WO2023002505A1

WO2023002505A1 - Assay for detection of epidemiologically important sars- cov-2 variants

Info

Publication number: WO2023002505A1
Application number: PCT/IN2022/050649
Authority: WO
Inventors: Shyam Sundar NANDI; Upendra LAMBE; Sonali Ankush Sawant; Jagadish DESHPANDE
Original assignee: Indian Council Of Medical Research
Priority date: 2021-07-19
Filing date: 2022-07-19
Publication date: 2023-01-26

Abstract

The present invention relates to novel oligonucleotide sequences for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein said oligonucleotide sequences acts as primers for amplification of sequences having Single-nucleotide polymorphisms (SNP)/mutations in the spike glycoprotein gene. The invention also relates to an assay for rapid screening of epidemiologically important variants of SARS-CoV-2 based upon using novel oligonucleotide sequences as primers. In particular, invention relates to oligonucleotide sequences of SEQ ID Nos. 1 – 37 for rapid screening of epidemiologically important variants of SARS-CoV-2.

Description

ASSAY FOR DETECTION OF EPIDEMIOLOGICALLY IMPORTANT

SARS- COV-2 VARIANTS

TECHNICAL FIELD OF THE INVENTION

[001] The present disclosure relates generally to the field of molecular biology. Specifically, the present disclosure provides for synthetic oligonucleotide sequences or primers and a screening assay for rapid and accurate detection of epidemiologically important SARS-CoV-2 variants employing said oligonucleotide sequences or primers. The present disclosure also discloses a kit comprising said primers for rapid and accurate detection of epidemiologically important SARS-CoV-2 variants. In case of detection of additional mutants in the variants of concern, the assay can be easily modified or updated based on the new mutants. The assay is modifiable and updatable in case occurrence of new variants (VOC/VOI) / reduced importance of older variants based on the need of the world.

BACKGROUND OF THE INVENTION

[002] Since the emergence of Coronavirus disease 2019 (COVID-19) pandemic, the scientific, public health and healthcare communities have come across the genetic variants of the new vims. The emerging variants are identified by increased transmissibility, increased morbidity and mortality, decreased susceptibility to antiviral drugs and antibodies which may cause significant impact on vaccine efficacy. Additionally, some variants might increase the risk of particular conditions such as multisystem inflammatory syndrome or post-acute sequel of COVID (CDC 2021). The variants that qualify one or more of these criteria are labeled as ‘variant under investigation’ or ‘variant of interest’ without confirmation of these properties. Many variants of SARS-CoV-2 are emerging, but the variants of concern (VOC) or the epidemiologically important SARS-CoV-2 variants should be identified for the better understanding of the severity of the disease. Once the variant is confirmed, it is termed as ‘variant of concern’ (Griffiths et ah, 2021).

[003] Worldwide, three most noteworthy VOCs have been identified. They were referred informally by associating with the name of the country where they were first identified. The names given to the variants are the “UK” (B.l.1.7), the “South Africa” (B.1.351) and the “Brazil” (PI) variants, along with the code given in brackets being their PANGO lineage nomenclature. [004] UK (B.1.1.7): Technically the UK variant is named as VUI-202012/01 and lineage B 1.1.7. (Chand et ah, 2020). The vims spread rapidly in south London and Southeast provinces in England. By December almost 60% of all newly detected COVID-19 cases (September through December) were attributed to this new variant (PHE 2020; Kupferschmidt 2020). The virus thus came to be recognized as a “super spreader” and other countries were alerted. Since then, its prevalence odds have doubled every 6.5 days, the presumed generational interval. It is correlated with a significant increase in the rate of COVID-19 infection in the United Kingdom, associated partly with the N501Y mutation (Mishra et ah, 2020). There are evidence suggesting that this variant has 40%-80% increased transmissibility (with most estimates lying around the middle to higher end of this range), and early analyses suggest an increase in lethality (Davis et ah, 2021; Horby et ah, 2021).

[005] South Africa (B.1.351): On 18 December 2020, the 501.V2 variant, also known as 501. V2, 20H/501Y.V2 (formerly 20C/501Y.V2), VOC-20DEC-02 (formerly VOC- 202012/02), or lineage B.1.351 was first detected in South Africa and reported by the country's health department (CDC 2020). It has been noted that the variant contains several mutations that allow it to attach more easily to human cells, and particularly, because of mutations in the receptor-binding domain (RBD) in the spike glycoprotein of the virus (Cosic et ah, 2021).

