WO2023119307A1 - Primer and probes for detection of omicron variant of sars-cov-2, methods and uses thereof - Google Patents

Primer and probes for detection of omicron variant of sars-cov-2, methods and uses thereof Download PDF

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WO2023119307A1
WO2023119307A1 PCT/IN2022/050748 IN2022050748W WO2023119307A1 WO 2023119307 A1 WO2023119307 A1 WO 2023119307A1 IN 2022050748 W IN2022050748 W IN 2022050748W WO 2023119307 A1 WO2023119307 A1 WO 2023119307A1
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seq
sequence
set forth
primer
variant
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French (fr)
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Girish KRISHNAMURTHY
Sunil Govekar
Satyaprakash PANDEY
Mohammad Azhar
Pramod Devanga
Vasanthapuram Ravi
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Krishnamurthy Girish
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The present invention provides synthetic DNA based labelled hybridization probes capable of particularly binding to Omicron variant of SARS-CoV-2 for Omicron variant specific detection using qRT-PCR method. The present invention also provides methods of detection of Omicron variant and kits comprising primers/probes and reagents for Omicron variant detection. The reagents and methods of detection of Omicron variant show high degree of specificity and no false positive or false negative results.

Description

PRIMER AND PROBES FOR DETECTION OF OMICRON VARIANT OF SARS-COV-2,
METHODS AND USES THEREOF
FIELD OF INVENTION
The present invention relates to the field of diagnostics. In particular, the present invention provides DNA based primers and probes for the detection of particular variant of SARS-CoV-2.
BACKGROUND OF THE INVENTION
Coronavirus causes infection in the nose, sinuses, or upper respiratory tract. Most coronaviruses are not dangerous. In early 2020, after a December 2019 outbreak in China, the World Health Organization (WHO) identified SARS-CoV-2 as a new type of coronavirus, that quickly spread around the world.
COVID-19, caused by SARS-CoV-2, can trigger a respiratory tract infection, and affect the upper respiratory tract (sinuses, nose, and throat) or lower respiratory tract (windpipe and lungs). It spreads the same way as other coronaviruses i.e., mainly through person-to-person contact. Infections range from mild to severe. Standard recommendations are followed to prevent the spread of infection.
Like other coronaviruses, the SARS-CoV-2 virus has been undergoing continuous mutations leading to multiple Variants of Concern (VOCs) such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.l), Delta (B.1.617.2) and most recently Omicron (B.1.1.529). The B.1.1.529 variant was first reported to WHO from South Africa on 26th November 2021. Omicron variant is designated as a VOC and hallmarked by presence of multiple mutations in the spike protein (S gene), that lead to increased transmissibility and decreased effectiveness of public health measures.
As per the advisory from WHO on 26th November 2021, Omicron poses an increased risk and infections have increased steeply across the globe. Screening for Omicron variant is thus critical and will ensure timely detection, intervention and control the spread of this variant.
As of 20th December 2021, more than 16000 Omicron sequences have been deposited by 75 countries demonstrating the highly transmissible and infectious nature of this variant. The standard method for diagnosis of SARS-CoV-2 is by Real Time Reverse Transcription PCR (rRT- PCR). Presence of multiple mutations pose a Herculean challenge to precisely detect Omicron. At present, the only reliable method for confirmation of Omicron variant is whole genome sequencing. This approach is costly, time-consuming and resource intensive making it impractical for routine testing.
Although, assays using a S gene dropout/ S gene target failure are being employed to detect the Omicron variant, these assays target the 69-70 deletion or specific point mutations that are observed in Omicron variant. However, both these strategies are not fool proof as these regions and mutations are not unique to Omicron and can be found in other VOCs (for example, 69-70 deletion is present in the Alpha VOC). Hence, these assays best serve as a surrogate marker of presence of Omicron variant. Additionally, a "stealth" version of Omicron lacks this deletion region altogether making it more challenging to design and develop an Omicron detection method.
Therefore, there is a need in the art to develop Omicron specific testing and detection schemes which are quick and economical while providing high level of confidence.
OBJECTS OF THE INVENTION
An object of the invention is to provide nucleotide-based primer and probes for specific detection of Omicron variant of SARS-CoV-2 in a sample suspected of comprising the Omicron variant.
Another object of the invention is to provide a multiplex assay system for identification of Omicron variant of SARS-CoV-2 in a sample suspected of comprising the Omicron variant.
Yet another object of the invention is to provide a uniplex and multiplex assay combining dropout strategy and detection strategy to identify presence of Omicron variant of SARS-CoV-2 in a sample suspected of comprising the Omicron variant.
SUMMARY OF THE INVENTION
In an aspect of the present invention, there is provided synthetic DNA based labelled probes having sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 9 capable of specifically hybridizing to a region of the Omicron variant of SARS-CoV-2 coronavirus.
