodiments, the composition of the each of the 25 mΐ of the reaction for RT-LAMP includes IX WarmStart Colorimetric LAMP Master Mix (Cat. No M18000L; New England Biolabs, Ipswich, MA 01938-2723, USA) and the one or more of the first and or the second primers at a concentrations of 1-6 mM FIP/BIP, 0-2 mM F3/B3, 0·4 mM FL/BL.

EXAMPLES

For verifying desired working of method and system of the present disclosure based on the RT-LAMP principle, a kit was devised that works under a single and constant temperature of 65°C and include using reverse transcriptase and Bst 2.0 Warm Start DNA polymerase. The kit converts the SARS-CoV-2 specific viral RNA to cDNA and amplifies the target sequence using six specific primers in an efficient, rapid, and robust manner, to achieve the purpose of rapid detection. The six primers target a segment of ORFlab gene responsible for production of nonstructural protein 2, also known as nsp2. The nsp2 protein varies among different Coronaviruses and in the SARS-CoV-2 genome, the nsp2 target is 100% conserved (Yip et ah, 2020). The reaction endpoint (positive or negative) can be visually observed as shown in FIG 1 making the kit usable in laboratories with minimum resources. The whole process from the conversion of viral RNA (template) to the cDNA, amplification of the cDNA, and detection are completed in a single reaction tube (i.e., tubes with a red color lid).

Sample Integrity and Human beta-actin Control

Furthermore, to verify the sample integrity and to ensure the proper functioning of the RT- LAMP reaction, a separate assay targeting human beta-actin mRNA has been included in the test kit. The RT-LAMP assay targeting human beta-actin mRNA comes in separate tubes with a grey color lid. The six LAMP primers targeting human beta-actin mRNA were developed in a way that the LAMP reaction will not yield a positive outcome by binding to the human beta-actin gene, but only to the cDNA coming from conversion of mRNA to cDNA with the RT-LAMP reaction. This indirectly confirms the presence and integrity of mRNA in the sample and the successful conversion of the mRNA into the cDNA. The human beta-actin RT-LAMP assay was carried out parallel to the SARS-CoV-2 RT-LAMP using the same processed biological sample obtained from the patient (i.e., extracted RNA). Also, the reaction mixture of the human beta-actin RT-LAMP is identical to SARS-CoV-2 RT-LAMP except for the primers and the primer functionality. The interpretation of the test outcome is provided below in Table 1. The positive agreement of 95% with RT-PCR samples with a CT value <32 achieved is good evidence of the sensitivity of the assay.

Table 1

Cross-Reactivity (Analytical Specificity) and Microbial Interference

To estimate the likelihood of cross -reactivity of the SARS-CoV-2 RT-LAMP primers with organisms that were not available for wet testing, In silico analysis using the Basic Local Alignment Search Tool (BLAST) managed by the National Center for Biotechnology Information (NCBI) was carried out to assess the specificity of SARS-CoV-2 primers and presented in Table 3 below. In addition, salvia samples obtained from 26 healthy individuals were tested with the LAMP test kit and all became negative. Table 3

Further, the LAMP test was carried out with five human total fecal DNA samples, collected and preserved at -20°C before November 2019 from Tuberculosis suspected patients, as the gut microbiome is the most diverse and complex microbial habitat in and on the human body comprising of many uncharacterized viruses and bacteria. All five samples gave a negative result. The data provides evidence of the high specificity of the LAMP assay.

