The present invention relates to the field of diagnosis of coronavirus disease. The Invention in particular provides efficient and sensitive primers for the detection of covid 19 or coronavirus infection in a sample.
Background of the invention:
The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Novel Coronavirus disease (COVID-19) is an infectious disease caused by a newly discovered coronavirus, which has spread from Wuhan city of China. The COVID-19 virus spreads primarily through droplets of saliva or discharge from the nose when an infected person coughs or sneezes. At this time, there are no specific vaccines or treatments for COVID-19 and only diagnosis can help to identify the COVID-19 cases so that they can be quarantined so that disease can be controlled from spreading widely. As there is an emergency around the world due to COVID-19, there is a dire need of rapid and reliable diagnostic kit for mass screening. Currently available RT-PCR are expensive and require skilled personnel and costly setup therefore they cannot be used for mass screening.

Presently, there are 12.7 million confirmed COVID-19 cases and more than 166000 deaths in India. It has been declared pandemic worldwide and there is no effective vaccine and drug for COVID-19 at present. In such situation only the diagnosis and quarantine of confirmed cases can control the spread of this dreadful disease. Presently used molecular test kits are highly costly with need of costly instruments with a high infrastructure cost. Our COVID-19 LAMP test can solve these issues and can be used for mass screening for COVID-19 cases detection.

In LAMP assay, the target sequence is amplified at a constant temperature of 60-65°C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. Shorter reaction time with visual judgment of positivity without requiring sophisticated equipment make this test applicable for mass screening. The positive samples produced a green colour almost immediately on addition of SYBR Green I while the negatives remained orange. LAMP has a higher tolerance for inhibitors therefore high purity of nucleic acid is not needed. It only needs a dry bath instrument which can be installed easily at any basic laboratory without any special requirements.

COVID-19 LAMP test is isothermal which eliminates the need for expensive thermal cyclers used in conventional PCR. It is particularly useful test for COVID-19 detection in low- or middle-income countries for mass screening purpose due to its simplicity, ruggedness and low cost. It detects viral RNA in a sensitive and specific manner and there is naked eye visual detection of COVID-19 virus RNA amplification. Currently, there is no LAMP assay based COVID-19 test kit is available in India.

As there is an emergency around the world due to COVID-19 pandemic, currently there are more than 132 million COVID cases worldwide and they are increasing rapidly due to highly contagious nature of this virus. There is a dire need of rapid and reliable diagnostic kit for mass screening. Currently available RT-PCR are expensive and require skilled personnel and costly setup therefore they cannot be used for mass screening.

There is a need of highly efficient molecular and diagnostic methods / test kits for the detection of Coronavirus disease. The Invention provides a rapid, sensitive and specific cost-effective test for mass screening of COVID-19.

Object/s of invention:
Primary object of the present invention is to overcome the limitation of prior art.
Another object of the present invention is to provide a rapid, sensitive and specific cost-effective test for mass screening of COVID-19.
Another object of the present invention is to provide efficient LAMP primers set for diagnostic methods / kits development for the detection of Coronavirus disease.
Another object of the present invention is to provide LAMP assay based COVID-19 test kit.

Detailed description of Drawing:
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:
Figure 1: illustrates the COVID-19 RT-LAMP assay using different primer sets (1 to 8, and 9th is negative control). Panel A is result under day light (green colored tubes are positive while orange tubes are negatives for amplification), while Panel B is result-using UV light (bright tubes are positives and dark tubes are negatives). Primer set 2nd, 4th, 7th and 8th are working well while 3rd is faint positive while rest are not amplifying the target.

Figure 2: illustrates COVID-19 RT-LAMP assay with using different primer sets (1-4), 5th tube is negative control.

Figure 3: illustrates COVID-19 RT-LAMP assay with different concentrations of Twist RNA control (1st at 2000 RNA copies/µl, 2nd at 1000 RNA copies/µl, 3rd at 500 RNA copies/µl and 4th at 250 RNA copies/µl and 5th is negative control having no RNA copies).

Figure 4: illustrates COVID-19 LAMP assay lyophilized tubes containing the LAMP reagents in dried form at bottom of the tube and SYBR Green I on the inner side of the cap of the tubes.