In another aspect of the present invention, there is provided an in-vitro method of detecting Omicron variant of SARS-CoV-2 coronavirus in a sample, said method comprising: (a) carrying out a DNA amplification reaction, said reaction comprising: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer sequence having sequence as set forth in SEQ ID NO: 2; and (ii) a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; and (b) detecting fluorescence level, wherein increase in fluorescence level is indicative of the sample being positive for Omicron variant of SARS-CoV-2; or (a) carrying out an amplification reaction, said reaction comprising: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 6 and a second primer sequence having sequence as set forth in SEQ ID NO: 8; and (ii) a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9; and (b) detecting fluorescence level, wherein increase in fluorescence level is indicative of the sample being positive for Omicron variant of SARS-CoV-2; or (a) carrying out an amplification reaction, said reaction comprising (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO:2; (ii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3; (iii) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; (b) detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample; or (a) carrying out an amplification reaction, said reaction comprising: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO:2; (ii) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 6 and a second primer having sequence as set forth in SEQ ID NO: 8; (iii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3; and (iv) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9; and (b) detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample; or (a) carrying out an amplification reaction, said reaction comprising: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 and a second primer having sequence as set forth in SEQ ID NO:6; (ii) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO: 2; (iii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and (iv) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; and (b) detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample; or (a) carrying out an amplification reaction, said reaction comprising: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 a second primer having sequence as set forth in SEQ ID NO:6; and a third primer having sequence as set forth in SEQ ID NO: 8; (ii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and (iii) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9; and (b) detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample.
In yet another aspect of the present invention, there is provided a kit comprising at least one of: (a) a first primer having sequence as set forth in SEQ ID NO: 1; (b) a second primer having sequence as set forth in SEQ ID NO: 2; and (c) a labelled probe having sequence as set forth in SEQ ID NO: 4; or (a) a first primer having sequence as set forth in SEQ ID NO: 6; (b) a second primer having sequence as set forth in SEQ ID NO: 8; and (c) a labelled probe having sequence as set forth in SEQ ID NO: 9; or (a) a first primer having sequence as set forth in SEQ ID NO: 1; (b) a second primer having sequence as set forth in SEQ ID NO: 2; (c) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 3; and (d) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4; or (a) a first primer having sequence as set forth in SEQ ID NO: 1; (b) a second primer having sequence as set forth in SEQ ID NO: 2; (c) a third primer having sequence as set forth in SEQ ID NO: 6; (d) a fourth primer having sequence as set forth in SEQ ID NO: 8; (e) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 3; and (f) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9; or (a) a first primer having sequence as set forth in SEQ ID NO: 5; (b) a second primer having sequence as set forth in SEQ ID NO: 6; (c) a third primer having sequence as set forth in SEQ ID NO: 1; (d) a fourth primer having sequence as set forth in SEQ ID NO: 2; (e) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4; and (f) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 7; or (a) a first primer having sequence as set forth in SEQ ID NO: 5; (b) a second primer having sequence as set forth in SEQ ID NO: 6; (c) a third primer having sequence as set forth in SEQ ID NO: 8; (d) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 7; and (f) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9.
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
Figure 1 depicts the schematic layout of a dropout strategy for detection of Omicron variant of SARS-CoV-2 in a sample, in accordance with an embodiment of the present invention.
Figure 2 depicts the schematic layout of the detection strategy to particularly identify presence of Omicron variant of SARS-CoV-2 in a sample, in accordance with an embodiment of the present invention.
SEQUENCES
SEQ ID NO: 1 depicts the first primer for S gene dropout 1 or for S gene detection 1.
SEQ ID NO: 2 depicts the second primer for S gene dropout 1 for S gene detection 1.
SEQ ID NO: 3 depicts the labelled probe for S gene dropout 1.
SEQ ID NO: 4 depicts the labelled probe for S gene detection 1.
SEQ ID NO: 5 depicts the first primer for S gene dropout 2.
SEQ ID NO: 6 depicts the second primer for S gene dropout 2 or first primer for S gene detection 2.
SEQ ID NO: 7 depicts the labelled probe for S gene dropout 2. SEQ ID NO: 8 depicts the second primer for S gene detection 2.
SEQ ID NO: 9 depicts the labelled probe for S gene detection 2.
SEQ ID NO: 10 depicts the first primer for N gene.
SEQ ID NO: 11 depicts the second primer for N gene.
SEQ ID NO: 12 depicts the labelled probe for N gene.
SEQ ID NO: 13 depicts the first primer for RdRp gene.
SEQ ID NO: 14 depicts the second primer for RdRp gene.
SEQ ID NO: 15 depicts the labelled probe for RdRp gene.
SEQ ID NO: 16 depicts the first primer for ORFlab gene.
SEQ ID NO: 17 depicts the second primer for ORFlab gene
SEQ ID NO: 18 depicts the labelled probe for ORFlab gene.
