Claims:
WE CLAIM:
1. An in-vitro diagnostic (IVD) kit for the rapid detection of an analyte from a test sample, comprising:
a Sample Tube (1) for collecting a test sample;
a Lysis Tube (2) comprising a lysis composition;
a Reconstitution Tube (3) that comprises reconstitution buffer for adding to a Reaction Mix;
a Reaction Tube (4) comprising a lyophilized Reaction Mix of primers, probes and PCR enzyme cocktail, wherein the said PCR tube is capable of being directly transferred to a PCR machine,
characterised in that the said kit does not require pipetting of the contents; and
does not require refrigeration.

2. The kit as claimed in claim 1, wherein the lysis composition is made using water, buffer, polyethylene glycol, detergent, lysis enzyme and/or salt or combinations thereof.

3. The kit as claimed in claim 1, wherein the detergent is selected from a group consisting of SDS, Triton-X, Proteinase K or Tween 20.

4. The kit as claimed in claim 1, wherein the optionally further comprises a swab for collecting the test sample.

5. The kit as claimed in claim 1, wherein the analyte is a nucleic acid or fragment thereof.

6. The kit as claimed in claim 1, wherein the analyte is SARS-Cov-2.

7. The kit as claimed in claim 1, wherein the detection of the analyte using rapid real-time RT-PCR test is obtained within 60 minutes.

8. The kit as claimed in claim 1, wherein the lysis composition comprises composition containing polyethylene glycol selected from the group consisting of PEG-100, PEG-200, PEG-300, PEG-400, PEG-500, PEG-1000, PEG-6000, PEG-8000.

9. The kit as claimed in claim 1, wherein the lysis composition comprises alkali selected from the group consisting of NaOH, KOH, NH4OH.

10. The kit as claimed in claim 1, wherein the Reaction Tube contains lyophilised Reaction Mix comprising primers and probes specific for ORF1ab, E, N and RdRp genes of SARS-CoV-2, wherein the said primers and probes are selected from oligonucleotide sequences having SEQ ID NO. 01 to SEQ ID NO. 15.

11. The kit as claimed in claim 1, wherein the Reaction Tube contains lyophilised Reaction Mix additionally comprising primers and probes specific for S gene of SARS-CoV-2, wherein the said primers and probes are selected from oligonucleotide sequences having SEQ ID NO. 13 to SEQ ID NO. 15.

12. The kit as claimed in claim 1, wherein the probes are labeled with different fluorescent reporter groups at their 5 'ends and a fluorescent quencher group at their 3' ends.

13. The probes as claimed in claim 12, wherein fluorescent reporter groups are selected from the group consisting of FAM, HEX, ROX, JOE, CY3, VIC, TET, TAXAS RED, NED, ALEXA, TAMRA, CY5.5, and CY5, and the fluorescence quencher is selected from the group consisting of BHQ1, BHQ2, BHQ3, MGB, and DABCYL.

14. The kit as claimed in claim 1, wherein the Reconstitution Buffer tube (3) contains buffer optionally comprising at least one cationic salt and deoxyribonucleotides;
a. the said buffer selected from a group comprising Tris, Tris-HCl, Tris chloride, HEPES or phosphate
b. the said cation selected from a group comprising Mg2+, NH4+, K+ or Na+ at a concentration of XX mM.
wherein the deoxyribonucleotides include dATP, dCTP, dGTP and dUTP / dTTP
each present in equimolar concentrations.

15. The kit as claimed in claim 1, wherein the enzyme cocktail comprises a DNA polymerase selected from at least one of Taq, Fast Taq, Tfl, Pfu, or Tth DNA polymerase

16. The kit as claimed in claim 1, wherein the enzyme cocktail comprises a reverse transcriptase selected from at least one of M-MLV reverse transcriptase or AMV reverse transcriptase.

17. An in-vitro diagnostic (IVD) kit for the rapid detection of an analyte from a test sample, comprising:
a tube with a partitioning element in the middle that isolates the reconstitution buffer from Reaction Mix;
characterised in that the said kit does not require pipetting of the contents; and
does not require refrigeration.

18. The kit as claimed in claim 17, wherein the partitioning element in the middle that isolates the reconstitution buffer from Reaction Mix may be vertical or horizontal.

19. The kit as claimed in claim 1, wherein the lyophilisation process for making lyophilized primers, probes, PCR enzyme cocktail and buffer salts is sequential or one-pot.

20. A method for the for the rapid detection of an analyte from a test sample, comprising:
a. collecting a sample in a sample tube;
b. lysing the sample by adding a lysis composition;
c. mixing the contents of step (b) by tapping;
d. incubating the mixture of step (c) at room temperature for up to 10 minutes to obtain a lysate;
e. adding a drop of the lysate of step (d) to a PCR tube containing lyophilised Reaction Mix/, the said mix being reconstituted with a reconstitution buffer tube prior to adding the lysate;
f. transferring the PCR tube to a PCR machine;
in order to rapidly screen for the analyte;
characterised in that the said method does not require pipetting of the contents; and
does not require refrigeration.

21. The method as claimed in claim 20, wherein the lysis composition is made using water, buffer, polyethylene glycol, detergent, lysis enzyme and/or salt or combinations thereof.

22. The method as claimed in claim 20, wherein the detergent is selected from a group consisting of SDS, Triton-X or Tween 20.

23. The method as claimed in claim 20, wherein the analyte is a nucleic acid or fragment thereof.

24. The method as claimed in claim 20, wherein the analyte is SARS-Cov-2.

25. The method as claimed in claim 20, wherein the detection of the analyte using rapid real-time RT-PCR test is obtained within 60 minutes.