However, the following gene sequences at the NCBI database gave a high likelihood of binding to the primers due to the similarity with SARS-CoV-2 genome. i. Bat coronavirus RaTG13 GenBank Accession No. MN996532 ii. Bat SARS-like coronavirus isolate bat-SL-CoVZXC21 GenBank Accession No. MG772934 iii. Bat SARS-like coronavirus isolate bat-SL-CoVZC45 GenBank Accession No. MG772933 iv. Pangolin coronavirus isolate MP789 GenBank Accession No. MT121216 Endogenous Interfering Substances

Both the SARS-CoV-2 RT-LAMP and the Human beta-actin RT-LAMP worked in the presence of 5pl of saliva spiked with the SARS-CoV-2 cDNA without using the extracted RNA. Unlike RT-PCR, the LAMP method of the present disclosure does not get inhibited by endogenous substances present in saliva. Ingredients

A typical RT-LAMP colorimetric master mix may include (Tris-HCl, (NH^SCL, KC1, MgSCL, Tween® 20, dNTPs, Bst 2.0 WarmStart DNA Polymerase, Antarctic Thermolabile UDG, WarmStart Reverse Transcriptase, pH indicator and glycerol), oligonucleotide primers and nuclease free water. Formulation

The following Table 4 provides the composition of each 20pl reaction of the product (after adding the 5m1 of template RNA, the total reaction volume is 25m1). For both the SARS- CoV-2 RT-LAMP and the Human beta-actin RT-LAMP the same following formulation is used except for the primers.

Table 4

Designing of LAMP Primers Designing specific and efficient primers is a key step in developing a diagnostic test in which the diagnosis is carried out primarily by specific amplification of nuclear acids of the disease- causing agent. Specific LAMP primers were designed using sequence data in public repositories. Over 200 SARS-CoV-2 sequences were aligned, and a consensus sequence was constructed. The consensus sequence was split into nearly less than 2000 bases and saved separately so that the bases can be used with the primer designing tool Primer Explorer Version 5 (Fujitsu Limited). Using various regions/segments of the consensus nucleic acid sequence, multiple sets of LAMP primers were designed. About 1000 primers were designed for each specific region using Primer Explorer Version 5 (Fujitsu Limited). Out of these six LAMP primers (denoted as F3, B3, FIP, BIP, LF, and LB) were manually selected for a region/segment considering the least possibility of making primer-dimers and hairpin structures using IDT OligoAnalyzer (Integrated DNA Technologies (Pvt) Ltd) and Multiple Primer Analyzer (ThermoFisher Scientific) tools. Finally, the specificity and sensitivity of the multiple sets of primers were compared and the best primer set; BRF1-LB1 was selected for the SARS-CoV-2 assay of the LAMP test kit. The details of the six primers namely, BRF1-F3, BRF1-B3, BRF1-FIP, BRF1-BIP, BRF1-LF, and BRF1-LB1 are provided in Table 5. This was followed by the sensitivity (in consonance with the PCR) and the specificity of the primers using RNA extracted from nasopharyngeal and / or nasal mid turbinate specimens collected into viral transport medium or lysis buffer from confirmed Covid-19 positive and negative individuals in parallel to PCR (Table 2) following the test procedure given below. This data is presented in the method performance section above.

Furthermore, to verify the sample integrity especially of RNA and to ensure the proper functioning of the reverse transcription process of the RT-LAMP reaction, a set of LAMP primers targeting the human beta-actin mRNA or subsequently producing cDNA have been designed and included in the test kit (Human beta-actin RT-LAMP assay in provided with the grey color tube). A primer critical for LAMP amplification SEQ. ID. No. 14 of which the details are provided in FIG. 1 and Table 5 below was developed to target/bind to a region of the cDNA corresponding to the exon-exon junction of the beta-actin mRNA created during the process of splicing of mRNA after transcription. This design allows a complete binding of the primer to the beta-actin mRNA or the cDNA, while there is a partial binding to the beta-actin gene with a hanging 3’ end. Also, it is developed in a way that a majority of the bases complementary to the 2nd exon region over the 5’ end of the primer while a few bases complementary to the 1st exon region over the 3’ end of the primer. This design only allows the amplification of the cDNA, but not the beta-actin genomic sequences or the genes. As a result, this assay can be used to confirm the presence of the beta-actin mRNA in the sample and the successful conversion of the mRNA into the cDNA. Hence this assay indirectly provides evidence on the integrity of the RNA in the sample ability for the reaction to successfully convert the RNA to the cDNA and amplify the cDNA. The Human Beta Actin RT-LAMP was carried out in parallel to the SARS-CoV-2 LAMP in two different tubes, but using the same processed biological sample obtained from the patient. Also, the reaction mixture of the Beta-actin RT-LAMP is identical to the above-described SARS- CoV-2 RT-LAMP except for the primers.