SEQ ID NO: 19 depicts the amplicon obtained from first and second primer for S gene dropout 1.
SEQ ID NO: 20 depicts the amplicon obtained from first and second primer for S gene detection 1.
SEQ ID NO: 21 depicts the amplicon obtained from first and second primer for S gene dropout 2.
SEQ ID NO: 22 depicts the amplicon obtained from first and second primer for S gene detection 2.
SEQ ID NO: 23 depicts the amplicon obtained from first and second primer for N gene.
SEQ ID NO: 24 depicts the amplicon obtained from first and second primer for RdRp gene.
SEQ ID NO: 25 depicts the amplicon obtained from first and second primer for ORFlab gene.
DETAILED DESCRIPTION OF THE INVENTION
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and all combinations of any two or more of said steps or features.
Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of the ordinary skills in the art to which the invention belongs. For further guidance, term definitions may be included to better appreciate the teaching of the present invention.
Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
It should be emphasized that the term "comprises/comprising" when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof.
The present invention relates to a synthetic DNA based labelled probe having sequence as set forth in SEQ ID NO: 4 capable of specifically hybridizing to a region of the Omicron variant of SARS-CoV-2 coronavirus.
The present invention relates to a synthetic DNA based labelled probe having sequence as set forth in SEQ ID NO: 9 capable of specifically hybridizing to a region of the Omicron variant of SARS-CoV-2 coronavirus.
The synthetic DNA based labelled probe label is any one of FAM-BHQ1, HEX-BHQ1, and Cy5- BHQ2. In an embodiment, the probe is FAM-BHQ1. In another embodiment, the label is HEX- BHQ1. In yet another embodiment, the label is Cy5-BHQ2. It is understood by a person skilled in the art that any other suitable labels known in the art can be used as substitutes.
The present invention relates to a first in-vitro method of detecting Omicron variant of SARS- CoV-2 in a sample suspected of comprising at least one variant of SARS-CoV-2, comprising:
(a) carrying out an amplification reaction, wherein the amplification reaction comprises: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer sequence; having sequence as set forth in SEQ ID NO: 2; and
(ii) a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3; and
(b) detecting fluorescence level wherein increase in fluorescence level is indicative of the sample being positive for at least one variant of SARS-CoV-2 and wherein said at least one variant is not Omicron variant.
The amplicon obtained from the amplification reaction of the first method is as set forth in SEQ ID NO: 19.
The present invention relates to a second in-vitro method detecting Omicron variant of SARS- CoV-2 in a sample suspected of comprising at least one variant of SARS-CoV-2, comprising:
(a) carrying out an amplification reaction, wherein the amplification reaction comprises:
(i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer sequence having sequence as set forth in SEQ ID NO: 2; and
(ii) a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; and
(b) detecting fluorescence level wherein increase in fluorescence level is indicative of the sample being positive for at least the Omicron variant.
The amplicon obtained from the amplification reaction of the second method is as set forth in SEQ ID NO: 20.
The present invention relates to a third in-vitro method of detecting Omicron variant of SARS- CoV-2 in a sample suspected of comprising at least one variant of SARS-CoV-2, comprising:
(a) carrying out an amplification reaction, wherein the amplification reaction comprises: (i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 and a second primer sequence having sequence as set forth in SEQ ID NO: 6; and
(ii) a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and
(b) detecting fluorescence level wherein increase in fluorescence level is indicative of the sample being positive for at least one variant of SARS-CoV-2 and wherein said at least one variant is not Omicron variant.
The amplicon obtained from the amplification reaction of the third method is as set forth in SEQ ID NO: 21.
The present invention relates to a fourth in-vitro method detecting Omicron variant of SARS-CoV- 2 in a sample suspected of comprising at least one variant of SARS-CoV-2, comprising:
(a) carrying out an amplification reaction, wherein the amplification reaction comprises:
(i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 6 and a second primer sequence having sequence as set forth in SEQ ID NO: 8;
(ii) a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9;
(b) detecting fluorescence level wherein increase in fluorescence level is indicative of the sample being positive for at least the Omicron variant.
The amplicon obtained from the amplification reaction of the fourth method is as set forth in SEQ ID NO: 22.
In an embodiment, the sample is a human sample isolated from at least one part of the human.
The first method of detection as described above is an indirect confirmation of absence of Omicron variant. The first method is a uniplex qRT-PCR reaction. The first method is a S-gene dropout assay. The second method of detection is a direct confirmation of presence of Omicron variant. The second method is a uniplex qRT-PCR reaction. The second method is a S-gene detection assay.
The third method of detection is an indirect confirmation of absence of Omicron variant. The third method is a uniplex qRT-PCR reaction. The third method is a S-gene dropout assay.
The fourth method of detection is a direct confirmation of presence of Omicron variant. The fourth method is a uniplex qRT-PCR reaction. The fourth method is a S-gene detection assay.