[006] Some variants are specific to Indian regions, which includes one called B.1.36. This variant is found to be present in a large number of cases tested in Bengaluru and other southern states. The specific mutation carried by the B.1.36 variant is N440K. Even though the data is insufficient, there are evidence that the B.1.36 variant may be responsible for some reinfections. Another, variant known as B.1.1.7 is also observed in the new cases in Punjab. Another variant, named B.1.617 is prominently observed in the sudden increase of cases in Maharashtra. This variant contains two specific mutations, called as E484Q and L452R. Both these mutations alter the spike region, allowing the variant to bind more easily to cells. This variant appears to have higher infectivity and transmissibility. But more disturbingly, recent studies show that the L452R mutation is also capable of immune escape, dodging both antibodies generated by a prior infection or a dose of vaccine as well as other forms of immunity that do not rely on antibodies. That the circulation of the new, potentially more infectious variants is responsible for the spike in cases after January 2021, seems to be increasingly inescapable. The parameters that enter models of how cases might increase now need to be changed by unrealistic amounts to account for the current rise (Menon 2021). Along with the single occurrence, there have been reports of double mutants from Pune or triple mutants from West Bengal. This raises alarming concerns regarding virulence of SARS-CoV- 2 among Indian population.

[007] Genomic surveillance of SARS-CoV-2 variants has largely focused on mutations in the spike glycoprotein, which mediates attachment to cells and is a major target of neutralizing antibodies. There is intense interest in whether mutations in the spike glycoprotein mediate escape from host antibodies and could potentially compromise vaccine effectiveness since spike is the major viral antigen in the current vaccines. The Ministry of Health & Family Welfare has formed Indian SARS-COV-2 Genomics Consortium (INSACOG) of laboratories having facilities of next generation sequencing to determine complete genome sequences of SARS-CoV-2 with NCDC, Delhi as the nodal agency and ICMR-NIV Pune as the apex laboratory along with eight other laboratories for sequence analysis. The INSACOG has a mandate of collecting 1-5% of all the positive COVID-19 samples in every state and 100% of all the positive samples from international travelers for whole genome sequencing analysis. The number of samples sequenced from January to March 18^th, 2021, in all over India are 7664. While the number of samples tested at ICMR-NIV from January to March 18^th, 2021, were 886. Because of the tedious process of whole sequencing analysis, it becomes difficult to sequence a larger number of samples. Looking at the increasing number of COVID-19 cases allover India, an easier technology is the need of an hour to screen the epidemiologically important variants of SARS-CoV-2. Apart from whole genome sequencing, Sanger sequencing assays for sequencing of Spike protein gene of SARS-CoV-2 and real time PCR based assays have been developed for the detection of epidemiologically important variants of SARS-CoV- 2. But till the date there is no report of direct detection of variant SNP/ mutations in SARS- CoV-2 by using multiplex primer extension (SNaPshot) based method. The advantage of using this method is that this assay enables multiplexing up to 10 single nucleotide polymorphisms in one reaction. This system can be used to assess and confirm SNPs/mutations with a better sensitivity than the standard capillary sequencing. Also, such assay reduces the complexity of the experiment, making it an easy to use and cost-effective method for genotyping, mutation detection and mapping.

[008] The present invention provides novel and synthetic oligonucleotide sequences for efficient screening assay which can be used at the COVID-19 diagnostic laboratories “to flag” samples most likely to contain the epidemiological important SARS-CoV-2 variants. These samples may be given priority for wide genome sequencing to confirm the presence of the variants and also to support studies on its evolution (molecular epidemiology).

OBJECTIVES OF THE INVENTION

[009] It is aim of the present invention to design and develop an assay(s) for screening of clinical samples for identification of epidemiologically important genetic variants of SARS- CoV-2 by:

• Designing of novel oligonucleotide primers for PCR amplification, sequencing, and site-specific nucleotide detection at several locations in the genome.

• Using the oligonucleotide primers, for multiplex detection of site- specific nucleotide in a concerted manner for easy presentation.