Figure 5: illustrates COVID-19 LAMP assay with using lyophilized tubes (incubated at 37 oC), giving good amplification after incubation

Figure 6: illustrates COVID-19 LAMP assay procedure.

Detailed description of the Invention:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components.

The Invention provides novel set of primers which can be used in development of Loop-mediated isothermal amplification (LAMP) assay based detection of COVID-19.

The Invention provides a diagnostic test, based on novel set of primers, that can be performed within 90 min. at a very basic laboratory for COVID-19 testing. About 90 tests can be performed within 2 hrs using one instrument with 96 wells.

The Invention provides new sets of LAMP primers designed using Primer ExplorerV.5 software and manually. For each set of primers there are 6/4 primers (F3, B3, FIP, BIP, FLP -/ BLP or F3, B3, FIP and BIP).

In an embodiment, the Invention provides different COVID-19 genomes sequences including Indian strains sequences for these sets designing. The Invention provides 4 primers (F3, B3, FIP and BIP) designed using Primer ExplorerV.5 software or manually and other 2 primers (FLP and BLP) of each set manually. There were many sequence alignment and searches for probability of getting a good efficiently working LAMP primer set that can detect the target in a sensitive and specific manner. There were lots of Basic Local Alignment Search Tool (BLAST), that finds regions of local similarity between sequences, were done to check the binding probability of primers for binding to nonspecific targets. There were many manipulations in LAMP primer designing.

The novel LAMP primer sets are able to detect COVID-19 in a specific and sensitive manner. The LAMP assay based detection of infectious agents is dependent on the proper binding/hybridization of primers and if the primer designing is not appropriate then they may bind to non-specific targets and may give false positive results. The Inventors have designed the novel LAMP primer sets which are specific for COVID-19 detection.

In an embodiment, the LAMP assay based method for detecting the presence of COVID-19 virus in a sample comprises following :
a) treating the sample with primers comprising nucleotide sequence/s represented by Id 2,4,7 and 8 along with reaction mixture, positive control DNA present in an amount of approx. 2 µl, dye comprising SYBR Green 1 10000x diluted to approx. 1/10;
b) Incubating the solution resulting from step (a) at temperature ranging from 67°C for approx. 40 min in a heating block;
c) Centrifuging the tube resulting from step (b) for mixing of dye comprising SYBR Green I with the amplified product
where green color shows presence of COVID-19 virus in a sample while the orange color in the sample shows the absence of COVID-19 virus.
In an embodiment, the reaction mixture comprises following components:
i) approx.1.4 mM dNTPs;
ii) approx.0.8 mM betaine,
iii) approx.20 mM Tris-HCl of pH comprising approx.8.8,
iv) approx.10 mM KCl,
v) approx.10 mM (NH4)2SO4,
vi) approx.8 mM MgSO4,
vii) approx.0.1% Triton X-100,
viii) approx.8 U of Bst DNA polymerase large fragment
ix) approx.5 U of Reverse Transcriptase
In an embodiment, the reaction mixture is placed at the bottom of PCR tube while the diluted SYBR Green 1 was placed on the inner side of the cap of PCR tube.

In an embodiment, the Loop-mediated isothermal amplification (LAMP) primers for detecting the presence of COVID-19 virus in a sample wherein said primers nucleotide sequence is represented by Id 2,4,7 and 8.

The details of the primer sequences are mentioned below:

Set 1 – referred as Sequence Id 1
F3 CCAAAAAGAGAAAGTCAACATCA (referred as sequence Id 1A)
B3 ACTCAGTATTGATTTCTGTTCAC (referred as sequence Id 1B)
FIP CTTGTGGAAGCAGAAAAAGATGCTGGTGACTTTAAACTTAATGAAGAG (referred as sequence Id 1C)
BIP TCAAACAAATTGTTGAATCCTGTGGGGCACCTTTTTTAGCTTTTCC (referred as sequence Id 1D)

Set 2 - referred as Sequence Id 2
F3 CCCGCACTCTTGAAACTG (referred as sequence Id 2A)
B3 CCACTGCGAAGTCAACTG (referred as sequence Id 2B)
FIP TCTCAGTGAATACTGTGAAATTCCACTCAAAATTCTGTGCGTGT (referred as sequence Id 2C)
BIP TGATGCTATGATGTTCACATCTGATCAACACCACCTGTAATGTAGG (referred as sequence Id 2D)
BLP TGGCTACTAACAATCTAGTTGTAATG (referred as sequence Id 2E)
FLP GTATTGTTATAGCGGCCTTCTGTA (referred as sequence Id 2F)