Referring to FIG 2, the figure illustrates the Beta Actin2 B3 primer of the Human Beta Actin RT-LAMP assay amplifying the beta-actin mRNA while avoiding the amplification of the gene. Beta Actin2 B3 is the critical primer out of the six primers controlling this whole process.

SEQ. ID NO. 25 (part of the gene)

CCGGGACCTGACTGACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGCT

TCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGC

TACGTCGCCCTGGACTTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTC

CCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATG

AGCGGTTCCGCTGCCCTGAGGCACTCTTCCAGCCTTCCTTCCTGGGTGAGTGG

AGACTGTCTCCCGGCTCTGCCTGACATGAGGGTTACCCCTCGGGGCTGTGCT

GTGGAAGCTAAGTCCTGCCCTCATTTCCCTCTCAGGCATGGAGTCCTGTGGCAT

CCACGAAA

Exon regions are not highlighted, and intron region is highlighted in the above DNA sequence

>SEQ. NO. 246 (corresponding to the above part of the gene and after removing of introns)

CCGGGACCTGACTGACTACCTCATGAAGATCCTCACCGAGCGCGGCTACAGCT TCACCACCACGGCCGAGCGGGAAATCGTGCGTGACATTAAGGAGAAGCTGTGC TACGTCGCCCTGGACTTCGAGCAAGAGATGGCCACGGCTGCTTCCAGCTCCTC CCTGGAGAAGAGCTACGAGCTGCCTGACGGCCAGGTCATCACCATTGGCAATG AGCGGTTCCGCTGCCCTGAGGCACTCTTCC AGCCTTCCTTCCTGGO > < <CATGG AGT CCTGT GGC AT CC AC G A A A After removing the intron, the exon-exon junction is marked as >><<

>SEQ. ID. No. 25 after the reverse transcription (corresponding to the above part of the mRNA. The primer binding regions are highlighted in the same color as the Beta actin2 primers listed in Table 5) Table 5

Exemplary Test Procedure

To use the kit of the present invention the sample can include nasopharyngeal and / or nasal mid-turbinate specimens collected into viral transport medium or lysis buffer by a trained person following standard practices (a specimen that is not collected correctly may lead to false-negative test results). Then, the RNA shall be extracted using an acceptable method by the health authorities for detection of SARS-CoV-2 by qRT-PCR following the manufacturer’s protocol (e.g., QIAamp Viral RNA Mini Kit or The Maxwell® HT Viral TNA Kit). Once the RNA samples are ready, the sample numbers should be noted in the sheet provided with the kit against the unique white and grey tube lid numbers. Followed by, switching on the heating device that can hold up to 64 PCR tubes provided in the kit and set the temperature to 65°C. Alternatively, any other heating device such as a heating block or a regular PCR machine that can hold 0.2ml PCR tubes at 65±loC can be used. Then, the required number of red (that amplifies SARS-CoV-2 RNA) and green (internal control that amplifies human beta-actin mRNA) color-coded tubes from the -20°C freezer and allow to thaw at room temperature for 2-3 minutes. Then, adding 5m of each sample (template RNA) separately to both the white and the grey color-coded tubes with the respective number on the lid (i.e., if 5m1 the first sample is added to the tube with the white color lid marked No. 01, then 5m1 of the same sample shall be added to the tube with the grey color lid marked No. 01). After adding all the samples to white and grey color-coded tubes, add 5m1 of nuclease-free water separately to a red and the respective green color-coded tubes serving as the no template control. Further, adding 5m1 of the SARS-CoV-2 positive control to a white color tube and the human beta-actin positive control to the respective grey color-coded tube to function as the positive controls. After adding the sample, ensuring that the reaction mix in the tube is at the bottom of the tube without any air bubbles, the PCR tubes should be placed in the heating device at 65°C. At 30 min from the start of incubating the assay tubes, removing the tubes from the heating device, and visually reading the test outcome concluded the assay. Positive controls and positive samples will turn yellow, while negative control and samples will remain in their original pink color before the incubation. The reaction end-point color will be visible immediately after removing the tubes from the heating device but can be intensified by allowing the tubes to cool down to room temperature. All sample integrity verification controls targeting human beta-actin mRNA (tubes with grey color lid) with added patient samples shall turn to yellow, and if a sample integrity verification control tube with an added patient sample/template does not provide a yellow color, the sample did not contain sufficient and / or quality RNA. The result can be photographed or scanned to record the colorimetric results, or simply kept at room temperature in the reaction tube for up to 4 hours. Incubating over 30 min should be avoided.