• Updatable assay for rapidly adapting it to evolving epidemiological situation.

SUMMARY OF THE INVENTION

[0010] The present invention relates to novel RT-PCR primers, which were designed to specifically amplify the receptor-binding domain (RBD) and region flanking the RBD, which belongs to the Spike protein gene of SARS-CoV-2. The number of important mutations in the SARS-CoV-2 genome are located in this region. This region was specifically selected in order to include the upcoming predicted future mutations also. For amplification of this region, four sets of primers (forward and reverse) have been designed. Along with these, the site-specific nucleotide detection primers (SNP primers) were designed specific to several locations of mutation in the RBD segment of S gene of SARS-CoV-2. These primers were attached with a non-specific tail of nucleotides (GACT) at 5’end of the SNP primers in an ascending concerted manner so as to get the easy presentable results. The primer belonging to the first nucleotide mutation has no tail, the second mutation has one tail, and the length of tail increases according to the location of mutation.

[0011] The present invention relates to amplification of these specific regions using single/multiplex reverse transcriptase polymerase chain reaction (RT-PCR). The amplified products were separated by agarose gel electrophoresis to produce one or more bands specific to the regions and the DNA is purified using gel extraction procedure. This DNA is later subjected to single or multiplex SNP reaction to identify mutations using SNaPshot (Thermo Scientific, USA) reagent in Genetic analyser (ABI Prism 3130x1). This was followed by analysis of results in Gene Mapper software (version 4). In addition, the present invention provides kit comprising said primer for rapid and accurate detection of epidemiologically important variants of SARS-CoV-2 such as Delta, Delta plus and Omicron variants. In case of detection of additional mutants in the variants of concern, the assay can be easily modified or updated based on the new mutants. The assay is modifiable and updatable in case occurrence of new variants (VOC/VOI) reduced importance of older variants based on the need of the world.

[0012] The present invention provides oligonucleotide sequences for rapid screening of epidemiologically important variants of SARS-CoV-2, said oligonucleotide sequences selected from the group consisting of SEQ ID Nos. 1 - 37.

[0013] In one of the aspects, the present invention provides oligonucleotide sequences for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein the oligonucleotide sequences of SEQ ID Nos. 1 - 24 are SNP primers for detection of mutations in the RBD region and regions flanking to the RBD domain of the spike protein of SARS-CoV-

2.

[0014] In one of the aspects, the present invention provides oligonucleotide sequences for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein the oligonucleotide sequences of SEQ ID Nos. 25 - 37 are RT-PCR amplification primers for variable region in the S gene of SARS-CoV-2.

[0015] In one of the aspects, the present invention provides oligonucleotide sequences for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein the oligonucleotide sequences of SEQ ID Nos. 1 - 24 are provided with a non-specific tail of nucleotides (GACT) in an ascending consorted manner so as to get the easy presentable results, wherein the oligonucleotide of the first nucleotide mutation has no tail, the second mutation has one tail, and the length of tail increases according to the location of mutation. [0016] In one of the aspects, the present invention provides oligonucleotide sequences for rapid screening of epidemiologic ally important variants of SARS-CoV-2, wherein the oligonucleotide sequences of SEQ ID Nos. 25 - 37 are grouped into four sets, the first set (SEQ ID Nos. 25 - 29) consisting of the region having five mutations, the second set (SEQ ID Nos. 30 and 31) consisting of three mutations, third set (SEQ ID Nos. 32 - 35) consisting of one mutation, and fourth set (SEQ ID Nos. 36 and 37) to amplify the complete target segment of S gene.

[0017] In one of the aspects, the present invention provides oligonucleotide sequences for rapid screening of epidemiologic ally important variants of SARS-CoV-2, wherein the oligonucleotide sequences are useful in rapid and accurate detection of Delta, Delta plus and Omicron variants of SARS-CoV-2.