Set 3 -- referred as Sequence Id 3
F3 AATTTTTCTTATTGTTGCGGC (referred as sequence Id 3A)
B3 TGATGATTCCTAAGAAAACAAGA (referred as sequence Id 3B)
FIP GCAGAAAGGCTAAAAAGCACAAATAGCTTCACACTCAAAAGAAAGAC (referred as sequence Id 3C)
BIP TGGTTCTCACTTGAACTGCAAGAATTTCATGTTCGTTTAGGCG (referred as sequence Id 3D)

Set 4 - referred as Sequence Id 4
F3 ACATTGCTGCTAATACTGTGA (referred as sequence Id 4A)
B3 ACCTTTAACACTACCTTCTGT (referred as sequence Id 4B)
FIP TGTCAGTCATAGAACAAACACCAATTCTGGGACTACAAAAGAGATG (referred as sequence Id 4C)
BIP CACCACTCACTGTCTTTTTTGATGAACACCATTACGGGCATT (referred as sequence Id 4D)
FLP CTCCAGCACATATATCTACT (referred as sequence Id 4E)
BLP AGAGTTGATGGTCAAGTAGACTTATT (referred as sequence Id 4F)

Set 5 - referred as Sequence Id 5
F3 GCTGCAATCGTGCTACAACT (referred as sequence Id 5A)
B3 TTGCTCTCAAGCTGGTTCAA (referred as sequence Id 5B)
FIP TGCGACTACGTGATGAGGAACGTTGCCAAAAGGCTTCTACGC (referred as sequence Id 5C)
BIP GGCAGCAGTAGGGGAACTTCTCTCTGTCAAGCAGCAGCAAAG (referred as sequence Id 5D)
FLP AGGCTTGACTGCCGCCTCTG (referred as sequence Id 5E)
BLP CTGCTAGAATGGCTGGCAATG(referred as sequence Id 5F)
Set 6 -- referred as Sequence Id 6
F3 TGGCTACTACCGAAGAGCT (referred as sequence Id 6A)
B3 TTGCAGCATTGTTAGCAGGA (referred as sequence Id 6b)
FIP TCTGGCCCAGTTCCTAGGTAGTGACGAATTCGTGGTGGTGAC (referred as sequence Id 6C)
BIP AGACGGCATCATATGGGTTGCAGCGGGTGCCAATGTGATC (referred as sequence Id 6D)
FLP GAAATACCATCTTGGACTGAGATCTTT (referred as sequence Id 6E)
BLP TGAGGGAGCCTTGAATACACCAAAA (referred as sequence Id 6F)

Set 7 - referred as Sequence Id 7
F3 GCTGTTTTGTAGATGATATCGTA (referred as sequence Id 7A)
B3 TGAAGTGTTATCATTAGTAAGCA (referred as sequence Id 7B)
FIP GGATGTTTAGTAAGTGGGTAAGCATAAAACAGATGGTACACTTATGATTG (referred as sequence Id 7C)
BIP AGGAGTATGCTGATGTCTTTCATTTATGTCTAACATGTGTCCTGT (referred as sequence Id 7D)
FLP CTATAGCTAAAGACACGAACCGTT (referred as sequence Id 7E)
BLP AATACATAAGAAAGCTACATGATGAGTTA(referred as sequence Id 7F)

Set 8- referred as Sequence Id 8
F3 GCACTGACTTGCTTTAGC (referred as sequence Id 8A)
B3 TGAAAGTTCAATCATTCTGTCTT (referred as sequence Id 8B)
FIP TAGGTGAAACTGATCTGGCACGTCAATTTGCTTTTGCTTGTCC (referred as sequence Id 8C)
BIP AGGAAGTTCAAGAACTTTACTCTCCTGAGTGTGAAGCAAAGTGT (referred as sequence Id 8D)

