WO2022084381A1 - Kit and method - Google Patents
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- WO2022084381A1 WO2022084381A1 PCT/EP2021/079071 EP2021079071W WO2022084381A1 WO 2022084381 A1 WO2022084381 A1 WO 2022084381A1 EP 2021079071 W EP2021079071 W EP 2021079071W WO 2022084381 A1 WO2022084381 A1 WO 2022084381A1
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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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
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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
Definitions
- the invention relates to novel kits and methods for extracting RNA from an RNA virus or obtaining viral RNA from a sample and for determining the presence or absence of an RNA virus, such as SARS-CoV-2.
- Coronaviruses are members of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales.
- the virus belongs to genera Betacoronavirus and has close similarities to severe acute respiratory syndrome-related coronaviruses (SARS-CoV).
- SARS-CoV severe acute respiratory syndrome-related coronaviruses
- the virus uses ACE2 as the entry receptor-like SARS-CoV.
- RNA virus comprising (a) an inactivation solution comprising t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced), (b) a dilution solution comprising TRIZMA® (2-Amino-2- (hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA) and (c) an optimisation solution comprising potassium chloride (KC1) and magnesium chloride (MgCl 2 ); a sample preparation buffer for obtaining viral RNA from a kit of RNA virus comprising (a) an inactivation solution comprising t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100
- Figure 1 shows the standard curve of SARS-Cov-2 whole genomic RNA (EvaG) using a real time RT-PCR assay (curves from left to right: 10 4 copies/pl, 10 3 copies/pl, 10 2 copies/pl and 10 copies/pl).
- Figure 2 shows the standard curve of SARS-Cov-2 whole genomic RNA (EvaG) using a real time RT-PCR assay (curves from left to right: 10 4 copies/pl, 10 2 copies/pl, 10 copies/pl and 0.59 copies/pl).
- Figure 3 shows the standard curve of SARS-Cov-2 whole genomic RNA (EvaG) using a real time RT-PCR assay (curves from left to right: 10 6 copies/pl, 10 4 copies/pl, 10 2 copies/pl, 10 copies/pl and 5 copies/pl).
- Figure 4 shows results using the sample preparation buffer of the invention.
- the control is the positive control template.
- Other curves represent samples amplifying at the limit of detection.
- Figure 5 shows results using the sample preparation buffer of the invention.
- the control is the positive control template.
- Other curves represent samples amplifying at the limit of detection.
- SEQ ID NO: 1 shows the forward primer used in the Examples.
- SEQ ID NO: 2 shows the reverse primer used in the Examples.
- SEQ ID NO: 3 shows the sequence of the probe used in the Examples.
- SEQ ID NO: 4 shows the cDNA amplified in the Examples.
- SEQ ID NO: 5 shows the reverse complement of SEQ ID NO: 3.
- SARS-CoV-2 is interchangeable with 2019-nCoV or COVID-19.
- extracting RNA from an RNA virus is interchangeable with inactivating an RNA virus.
- the new kits and methods of the invention may be for inactivating an RNA virus.
- extracting RNA from an RNA virus is interchangeable with obtaining viral RNA from a sample.
- the new kits and methods of the invention may be for obtaining viral RNA from a sample.
- extracting RNA from an RNA virus is interchangeable with preparing the RNA in an RNA virus for reverse-transcription polymerase chain reaction (RT-PCR).
- RT-PCR reverse-transcription polymerase chain reaction
- determining is interchangeable with detecting.
- the new kits and methods of the invention may be for detecting the presence or absence of an RNA virus, such as SARS- CoV-2, in a sample or patient. Also, in all instances, the new kits and methods of the invention may be for determining/detecting/identifying whether or not a sample contains or comprises an RNA virus, such as SARS-CoV-2, or whether or not a patient is infected with an RNA virus, such as SARS-CoV-2.
- kits and methods for extracting RNA from a variety of RNA viruses in a variety of samples have several advantages.
- the new kits and methods are capable of preparing the RNA from an RNA virus for RT- PCR in a safe manner.
- the inactivation solution inactivates the virus by disrupting the viral envelope and reducing its ability to infect the person using the kit or conducting the method.
- kits and methods do not require complicated inactivation steps (for instance using heat) and/or RNA extraction steps (for instance using magnetic beads).
- the kits and methods of the invention are not only safe, but also straightforward and quick to use. This further increases the safety of the kits and the methods of the invention by minimising exposure of the user to infective virus. It also means the presence or absence of the virus can be detected/determined more quickly.
- kits and methods are optimised to allow the RNA in an RNA virus to be detected using RT-PCR with a high degree of sensitivity without complicated RNA extraction steps, such as using magnetic beads. This facilitates the speed and ease with which the kits and methods can be used to detect low viral titres. As shown in the Examples, the method is capable of detecting 10 of fewer copies of the SARs-CoV-2 RNA genome per uL of sample.
- the invention provides a kit for extracting RNA from an RNA virus.
- the kit comprises an inactivation solution comprising t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
- the kit also comprises a dilution solution comprising TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA).
- the kit further comprises an optimisation solution comprising potassium chloride (KC1) and magnesium chloride (MgCl 2 ).
- the three different solutions are typically provided in separate containers so they may be used in the method of the invention.
- the containers are typically sealed.
- the containers are typically labelled to indicate their contents.
- the kit may be for extracting RNA from one or more RNA viruses as discussed below with reference to the method of the invention.
- the invention also provides a sample preparation buffer for obtaining viral RNA from a sample.
- the viral RNA is typically from an RNA virus.
- the sample preparation buffer may be for extracting RNA from an RNA virus.
- the sample preparation buffer comprises (a) t- octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced) and (b) TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA).
- the sample preparation buffer comprises the components of the inactivation solution and the dilution solution discussed above.
- the sample preparation buffer may be for obtaining viral RNA from one or more RNA viruses from a sample or extracting RNA from one or more RNA viruses as discussed below with reference to the method of the invention.
- the invention also provides a kit for determining the presence or absence of an RNA virus.