26. The method as claimed in claim 20, wherein the lysis composition comprises composition containing polyethylene glycol selected from the group consisting of PEG-100, PEG-200, PEG-300, PEG-400, PEG-500, PEG-1000, PEG-6000, PEG-8000.

27. The method as claimed in claim 20, wherein the lysis composition comprises alkali selected from the group consisting of NaOH, KOH, NH4OH.

28. The method as claimed in claim 20, wherein the Reaction Tubecontains lyophilised Reaction Mix comprising primers and probes specific for ORF1ab, E, N and RdRp genes of SARS-CoV-2, wherein the said primers and probes are selected from oligonucleotide sequences having SEQ ID NO. 01 to SEQ ID NO. 15.

29. The method as claimed in claim 20, wherein the Reaction Tube tube contains lyophilised PCR Reaction Mix mix additionally comprising primers and probes specific for S gene of SARS-CoV-2, wherein the said primers and probes are selected from oligonucleotide sequences having SEQ ID NO. 13 to SEQ ID NO. 15.

30. The method as claimed in claim 20, wherein the probes are labeled with different fluorescent reporter groups at their 5 'ends and a fluorescent quencher group at their 3' ends.

31. The method as claimed in claim 20, wherein fluorescence reporter groups are selected from the group consisting of FAM, HEX, ROX, JOE, CY3, VIC, TET, TAXAS RED, NED, ALEXA, TAMRA, CY5.5, and CY5, and the fluorescence quencher is selected from the group consisting of BHQ1, BHQ2, BHQ3, MGB, and DABCYL.

32. The method as claimed in claim 20, wherein the reconstitution buffer optionally comprises at least one cationic salt and deoxyribonucleotides;
a. the said buffer selected from a group comprising Tris, Tris-HCl, Tris chloride, HEPES or phosphate
b. the said cation selected from a group comprising Mg2+, NH4+, K+ or Na+
wherein the deoxyribonucleotides include dATP, dCTP, dGTP and dUTP / dTTP present at equimolar concentrations.

33. The method as claimed in claim 20, wherein the enzyme cocktail comprises a DNA polymerase selected from at least one of Taq, Fast Taq, Tfl, Pfu, or Tth DNA polymerase

34. The method as claimed in claim 20, wherein the enzyme cocktail comprises a reverse transcriptase selected from at least one of M-MLV reverse transcriptase or AMV reverse transcriptase.

35. The kit as claimed in claim 1, coupled with any PCR machine, such that the results may be directly uploaded / made accessible via cloud.

36. The kit as claimed in claim 1, wherein the sample tube (1) is designed to prevent spilling of sample or lysate by means of a conical shape optionally appended with a dropper (5) having a nozzle through which a measured quantity of sample or lysate is dispensed.

Dated this 08th Day of January 2022

Priyank Gupta
Agent for the Applicant
IN/PA-1454
, Description:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003

COMPLETE SPECIFICATION

(See Section 10 and Rule 13)

Title of invention:
A KIT FOR THE RAPID DETECTION OF AN ANALYTE AND METHOD THEREOF

MYLAB DISCOVERY SOLUTIONS PRIVATE LIMITED
An Indian entity having address as:
PLOT NO 99-B, LONAVALA INDUSTRIAL CO-OPERATIVE ESTATE LTD,
NANGARGAON, LONAVALA, PUNE – 410401
MAHARASHTRA, INDIA

The following specification describes the invention and the manner in which it is to be performed.
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY
The present application does not claim priority from any other patent application.

TECHNICAL FIELD
The present subject matter described herein, in general, relates to a field of molecular biological detection of viruses. More particularly, the present subject matter relates to a kit for the detection of SARS-Cov-2 and thereof.

BACKGROUND

Present times have thrown up the need for devising rapid detection protocols that may be carried out by untrained personnel in an accurate manner. Complicated multi-step tests require highly trained personnel for obtaining reliable results. Moreover, infectious samples require proper handling to avoid spread as well as sample contamination which may alter results. Due to the unavailability of such trained personnel and protocols that require multiple handling steps, the time required for such tests is very high.
Shorter quicker tests that require less handling, such as reduced or no pipetting steps, that may be automated by high-throughput, which are easy to perform without requiring exhaustive trainings are the need of the hour.
Ordinarily, additional obstacles in such rapid detections may arise from restricted availability of resources owing to their specific requirements for transport and storage. Many reagents need cold supply chain such as ice packs or dry ice transport and storage facilities to maintain their activity which adds up to space and cost.
Thus, there is a requirement to develop methods and tests that may be run in a short time, may be performed by untrained persons that are easy to operate and require fewer handling steps. Additionally, it is imperative to devise ways to take the tests far and wide, and make them available in a manner that is easy to transport and store.

Presently the world is facing a pandemic – an acute coronavirus-associated respiratory disease called SARS (severe acute respiratory syndrome) or coronavirus disease 19 (COVID-19). International Committee on Taxonomy of Viruses has placed the human pathogen, named 2019-nCoV, within Coronaviridae a family of large, enveloped RNA viruses with a “crown-like” appearance that affect humans, and domestic animals.

Based on taxonomy and phylogeny, 2019-nCoV is closely related to the prototype human and bat severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and is thus designated as SARS-CoV-2.