Set 9 - referred as Sequence Id 9
F3 TGGCTACTACCGAAGAGCT (referred as sequence Id 9A)
B3 TTGCAGCATTGTTAGCAGGA (referred as sequence Id 9B)
FIP TCTGGCCCAGTTCCTAGGTAGTGACGAATTCGTGGTGGTGAC (referred as sequence Id 9C)
BIP AGACGGCATCATATGGGTTGCAGCGGGTGCCAATGTGATC (referred as sequence Id 9D)
FLP GAAATACCATCTTGGACTGAGATCTTT (referred as sequence Id 9E)
BLP TGAGGGAGCCTTGAATACACCAAAA (referred as sequence Id 9F)

The novel LAMP primers are evaluated for the specific detection of synthetic DNA having the sequences of COVID-19 virus. The Invention has also applied test with standard positive and negative controls (using synthetic standards and healthy controls). After amplification with positives controls tubes become green colored while negatives remained orange colored after addition of SYBR Green 1(Reference Figure 1 a).

The present invention provides a sensitive and specific LAMP primer sets to detect COVID-19 using LAMP assay technology. LAMP assay is based on DNA amplification by strand displacement activity Bst DNA polymerase and the formation of Loops (due to the specific primers-FIP & BIP) which causes the doubling of target DNA during incubation at high temperature (60-70oC) during each cycle. When the target DNA, 4 primers (F3, B3, FIP & BIP; FIP/BIP are comprised of two primers together) and the reagents are incubated at a constant temperature between 60-70°C. As double stranded DNA is in the condition of dynamic equilibrium at the temperature around 60-70°C, one of the LAMP primers can anneal to the complimentary sequence of double stranded target DNA, then initiates DNA synthesis using the DNA polymerase with strand displacement activity, displacing and releasing a single stranded DNA. With the LAMP method, unlike with PCR, there is no need for heat denaturation of the double stranded DNA into a single strand.

These LAMP primer sets were designed for amplification of COVID-19 RNA by analysis of COVID-19 sequences available at www.ncib.nlm.nih/gov/genbank with Accession noMT019531.1using PrimerExplorer software. A total of 9 sets were designed. These primers were used for COVID-19 detection by LAMP assay, using the reaction mixtures and conditions for amplification of synthetic positive controls containing target gene sequences as follows:

In an embodiment, 25 µl LAMP reaction mixture consisting of 6 primers [40 pmol each of the forward inner primer (FIP) and backward inner primer (BIP), 5 pmol each of the F3 and B3 primers, 20 pmol each of the forward loop primer (FLP) and backward loop primer (BLP)], 1.4 mM dNTPs, 0.8 mM betaine, 20 mM Tris-HCl (pH8.8), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Triton X-100, 8 U of Bst DNA polymerase large fragment and 2 µl of positive control DNA was placed at the bottom of PCR tube, and 1 µl of diluted (1/10) SYBR Green 1 was placed on the inner side of the cap of PCR tube. The tube was incubated at 67°C for 40 min in a heating block and after incubation the tube was centrifuged to allow mixing of SYBR Green I with the amplified product. All positive samples produced a green colour almost immediately on addition of SYBR Green I after centrifugation, while the negatives remained orange (Figure 1a). The assay was specific to COVID-19 specific target detection and was negative with healthy human cDNA.
In an embodiment, the LAMP assay based test kit for detecting the presence of COVID-19 virus in a sample comprising:
a) LAMP reaction mixture present in an amount of 25 µl wherein said reaction mixture comprises:
i) 40 pmol each of the forward inner primer (FIP) and backward inner primer (BIP), as claimed in claims 1 to 8;
ii) 5 pmol each of the F3 and B3 primers, as claimed in claims 1 to 8;
iii) 20 pmol each of the forward loop primer (FLP) and backward loop primer (BLP), as claimed in claims 1 to 8;
iv) 1.4 mM dNTPs;
v) 0.8 mM betaine,
vi) 20 mM Tris-HCl of pH comprising approx.8.8,
vii) 10 mM KCl,
viii) 10 mM (NH4)2SO4,
ix) 8 mM MgSO4,
x) 0.1% Triton X-100,
xi) 8 U of Bst DNA polymerase large fragment
xii) 5 U Reverse Transcriptase
b) positive control present in an amount of 2-5 µl;
c) dye comprising SYBR Green 1, placed on the inner side of the cap of PCR tube, where said dye is diluted to approx. 1/10.
In an embodiment, the method of testing a sample for the presence of COVID-19 virus by the kit as describe above, comprises following steps:
a) Adding sample to lyophilized LAMP tube comprising primers represented by nucleotide sequence Id 2, 4 7 and 8 and reaction mixture of claim 2;
b) Adding water to solubilize the lyophilized components in the LAMP tube of step (a);
c) Incubating the tube at temperature ranging from 67°C for approx. 40 min for amplification where green color shows presence of COVID-19 virus in a sample while the orange color in the sample shows the absence of COVID-19 virus.
¬
The Invention is further described with the help of non-limiting examples:
Example 1:
The Invention provides novel set of primers which can be used in development of Loop-mediated isothermal amplification (LAMP) assay based detection of COVID-19. LAMP assay is a DNA amplification technique in which DNA amplification under isothermal conditions occurs in a rapid and specificity manner. In LAMP assay, the target sequence is amplified at a constant temperature of 60-70 °C using either two or three sets of primers and a polymerase with high strand displacement activity in addition to a replication activity. When the Reverse Transcriptase (RT) enzyme is added (which converts RNA to DNA), in the LAMP assay, RNA can be detected by LAMP assay due to RNA to DNA conversion through RT enzyme and then this DNA work as starting material for DNA amplification for LAMP assay. This RNA detection through addition of RT in LAMP assay is known as Reverse Transcriptase-LAMP (RT-LAMP) assay.
Example 2:
The Inventors designed and selected 8 primer sets using Primer Explorer V.5 software and manually targeting different genes of COVID-19 virus genome like ORF1a, N gene and RdRp genes. These primer sets were screened for their sensitivity and specificity using synthetic control available at Twist Biosciences (Twist Synthetic SARS-CoV-2 RNA Control). It was found that 3 sets are not working (May be Twist synthetic RNA is not complete for these sets or primers are not working) while 4 out of 6 sets are working well with the following conditions:
A 25 µl RT-LAMP reaction mixture consisting of primer mixture, (40 pmol each of the FIP and BIP, 5 pmol each of the F3 and B3 primers, 20 pmol each of the FLP and BLP), 1.4 mM dNTPs, 0.8 mM betaine, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 8 mM MgSO4, 0.1% Triton X-100, Bst DNA polymerase large fragment, Reverse Transcriptase, and positive control (Twist RNA). The reaction mixture was placed at the bottom of PCR tube and diluted SYBR Green 1 was placed on the inner side of the cap of PCR tube. The tube was incubated at 67°C for 40 min in a heating block and after incubation the tube was centrifuged to allow mixing of SYBR Green I with the amplified product. All positive samples produced a green colour almost immediately on addition of SYBR Green I after centrifugation, while the negatives remained orange (Figure 1). Result can be seen under UV light also which enhances the differentiation of positives and negatives better than visual day light result analysis.
Example 3:
The Invention provides designed novel LAMP primer sets that can detect COVID-19 in a specific and sensitive manner. The LAMP assay based detection of infectious agents is dependent on the proper binding/hybridization of primers and if the primer designing is not appropriate then they may bind to non-specific targets and may give false positive results.
After screening the working primer sets using Twist synthetic controls, the primer sets are selected which are working good for COVID-19 detection. The RT-LAMP is standardized in which both cDNA synthesis and DNA amplification occurs in the same tube for making the test simpler and field applicable (Figure 2). Figure 2 illustrates COVID-19 RT-LAMP assay with using different primer sets (1-4), 5th tube is negative control.
Example 4:
Experiment was performed to check the limit of detection using different RNA concentration of Twist control with COVID-19 RT-LAMP assay. The experiment results show good sensitivity up to 500 copies using Twist RNA control (Figure 3). Figure 3 illustrates COVID-19 RT-LAMP assay with different concentrations of Twist RNA control (1st at 2000 RNA copies/µl, 2nd at 1000 RNA copies/µl, 3rd at 500 RNA copies/µl and 4th at 250 RNA copies/µl and 5th is negative control having no RNA copies).
The Inventors also standardized the lyophilization of the RT-LAMP components in the single tube with SYBR Green on the inner side of the cap of the tube so that these tubes can be stored at room temperature or 2-8 oC storage to avoid the cost of cold storage and cold chain for transport (Figure 4). Figure 4 illustrates COVID-19 LAMP assay lyophilized tubes containing the LAMP reagents in dried form at bottom of the tube and SYBR Green I on the inner side of the cap of the tubes.