- the kit comprises (i) a sample preparation buffer solution of the invention and (ii) a PCR reaction mix comprising potassium chloride (KC1) and magnesium chloride (MgCT). Parts (i) and (ii) are typically provided in separate containers so they may be used in the method of the invention.
- the containers are typically sealed.
- the containers are typically labelled to indicate their contents.
- the inactivation solution comprises t-octylphenoxypoly ethoxy ethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
- the inactivation solution may comprise Triton® X-100, Triton® X-100 reduced or a mixture of both.
- Triton® X-100 and Triton® X-100 reduced are detergents which disrupt viral envelopes and inactivate RNA viruses.
- Triton® X-100 is also known as t-octylphenoxypoly ethoxy ethanol and oly ethylene glycol tertoctylphenyl ether. These terms are interchangeable.
- Triton® X-100 reduced is also known as polyoxyethylene (10) isooctylcyclohexyl ether.
- the inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 and/or Triton® X-100 reduced by volume.
- the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 and/or Triton® X- 100 reduced by volume.
- the inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 and/or Triton® X-100 reduced.
- the inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 by volume.
- the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 by volume.
- the inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 by volume.
- the inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 reduced by volume.
- the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 reduced by volume.
- the inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 reduced by volume.
- the inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 and Triton® X-100 reduced by volume.
- the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 and Triton® X-100 reduced by volume.
- the inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 and Triton® X-100 reduced.
- the amounts by volume of each add together to give the relevant values. For instance, 2.5% of Triton® X-100 and 2.5% Triton® X- 100 reduced give 5% in total.
- Triton® X-100 and Triton® X-100 reduced may be present in any ratio, such as 1: 10, 1 :5, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 5:1 or 10: 1.
- the inactivation solution preferably comprises only Triton® X-100.
- the inactivation solution preferably comprises universal transport medium (UTM), virus transport medium (VTM), PBS or saline.
- UTM universal transport medium
- VTM virus transport medium
- PBS saline
- the Triton® X-100 and/or Triton® X-100 reduced are preferably present in UTM, VTM, PBS or saline.
- UTM is commercially available from a variety of sources, such as Copan diagnostics (catalogue number UTM - 366C).
- VTM is commercially available from a variety of sources, such as Sigma- virucult VTM (catalogue number MW951S).
- PBS can be prepared using standard methods. For instance, PBS can be prepared using PBS tablets (P32080) supplied by Melford laboratories.
- lOOmL of PBS contains 137mM NaCl, 11.9mM Phosphate buffer and 2.7mM KC1.
- Saline can be prepared using standard methods.
- the saline could be STERETS Normasol (Ref: 99766774), which is sodium chloride solution 0.9%w/v.
- the PBS and saline are typically prepared using nuclease-free water as discussed below.
- the inactivation solution preferably comprises or consists of PBS or saline with from about 1% to about 5.0% of Triton® X-100 and/or Triton® X-100 reduced by volume. This solution may comprise any of the amounts of Triton® X-100 and/or Triton® X-100 reduced discussed above.
- the inactivation solution more preferably comprises or consists of PBS or saline with from about 1.0% to about 5.0% of Triton® X-100 by volume. This solution may comprise any of the amounts of Triton® X-100 discussed above.
- the inactivation solution preferably comprises or consists of PBS or saline with about 1% or about 5.0% of Triton® X-100 and/or Triton® X-100 reduced by volume. This solution may comprise any of the amounts of Triton® X-100 and/or Triton® X-100 reduced discussed above.
- the inactivation solution more preferably comprises or consists of PBS or saline with about 1.0% or about 5.0% of Triton® X-100 by volume. This solution may comprise any of the amounts of Triton® X-100 discussed above.
- the kit may comprise any volume of inactivation solution, such as at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2 mL, at least about 5mL, at least about lOmL, at least about 20mL, or at least about 50mL.
- the kit may comprise higher volumes of the inactivation solution, such as at least about lOOmL, at least about 500mL, at least about IL or more.
- the kit typically comprises sufficient inactivation solution to allow an RNA virus in any of the samples discussed below, such as a saliva swab (also known as an oropharyngeal swab) or a nasopharyngeal swab, to be inactivated.
- a saliva swab also known as an oropharyngeal swab
- a nasopharyngeal swab to be inactivated.
- the inactivation solution is typically nuclease-free. This can be achieved by obtaining nuclease-free components or preparing nuclease free components using nuclease-free water. Nuclease-free water is commercially available, such as from Fisher Scientific (Cat number: BP561) and Sigma Aldrich (Cat number W4502). Nuclease-free water in the context of the invention typically means DNAase-free and RNAase-free water.
- the dilution solution comprises TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA).
- TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol) is well known in the art and can be prepared using standard methods. For instance, it can be prepared to pH8.5, IM using 4.42g Trizma hydrochloride (Sigma- Aldrich, Cat number: T3253) and 8.72g Trizma base (Sigma- Aldrich, Cat number: T1503). Glycerol is commercially available, such as bidistilled 99.5% (Supplier: VWR (Cat number: 24388.295). EDTA is also commercially available, such as 0.5M, pH8.0 from Thermo Fisher Scientific (Cat number: AM9260G).
- the dilution solution typically comprises or consists of the three components in nuclease-free water.
- the dilution solution preferably comprises from about 5.0mM to about 20.0mM TRIZMA®.
- the dilution solution more preferably comprises from about 8.0mM to about 15.0mM TRIZMA®.
- the dilution solution more preferably comprises about lO.OmM TRIZMA®.
- the dilution solution preferably comprises from about 0.5mM to about 3mM glycerol.
- the dilution solution more preferably comprises from about l.OmM to about 2.0mM glycerol.
- the dilution solution more preferably comprises about 1.4mM, such as about 1.368mM, glycerol.