The genome of SARS-CoV-2 is positive-sense single-stranded RNA [(+)ssRNA] with a 5’cap, 3’UTR poly(A) tail, comprising 14 open reading frames (ORFs) and non-structural proteins (NSPs) for virus replication and assembly processes. It also comprises structural proteins including spike (S), envelope (E), membrane/matrix (M) and nucleocapsid (N), and accessory proteins. The S protein is a transmembrane protein that attaches to the ACE2 receptors on host cell surfaces while the N protein attaches to the viral genome and is involved in the RNA replication, virion formation and immune evasion. The M protein is well-conserved and the most abundant protein in the virion structure.

COVID-19 symptoms are observed approximately 5 days after incubation and those affected display symptoms for 11.5 days. (Lauer SA, Grantz KH, Bi Q, et al. The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: estimation and application. Ann Intern Med. 2020; 172:577-582) Early symptoms like fever, chills, headache, cough, sore throat, and runny nose may also include gastrointestinal disturbances like diarrhoea, vomiting and anorexia. (Adhikari SP, Meng S, Wu Y-J, et al. Epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (COVID-19) during the early outbreak period: a scoping review. Infect Dis Poverty. 2020; 9:29) After 2 to 7 days, SARS patients generally develop a dry, non-productive cough. In some cases, there may be rapid deterioration of conditions, with low oxygen saturation and acute respiratory distress.

RNA viruses have a high mutational load. Mutation in SARS-CoV-2 involves a change in genetic sequence compared to a reference sequence such as Wuhan-Hu 1 or USA-WA1/2020. Current variants of SARS-CoV-2 include alpha, beta, gamma, epsilon, eta, iota, kappa, mu, zeta, delta, omicron and so on of which, delta and omicron have been classified by WHO as variant of concern (VOC) since they have a high transmission rate.

Thus, tracing the path/progression of SARS-CoV-2 is crucial to containing its spread. Present methods are focused on epidemiological data, clinical symptoms, and some advanced molecular diagnostic technologies such as nucleic acid detection and immunological assays.
Molecular diagnostic methods lend an advantage on account of detecting minimal amounts of nucleic acid, accurately differentiating different pathogens, and a possibility for automation. Therefore, real-time RT-PCR (rRT-PCR) and high-throughput sequencing are promising for the detection of SARS-CoV-2 nucleic acid; albeit sequencing demonstrates limited use in clinical diagnosis owing to laborious operations, comparatively low reliance and high costs.

Real-time RT-PCR displays high sensitivity and specificity and is the gold standard assay for the final diagnosis of SARS-CoV-2. Further, to avoid cross-reaction with other human coronaviruses and prevent the potential genetic drift of SARS-CoV-2, regulatory bodies recommend the use of at least two molecular targets in nucleic acid detection: one non-specific for detecting other CoVs, and one specific for SARS-CoV-2. Thus, the CDC-recommended assays rely on two nucleocapsid proteins N1 and N2 while, the WHO recommends E gene assay as a first-line screening, followed by RdRp gene assay as a confirmatory test.

The sensitivity of the assays may vary depending on viral load, RNA extraction technique, sampling source and disease stage during the time of sampling. Further, RT-PCR false-positives may also be due to sample cross-contamination and handling errors whereas false-negatives may occur due to improper sample collection, storage and processing.

In the present circumstances, the sheer pace and numbers for testing necessitate use of kits that provide all the requisite chemicals for conducting above assay.
Most available kits for real-time RT-PCR based detection of SARS-Cov-2 involve liquid reagents that need a cold supply chain of sub-zero temperatures such as -20°C for transport and storage which increases the cost of detection per test. Moreover, liquid reagents particularly biologicals may be thermolabile and susceptible to loss of efficacy leading to plausibly false test outcomes.
Further, owing to the high sensitivity of the assays, the reagents are required to be prepared fresh prior to use, thus needing highly trained personnel with adequate skills and expertise.

Additionally, classic nucleic acid detection samples require purification steps that increase the risk of infection for operators. Also conventionally, nearly 6-8 hours are required for obtaining the result owing to the time consumed in the multiple steps involved such as processing of the sample for RNA purification, reverse transcription and PCR which is far from ideal. Moreover, the turnaround time for reporting test outcomes is vital, particularly in crowded places that see mass movement of people, such as airports, where test results may govern further movement.

Thus, there is an urgent need for a lab-free, sensitive, specific assay method and/or a point of care test that is easy to transport, store, automate and use by untrained personnel who are unfamiliar with highly specific laboratory equipment like pipettes. Automation of the entire process from extraction to detection or even to reporting of results may reduce the manual errors and shorten the time required for end-to-end processing of the samples.

A Point of Care test kit (POCT) of the present invention as shown in Fig. 3 solves all the above problems by providing all the material required for detecting SARS-Cov-2 in test samples by real-time RT-PCR based assay. Some of the kit components are lyophilised, thus imparting thermostability, reducing volumes, and allowing for transport and storage at room temperature, reducing overall detection costs.

All material components are packed in separate containers, comprising – a Sample Tube (1) shown in Fig. 1, having a dropper for sample collection; Lysis Buffer Tube (2) comprising extraction composition for nucleic acid extraction; Reconstitution Buffer Tube (3) comprising buffer for reconstituting lyophilised Reaction Mix; Lyophilised PCR Tube (4) containing lyophilised Reaction Mix comprising primers, probes, PCR buffer and salts; allowing easy retrieval and usage. The kit packet carries a “User” manual that is easy to follow by untrained persons.

Another aspect is that the isolation of SARS-CoV-2 requires biosafety level-3 to ensure personnel safety. The kit packet further contains a swab designed to break at a specified length by means of a ‘breakpoint’ in order to contain the likely infectious material within the sample tube to avoid possible contamination or spread.