The present invention also provides a fifth in-vitro method of detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, which is a multiplex assay, said method comprising:
(a) carrying out an amplification reaction, wherein amplification reaction comprises:
(i) a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO:2;
(ii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3;
(iii) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4
(b) detecting the fluorescence level of first and second fluorescent labeled probe; and
(c) comparing the fluorescence level of first relative to the second fluorescent labeled probe, wherein the fluorescent label of the first probe is different from the fluorescent label of the second probe; wherein increase in fluorescence level of first fluorescent probe in the absence of increase in fluorescence level of second fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of second fluorescent probe in the absence of increase in fluorescence level of first fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level of first or second fluorescent probe is indicative of absence of SARS-CoV-2. The present invention also provides a sixth in-vitro method of detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, which is a multiplex assay, said method comprising:
(a) carrying out an amplification reaction, wherein the amplification reaction comprises:
(i) a first primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer sequence having sequence as set forth in SEQ ID NO: 2;
(ii) a second primer pair consisting of a first primer pair having sequence as set forth in SEQ ID NO: 6 and a second primer sequence having sequence as set forth in SEQ ID NO: 8;
(iii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3; and
(iv) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9;
(b) detecting the fluorescence level of first and second fluorescent labeled probe; and
(c) comparing the fluorescence level of first relative to the second fluorescent labeled probe, wherein the fluorescent label of the first probe is different from the fluorescent label of the second probe; wherein increase in fluorescence level of first fluorescent probe in the absence of increase in fluorescence level of second fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of second fluorescent probe in the absence of increase in fluorescence level of first fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level of first or second fluorescent probe is indicative of absence of SARS-CoV-2.
The present invention also provides a seventh in-vitro method of detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, which is a multiplex PCR reaction, said method comprising: (a) carrying out an amplification reaction, wherein the amplification reaction comprises:
(i) a first primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 and a second primer sequence having sequence as set forth in SEQ ID NO: 6;
(ii) a second primer pair consisting of a first primer pair having sequence as set forth in SEQ ID NO: 1 and a second primer sequence having sequence as set forth in SEQ ID NO: 2;
(iii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and
(iv) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4;
(b) detecting fluorescence level of first and second fluorescent labeled probe; and
(c) comparing the fluorescence level of first relative to the second fluorescent labeled probe, wherein the fluorescent label of the first probe is different from the fluorescent label of the second probe; wherein increase in fluorescence level of first fluorescent probe in the absence of increase in fluorescence level of second fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of second fluorescent probe in the absence of increase in fluorescence level of first fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level of first or second fluorescent probe is indicative of absence of SARS-CoV-2.
The present invention also provides an eight in-vitro method of detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, which is a multiplex PCR reaction, said method comprising:
(d) carrying out an amplification reaction, wherein the amplification reaction comprises: (v) a first primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 and a second primer sequence having sequence as set forth in SEQ ID NO: 6;
(vi) a second primer pair consisting of a first primer pair having sequence as set forth in SEQ ID NO: 6 and a second primer sequence having sequence as set forth in SEQ ID NO: 8;
(vii) a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and
(viii) a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9
(e) detecting fluorescence level of first and second fluorescent labeled probe; and
(f) comparing the fluorescence level of first relative to the second fluorescent labeled probe, wherein the fluorescent label of the first probe is different from the fluorescent label of the second probe; wherein increase in fluorescence level of first fluorescent probe in the absence of increase in fluorescence level of second fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of second fluorescent probe in the absence of increase in fluorescence level of first fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level of first or second fluorescent probe is indicative of absence of SARS-CoV-2.
In any of the in-vitro methods of detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2 as described herein (uniplex or multiplex), the method optionally further comprises carrying out a separate amplification reaction for detection of at least one of RdRp (RNA-dependent RNA polymerase) gene, envelope Orflab gene, or nucleocapsid gene (N). This particular amplification serves the purpose of positive control to confirm presence of SARS-CoV-2 in the sample. The primer pair for amplification of the N gene is as set forth in SEQ ID NO: 10 and SEQ ID NO: 11. The fluorescent labeled probe capable of hybridizing to a complementary sequence in the N gene is as set forth in SEQ ID NO: 12. The amplicon sequence obtained by using the said primer pair is as set forth in SEQ ID NO: 23. Increase in fluorescence level is indicative of presence of SARS-CoV-2 in the sample.
The primer pair for amplification of the RdRp gene is as set forth in SEQ ID NO: 13 and SEQ ID NO: 14. The fluorescent labeled probe capable of hybridizing to a complementary sequence in the RdRp gene is as set forth in SEQ ID NO: 15. The amplicon sequence obtained by using the said primer pair is as set forth in SEQ ID NO: 24. Increase in fluorescence level is indicative of presence of SARS-CoV-2 in the sample.