- the dilution solution preferably comprises from about 0.1 to about l.OmM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution more preferably comprises from about 0.2 to about 0.8mM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution more preferably comprises about 0.5mM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution preferably comprises from about 5.0mM to about 20.0mM TRIZMA®, from about 0.5mM to about 3mM glycerol and from about 0.1 to about l.OmM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution more preferably comprises from about 8.0mM to about 15.0mM TRIZMA®, from about l.OmM to about 2.0mM glycerol and from about 0.2 to about 0.8mM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution more preferably comprises about lO.OmM TRIZMA®, about 1.4mM, such as about 1.368mM, glycerol and about 0.5mM ethylenediaminetetraacetic acid (EDTA).
- EDTA ethylenediaminetetraacetic acid
- the dilution solution preferably comprises or consists of nuclease-free water with from about 5.0mM to about 20.0mM TRIZMA®, from about 0.5mM to about 3.0mM glycerol and from about 0.1 to about l.OmM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution more preferably comprises or consists of nuclease-free water with from about 8.0mM to about 15.0mM TRIZMA®, from about l.OmM to about 2.0mM glycerol and from about 0.2 to about 0.8mM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution more preferably comprises or consists of nuclease-free water with about lOrnM TRIZMA®, about 1.4mM, such as about 1.368mM, glycerol and about 0.5mM ethylenediaminetetraacetic acid (EDTA).
- lOrnM TRIZMA® nuclease-free water with about lOrnM TRIZMA®, about 1.4mM, such as about 1.368mM, glycerol and about 0.5mM ethylenediaminetetraacetic acid (EDTA).
- the dilution solution most preferably has the composition shown in Table 6.
- the kit may comprise any volume of dilution solution, such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
- the kit may comprise higher volumes of the dilution solution, such as at least about lOOmL, at least about 500mL, at least about IL or more.
- the kit typically comprises sufficient dilution solution to dilute the inactivated sample as discussed below with reference to the method of the invention.
- the dilution solution is typically nuclease-free. This can be achieved by obtaining or preparing nuclease-free components. Nuclease-free water is discussed above.
- the optimisation solution comprises potassium chloride (KC1) and magnesium chloride.
- KC1 is commercially available, for instance from Sigma-Aldrich (Cat number: P9541).
- MgCl 2 such as MgC12.6H 2 O, is commercially available, such as from VWR (Cat number: 25108.925).
- the optimisation solution typically comprises or consists of KC1 and MgCl 2 in nuclease-free water.
- the optimisation solution preferably comprises from about 0.1M to about 1.0M KC1.
- the optimisation solution more preferably comprises from about 0.2M to about 0.8M KC1 or from about 0.3M to about 0.6M KC1.
- the optimisation solution more preferably comprises about 0.5M, about 0.58M or about 0.5749M KC1.
- the optimisation solution preferably comprises from about 0.5M to about 10M MgCl 2 .
- the optimisation solution more preferably comprises from about 1.0 to about 7.0M MgCl 2 or from about 1.5M to about 5.0M MgCl 2 .
- the optimisation solution more preferably comprises about 3M, about 2.9M or about 2.91622M MgCl 2 .
- the optimisation solution preferably comprises from about 0.1M to about 1.0M KC1 and from about 0.5M to about 10M MgCl 2 .
- the optimisation solution more preferably comprises from about 0.2M to about 0.8M KC1 and from about 1.0M to about 7.0M MgCl 2 .
- the optimisation solution more preferably comprises from about 0.3M to about 0.6M KC1 and from about 1.5M to about 5.0M MgCl 2 .
- the optimisation solution more preferably comprises about 0.5M, about 0.58M or about 0.5749M KC1 and about 3M, about 2.9M or about 2.91622M MgCl 2 .
- the optimisation solution preferably comprises or consists of nuclease-free water with from about 0. IM to about 1.0M KC1 and from about 0.5M to about 10M MgCl 2 .
- the optimisation solution more preferably comprises or consists of nuclease-free water with from about 0.2M to about 0.8M KC1 and from about 1.0M to about 7.0M MgCl 2 .
- the optimisation solution more preferably comprises or consists of nuclease-free water with from about 0.3M to about 0.6M KC1 and from about 1.5M to about 5.0M MgCl 2 .
- the optimisation solution more preferably comprises or consists of nuclease-free water with about 0.5M, about 0.58M or about 0.5749M KC1 and about 3M, about 2.9M or about 2.91622M MgCl 2 .
- the optimisation solution most preferably has the composition shown in Table 7.
- the kit may comprise any volume of optimisation solution, such as at least about 2uL, at least about 5uL, lOuL, at least about 20uL, at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2 mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
- the kit may comprise higher volumes of the dilution solution, such as at least about lOOmL, at least about 500mL, at least about IL or more.
- the kit typically comprises sufficient optimisation solution to optimise the diluted sample as discussed below with reference to the method of the invention.
- the optimisation solution is typically nuclease-free. This can be achieved by obtaining or preparing nuclease-free components.
- the sample preparation buffer of the invention comprises (a) t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
- the sample preparation buffer preferably comprises polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
- the sample preparation buffer more preferably comprises about 0.5% t- octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
- the sample preparation buffer most preferably comprises about 0.5% polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
- the sample preparation buffer of the invention also comprises (b) TRIZMA® (2-Amino-2- (hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA). Any of the embodiments discussed above with reference to the dilution solution, especially the concentrations of the components, equally apply to (a) of the sample preparation buffer.
- the sample preparation buffer is effectively a TRIZMA®-EDTA buffer comprising t-octylphenoxypoly ethoxy ethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced) and glycerol.
- the sample preparation buffer is typically nuclease-free. This can be achieved by obtaining or preparing nuclease-free components.
- the sample preparation buffer typically comprises or consists of the four or five components in nuclease-free water.
- the sample preparation buffer preferably comprises about 1% IM or 0.01M TRIZMA® (2- Amino-2-(hydroxymethyl)-l,3-propanediol).
- the sample preparation buffer preferably comprises about 0.1% 0.5M or 0.5mM EDTA.
- the sample preparation buffer preferably comprises about 10% glycerol.