[0018] In another aspect, the present invention provides an assay for rapid screening of epidemiologically important variants of SARS-CoV-2, said assay comprising of: (i) amplifying the sample RNA with RT-PCR amplification primers/ oligonucleotide sequences selected from the group consisting of SEQ ID Nos. 25 - 37; (ii) separating and purifying PCR amplicon obtained from above steps by conventional methods known in the art, and wherein the conventional methods known in the art for separating and purifying PCR amplicon comprises of separation of amplicon using agarose gel electrophoresis and separate extraction of different bands of gel using extraction reagents, or PCR purification kits or any other suitable method; (iii) subjecting the purified PCR amplicon obtained in above step to a multiplex SNP/ mutation reaction using SNP primers/ oligonucleotide sequences selected from the group consisting of SEQ ID Nos. 1 - 24; (iv) treating the reaction mixture obtained in above step with equal volumes of an alkaline phosphatase enzyme and buffer and incubating at 37°C for 15 minutes followed by deactivation at 72°C for 10 minutes; (v) mixing lpl of the reaction mixture obtained in above step with 8.9pl highly deionized formamide and O.ΐmΐ GeneScan 120 LIZ, and heating to 95°C for 5 minutes, followed by snap chilling on ice; and (vi) running the above reaction mixture on a genetic analyzer at 60°C at 15KV and 5mA current for 45 min and identifying the variants.

[0019] In one of the aspects, the present invention provides an assay for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein the sample RNA is a total viral RNA, extracted from the deactivated samples from suspected COVID-19 patient in viral transport medium (VTM) or Molecular transport medium (MTM).

[0020] In one of the aspects, the present invention provides an assay for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein the identification of the variants in step (vi) is carried out using Gene Mapper software V4.

[0021] In one of the aspects, the present invention provides an assay for rapid screening of epidemiologically important variants of SARS-CoV-2, wherein SNP primers/ oligonucleotide sequences of SEQ ID Nos. 1, 2 and 10 are used as forward primers, while SEQ ID Nos. 16, 18, 19 and 20 were used as reverse primers for detection of delta variant of SARS-CoV-2 and wherein SNP primers/ oligonucleotide sequences of SEQ ID Nos. 1, 2, 6, 7, 8 and 10 are used as forward primers, while SEQ ID No. 16 was used as reverse primers for detection of omicron variants of SARS-CoV-2.

DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0022] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings wherein:

[0023] Figure 1: Shows results of multiplex SNP assay for detection of epidemiologically important SARS-CoV-2 Delta variant. The figure shows significant sites mutations in the Spike protein gene. These mutations are analysed by using Gene Mapper Software version 4 to identify the Delta and Delta plus variants of SARS-CoV-2.

[0024] Figure 2: Shows results of multiplex SNP assay for detection of epidemiologically important SARS-CoV-2 Omicron variant. The figure shows significant sites mutations in the Spike protein gene. These mutations are analysed by using Gene Mapper Software version 4 to identify the Omicron variants of SARS-CoV-2.

DETAILED DESCRIPTION OF THE INVENTION [0025] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the invention, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

[0026] Definitions: For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person skilled in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0027] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purposes of exemplification only. Functionally equivalent products and methods are clearly within the scope of the disclosure, as described herein.

[0029] The present invention relates to novel oligonucleotide sequences/primers against multiplex mutation detection, which were identified in the spike glycoprotein gene from various published meta-analysis studies. To amplify the variable region in the S gene of SARS- CoV-2, four sets of multiplex RT-PCR primers were designed. The first set of RT-PCR primers consist of the region having five mutations, the second set consisted of three mutations and the third set of primers consisted of one mutation. In addition to these, one more comprehensive set of primers were designed to amplify the complete targeted segment of S gene in which all the mutations were observed (CoV-SetA_Fl/Rl).

[0030] Variants of SARS-CoV-2 are especially important to be detected to study the molecular evolution of the virus. In this invention, epidemiologic ally important mutations were identified in the spike glycoprotein gene from various published meta-analysis studies. The site-specific nucleotide detection primers (SNP primers) were designed specific to several locations of mutation in the RBD region and regions flanking to the RBD domain of Spike protein. These primers were added a non-specific tail of nucleotides (GACT) in an ascending consorted manner so as to get the easy presentable results. The primer belonging to the first nucleotide mutation has no tail, the second mutation has one tail, and the length of tail increases according to the location of mutation. A multiplexed SNP reaction was set up for detecting nucleotides at specific positions in the amplified DNA product. The reaction is run in ABI Genetic Analyzer. The results were recorded using Gene Mapper software which is already installed in the machine. Combination of the nucleotides at the defined positions will inform whether the sample contains the epidemiologic ally important variants.