The primer pair for amplification of the Orflab gene is as set forth in SEQ ID NO: 16 and SEQ ID NO: 17. The fluorescent labeled probe capable of hybridizing to a complementary sequence in the Orflab gene is as set forth in SEQ ID NO: 18. The amplicon sequence obtained by using the said primer pair is as set forth in SEQ ID NO: 25. Increase in fluorescence level is indicative of presence of SARS-CoV-2 in the sample.
In any of the methods of detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2 as described herein, the method optionally further comprises a human RNase P gene amplification as an internal control.
It is to be understood by a person skilled in the art that the amplification step is preceded by a reverse transcription step as SARS-CoV-2 is (+strand) RNA virus. In an embodiment, the reverse transcription step is not essential if the starting sample provided is DNA and not RNA.
Limit of Detection (LOD) is generally defined as the lowest amount of analyte, which can be detected with at least a stated level of confidence, though LOD is not necessarily quantified as an exact value. Another alternate definition of LOD can be the minimum concentration of nucleic acid which always gives a positive PCR result in all replicates tested.
In a preferred embodiment, the limit of detection (LOD) of DNA template (post reverse transcription of viral RNA) is 40 copies per reaction. In an embodiment, the limit of detection is 20-50 copies per reaction. The confidence level is at least 95%.
In a preferred embodiment, the concentration of primers in amplification reaction in any of the methods as described herein is in the range of 200-900nM. In a preferred embodiment, the concentration of fluorescent labelled probes for hybridization in any of the methods as described herein is in the range of 50-300nM.
In a preferred embodiment, the PCR reaction comprises 45 cycles of denaturing-annealing- extension. In an embodiment, the PCR reaction comprises 40-45 cycles. In another embodiment, the PCR reaction comprises 45-50 cycles
In a preferred embodiment, the denaturation step is carried out at 95°. In an embodiment, the denaturation step is carried out a 90-98°C. In a preferred embodiment, the denaturation step is carried out for 15s. In an embodiment, the denaturation step is carried out for 10-20 seconds.
In a preferred embodiment, the annealing+extension step is carried out at 58°C. In an embodiment, the step is carried out at 56-60°C. In a preferred embodiment, the step is carried out for 30 seconds. In an embodiment, the annealing step is carried out for 25-40 seconds.
In a preferred embodiment, the PCR reaction comprises an initial step of 1 cycle of reverse transcription caried out 50°C. In an embodiment, the temperature is in the range of 42-55°C. The step of reverse transcription is followed by 1 cycle of activation carried out preferably at a temperature in the range of 90-98°C, preferably 95°C and 2-10 minutes, preferably for preferably 2mins.
The present invention provides a first kit for detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, said kit comprising:
(a) a first primer having sequence as set forth in SEQ ID NO: 1;
(b) a second primer having sequence as set forth in SEQ ID NO: 2; and
(c) a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4.
The present invention provides a second kit for detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, said kit comprising:
(a) a first primer having sequence as set forth in SEQ ID NO: 6;
(b) a second primer having sequence as set forth in SEQ ID NO: 8; and
(c) a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9. In an embodiment, the first or second kit comprises sufficient concentration of primers and probes to carry out at least 1 uniplex PCR amplification reaction. In another embodiment, the first or second kit comprises sufficient concentration of primers and probes to carry out at least 5 uniplex PCR amplification reaction. In another embodiment, the first or second kit comprises sufficient concentration of primers and probes to carry out at least 10 uniplex PCR amplification reaction.
The present invention provides a third kit for detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, said kit comprising:
(a) a first primer having sequence as set forth in SEQ ID NO: 1;
(b) a second primer having sequence as set forth in SEQ ID NO: 2;
(c) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 3; and
(d) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4 where the fluorescent label of the first probe is different from that of the second probe.
The present invention provides a fourth kit for detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, said kit comprising:
(a) a first primer having sequence as set forth in SEQ ID NO: 1;
(b) a second primer having sequence as set forth in SEQ ID NO: 2;
(c) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 3;
(d) a third primer having sequence as set forth in SEQ ID NO: 6;
(e) a fourth primer having sequence as set forth in SEQ ID NO: 8; and
(f) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9 where the fluorescent label of the first probe is different from that of the second probe.
The present invention provides a fifth kit for detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, said kit comprising:
(a) a first primer having sequence as set forth in SEQ ID NO: 5; (b) a second primer having sequence as set forth in SEQ ID NO: 6;
(c) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 7;
(d) a third primer having sequence as set forth in SEQ ID NO:1;
(e) a fourth primer having sequence as set forth in SEQ ID NO: 2; and
(f) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4 where the fluorescent label of the first probe is different from that of the second probe.