- the sample preparation buffer preferably comprises about 1% IM or O.OlmM TRIZMA® (2-Amino-2- (hydroxymethyl)-l,3-propanediol), about 0.1% 0.5M or 0.5mM EDTA and about 10% glycerol.
- the sample preparation buffer may be present as any volume, such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
- the sample preparation buffer may be present in higher volumes, such as at least about lOOmL, at least about 500mL, at least about IL or more.
- the sample preparation buffer is preferably present in about ImL.
- the invention also provides a kit for determining the presence or absence of an RNA virus comprising (i) a sample preparation buffer of the invention and (ii) a PCR reaction mix comprising potassium chloride (KC1) and magnesium chloride (MgCl 2 ).
- PCR reaction mixes are known in the art and are discussed below and in the Examples.
- the PCR reaction mix may be any of those. Any of the embodiments discussed above with reference to the optimisation solution, especially the concentrations of the components, equally apply to (ii) of the kit comprising the sample preparation buffer.
- the PCR reaction mix preferably comprises from about 5mM to about 25mM potassium chloride (KC1) and from about 5mM to about 25mM magnesium chloride (MgCl 2 ), such as from about 6mM to about 24mM, from about 7mM to about 23mM, from about 8mM to about 22mM, from about 9mM to about 21mM or from about lOmM to about 20mM potassium chloride (KC1) and from about 6mM to about 24mM, from about 7mM to about 23mM, from about 8mM to about 22mM, from about 9mM to about 21mM or from about lOmM to about 20mM magnesium chloride (MgCl 2 ).
- KC1 5mM to about 25mM potassium chloride
- MgCl 2 magnesium chloride
- the PCR reaction mix preferably comprises about 18.8mM potassium chloride (KC1) and about 18.73mM magnesium chloride (MgCl 2 ).
- KC1 18.8mM potassium chloride
- MgCl 2 18.73mM magnesium chloride
- 5uL of sample preparation buffer is mixed with 15uL of the PCR reaction mix giving a final potassium chloride (KC1) concentration of 14. ImM and a final magnesium chloride (MgCl 2 ) concentration of 14.05mM in the 20uL RT-PCR reaction.
- the kit may comprise any volume of sample preparation buffer, such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
- the kit may comprise higher volumes of the sample preparation buffer, such as at least about lOOmL, at least about 500mL, at least about IL or more.
- the kit typically comprises about ImL of sample preparation buffer.
- the kit may comprise any volume of PCR reaction mix, such as such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
- the kit of the invention preferably further comprises an internal extraction control (IEC) RNA.
- IEC internal extraction control
- RNA extraction and RT-PCR it is advantageous to have an exogenous source of RNA template that is spiked into the extraction.
- This control RNA is then co-purified with the sample RNA and can be detected as a positive control for the extraction process (internal extraction control).
- Successful co-purification and RT- PCR amplification of the control RNA also indicates that PCR inhibitors are not present at a high concentration.
- IEC RNA can be prepared or is commercially available.
- the kit may comprise any volume of IEC, such as at least about 20uL, at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
- the kit may comprise higher volumes of the dilution solution, such as at least about lOOmL, at least about 500mL, at least about IL or more.
- the kit typically comprises sufficient IEC to conduct the method of the invention.
- the kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the methods below to be carried out.
- reagents or instruments include, but are not limited to, one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a patient (such as a vessel or an instrument comprising a needle or a swab) or tubes in which quantitative reactions can be done.
- suitable buffer(s) aqueous solutions
- means to obtain a sample from a patient such as a vessel or an instrument comprising a needle or a swab
- the kit may, optionally, comprise instructions to enable the kit to be used in the methods of the invention or details regarding which patients may be tested.
- the kit may further comprise any of the reagents required to conduct RT-PCR discussed below, including primers, probes, a reverse transcriptase, heat-stable DNA polymerase and negative control, such as nuclease-free water.
- the invention provides a method of extracting RNA from an RNA virus, if present, in a sample.
- the method can only extract the RNA from the RNA virus if the virus is present and, as explained in more detail below, the sample may not contain the virus.
- the method comprises using a kit of the invention.
- First (a), the method comprises contacting the sample with an inactivation solution comprising Triton® X-100 and/or Triton® X-100 reduced.
- Second (b), the method comprises diluting the inactivated sample with a dilution solution comprising TRIZMA®, glycerol and EDTA.
- Third (c) the method comprises adding to the diluted sample an optimisation solution comprising KC1 and MgCl 2 .
- the inactivation solution may be any of those discussed above with reference to the kits of the invention.
- the dilution solution may be any of those discussed above with reference to the kits of the invention.
- the optimisation solution may be any of those discussed above with reference to the kits of the invention.
- any amount of the inactivation solution may be contacted with the sample, such as about ImL, about 2mL, about 5mL or more.
- a saliva swab also known as an oropharyngeal swab
- a nasopharyngeal swab is preferably contacted with about ImL inactivation solution.
- a saliva swab also known as an oropharyngeal swab
- a nasopharyngeal swab is more preferably contacted with about ImL inactivation solution comprising PBS or saline.
- the inactivated sample in (a) may be diluted in step (b) by about 1:5, about 1:10, about 1 : 15 or about 1:25. Dilution of 1:5 means 1 part (in terms of volume) of the inactivated sample is combined with 4 parts dilution sample (to form 5 parts in total).
- the inactivation solution comprises UTM or VTM
- the inactivated sample in (a) is preferably diluted in step (b) by about 1:15.
- 6uL of inactivated sample is combined with 84uL of dilution solution.
- the inactivation solution comprises PBS or saline
- the inactivated sample in (a) is preferably diluted in step (b) by about 1:5.
- Step (c) preferably comprises adding 1 part (by volume) of the optimisation solution to 30 parts or 45 parts of the diluted sample produced in (b).