Example 5:
Different combinations of temperature, pressure and lyo-protectant was used for obtaining good activity of the lyophilized enzymes and components by doing stability testing (Figure 5). The lyophilized tubes were able to be stored for 5-6 days at Room temperature (25 oC). These accelerated studies show that the kits will remain stable for 5-6 months as per Arrhenius equations. Figure 5 illustrates COVID-19 LAMP assay with sing lyophilized tubes (incubated at 37 oC), giving good amplification after incubations.
Example 6:
The Invention employs COVID-LAMP test for solving the COVID-19 mass screening problem. The customized/ modified COVID-LAMP provided by the present invention test is an easy to use, rapid and reliable cost-effective molecular diagnostic solution. It will solve the sensitivity issue with a simpler solution, so that it can be easily used in peripheral settings with a minimal requirement for technical expertise etc.
The procedure for COVID-LAMP test is described in Figure 4. Briefly, RNA (50-60 min) or lysed saliva sample (within 10 min.) can be added to lyophilized LAMP tube (containing all the reagents at the bottom of tube) and water will be added to solubilize the lyophilized components. After this, tube will be incubated at 67oC for 40 min. for amplification. After amplification step, tube will be flicked up and down and then color will be checked. Positive sample will give green color while negatives will remain orange. For better results, tube can be seen under UV light (positives will seem brighter while negatives will remain dark)
The currently available RT-PCR is expensive and requires skilled personnel and costly setup; therefore, they cannot be used for mass screening. The COVID-19 LAMP test, provided by the present Invention, is isothermal which eliminates the need for expensive thermal cyclers used in conventional PCR. It is particularly useful test for COVID-19 detection in low- or middle-income countries for mass screening purpose due to its simplicity, ruggedness and low cost. It detects viral RNA in a sensitive and specific manner and there is naked eye visual detection of COVID-19 virus RNA amplification. There is a dire need of rapid and reliable molecular diagnostic test for mass screening purpose.

We Claim

1. A LAMP assay based method for detecting the presence of COVID-19 virus in a sample comprising:
a) treating the sample with primers comprising nucleotide sequence/s represented by Id 2,4,7 and 8 along with reaction mixture, positive control DNA present in an amount of approx. 2 µl, dye comprising SYBR Green 1 10000x diluted to approx. 1/10;
b) Incubating the solution resulting from step (a) at temperature ranging from 67°C for approx. 40 min in a heating block;
c) Centrifuging the tube resulting from step (b) for mixing of dye comprising SYBR Green I with the amplified product
where green color shows presence of COVID-19 virus in a sample while the orange color in the sample shows the absence of COVID-19 virus.

2. The method as claimed in claim 1, wherein said reaction mixture comprises:
i) approx.1.4 mM dNTPs;
ii) approx.0.8 mM betaine,
iii) approx.20 mM Tris-HCl of pH comprising approx.8.8,
iv) approx.10 mM KCl,
v) approx.10 mM (NH4)2SO4,
vi) approx.8 mM MgSO4,
vii) approx.0.1% Triton X-100,
viii) approx.8 U of Bst DNA polymerase large fragment
ix) approx.5 U of Reverse Transcriptase

3. The method as claimed in claim 1, wherein the reaction mixture is placed at the bottom of PCR tube while the diluted SYBR Green 1 was placed on the inner side of the cap of PCR tube.