The present invention provides a sixth kit for detection of Omicron variant in a sample suspected of comprising at least one variant of SARS-CoV-2, said kit comprising:
(a) a first primer having sequence as set forth in SEQ ID NO: 5;
(b) a second primer having sequence as set forth in SEQ ID NO: 6;
(c) a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 7;
(d) a third primer having sequence as set forth in SEQ ID NO:6;
(e) a fourth primer having sequence as set forth in SEQ ID NO: 8; and
(f) a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9 where the fluorescent label of the first probe is different from that of the second probe.
In an embodiment, any one of the third-sixth kit comprises sufficient concentration of primers and probes to carry out at least 1 multiplex PCR amplification reaction. In an embodiment, the kits comprise sufficient concentration of primers and probes to carry out at least 5 multiplex PCR amplification reaction. In an embodiment, the kits comprise sufficient concentration of primers and probes to carry out at least 10 multiplex PCR amplification reaction. The fluorescent label of the labelled probe can be any one of FAM-BHQ1, HEX-BHQ1, Cy5-BHQ2. It is understood by a person skilled in the art that other known labels can also be used. In a preferred embodiment, the probe having sequence as set forth in SEQ ID NO: 3 or 7 is labelled with FAM-BHQ1. In a preferred embodiment, the probe having sequence as set forth in SEQ ID NO: 4 or 9 is labelled with HEX-BHQ1. In a preferred embodiment, the probe having sequence as set forth in SEQ ID NO: 12, 15, or 18 is labelled with Cy5-BHQ2. In an embodiment, any of the kits comprise reagents required to carry out a RT-PCR reaction. In an embodiment, any of the kits optionally further comprise sufficient quantity of DNA polymerase and/or reverse-transcriptase. In an embodiment, any of the kits comprise an instruction manual.
In an embodiment, any of the kits optionally further comprises at least one of (a) a first primer having sequence as set forth in SEQ ID NO: 10; a second primer having sequence as set forth in SEQ ID NO: 11; and a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 12, wherein said primers and probes are useful in amplification and detection of N gene region; (b) a first primer having sequence as set forth in SEQ ID NO: 13; a second primer having sequence as set forth in 14; and a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 15, wherein said primers and probes are useful in amplification and detection of RdRp gene region; and (c) a first primer having sequence as set forth in SEQ ID NO: 16; a second primer having sequence as set forth in 17; and a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 18, wherein said primers and probes are useful in amplification and detection of ORFlab gene region.
The contents of any of the kits as described herein can be combined in any combination to give a new kit.
The present invention provides a fluorescent labelled probe for use in specific detection of Omicron variant of SARS-CoV-2, said probe having sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 9.
The probe having sequence as set forth in SEQ ID NO: 4 may be used in conjunction with primer pair having sequences as set forth in SEQ ID NO: 1 and SEQ ID NO: 2 for specific detection of Omicron variant of SARS-CoV-2 using qRT-PCR.
The probe having sequence as set forth in SEQ ID NO: 9 may be used in conjunction with primer pair having sequences as set forth in SEQ ID NO: 6 and SEQ ID NO: 8 for specific detection of Omicron variant of SARS-CoV-2 using qRT-PCR.
The present invention also provides use of primers having sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2; and a hybridizing probe having sequence as set forth in SEQ ID NO: 4 in particularly detecting Omicron variant of SARS-CoV-2 to the exclusion of other variants in a qRT- PCR assay. The present invention also provides use of primers having sequence as set forth in SEQ ID NO: 6 and SEQ ID NO: 8; and a hybridizing probe having sequence as set forth in SEQ ID NO: 9 in particularly detecting Omicron variant of SARS-CoV-2 to the exclusion of other variants in a qRT- PCR assay. The present invention also provides use of the primers and probes as substantially described in the present invention in uniplex or multiplex qRT-PCR to detect Omicron variant of SARS-CoV-2 to the exclusion of other variants or both Omicron and other variant of SARS-CoV-2 in a sample.
The present invention also provides use of the kits as substantially described in the present invention for use in detecting Omicron variant of SARS-CoV-2 to the exclusion of other variants in a qRT-PCR assay.
EXAMPLES
Table 1 below depicts a representative qRT-PCR assay scheme for detection of SARS-CoV-2 in a sample using the primers and probes of the present invention.
Table 1
Figure imgf000020_0001
Table 2 below depicts the Ct cutoff values for the assays.
Table 2
Figure imgf000020_0002
It is to be noted that (a) Cq Cutoff Values are uniform across all the targets and internal controls, and are based on the limit of detection and sensitivity and specificity parameters of the primerprobe and standard mastermix.
Commercially synthesised gene constructs were quantified in-house. Determination of sensitivity and specificity is reiterative process. In the first step, the proof of concept of the designed primerprobe mixes was validated. Multiple primer and probe sets for each gene were designed. Any primer and probe set that failed in this step was discarded. The primer and probe set which gave accurate results in this evaluation were used in all subsequent experiments.