- step (c) preferably comprises adding 3uL of the optimisation solution to the 90uL of the diluted sample produced in (b) or 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
- step (c) preferably comprises adding 1 part (by volume) of the optimisation solution to 45 parts of the diluted sample produced in (b), such as adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
- step (c) preferably comprises adding 1 part (by volume) of the optimisation solution to 30 parts or 45 parts of the diluted sample produced in (b), such as adding 2uL or 3uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
- Step (c) most preferably comprises adding 1 part (by volume) of the optimisation solution to 45 parts of the diluted sample produced in (b), such as adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
- the method preferably further comprises step (d) further diluting the optimised sample produced in step (c).
- the inactivation solution in step (a) comprises PBS or saline
- step (b) comprises diluting the inactivated sample produced in (a) 1:5 with the dilution solution
- step (c) comprises adding 1 part of the optimisation solution to 45 parts of the diluted sample produced in (b).
- the inactivation solution in step (a) comprises PBS or saline
- step (b) comprises diluting 18uL of inactivated sample produced in (a) with 72uL of the dilution solution
- step (c) comprises adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
- the inactivation solution in step (a) comprises UTM or VTM
- step (b) comprises diluting the inactivated sample produced in (a) 1:15 with the dilution solution
- step (c) comprises adding 1 part of the optimisation solution to 45 parts of the diluted sample produced in (b).
- the inactivation solution in step (a) comprises UTM or VTM
- step (b) comprises diluting 6uL of the inactivated sample produced in (a) with 84uL dilution solution
- step (c) comprises adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
- the method preferably further comprises (d) adding 5 parts of nuclease free water or IEC to 23 parts of the optimised sample produced in (c).
- the method preferably further comprises (d) adding 20uL of nuclease free water or IEC RNA to the 92uL of the optimised sample produced in (c).
- the method preferably comprises (a) contacting the sample with an inactivation solution comprising or consisting of PBS or saline with Triton® X-100 and/or Triton® X-100 reduced, (b) diluting 18uL of inactivated sample produced in (a) with 72uL of the dilution solution, (c) adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b) and (d) adding 20uL of nuclease free water or IEC RNA to 92uL of the optimised sample produced in (c).
- the method preferably comprises (a) contacting the sample with an inactivation solution comprising or consisting of UTM or VTM with Triton® X-100 and/or Triton® X-100 reduced, (b) diluting 6uL of inactivated sample produced in (a) with 84uL of the dilution solution, (c) adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b) and (d) adding 20uL of nuclease free water or IEC RNA to 92uL of the optimised sample produced in (c).
- the invention also provides a method of obtaining viral RNA, if present, from a sample, comprising contacting the sample with a sample preparation buffer of the invention and thereby obtaining the viral RNA, if present, from the sample.
- the method can only obtain the viral RNA if the virus is present and, as explained in more detail below, the sample may not contain the virus.
- the sample preparation buffer may be any of those discussed above. Any amount of the sample preparation buffer may be contacted with the sample, such as about ImL, about 2mL, about 5mL or more. An oropharyngeal swab or a nasopharyngeal swab is preferably contacted with about ImL of the sample preparation buffer.
- the method may involve the use of the kit of the invention comprising the sample preparation buffer of the invention.
- the method of the invention preferably does not comprise any additional methods of inactivating the virus and/or extracting RNA from the virus.
- the method of the invention is sufficient to inactivate the virus and extract the RNA from the virus.
- the method of the invention is sufficient to obtain viral RNA from the sample.
- the method of the invention preferably does not comprise heat inactivation or using a viral inactivation kit.
- the method preferably does not comprise using an RNA extraction kit.
- kits are well known in the art, such as QIAamp® virus RNA mini kit (Qiagen).
- the invention may be carried out with any RNA virus.
- the RNA virus is typically from the realm Riboviria.
- the RNA virus may be from the kingdom of Orthornavirae or Pararnavirae.
- the RNA virus is preferably from the kingdoms of Orthornavirae.
- the RNA virus is preferably from the family of Coronaviridae .
- the virus may be the severe acute respiratory syndrome (SARS) coronavirus (also known as SARS-CoV or SARS-CoV-1).
- SARS-CoV severe acute respiratory syndrome
- SARS-CoV-1 severe acute respiratory syndrome coronavirus
- the virus may be the Middle East respiratory syndrome (MERS) coronavirus, also known as camel flu.
- the virus is preferably SARS-CoV-2.
- the RNA virus is preferably from the family of Orthomyxoviridae .
- the virus may an influenza virus.
- the influenza virus is preferably an influenza A virus, such as H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1,H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or H10N7.
- Influenza A viruses infect humans, pigs, birds, horses and bats.
- the influenza virus is preferably an influenza B virus, which infects humans and seals.
- the influenza virus is preferably an influenza D virus, which infects pigs and cattle.
- the influenza virus is preferably an influenza C virus, which infects humans, pigs and dogs.
- the influenza virus is preferably an infectious salmon anemia virus, which infects Atlantic salmon.
- the influenza virus is preferably Thogotovirus or Dhori virus, which infects ticks, mosquitos and mammals, includeing humans.
- the invention may extract the RNA from multiple RNA viruses or two or more RNA viruses, such as 3 or more, 4 or more, 5 or more or 10 or more viruses.
- the invention may extract RNA from one or more of (i) SARS-CoV, (ii) MERS coronavirus and (iii) SARS- CoV-2, such as (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii).
- the invention may extract RNA from a coronavirus and an influenza virus.
- the invention may extract RNA from one or more of (i) SARS-CoV, (ii) MERS coronavirus and (iii) SARS- CoV-2 (including as defined above) and an influenza virus.
- the invention may extract RNA from one or more of (i) SARS-CoV, (ii) MERS coronavirus and (iii) SARS-CoV-2 (including as defined above) and one or more of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1,H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 and H10N7.
- the invention provides various methods of determining the presence or absence of an RNA virus, such as SARS-CoV-2, in a sample. Any number of RNA viruses may be detected as discussed above. All of the methods comprise extracting RNA from the RNA virus in the sample, if present, using a method of the invention. Any of the methods and kits discussed above may be used.