EXAMPLES:

[0031] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

Example 1:

[0032] Methodology: Since emergence of SARS-CoV-2, many variants have been identified. In this invention, a screening assay has been disclosed for detection of epidemiologically important SARS-CoV-2 variants. Some of them are identified by increased transmissibility, increased morbidity and mortality, decreased susceptibility to antiviral drugs and antibodies which may cause plausible impact on vaccine efficacy. These variants are especially important to be detected to study the molecular evolution of the virus. In this invention, epidemiologically important mutations were identified in the spike glycoprotein gene from various published meta-analysis studies. The identified mutation and their positions in the SARS-CoV-2 genome are described in table 1.

[0033]

Table 1: Epidemiologically important mutation of SARS-CoV-2 and their nucleotide position

[0034] Primer designing: The site-specific SNP detection oligonucleotide sequences/primers for each mutation were designed in both sense and antisense directions. The site- specific nucleotide detection primers were designed specific to several locations of mutation in the RBD region of Spike protein. These primers were added a non-specific tail of nucleotides (GACT) in an ascending consorted manner so as to get the easy presentable results. The primer belonging to the first nucleotide mutation has no tail, the second mutation has one tail, and the length of tail increases according to the location of mutation.

[0035] To amplify the variable region in the S gene of SARS-CoV-2, various sets of multiplex RT-PCR primers were designed. The first set of RT-PCR primers consist of the region having five mutations, the second set consisted of three mutations and the third set of primers consisted of one mutation. In addition to these, one more comprehensive set of primers were designed to amplify the complete targeted segment of S gene in which all the mutations were observed (CoV-SetA_Fl/Rl). All the primers were self-designed and were synthesized from SIGMA Chemical (USA). The list of PCR primers and SNP primers is provided below (Table 2 and 3).

[0036]

Table 2: List of SNP primers

[0037]

Table 3: Details of primers used in RT-PCR

Example 2:

Methodology for detection of epidemiologically important variants of concern:

[0038] Sample acquisition: The Nasopharyngeal and / or oropharyngeal swab samples collected from healthcare centers are brought to the ICMR-NIV, Mumbai unit. These samples from suspected COVID-19 patient were received in viral transport medium (VTM) or Molecular transport medium (MTM).

[0039] RNA Extraction: The samples were processed in the BSL-III facility and deactivated by addition of lysis buffer. The samples were then further processed in BSL-II facility for further processing. Total viral RNA was extracted from the deactivated samples using commercial viral RNA extraction kit according to the manufacturer’s instructions (Cat No./ID: 52906; QIAamp Viral RNA Mini Kit).

[0040] RT-PCR: The RT-PCR was performed by using S gene specific primers. The present invention relates to novel RT-PCR primers which were designed to specifically amplify the receptor binding domain (RBD) and region upstream of the Spike protein gene of SARS-CoV- 2. The number of important mutations in the SARS-CoV-2 genome are located in this region. This region was specifically selected in order to include the upcoming predicted future mutations also. The extracted RNA was subjected to multiplex RT-PCR using novel sets of oligonucleotide sequences/primers designed for Spike protein gene of SARS-CoV-2. Four sets of primers (forward and reverse) were used to amplify the genomic region targeted for detection of variants. The details of oligonucleotide sequences/primers are mentioned in table

3.

[0041] One step RT-PCR kit was used to perform RT-PCR using known SARS-CoV-2 positive RNA. The reaction mix was prepared as per the manufacturer’s instructions (Table 4). The reaction was set up and incubated at cycling temperature conditions in ABI thermal cycler (ABI 9700). The reverse transcription step was performed at 50 °C for 30 minutes followed by initial denaturation step was done at 95°C for 5 min, three step cyclic (40 cycles) denaturation at 95°C for 30 seconds, extension step was at 72°C for 90 seconds and final extension at 72°C for 7 min, annealing temperature was 60°C for 30 sec.