Storage Conditions

The kit should be stored at or below -20°C to prevent degradation of the ingredients in the LAMP reaction mix, remove the required number of tests from the freezer just before each use to minimize unnecessary freeze-thaw cycles. The test kit shall NOT be transported under dry ice.

Shelf-Life

The shelf-life of the colorimetric LAMP master mixture and the DNA oligonucleotides used in the product are up to one year and two years, respectively. However, the proper functionality of the test kit has only been experimentally verified up to six months as of now.

INDUSTRIAL APPLICABILITY

The present disclosure provides the method and system for identifying a subject infected with a SARS-CoV-2 virus. One of the advantages of the present disclosure is that the method is very rapid and takes less than 50% of the time required to carry out a PCR and thus enables, test report generation in a shorter time. Further, in places with auto-RNA extractors, the results can be provided within an hour.

Another advantage of the LAMP test kit of the present disclosure is that the kit has a high specificity of up to 100% and a sensitivity of about 95% (CT value < 32) compared to that of PCR. Further, the LAMP test kit is equipped with an internal control assay based on the human beta-actin gene to ensure the sample integrity and can detect all the new strains/variants of the virus. The LAMP primers used in the LAMP test kit are unique and in a comparison study with six other sets of LAMP primers the SARS-CoV-2 primer set in the present disclosed test kit was found to have the highest sensitivity.

Yet, another advantage of the method presented in this disclosure is that the method can be made into a commercially available mass-produced rapid test kit for the diagnosis of SARS- CoV-2. Such rapid, and cost-effective test kits are an essential part of the diagnosis and isolation of COVID-19 infected patients all over the world during this COVID-19 pandemic until a successful vaccine coverage is provided to the susceptible population which could potentially take many years. Further, in a post-pandemic situation where the virus could be spreading in a lower intensity in certain community pockets, rapid diagnostic tests will be extremely helpful for routine screening of communities or workers at risk or to protect borders of a country to avoid reintroduction of the variant strains of the virus to a country targeting conserved genes of the virus such as with this LAMP test kit.

Another advantage of the system of the present disclosure is that the system allows the specific identification of SARS-CoV-2 while eliminating a separate viral RNA extraction step by incorporating the RNA extraction step into the DNA amplification process itself.

Yet another advantage of the present method and system is that less technical expertise is required to run the assays as the procedure is simple, portable, and user-friendly.

While aspects of the present disclosure have been particularly shown and described regarding the embodiments above, it will be understood by those skilled. In the art that the examples, embodiments, and teachings presented in this application are described merely for illustrative purposes. Any variations or modifications thereof are to be included within the scope of the present application as discussed.