- the method comprises (i) extracting RNA from the RNA virus in the sample, if present, or obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
- RT-PCR reverse-transcription polymerase chain reaction
- the method may comprise (i) extracting RNA from the RNA virus in the sample, if present, using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
- the method may comprise (i) obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
- the method comprises conducting a RT-PCR assay on the sample that has been subjected to the extraction method of the invention.
- one 14th of the sample is preferably used in the RT-PCR.
- Various embodiments discussed above result in final volume of 112uL.
- 8uL of the extracted sample is used in RT-PCR.
- the RT-PCR is preferably direct RT-PCR.
- direct RT-PCR the sample, preferably 8uL, is directly added to the RT-PCR assay after conducting the extraction method of the invention without any intervening steps.
- the method comprises conducting a RT-PCR assay on the viral RNA obtained from the sample using the sample preparation buffer of the invention.
- the method preferably comprises contacting an oropharyngeal swab or a nasopharyngeal swab with about ImL sample preparation buffer and then adding about 5uL of the sample preparation buffer to about 15uL of the PCR reaction mix to form a total RT-PCR reaction volume of about 20uL.
- the PCR reaction mix comprises potassium chloride (KC1) and magnesium chloride (MgCl 2 ).
- Reverse transcription involves the use of the enzyme reverse transcriptase to convert viral RNA into cDNA.
- PCR involves using the primers to amplify a specific portion of the cDNA, namely the cDNA amplicon, which typically contains the target cDNA of interest.
- reverse transcriptases are commercially available (e.g. Superscript® II reverse transcriptase (Invitrogen) and Affinity script (Agilent)). Methods typically construct cDNA from random cDNA hexamers included in the reaction mixture.
- PCR involves amplifying a cDNA amplicon using a heat-stable DNA polymerase and a pair of forward and reverse polynucleotide primers. Because the newly synthesized cDNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence.
- Many polymerase chain methods are known to those of skill in the art and may be used in the methods of the invention.
- cDNA can be subjected to 20 to 40 cycles of amplification in a thermocycler as follows: 95°C for 30 sec, 52° to 60°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min.
- cDNA can be subjected to 20 to 40 polymerase chain reaction cycles in a thermocycler at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54°C to 58°C for 1 min, an extension step at 70°C for 1 min, with a final extension step at 70°C for 5 min.
- Heat stable DNA polymerases are commercially available (such as GoTaq® G2 (Promega)).
- the RT-PCR assay may be conducted using the conditions set out in the Examples.
- the reverse transcriptase is preferably Affinity script (Agilent).
- the DNA polymerase is preferably GoTaq® G2 (Promega). Both enzymes are preferably present in a 2x mastermix.
- the reaction mix is preferably lOuL 2x oasig mastermix, 2uL primer/probe mix (resuspended in template preparation buffer) and 8uL sample.
- the reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
- the RT-PCR method is preferably real time RT-PCR. This method is known in the art.
- the RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one-step reactions are routine in the art.
- Step (ii) of the method preferably comprises reverse transcribing any RNA in the sample, contacting any cDNA produced with a pair of primers under conditions which allow the primers to amplify their cDNA amplicon and determining the presence or absence of the cDNA amplicon.
- Detection of the cDNA amplicon indicates that the sample contains the RNA virus (i.e. indicates the presence of RNA virus in the sample).
- a lack of detection of the cDNA amplicon indicates that the sample does not contain the RNA virus (i.e. indicates the absence of RNA virus from the sample).
- probes and primers for use in the method of the invention are known in the art. Specific types of probes and primers that may be used in the invention are discussed below with reference to the preferred probes and primers for use in the invention. Probes and primers for detecting SARS-CoV, MERS coronavirus and influenza viruses are known in the art. As a result, the skilled person can detect any RNA virus or any combination of RNA viruses.
- the method of the invention is for determining the presence or absence of SARS-CoV -2 in a sample.
- the invention provides a method of determining the presence or absence of SARS- CoV-2 in a sample, wherein the method comprises (i) extracting RNA from the SARS-CoV -2 in the sample, if present, or obtaining SARS-CoV -2 RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
- RT-PCR reverse-transcription polymerase chain reaction
- the method may comprise (i) extracting RNA from the SARS-CoV-2 in the sample, if present, using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
- the method may comprise (i) obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
- step (ii) comprises using a preferred pair of primers discussed below and detecting the cDNA amplicon amplified by the primers if present.
- Any method may be used to detect the cDNA amplicon.
- the method may use SYBR Green to detect the cDNA amplicon. When the SYBR Green binds to the cDNA, it emits light and the intensity of the fluorescence increases as the cDNA amplicons accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding.
- the method preferably uses a preferred polynucleotide probe discussed below which specifically hybridises to the cDNA amplicon. The probe may be any of those discussed below. The use of the probe allows the specific cDNA amplicon to be identified if present.
- the probe is preferably a TaqMan® probe.
- step (ii) comprises using a pair of primers that amplifies a cDNA amplicon comprising (or consisting of) a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3 if present.
- the cDNA amplicon may be any of those discussed below.
- the cDNA amplicon preferably comprises or consists of SEQ ID NO: 4.
- the cDNA amplicon preferably comprises or consists of SEQ ID NO: 5.
- the method also comprises using a preferred polynucleotide probe discussed below which comprises the sequence shown in SEQ ID NO: 3 or a variant thereof as defined above to detect the cDNA amplicon if present.
- the polynucleotide probe may be any of those discussed below.
- the probe is preferably a TaqMan® probe.
- the polynucleotide probe specifically hybridises to cDNA amplicon if present.
- step (ii) comprises using a preferred a pair of primers discussed below and a preferred polynucleotide probe discussed below.
- the pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2.
- the polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3. The pair of primers amplifies a cDNA amplicon as discussed below and then this is specifically detected using the polynucleotide probe discussed below.
- detection of the cDNA amplicon indicates that the sample contain SARS-CoV-2 (i.e. indicates the presence of SARS-CoV-2 in the sample).
- a lack of detection of the cDNA amplicon indicates that the sample does not contain SARS-CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample).