SUMMARY
This summary is provided to introduce a POCT kit for the rapid detection of an analyte from a test sample and a method thereof which are further described in the detailed description. This summary is not intended to identify all the essential features of the claimed subject matter, nor it is intended to use in determining or limiting the scope of claimed subject matter.

In an exemplary embodiment, a POCT kit for the rapid detection of an analyte from a test sample includes a Sample Tube (1) for collecting a test sample, a Lysis Tube (2) comprising a lysis composition, a Reconstitution Tube (3) that comprises reconstitution buffer for adding to a reaction mix , a Reaction Tube (4) comprising a lyophilized Reaction Mix of primers, probes and PCR enzyme cocktail, wherein the said PCR tube is capable of being directly transferred to a PCR machine, characterised in that the said kit does not require pipetting of the contents and does not require refrigeration.

In another embodiment, a method for the detection of SARS-Cov-2 comprises collecting a sample in a sample tube; lysing the sample by adding a lysis composition; mixing the contents of step (b) by tapping; incubating the mixture of step (c) at room temperature for up to 10 minutes to obtain a lysate; adding a drop of the lysate of step (d) to a PCR tube containing lyophilised Reaction Mix, the said mix being reconstituted with a reconstitution buffer tube prior to adding the lysate; transferring the PCR tube to a PCR machine in order to rapidly screen for the analyte; characterised in that the said method does not require pipetting of the contents; and does not require refrigeration.

BRIEF DESCRIPTION OF DRAWINGS
The detailed description is described with reference to the accompanying figures. In the Figures, the left-most digit(s) of a reference number identifies the Figure in which the reference number first appears. The same numbers are used throughout the drawings to refer like features and components.

Figure 1 illustrates a Sample Tube (1) with dropper (5) having a nozzle, in accordance with an embodiment of a present subject matter.

Figure 2 illustrates another embodiment tube with partitioning element, in accordance with the present disclosure.

Figure 3 illustrates components included in CoviSwiftTM COVID-19 Rapid PCR Kit, in accordance with the present disclosure.

DETAILED DESCRIPTION
Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
Further, the technical solutions offered by the present invention are clearly and completely described below. Examples in which specific reagents or conditions may not have been specified, have been conducted under conventional conditions or in a manner recommended by the manufacturer.

The present invention relates to an in-vitro diagnostic (IVD) kit for rapid detection of an analyte from a test sample includes a Sample Tube (1) for collecting a test sample, a Lysis Tube (2) comprising a lysis composition, a Reconstitution Tube (3) that comprises reconstitution buffer for adding to a Reaction Mix, a Reaction Tube (4) comprising a lyophilized Reaction Mix of primers, probes and PCR enzyme cocktail, wherein the said PCR tube is capable of being directly transferred to a PCR machine, characterised in that the said kit does not require pipetting of the contents and does not require refrigeration.

The present disclosure also relates to a kit and assay method for the rapid detection of SARS-CoV-2 using real-time RT-PCR. The exemplary embodiment pertains to a kit comprising: a Sample Tube (1) for collecting a sample, preferably from lower or upper respiratory tract; a Lysis Tube (2) containing lysis composition comprising polyethylene glycol; a Reconstitution Buffer tube (3) containing suitable buffer for adding to a Reaction Tube(4) containing lyophilized Reaction Mix comprising lyophilized primers, probes, PCR enzyme cocktail and buffer salts, capable of being directly transferred to a PCR machine, in order to rapidly detect SARS-CoV-2 virus using real-time RT-PCR.

In a first aspect, the present disclosure provides a kit for the rapid detection of SARS-CoV-2 comprising:
a Sample Tube (1) for collecting a sample, preferably from lower or upper respiratory tract by means of a swab;
a Lysis Tube (2) containing lysis composition comprising polyethylene glycol;
a Reconstitution Buffer tube (3) containing suitable buffer for adding to a Reaction Tube (4); the Reaction Tube (4) containing lyophilized Reaction Mix comprising lyophilized primers, probes, PCR enzyme cocktail and buffer salts, capable of being directly transferred to a PCR machine for rapidly detecting SARS-CoV-2 virus using real-time RT-PCR.

SARS CoV-2 RNA has been detected in faeces, blood, urine, and breast milk, although the transmissibility of the virus from these fluids is unknown. The most commonly used samples for checking SARS-CoV-2 presence are nasal or oral specimen, respiratory secretions collected from the upper (nasopharyngeal, oropharyngeal, nasal swabs, saliva) or lower (sputum, tracheal aspirate, BAL) respiratory tract. In a preferred embodiment, a sample comprising nasal or oral specimen is collected in the Sample Tube (1) by means of a swab.

The swab is designed to break at a specific length by means of a ‘breakpoint’ in order to contain the likely infectious material within the sample tube to avoid possible contamination or spread.

In one embodiment, the Sample Tube (1) may be empty or comprise any one of a buffer solution, nuclease free water, viral transport medium, molecular transport medium, a solution, a medium and a lysis composition, preferably a viral transport medium or molecular transport medium.

In an embodiment, Sample Tube (1) is designed to prevent spilling of sample or lysate by means of a conical shape optionally appended with a dropper (5) having a nozzle through which a measured quantity of sample or lysate is dispensed.