[0042]

Table 4: RT-PCR reaction mixture

[0043] Agarose Gel Electrophoresis: The PCR products were resolved in 1-2 % agarose (Life Tech) gel containing Gel red dye (Biotium) in tris-acetate-EDTA (TAE) buffer along with 1000 bp DNA ladder (Thermo Fisher scientific). The 40 pi of RT-PCR product was mixed with 6 mΐ of 6X loading dye and was loaded in the wells. The electrophoresis was carried out at 12 V/cm of gel in IX TAE running buffer in horizontal electrophoresis unit (Biometra, USA) and power supply (Pharmacia) till the indicator 6X loading dye reached end of the gel. The gels were visualized under UV transilluminator (Biovis) and photographed. The expected size of PCR products was estimated by comparison with that of standard DNA ladder. The expected band was cut, and DNA was purified using gel extraction kit (QIAquick Gel Extraction Kit - QIAGEN). The gel purified DNA was used in further experiments. [0044] Multiplex SNP reaction: The multiplex SNP reaction was set up using SNP (10 mM working stock) primers mixed in equal proportions. For delta variants forward primers of K417N, N440K and D614G while reverse primers for T478K, Q493R, N501Y and T547K were used. On the other hand, for Omicron variants forward primers of K417N, N440K, Q493R, N501Y, T547K and D614G while reverse primers for T478K were used. The reaction contained SNaPshot® Multiplex master mix 2.5 pi, while template (gel purified) DNA and SNP primer mix (or individual SNP primer) were added 1.25 mΐ each. The reaction was set up and incubated at cycling temperature conditions in ABI thermal cycler (ABI 9700). Three step cyclic denaturation at 95°C for 10 seconds, extension step was at 60°C for 30 seconds and annealing temperature was 52°C for 10 sec with 25 cycle was performed.

[0045] The SNP multiplex reaction was treated with alkaline phosphate by mixing equal volumes of FastAP Thermosensitive Alkaline Phosphatase (EF0651 Thermo Fisher Scientific) with FastAP buffer. The reaction was incubated at 37°C for 15 minutes and deactivated at 72°C for 10 minutes to stop the reaction.

[0046] Genetic Analyzer: Imΐ of the SNP reaction product was mixed with 8.9m1 highly deionized Formamide and O.ΐmΐ GeneScan 120 LIZ (Applied Biosystems). This mixture was heated at 95 °C for 5 minutes and snap chilled on ice. This was followed by electrophoresis at 60°C at 15KV and 5mA current for 45 min in ABI 3130x1 Genetic Analyzer (Applied Biosystems). Results were analyzed using Gene Mapper software V4. The GeneScan 120 LIZ is Size Standard which is designed for sizing DNA fragments in the 15-120 nucleotides range and provides nine single- stranded labeled fragments of: 15, 20, 25, 35, 50, 62, 80, 110 and 120 nucleotides. The sizing curve generated from these short fragments make the GeneScan™ 120 LIZ™ Size Standard ideal for use with the ABI Prism SNaPshot™ Multiplex Kit as well as other short fragment analysis applications. Each of the DNA fragments is labeled with the LIZ™ fluorophore which results in a single peak when run under denaturing conditions. With the 5th dye LIZ™ your marker fragments can be labeled with the dyes FAM™, VIC™, NED™ or PET™. Since the standard is labeled with the fifth dye, one can genotype a greater number of markers in a given lane, compared to the four-dye system.

[0047] Analysis: The results were analysed using Gene mapper (V4) and the variants identified based on multiplex SNP reactions are reported. For the purpose of validation of this technology, a sum total of 30 known samples of circulating variants (15 samples of Delta and 15 samples of Omicron variants) were tested. The results are displayed in the Table 5 and 6 as well as Figure 1 and 2.

[0048]

Table 5: List of known positive delta variant of SARS-CoV-2 samples tested using SNP assay and analysis of their results.

[0049]

Table 6: List of known positive omicron variant of SARS-CoV-2 samples tested using SNP assay and analysis of their results

[0050] Confirmation by sequencing: To confirm the results shown by multiplex SNP reaction, the same gel extracted DNA samples were sequenced by in ABI 3130x1 Genetic Analyzer (Applied Biosystems). The sequences were analyzed on Sequencher V5.4. The locations of SNPs were identified in the sequence and the results were matched with multiplex SNP assay results.

[0051] Advantages of the present invention:

• The assay can be performed using a regular capillary sequencer device and the high throughput next generation sequencing machine is not required.