- Detection of multiple copies of the cDNA amplicon indicates that the sample contain SARS-CoV-2 (i.e. indicates the presence of SARS-CoV-2 in the sample).
- a lack of detection of multiple copies of the cDNA amplicon indicates that the sample does not contain SARS- CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample).
- the method preferably comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the pair of primers under conditions which allow the primers to amplify their cDNA amplicon and (c) determining the presence or absence of the cDNA amplicon.
- Step (c) preferably uses a polynucleotide probe as described below.
- the RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one-step reactions are routine in the art.
- Preferred primers for use in detecting SARS-CoV-2 are preferred primers for use in detecting SARS-CoV-2
- the forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length.
- the reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 80% homology to SEQ ID NO: 2 based on sequence identity over its entire length.
- a polynucleotide such as a nucleic acid, is a polymer comprising two or more nucleotides.
- the nucleotides can be naturally occurring or artificial.
- a nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2’0-methyl, 2’ methoxy-ethyl, phosphorami date, methylphosphonate or phosphorothioate group.
- the nucleobase is typically heterocyclic.
- Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).
- the sugar is typically a pentose sugar.
- Nucleotide sugars include, but are not limited to, ribose and deoxyribose.
- nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine.
- the nucleosides are most preferably adenosine, guanosine, uridine and cytidine
- the nucleotides are typically ribonucleotides or deoxyribonucleotides.
- the nucleotides are preferably deoxyribonucleotides.
- the nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5’ or 3’ side of a nucleotide.
- Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5- methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic
- the nucleotides are preferably selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP.
- the nucleotides are preferably selected from dAMP, dTMP, dGMP or dCMP.
- nucleotides may contain additional modifications.
- suitable modified nucleotides include, but are not limited to, 2’ amino pyrimidines (such as 2’-amino cytidine and 2’- amino uridine), 2’-hyrdroxyl purines (such as , 2’ -fluoro pyrimidines (such as 2’ -fluorocytidine and 2’fluoro uridine), hydroxyl pyrimidines (such as 5’-a-P-borano uridine), 2’-O-methyl nucleotides (such as 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine and 2’-O-methyl uridine), 4’- thio pyrimidines (such as 4’ -thio uridine and 4’ -thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentyl
- One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag.
- the label may be any suitable label which allows the polynucleotide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125 1, 35 S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.
- the nucleotides in the polynucleotide may be attached to each other in any manner.
- the nucleotides may be linked by phosphate, 2’0-methyl, 2’ methoxy-ethyl, phosphorami date, methylphosphonate or phosphorothioate linkages.
- the nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids.
- the nucleotides may be connected via their nucleobases as in pyrimidine dimers.
- the polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
- the polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LN A), morpholino nucleic acid or other synthetic polymers with nucleotide side chains.
- PNA peptide nucleic acid
- GNA glycerol nucleic acid
- TAA threose nucleic acid
- LN A locked nucleic acid
- morpholino nucleic acid or other synthetic polymers with nucleotide side chains.
- the polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.
- the polynucleotide may be DNA or RNA.
- a primer polynucleotide that is RNA has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 if all of the instances of dAMP, dTMP, dGMP and dCMP are replaced with AMP, UMP, GMP and CMP respectively.
- the DNA nucleotides are replaced with their corresponding RNA nucleotides and T is replaced with U.
- the polynucleotide is preferably DNA.
- the polynucleotide may be single stranded or double stranded.
- the primer polynucleotide is preferably single stranded.
- any of the polynucleotides discussed herein may be isolated, substantially isolated, purified or substantially purified.
- the polynucleotide is isolated or purified if it is completely free of any other components, such as buffer, other polynucleotides, virus material or cells.
- the polynucleotide is substantially isolated or substantially purified if it is only mixed with carriers or diluents, such as buffers or excipients, which will not interfere with its intended use, such as in a RT-PCR assay.
- the primer polynucleotides are not naturally occurring.
- the primer may comprise a variant sequence based on SEQ ID NO: 1 or 2.
- a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 1 or 2.
- a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2 and which retains its ability to specifically hybridise to a sequence that is complementary to SEQ ID NO: 1 or 2.
- a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2 and which retains its ability to amplify the complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNA by SEQ ID NOs: 1 and 2 (i.e. SEQ ID NO: 4).
- cDNA complementary DNA
- amplified refers to the process of making multiple copies of the polynucleotide, such as cDNA, from a single polynucleotide or fewer polynucleotides.
- Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al. , 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley- Interscience, New York (1995)).
- Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C.
- Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCl, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C.
- Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37 °C, followed by a wash in 0. IX (0.0165 M Na+) SSC at 60 °C.
- a variant “specifically hybridises” if it hybridises to its partner with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotides.
- Tm melting temperature
- the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotides.
- the variant typically hybridises to its target sequence with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C.
- Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.
- the variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides.
- the variant sequence is typically the same length as SEQ ID NO: 1 or 2, but may be longer or shorter.
- a variant of SEQ ID NO: 1 is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- a variant of SEQ ID NO; 2 is preferably at least 20 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
- a variant sequence is at least about 80% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence has at least about 80% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 1.
- a variant sequence is preferably at least about 84% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 84% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 1. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 88% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 88% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 1.
- a variant sequence is preferably at least about 92% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 92% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 1.
- a variant sequence is preferably at least about 96% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 96% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 1.
- a variant sequence is preferably at least about 79% or at least about 79. 17% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 79% or at least about 79. 17% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 2.
- a variant sequence is preferably at least about 80% or at least about 83.3% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 80% or at least about 83.3% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 2.
- a variant sequence is preferably at least about 85% or at least about 87.5% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 85% or at least about 87.5% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 2.
- a variant sequence is preferably at least about 90% or at least about 91.67% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 90% or at least about 91.67% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 2.
- a variant sequence is preferably at least about 95% or at least about 95.8% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 95% or at least about 95.8% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 2.
- the PILEUP and BLAST algorithms can also be used to calculate identity, homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
- HSPs high scoring sequence pair
- Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
- the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787.