Another embodiment relates to preparation of the sample lysate comprising nucleic acids using lysis composition contained in Lysis Tube (2); the said lysis composition is made using water, buffer, polyethylene glycol, detergent, lysis enzyme and/or salt or combinations thereof that are known to a skilled person in the art. In a related embodiment the lysis composition comprises buffer selected from Tris, phosphate, citrate and/or combinations thereof. In a further embodiment the lysis composition lysis enzyme selected from denaturing enzymes such as proteinase K. An embodiment discloses lysis composition comprising detergent selected from the group comprising SDS, Tween-20, Triton-X and any other known to a skilled artisan.
In a preferred embodiment, the lysis composition comprises polyethylene glycol; the said polyethylene glycol selected from the group consisting of PEG-100, PEG-200, PEG-300, PEG-400, PEG-500, PEG-1000, PEG-6000 or PEG-8000. In a preferred embodiment, the polyethylene glycol is PEG-200. The concentration of PEG-200 is in the range of 10% to 50% (v/v), preferably from 20% to 40% (v/v).

In a related embodiment, the lysis composition further comprises an alkali/a base selected from the group consisting of sodium hydroxide, potassium hydroxide or ammonium hydroxide at a concentration ranging from 1 to 20 mM. In a preferred embodiment, the lysis composition comprises sodium hydroxide at a concentration ranging from 5 to 15 mM. Further, the pH of the lysis composition is alkaline ranging from pH 8 to 14, preferably from pH 10 to 13 and more preferably from pH 12 to 13.

In another embodiment, the sample lysate is prepared by adding the lysis composition to Sample Tube (1) to release the nucleic acids.
Alternatively, the sample lysate may be prepared by adding the sample to the Lysis Tube (2) via dropper (5).

In another embodiment, a Reconstitution Buffer tube (3) comprises buffers selected from the group consisting of Tris, Tris-HCl, Tris chloride, HEPES or phosphate. The concentration of the buffer ranges from 10 to 500 mM, preferably from 50 to 300 and more preferably 100-200 mM. The pH of the reconstitution buffer ranges from pH 6 to 9.
Optionally, the reconstitution buffer may further comprise ethylene diamine tetraacetate (EDTA) at a concentration ranging from 1-10 mM.
In a preferred embodiment, the reconstitution buffer comprises Tris-EDTA having pH 8-9.

In a related embodiment, the reconstitution buffer may optionally comprise at least one cationic salt selected from a group comprising Mg2+, NH4+, K+ or Na+, preferably Mg2+; wherein the concentration of the said salt ranges from 5 mM to 100 mM, preferably from 20 mM to 80 mM and more preferably from 40 mM to 60 mM per reaction.

In another related embodiment, the reconstitution buffer may optionally comprise deoxyribonucleotides including dATP, dCTP, dGTP, dUTP and/or dTTP; wherein the concentrations of the said deoxyribonucleotides ranges from 0.1 mM to 1 mM, preferably from 0.2 mM to 0.7 mM and more preferably from 0.4 mM to 0.6 mM per reaction.

The reconstitution buffer is added to Reaction Tube (4) prior to addition of the sample lysate.
In a further embodiment, the Reaction Tube (4) contains lyophilised Reaction Mix comprising primers and probes specific for genes of SARS-CoV-2 that may be selected from the group consisting of ORF1ab, E, N, S and RdRp, wherein the said primers and probes are selected from oligonucleotide sequences having SEQ ID NO. 01 to SEQ ID NO. 18.

In a first aspect, oligonucleotide sequences for detecting SARS-CoV-2, comprises at least four combinations:
a first combination of oligonucleotide sequences comprises: a first primer pair having SEQ ID NO. 01 and SEQ ID NO. 02, and a first probe having SEQ ID NO. 03;

a second combination of oligonucleotide sequences comprises: a second primer pair having SEQ ID NO. 04 and SEQ ID NO. 05, and a second probe having SEQ ID NO. 06;

a third combination of oligonucleotide sequences comprises: a third primer pair having SEQ ID NO. 07 and SEQ ID NO. 08, and a third probe having SEQ ID NO. 09;

a fourth combination of oligonucleotide sequences comprises: a fourth primer pair having SEQ ID NO. 10 and SEQ ID NO. 11, and a fourth probe having SEQ ID NO. 12.

Alternatively, in another aspect, oligonucleotide sequences for detecting SARS-CoV-2, additionally comprises:
a fifth combination of oligonucleotide sequences comprises: a fifth primer pair shown in SEQ ID NO. 13 and SEQ ID NO. 14, and a fifth probe shown in SEQ ID NO. 15.

In a further aspect, oligonucleotide sequences for detecting SARS-CoV-2 comprises a sixth combination of oligonucleotide sequences: a sixth primer pair having SEQ ID NO. 16 and SEQ ID NO. 17, and a sixth probe having SEQ ID NO. 18.

The sixth combination of oligonucleotide sequences may be used as an endogenous internal control, wherein the control gene could be RNAse P from humans, to enhance the accuracy of the analysis such that PCR products/ amplification output from all combinations are mutually non-interfering, devoid of nonspecific amplification, and add to the reliability of the result.

Thus, the present invention provides five oligonucleotide sequence combinations specially designed to hybridize with conserved gene fragments in SARS-CoV-2 namely, ‘ORF1ab’, ‘RdRp’, ‘N’ ‘E’ and optionally ‘S’, which differ from other coronaviruses, thus giving no cross reactivity.

In some embodiments of the present invention, the probes are labeled with different fluorescent reporter groups at their 5 'ends and fluorescent quencher groups at their 3' ends.
Optionally, in some embodiments, the fluorescent reporter groups are selected from a group consisting of FAM, HEX, ROX, JOE, CY3, VIC, TET, TAXAS RED, NED, ALEXA, TAMRA, CY5.5 and CY5. Further, the fluorescence quencher group may be selected from a group consisting of BHQ1, BHQ2, BHQ3, MGB and DABCYL.