• The time required for the assay is only 3 to 4 hours to perform.

• The results can be interpreted by observing peaks generated by Gene mapper software.

• It is cost effective and less time-consuming technology.

• This assay could enable screening of a large number of samples.

Claims

WE CLAIM:

1. An oligonucleotide sequences for rapid screening of epidemiologically important variants of SARS-CoV-2, said oligonucleotide sequences selected from the group consisting of SEQ ID Nos. 1 - 37.

2. The oligonucleotide sequences as claimed in claim 1, wherein the oligonucleotide sequences of SEQ ID Nos. 1 - 24 are SNP primers for detection of mutations in the RBD region of the spike protein of SARS-CoV-2.

3. The oligonucleotide sequences as claimed in claim 1, wherein the oligonucleotide sequences of SEQ ID Nos. 25 - 37 are RT-PCR amplification primers for variable region in the S gene of SARS-CoV-2.

4. The oligonucleotide sequences as claimed in claim 1, wherein the oligonucleotide sequences of SEQ ID Nos. 1 - 24 are provided with a non-specific tail of nucleotides (GACT) in an ascending consorted manner so as to get the easy presentable results, wherein the oligonucleotide of the first nucleotide mutation has no tail, the second mutation has one tail, and the length of tail increases according to the location of mutation.

5. The oligonucleotide sequences as claimed in claim 1, wherein the oligonucleotide sequences of SEQ ID Nos. 25 - 37 are grouped into four sets, the first set (SEQ ID Nos. 25 - 29) consisting of the region having five mutations, the second set (SEQ ID Nos. 30 and 31) consisting of three mutations, third set (SEQ ID Nos. 32 - 35) consisting of one mutation, and fourth set (SEQ ID Nos. 36 and 37) to amplify the complete targeted segment of S gene.

6. The oligonucleotide sequences as claimed in claim 1, wherein the oligonucleotide sequences are useful in rapid and accurate detection of Delta, Delta plus and Omicron variants of SARS-CoV-2.

7. An assay for rapid screening of epidemiologically important variants of SARS-CoV-2, said assay comprising of: (i) amplifying the sample RNA with RT-PCR amplification primers/ oligonucleotide sequences selected from the group consisting of SEQ ID Nos. 25 - 37;

(ii) separating and purifying PCR amplicon obtained from above steps by conventional methods known in the art, and wherein the conventional methods known in the art for separating and purifying PCR amplicon comprises of separation of amplicon using agarose gel electrophoresis and separate extraction of different bands of gel using extraction reagents, or PCR purification kits or any other suitable method;

(iii) subjecting the purified PCR amplicon obtained in above step to a multiplex SNP/ mutation reaction using SNP primers/ oligonucleotide sequences selected from the group consisting of SEQ ID Nos. 1 - 24;

(iv) treating the reaction mixture obtained in above step with equal volumes of an alkaline phosphatase enzyme and buffer and incubating at 37°C for 15 minutes followed by deactivation at 72°C for 10 minutes;

(v) mixing lpl of the reaction mixture obtained in above step with 8.9pl highly deionized formamide and O.ΐmΐ GeneScan 120 LIZ, and heating to 95°C for 5 minutes, followed by snap chilling on ice; and

(vi) running the above reaction mixture on a genetic analyzer at 60°C at 15KV and 5mA current for 45 min and identifying the variants.

8. The assay as claimed in claim 7, wherein the sample RNA is a total viral RNA, extracted from the deactivated samples from suspected COVID-19 patient in viral transport medium (VTM) or Molecular transport medium (MTM).

9. The assay as claimed in claim 7, wherein the identification of the variants in step (vi) is carried out using Gene Mapper software V4.

10. The assay as claimed in claim 7, wherein SNP primers/ oligonucleotide sequences of SEQ ID Nos. 1, 2 and 10 are used as forward primers, while SEQ ID Nos. 16, 18, 19 and 20 were used as reverse primers for detection of delta variant of SARS-CoV-2 and wherein SNP primers/ oligonucleotide sequences of SEQ ID Nos. 1, 2, 6, 7, 8 and 10 are used as forward primers, while SEQ ID No. 16 was used as reverse primers for detection of omicron variants of SARS-CoV-2.