Claims

CLAIMS What is claimed is:

1. A method for identifying a subject infected with a SARS-CoV-2 virus, the method comprising: extracting SARS-CoV-2 RNA and human beta-actin mRNA from a sample obtained from the subject, reverse transcribing the SARS-CoV-2 RNA to synthesize cDNA; amplifying the SARS-CoV-2 cDNA, wherein a set of one or more first primers hybridize to genome of the SARS-CoV-2 or the cDNA thereof; reverse transcribing the human beta-actin mRNA to synthesize cDNA; amplifying the human beta-actin cDNA, wherein a set of one or more second primers amplify the cDNA; and wherein, the set of one or more second primers are completely bound to the beta-actin cDNA and partially bound to beta-actin gene with a hanging 3 ’ end.

2. The method according to claim 1, wherein 55-95% bases in 5’ end of the set of one or more second primers are complementary to one exon region of the mRNA or the cDNA thereof.

3. The method according to claim 1, wherein 5-45% bases in the 3’ end of the second primers are complementary to an adjacent exon.

4. The method according to claim 1 further comprising, a reverse transcriptase to generate the cDNA and a DNA polymerase enzyme as part of a reaction mixture to amplify the cDNA.

5. The method according to preceding claims 1 to 4, wherein the amplification of the SARS- CoV-2 and the human beta-actin is performed separately. 6. The method according to claim 1 , wherein the set of one or more first primers include SEQ. ID No. 1, 2, 3, 4, 5,

6, 7, 8, 9, 10, 11, or 12.

7. The method according to claim 1, wherein the set of one or more second primers include SEQ.ID No.13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

8. The method according to any of the preceding claims 1 to 7, wherein the SARS-CoV-2 virus infection identification in the sample is performed via Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP).

9. The method according to claim 10, wherein the RT-LAMP is a colorimetric RT-LAMP assay.

10. The method according to the preceding claims 1 to 9, wherein the sample is a tissue, or a body fluid obtained from a subject.

11. The method according to claim 12, wherein the body fluid is blood, saliva, nasal drops, nasal swab, throat swab, pharyngeal swab, alveolar lavage fluid, plasma, teardrops, urine, stool, semen, or a tissue sample.

12. A system for detecting presence of a SARS-CoV-2 in a sample from a subject, the system comprising: one or more extraction tubes configured to receive the sample from the subject comprising an open top portion and a closed bottom portion, and sidewalls encompassing a sample void disposed with a straining apparatus inside each of the tubes; one or more of first set of tubes configured to receive the sample from the subject comprising an open top portion and a closed bottom portion, and sidewalls encompassing a sample void; a cover configured to couple with the top portion of each of the first set of tubes, wherein the cover is connected to the of first set of tube body by a flexible joint; a plurality of racks configured to accommodate the one or more of first set of tubes and a set of PCR tubes for RT-LAMP assay; wherein a first rack channel encompasses the first set of tubes comprising an inner volume and a second channel encompasses the PCR tubes having a closed end, an open end and an inner diameter and comprising an inner volume; a set of one or more first primers to hybridize to genome of the SARS-CoV-2 or the cDNA thereof present in the one or more PCR tubes; a set of one or more second primers to amplifying the human beta-actin cDNA present in the one or more first set of tubes; and a reverse transcriptase and a DNA polymerase as part of a reaction mixture present in the first set of tubes and the PCR tubes and an RNAse inhibiting preparation present in the extraction tubes.

13. The system according to claim 12, wherein the sample is a tissue or body fluid obtained from the subject.

14. A kit for performing loop-mediated isothermal amplification (LAMP) of a target nucleic acid sequence for identifying a subject infected with the SARS-CoV-2 virus, the kit comprising the sets of first and second primers, a DNA polymerase and a reverse transcriptase according to claim 1.

15. The kit according to claim 14, wherein the RT-LAMP assay has a specificity of up to 100% and a sensitivity of about 95% (CT value < 32).