It should be noted that, those skilled in the art may select other types of fluorescence reporters and quenchers according to actual needs, and whatever fluorescence reporters and quenchers are selected, they are within the scope of the present invention.

In a related embodiment, the Reaction Mix comprises oligonucleotide sequences at a concentration ranging from 5 to 30 pmol/reaction (please confirm), preferably from 10 to 20 pmol/reaction.

Optionally, in some embodiments of the invention, the Reaction Mix comprises a PCR enzyme cocktail comprising a DNA polymerase and a reverse transcriptase.
Alternatively, in some embodiments of the invention, the DNA polymerase may be at least one of Taq, Fast Taq, Tfl, Pfu, or Tth DNA polymerase, preferably Fast Taq DNA polymerase. In a further embodiment, the concentration of DNA polymerase ranges from is 1-10 U/reaction.
Optionally, in some embodiments of the invention, the reverse transcriptase is an M-MLV reverse transcriptase or an AMV reverse transcriptase, preferably M-MLV reverse transcriptase; wherein the concentration of reverse transcriptase ranges from 100-200 U/reaction.

Alternatively, in some embodiments, the Reaction Mix may comprise an RNAse inhibitor; wherein the concentration of the said inhibitor ranges from 5 to 50 U/reaction, more preferably from 15 to 25 U/reaction.

Additionally, Tris, EDTA, Mg2+ such as MgCl2, concentrations of dATP, dCTP, dGTP, dTTP, DNA polymerase, reverse transcriptase and RNAse inhibitor may also be adjusted as required, and any concentration present in the kit falls within the scope of the present invention.

Optionally in an embodiment of the present invention, the Reaction Mix may be a liquid or lyophilised powder. Lyophilization or freeze-drying is a benign process for removing water from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The process consists of three separate, unique, and interdependent processes; freezing, primary drying (sublimation), and secondary drying (desorption). The advantages of lyophilization include ease of processing a liquid, which simplifies aseptic handling, enhanced stability of a dry powder, removal of water without excessive heating of the product, enhanced product stability in a dry state and rapid and easy dissolution of reconstituted product.
In a preferred embodiment, the Reaction Mix is lyophilised.

Further, the method of the present invention detects exclusively the SARS-CoV-2 virus from a sample with high sensitivity and specificity at a concentration as low as 100 copies/mL.

In another embodiment, the kit comprises a tube with a partitioning element in the middle that isolates the reconstitution buffer from Reaction Mix; characterised in that the said kit does not require pipetting of the contents; and does not require refrigeration; the said tube selected from an Eppendorf, a PCR tube and any other available and known to a skilled person in the art.

The present disclosure further relates to a method for the rapid detection of SARS-CoV-2 using real-time RT-PCR.

In another embodiment, the present invention discloses a method for the for the rapid detection of an analyte from a test sample, comprising:
a. collecting a sample in a sample tube;
b. lysing the sample by adding a lysis composition;
c. mixing the contents of step (b) by tapping;
d. incubating the mixture of step (c) at room temperature for up to 10 minutes to obtain a lysate;
e. adding a drop of the lysate of step (d) to a PCR tube containing lyophilised Reaction Mix, the said mix being reconstituted with a reconstitution buffer tube prior to adding the lysate;
f. transferring the PCR tube to a PCR machine;
in order to rapidly screen for the analyte;
characterised in that the said method does not require pipetting of the contents; and
does not require refrigeration.

In another exemplary embodiment, the present invention provides a method for the rapid detection of SARS-CoV-2 from a test sample, comprising the following steps:
a. collecting a test sample in a sample tube;
b. lysing the sample by adding a lysis composition comprising polyethylene glycol;
c. mixing the contents by tapping;
d. incubating the tube of step (c) at room temperature up to 10 minutes to obtain a lysate;
e. adding a drop of the lysate of step (d) to a PCR tube containing lyophilised Reaction Mix, the said mix being reconstituted with a buffer prior to adding the lysate;
f. transferring the PCR tube to a PCR machine;
g. running a real time RT-PCR that may give a fluorescence signal;
in order to rapidly screen for SARS-CoV-2 in the test sample.

In another aspect, the present invention provides a method for detecting SARS-Cov-2 by real-time RT-PCR using the oligonucleotide sequences as described in any of the embodiments above, or by using the kit as described in any of the above embodiments.

In another embodiment, the method of the present invention for detecting SARS-Cov-2 using the kit of the present invention may be coupled with any PCR machine, such that the results may be directly uploaded / made accessible via cloud.

Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the principles and features described herein.

The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure.

The embodiments, examples and alternatives of the preceding paragraphs or the description and drawings, including any of their various aspects or respective individual features, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments, unless such features are incompatible.

Detailed Description
The features and properties of the present invention are described in further detail below with reference to examples.

Example 1
The primers and probes for detecting SARS-CoV-2 provided herein are depicted in Table 1:
a first combination of oligonucleotide sequences comprising:
a first primer pair for ORF1ab or ORF1ab gene shown in SEQ ID NO. 01 and SEQ ID NO. 02, and a first probe shown in SEQ ID NO. 03;
a second primer pair for RdRp gene shown in SEQ ID NO.04 and SEQ ID NO.05, and a second probe shown in SEQ ID NO. 06;
a third primer pair for N gene shown in SEQ ID NO. 07 and SEQ ID NO. 08, and a third probe shown in SEQ ID NO. 09;
a fourth primer pair for E gene shown in SEQ ID NO.10 and SEQ ID NO.11, and a second probe shown in SEQ ID NO. 12;
a fifth primer pair for S gene shown in SEQ ID NO.13 and SEQ ID NO.14, and a second probe shown in SEQ ID NO. 15;
and, optionally, a sixth combination of oligonucleotide sequences for RNAse P gene that could be used as an endogenous internal control comprising:
a sixth primer pair shown in SEQ ID NO.16 and SEQ ID NO. 17, and a sixth probe shown in SEQ ID NO. 18;
wherein, the 5' end of the first probe is marked with a fluorescence reporter group, and the 3' end is marked with a fluorescence quenching group.