4. A Loop-mediated isothermal amplification (LAMP) primers for detecting the presence of COVID-19 virus in a sample wherein said primers nucleotide sequence is represented by Id 2,4,7 and 8.
5. The primer as claimed in claim 4, wherein said sequence Id 2 comprises:
a) F3 CCCGCACTCTTGAAACTG (referred as sequence Id 2A)
b) B3 CCACTGCGAAGTCAACTG (referred as sequence Id 2B)
c) FIP TCTCAGTGAATACTGTGAAATTCCACTCAAAATTCTGTGCGTGT (referred as sequence Id 2C)
d) BIP TGATGCTATGATGTTCACATCTGATCAACACCACCTGTAATGTAGG (referred as sequence Id 2D)
e) BLP TGGCTACTAACAATCTAGTTGTAATG (referred as sequence Id 2E)
f) FLP GTATTGTTATAGCGGCCTTCTGTA (referred as sequence Id 2F)

6. The primer as claimed in claim 4, wherein said sequence Id 4 comprises:
a) F3 ACATTGCTGCTAATACTGTGA (referred as sequence Id 4A)
b) B3 ACCTTTAACACTACCTTCTGT (referred as sequence Id 4B)
c) FIP TGTCAGTCATAGAACAAACACCAATTCTGGGACTACAAAAGAGATG (referred as sequence Id 4C)
d) BIP CACCACTCACTGTCTTTTTTGATGAACACCATTACGGGCATT (referred as sequence Id 4D)
e) FLP CTCCAGCACATATATCTACT (referred as sequence Id 4E)
f) BLP AGAGTTGATGGTCAAGTAGACTTATT (referred as sequence Id 4F)

7. The primer as claimed in claim 4, wherein said sequence Id 7 comprises:
a) F3 GCTGTTTTGTAGATGATATCGTA (referred as sequence Id 7A)
b) B3 TGAAGTGTTATCATTAGTAAGCA (referred as sequence Id 7B)
c) FIP GGATGTTTAGTAAGTGGGTAAGCATAAAACAGATGGTACACTTATGATTG (referred as sequence Id 7C)
d) BIPAGGAGTATGCTGATGTCTTTCATTTATGTCTAACATGTGTCCTGT (referred as sequence Id 7D)
e) FLP CTATAGCTAAAGACACGAACCGTT (referred as sequence Id 7E)
f) BLP AATACATAAGAAAGCTACATGATGAGTTA (referred as sequence Id 7F)
8. The primer as claimed in claim 4, wherein said sequence Id 8 comprises:
a) F3 GCACTGACTTGCTTTAGC (referred as sequence Id 8A)
b) B3 TGAAAGTTCAATCATTCTGTCTT (referred as sequence Id 8B)
c) FIP TAGGTGAAACTGATCTGGCACGTCAATTTGCTTTTGCTTGTCC (referred as sequence Id 8C)
d) BIP AGGAAGTTCAAGAACTTTACTCTCCTGAGTGTGAAGCAAAGTGT (referred as sequence Id 8D)
9. A LAMP assay based test kit for detecting the presence of COVID-19 virus in a sample comprising:
d) LAMP reaction mixture present in an amount of 25 µl wherein said reaction mixture comprises:
xiii) 40 pmol each of the forward inner primer (FIP) and backward inner primer (BIP), as claimed in claims 1 to 8;
xiv) 5 pmol each of the F3 and B3 primers, as claimed in claims 1 to 8;
xv) 20 pmol each of the forward loop primer (FLP) and backward loop primer (BLP), as claimed in claims 1 to 8;
xvi) 1.4 mM dNTPs;
xvii) 0.8 mM betaine,
xviii) 20 mM Tris-HCl of pH comprising approx.8.8,
xix) 10 mM KCl,
xx) 10 mM (NH4)2SO4,
xxi) 8 mM MgSO4,
xxii) 0.1% Triton X-100,
xxiii) 8 U of Bst DNA polymerase large fragment
xxiv) 5 U Reverse Transcriptase
e) positive control present in an amount of 2-5 µl;
f) dye comprising SYBR Green 1, placed on the inner side of the cap of PCR tube, where said dye is diluted to approx. 1/10.

10. A method of testing a sample for the presence of COVID-19 virus by the kit as claimed in claim 9, comprising:
d) Adding sample to lyophilized LAMP tube comprising primers represented by nucleotide sequence Id 2, 4 7 and 8 and reaction mixture of claim 2;
e) Adding water to solubilize the lyophilized components in the LAMP tube of step (a);
f) Incubating the tube at temperature ranging from 67°C for approx. 40 min for amplification where green color shows presence of COVID-19 virus in a sample while the orange color in the sample shows the absence of COVID-19 virus.