Specificity of each of the designed primer and probe set was ascertained in silica during the stage of designing. Any primer and probe having E-value greater than 0.9 were omitted. Specificity of the selected primer and probe sets was ascertained in uniplex assay. Here, the selected primer and probe set was used to amplify the nucleic acid extracted from all the known viruses of Coronaviridae and other viruses that can cause similar respiratory infections like RSV and Influenza. Further, the clinical specificity of the assay was ascertained using 50 well characterised clinical specimens. All the 50 negatives did not show any amplification with any of the primer and probe set that are claimed herewith.
The primer and probe sets described in this invention are SARS-CoV-2 specific and do not amplify nucleic acid of any other viruses.
Table 3 below depicts the various outcomes of the uniplex or multiplex assay.
Table 3
Figure imgf000021_0001
Figure imgf000022_0001
Towards validation of the specificity of the present invention, 88 COVID-19 positive samples and 80 negative samples were tested. Among the 88 COVID-19 positive samples, 40 were confirmed as Omicron positive based on sequencing data, 35 were confirmed as Delta positive, and 13 were other variants. Each of the 40 Omicron positive samples (Omicron positive based on sequencing data) also tested positive using uniplex or multiplex qRT-PCR, which comprise the primers and probes as described in the present invention. A positive result (presence of Omicron variant) was based on enhanced fluorescence levels of the fluorophore attached to the probe that specifically hybridizes to a sequence only from the Omicron variant of SARS-CoV-2 under conditions as described herein. These data indicate that the primers/probes and the methods of the present invention are 100% accurate and 0% rate of false negatives.
Additionally, the 35 samples that were Delta variant positive and Omicron variant negative were also correctly identified by the primers/probes of the present invention using multiplexed qRT- PCR. Enhanced fluorescence levels were seen only for the fluorophore attached to the probe that specifically does not hybridizes to a sequence only from the Omicron variant of SARS-CoV-2 but binds to other variants, including Delta variant under conditions as described herein. Similar results were obtained for the 13 other variants (not being Delta or Omicron variant). No enhancement of fluorescent was seen for any of the 80 negative samples, either for Omicron specific fluorophore or other variant specific fluorophore. These data indicate that the primers/probes and the methods of the present invention are 100% accurate and 0% rate of false positive.
Overall, these data indicate the primers/probes and methods of the present invention are highly specific for the Omicron variant of SARS-CoV-2 and can be reliably used to detect Omicron variant in a sample with highest reliability and confidence. qRT-PCR using the same reagents is also significantly financially economical and rapidly scalable compared to present sequencing methods that are expensive, time consuming, and not scalable.
It must further be noted that the above disclosure is non-limiting and modifications and variations are possible without departing from the spirit and scope of the invention.

Claims

l/We claim:
1. Synthetic DNA based labelled probes having sequence as set forth in SEQ ID NO: 4 or SEQ ID NO: 9 capable of specifically hybridizing to a region of the Omicron variant of SARS-CoV-2 coronavirus.
2. The probes as claimed in claim 1, wherein said probes are labelled with any one of FAM- BHQ1, HEX-BHQ1, and Cy5-BHQ2.
3. An in-vitro method of detecting Omicron variant of SARS-CoV-2 coronavirus in a sample, said method comprising: a. carrying out a DNA amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer sequence having sequence as set forth in SEQ ID NO: 2; and ii. a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; and b. detecting fluorescence level, wherein increase in fluorescence level is indicative of the sample being positive for Omicron variant of SARS-CoV-2;
OR a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 6 and a second primer sequence having sequence as set forth in SEQ ID NO: 8; and ii. a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9; and b. detecting fluorescence level, wherein increase in fluorescence level is indicative of the sample being positive for Omicron variant of SARS-CoV-2;
23 OR a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO:2; ii. a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3; iii. a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; b. detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample;
OR a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO:2; ii. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 6 and a second primer having sequence as set forth in SEQ ID NO: 8; iii. a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 3; and iv. a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9; and b. detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample;
OR a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 and a second primer having sequence as set forth in SEQ ID NO:6; ii. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 1 and a second primer having sequence as set forth in SEQ ID NO: 2; iii. a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and iv. a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 4; and b. detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample;
OR a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 5 a second primer having sequence as set forth in SEQ ID NO:6; and a third primer having sequence as set forth in SEQ ID NO: 8; ii. a first fluorescent labeled probe having sequence as set forth in SEQ ID NO: 7; and iii. a second fluorescent labeled probe having sequence as set forth in SEQ ID NO: 9; and b. detecting the fluorescence level, wherein increase in fluorescence level of fluorophore of the first fluorescent probe is indicative of presence of at least a SARS-CoV-2 variant, which is not the Omicron variant; or wherein increase in fluorescence level of fluorophore of the second fluorescent probe is indicative of presence of Omicron variant only; or wherein increase in fluorescence level of fluorophore of both first and second fluorescent probe is indicative of presence of Omicron variant and at least one other variant of SARS-CoV-2; or wherein no increase in fluorescence level is indicative of absence of SARS-CoV-2 in the sample.