Table 1: Fluorescence reporter groups used in CoviSwiftTM COVID-19 Rapid PCR Kit and CoviSwiftTM COVID-19 S Plus Rapid PCR Kit
Gene Fluorescence reporter group for CoviSwiftTM COVID-19 Rapid PCR Kit Fluorescence reporter group for CoviSwiftTM COVID-19 S Plus Rapid PCR Kit
ORF1ab FAM VIC
RdRp -
N gene FAM
E gene Cy5 -
S gene - Cy5
RNAse P VIC/HEX ROX

Example 2: Process for lyophilization of Reaction Mix
This example demonstrates the process for lyophilization of the cocktail of Reaction Mix and oligonucleotides. The freeze-drying process consists of multiple consecutive phases.
1. First, the cocktail of Reaction Mix (RT and Taq pol) and oligonucleotides are prepared and aliquoted in a 0.2ml PCR tube.
2. Then, the PCR tubes containing the reagents are loaded into the -80°C freezers for freezing for at least 3hours. Alternatively, freeze dryer with freezing parameter (-80°C-110°C) can be used for gradual freezing until -80°C.
3. Next, the frozen PCR tubes should be transferred immediately to lyophilizer without any liquefaction.
4. Then, the chamber pressure was decreased gradually (from =1hpa to =0.05hpa) to establish the primary drying phase, enabling the sublimation of all ice and the formation of dried cake.
5. The procedure is as follows:
-80°C for =3hours,
=-80°C for =2.5hours,
And 25°C for 10-60minutes.
6. The procedure for freeze-drying chamber is maintained at less than =1 hpa throughout the freeze-drying.
Once the freeze-drying is complete, packaging of the PCR tubes with dried cake is performed into an aluminium foil maintaining the humidity of less than 10%.

Example 3: Kit components for rapid detection SARS-CoV-2
CoviSwiftTM COVID-19 Rapid PCR Kit:
For qualitative rapid detection of SARS-CoV-2 by real time PCR amplification. Assay involves detection of E gene for screening, and ORF1(RdRp)&N gene for confirmation in a single test along with RNaseP as internal control. The kit contains all the reagents from sample to result along with the quality controls as shown in Table 2.

Table 2: Components in CoviSwiftTM COVID-19 Rapid PCR Kit
Contents Description
Sample tube Molecular transport medium
Lysis tube Lysis composition comprising PEG
COVID-19 Reaction mix Lyophilized reaction mix containing reverse transcriptase enzyme, Taq polymerase, RNAse Inhibitor, and specific primers and probes for E, ORF1(RdRp)/N and RNAse P targets
Reconstitution Buffer Buffer components for reconstitution of lyophilized Reaction Mix comprising Tris EDTA
COVID-19 Positive Control In vitro transcripts for SARS-CoV-2 specific E, ORF1(RdRp)/N and Internal control RNase P gene targets

CoviSwiftTM COVID-19 S Plus Rapid PCR Kit:
For qualitative rapid detection of SARS-COV-2 by real time PCR for amplification and detection of COVID-19. Detection based on ORF1ab and N gene along with S gene covering the region for major variants of concern (VOC). The kit contains all the reagents (see Table 3) from sample to result along with the quality controls. The S gene dropout or S gene failure due to deletion in spike protein at 69-70 amino acid position has been reported in Omicron as in Alpha. Thus, the kit will help to identify the VOC Omicron within an hour time and thus limits the sequencing approach.

Table 3: Components in CoviSwiftTM COVID-19 S Plus Rapid PCR Kit
Contents Description
Sample tube Molecular transport medium
Lysis tube Lysis composition comprising PEG
COVID-19 Reaction Mix Lyophilized reaction mix containing reverse transcriptase enzyme, Taq polymerase, RNAse Inhibitor, and specific primers and probes for ORF1, N, S and RNAseP targets
Reconstitution Buffer Buffer components for reconstitution of lyophilized PCR mix comprising Tris EDTA
COVID-19 Positive Control In vitro transcripts for SARS-CoV-2 specific ORF1ab, N, S and Internal control RNase P gene targets

Example 4: Preparation of sample lysate and RT-PCR
The present example provides a method for the preparation of sample lysate and PCR sample for detecting SARS-CoV-2 using the above primers and probes, wherein the method comprises the rapid protocol steps:

1. Sample collection using provided swab and Sample Tube (1) which contains viral transport medium (VTM)
2. Lysate preparation in 5 minutes:
Adding sample to lysis tube through dropper (5); mixing the sample by tapping; incubating for five minutes incubation at room temperature to obtain crude lysate which is PCR-ready.

3. Real time PCR detection of SARS-CoV-2 within 50 minutes:
using gold standard real time PCR using hydrolysis probe technology;
lyophilized Reaction Mix comprising lyophilized Reaction Mix of primers, hydrolysis probes, PCR enzyme cocktail comprising RT enzyme, Fast Taq pol, in a single Reaction Tube;
optionally adding RNAse inhibitors to avoid interference with PCR, adding to speed of reaction; performed with quality controls - COVID-19 positive control and no template control for batch validity.
wherein the rapid PCR program is as follows:

Parameter Cycles Temperature Time
RT incubation Hold 50°C 15 mins
Enzyme inactivation Hold 95°C 1 min
PCR amplification 37-45 95°C 5 sec
60°C 10 secs

Thus, the PCR run time for is significantly reduced as the testing eliminates nucleic acid isolation steps by directly using the lysate for PCR amplification. Further, the as the kit components are provided in a easily usable format that is in sterile disposable dropper tubes, the process is becomes easy to operate and does not need highly trained personnel.