4. The method as claimed in claim 3, further comprising at least one or more amplification reaction that can be any one or more of: a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 10 and a second primer having sequence as set forth in SEQ ID NO: 11; ii. a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 12; and b. detecting the fluorescence level,
26 wherein increase in fluorescence level is indicative of presence of SARS-CoV-2 in the sample;
OR a. carrying out an amplification reaction, said reaction comprising: i. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 13 and a second primer having sequence as set forth in SEQ ID NO: 14; ii. a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 15; and iii. detecting the fluorescence level, wherein increase in fluorescence level is indicative of presence of SARS-CoV-2 in the sample;
OR a. carrying out an amplification reaction, said reaction comprising: iv. a primer pair consisting of a first primer having sequence as set forth in SEQ ID NO: 16 and a second primer having sequence as set forth in SEQ ID NO: 17; v. a fluorescent labeled probe having sequence as set forth in SEQ ID NO: 18; and b. detecting the fluorescence level, wherein increase in fluorescence level is indicative of presence of SARS-CoV-2 in the sample.
5. The method as claimed in claim 4, wherein said any one or more amplification reactions are not multiplexed.
6. The method as claimed in claim 4, wherein said any one or more amplification reaction is not multiplexed with any one of amplification reaction of claim 3.
27 The method as claimed in claims 3-4, wherein said probes are labelled with any one of FAM- BHQ1, HEX-BHQ1, and Cy5-BHQ2, and wherein any two different probes in the same amplification reaction are not labelled with the same fluorophore. The method as claimed in claim 7, wherein cleavage of said labelled probe results in increase in fluorescence level of fluorophore. The method as claimed in claims 3-4, optionally comprising a reverse transcription step preceding the DNA amplification step to obtain DNA template from the genetic material of the suspected SARS-Cov-2 virus present in the sample. A kit comprising at least one of: a. a first primer having sequence as set forth in SEQ ID NO: 1; b. a second primer having sequence as set forth in SEQ ID NO: 2; and c. a labelled probe having sequence as set forth in SEQ ID NO: 4;
OR a. a first primer having sequence as set forth in SEQ ID NO: 6; b. a second primer having sequence as set forth in SEQ ID NO: 8; and c. a labelled probe having sequence as set forth in SEQ ID NO: 9;
OR a. a first primer having sequence as set forth in SEQ ID NO: 1; b. a second primer having sequence as set forth in SEQ ID NO: 2; c. a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 3; and d. a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4;
OR a. a first primer having sequence as set forth in SEQ ID NO: 1; b. a second primer having sequence as set forth in SEQ ID NO: 2; c. a third primer having sequence as set forth in SEQ ID NO: 6;
28 d. a fourth primer having sequence as set forth in SEQ ID NO: 8; e. a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 3; and f. a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9;
OR a. a first primer having sequence as set forth in SEQ ID NO: 5; b. a second primer having sequence as set forth in SEQ ID NO: 6; c. a third primer having sequence as set forth in SEQ ID NO: 1; d. a fourth primer having sequence as set forth in SEQ ID NO: 2; e. a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 4; and f. a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 7;
OR a. a first primer having sequence as set forth in SEQ ID NO: 5; b. a second primer having sequence as set forth in SEQ ID NO: 6; c. a third primer having sequence as set forth in SEQ ID NO: 8; d. a first fluorescent labelled probe having sequence as set forth in SEQ ID NO: 7; and e. a second fluorescent labelled probe having sequence as set forth in SEQ ID NO: 9. as claimed in claim 10, optionally further comprising at least one of: a. a first primer having sequence as set forth in SEQ ID NO: 10; b. a second primer having sequence as set forth in SEQ ID NO: 11; and c. a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 12;
OR a. a first primer having sequence as set forth in SEQ ID NO: 13; b. a second primer having sequence as set forth in SEQ ID NO: 14; and c. a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 15;
29 OR a. a first primer having sequence as set forth in SEQ ID NO: 16; b. a second primer having sequence as set forth in SEQ ID NO: 17; and c. a fluorescent labelled probe having sequence as set forth in SEQ ID NO: 18. 12. The kit as claimed in claims 10-11, wherein said probes are labelled with any one of FAM-
BHQ1, HEX-BHQ1, and Cy5-BHQ2.
13. The kit as claimed in claims 10-11, wherein said kit contains sufficient quantity to primer(s) and probe(s) to carry out at least one DNA amplification reaction.
14. The kit as claimed in claims 10-11, optionally further comprising at least one of: a. instruction manual; b. reagents to carry out DNA amplification reaction(s); and c. reagents to carry out reverse transcription.
15. The probes as claimed in claim 1 or kit as claimed in claim 10 for use in detection of Omicron variant of SARS-CoV-2 in a sample.
30
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