Example 5: Results and Interpretation
The present example provides a method for result analysis and interpretation of the data obtained post the PCR run.

Table 5: Controls for CoviSwiftTM COVID-19 Rapid PCR Kit
ORF1 E(Cy5) RNaseP (VIC/HEX) Ct
Positive control (PC) + + + 35
No template control (NTC) - - - -

NTC should be negative and not exhibit amplification curves that cross the threshold line but in the event of high background noise RNaseP may show late amplification which may not be of significance.
PC reaction should produce a positive result for each target included in the test.
RNaseP should be positive at or before 35 cycles for all clinical samples thus indicating the presence of sufficient nucleic acid and the specimen is of acceptable quality.
When all the controls meet the stated requirements, a specimen is considered positive for SARS-CoV-2 in case of amplification curves cross the threshold line for N/ORF1(RdRp) and E gene target.
When all controls exhibit the expected performance, a specimen is considered negative if E and N/ORF1ab (RdRP) gene amplification curves are absent and the RNaseP growth curve DOES cross the threshold line.

Table 6: Interpretation of PCR results using CoviSwiftTM COVID-19 Rapid PCR Kit
ORF1(RdRP) (RdRp) (FAM) E (Cy5) RnaseP (VIC/HEX) Results
Positive Positive Positive SARS-CoV-2 RNA is detected.
Negative Negative Positive SARS-CoV-2 RNA not detected
Positive Negative Positive SARS-CoV-2 RNA is detected.
Negative Positive Positive SARS-CoV-2 Presumptive Positive
Repeat the test from original sample and repeating
the RT PCR.
If repeat test shows inconclusive, additional
confirmation testing with new specimen is
recommended*
Negative Negative Negative Inhibition of PCR reaction, repeat the test

Table 7: Interpretation of PCR results using CoviSwiftTM COVID-19 S Plus Rapid PCR Kit

ORF1ab
(VIC/HEX) N (FAM) S
(Cy5) RnaseP (ROX) Results
Positive Positive Positive Positive SARS-CoV-2 RNA is detected.
Negative Negative Negative Positive SARS-CoV-2 RNA not detected
Positive Positive Negative Positive In case of negative S gene target but with
positive ORF1ab and N gene targets, S
gene drop out may indicate chances of
Variant
Only one SARS-CoV-2 targets Positive Positive SARS-CoV-2 Inconclusive.
Repeat the test from original sample and
repeating the RT PCR.
If repeat test shows inconclusive,
additional confirmation testing with new
specimen is recommended
Negative Negative Negative Negative Inhibition of PCR reaction, repeat the test

Example 6: Time and temperature required for sample lysis to release nucleic acids was determined as per Table 8 using CoviSwiftTM COVID-19 Rapid PCR Kit and comparing the Ct values for ORF1ab(RdRp/N), E, RNAse P genes.

Table 8: Incubation time and temperature for sample lysis
Time 15mins 10 mins 5 mins
Temp. ORF1/
RdR P/
N E gene RNas e P ORF1/
RdR P/
N E gene RNaseP ORF1/ RdR P/
N E gene RNas e P
56 ºC 24.52 24.22 29.03 24.68 24.42 29.23 23.98 24.33 29.52
RT 23.54 24.17 28.98 23.69 24.36 29.23 23.52 24.63 28.63

Experiment was performed with a SARS-CoV-2 positive sample with lysis incubation for 15minutes which is a general standard practice, followed by 10minutes and 5minutes wherein incubation was performed at 56 ºC and at room temperature, judging the detection result according to Ct value displayed by a fluorescent PCR amplification instrument. From the above table it was observed that the incubation at room temperature for 5minutes showed optimum results.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING
SEQ ID No. Gene Feature Sequence (5’? 3’)
1 ORF1ab F-Primer CCCTGTGGGT TTTACACTTA A
2 ORF1ab R-Primer ACGATTGTGC ATCAGCTGA
3 ORF1ab Probe CCGTCTGCGG TATGTGGAAA GGTTATGG
4 RdRp F-Primer GTGARATGGT CATGTGTGGC GG
5 RdRp R-Primer CARATGTTAA ASACACTATT AGCATA
6 RdRp Probe CAGGTGGAAC CTCATCAGGA GATGC
7 N gene F-Primer AAATTTTGGG GACCAGGAAC
8 N gene R-Primer TGGCAGCTGT GTAGGTCAAC
9 N gene Probe ATGTCGCGCA TTGGCATGGA
10 E gene F-Primer ACAGGTACGT TAATAGTTAA TAGCGT
11 E gene R-Primer ATATTGCAGC AGTACGCACA CA
12 E gene Probe ACACTAGCCA TCCTTACTGC GCTTCG
13 S gene F-Primer TCAACTCAGG ACTTGTTCTT AC
14 S gene R-Primer TGGTAGGACA GGGTTATCAA AC
15 S gene Probe TGGTCCCAGA GACATGTATA GCAT
16 RNAseP F-Primer AGATTTGGAC CTGCGAGCG
17 RNAseP R-Primer GAGCGGCTGT CTCCACAAGT
18 RNAseP Probe TTCTGACCTG AAGGCTCTGC GCG