- One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- P(N) the smallest sum probability
- a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
- a variant is typically identical to SEQ ID NO: 1 over at least about 20, at least about 21, at least about 22, at least about 23, at least about 24 or at least about 25 consecutive nucleotides.
- a variant is typically identical to SEQ ID NO: 2 over at least about 20, at least about 21, at least about 22, at least about 23 or at least about 24 consecutive nucleotides.
- Each polynucleotide in the primer may be any length.
- the forward primer is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- the reverse primer is at least 20 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
- the forward primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 1.
- the reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 2.
- the forward primer preferably is the exact sequence shown in SEQ ID NO: 1.
- the reverse primer preferably is the exact sequence shown in SEQ ID NO: 2.
- the forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 1.
- the reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 2.
- the pair of primers may comprise any combination of primers discussed above.
- the pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.
- the pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 2.
- the pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.
- the pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 2.
- the pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is the exact sequence shown in SEQ ID NO: 2.
- the pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 2.
- Primers having any of the sequences above may be manufactured using standard techniques. Custom primers having specific sequences are commercially available from various suppliers (such as Biolegio).
- the cDNA amplicon is amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. Specific hybridisation is defined above with reference to the preferred primers.
- the cDNA amplicon is preferably amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 3.
- SARS-CoV-2 cDNA is cDNA produced by the reverse transcription of SARS-CoV-2 RNA.
- the sequence of SARS-CoV-2 cDNA is published (see above) and methods for reverse transcribing it are known in the art and discussed in the Example.
- the pair of primers discussed above may be used to amplify the cDNA amplicon using polymerase chain reaction (PCR). Methods for conducting PCR using primers are known in the art and discussed below and in the Examples.
- a cDNA amplicon is amplified from SARS-CoV-2 cDNA if it comprises a portion/part of the SARS-CoV-2 cDNA. In other words, it comprises a portion/part of the sequence of the SARS-CoV-2 cDNA.
- amplified refers to the process of making multiple copies of the cDNA from a single polynucleotide or fewer polynucleotides.
- An amplicon is the cDNA that results from amplification.
- primers such as the primers discussed above, may be used to amplify a part of the SARS-CoV-2 cDNA resulting in the shorter cDNA amplicons. These amplicons typically contain the cDNA target sequence being used in the context of the invention.
- the cDNA amplicon is typically a polynucleotide comprising nucleotides selected from dAMP, dTMP, dGMP or dCMP as discussed above.
- the cDNA may be double stranded or single stranded.
- the cDNA is preferably single stranded.
- the cDNA amplicon may be any length, such as from about 20 to about 100 nucleotides in the length, such as from about 25 to about 90, from about 30 to about 95 or from about 40 to about 90 nucleotides in length.
- the cDNA amplicon is preferably 82 nucleotides in length.
- the cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 4.
- the cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 5.
- the cDNA amplicon may be isolated, substantially isolated, purified or substantially purified as discussed above.
- the cDNA amplicon is not naturally occurring.
- polynucleotide probes for determining the presence or absence of SARS-CoV-2 in a sample.
- the polynucleotide probe specifically hybridises to the cDNA amplicon described above.
- the polynucleotide probe comprises or consists of a sequence which specifically hybridises to the cDNA amplicon described above.
- the polynucleotide probe preferably comprises or consists of a sequence which specifically hybridises to a target sequence on the cDNA amplicon described above.
- the target sequence may be any length, such as from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- the probe polynucleotide may be any of the polynucleotides discussed above.
- the polynucleotide probe is preferably DNA.
- the polynucleotide probe is preferably single stranded DNA.
- the polynucleotide probe may be any length, such as from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- the polynucleotide probe is preferably the same length as the target sequence it detects.
- the target sequence is preferably specific for SARS- CoV-2.
- the target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus.
- the target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus or in any human RNA.
- the target sequence is preferably found/present in the cDNA amplicon described above.
- the target sequence is preferably part of SEQ ID NO: 4.
- the target sequence is preferably the sequence shown in SEQ ID NO: 5.
- the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 3 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 based on sequence identity over its entire length.
- Polynucleotides are defined above with reference to the preferred primers.
- the probe polynucleotide may be any of the polynucleotides discussed above.
- the polynucleotide probe is preferably DNA.
- the polynucleotide probe is preferably single stranded DNA.
- the polynucleotide probe may be any length, such as from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- a variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 3 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 5. Specific hybridisation is discussed above. The variant sequence must not recognise or hybridise to any cDNA sequence from any other virus.
- the variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides.
- the variant sequence is typically the same length as SEQ ID NO: 3, but may be longer or shorter.
- a variant of SEQ ID NO: 3 is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- a variant sequence is at least about 80% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence has at least about 80% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 3.
- a variant sequence is preferably at least about 84% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 84% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 3.
- a variant sequence is preferably at least about 88% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 88% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 3.
- a variant sequence is preferably at least about 92% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 92% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 3.
- a variant sequence is preferably at least about 96% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 96% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 3.
- the polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3.
- the polynucleotide probe may be isolated, substantially isolated, purified or substantially purified as discussed above.
- the polynucleotide probe is not naturally occurring.
- the polynucleotide probe is preferably DNA probe, a TaqMan® probe, a molecular beacon or a scorpion probe.
- DNA probes hybridise to the, typically complementary, target sequence and then can be detected.
- TaqMan® probes are known in the art and are polynucleotides that have a fluorescent dye attached to the 5' end and a quencher to the 3' end. The polymerase used in PCR cleaves hybridised probes freeing the fluorescent dye from quenching such it can be detected.
- Molecular beacon probes are known in the art and are similar to TaqMan® probes except (rather than using cleavage to separate the dye from the quencher) hybridisation to the target sequence separates the dye from the quencher.
- Scorpion probes are known the art and are similar to molecular beacons except the 3' end also contains a sequence that is complementary to the extension product of the primer on the 5' end which opens the probe on hybridisation and allows the dye to be detected.
- the polynucleotide probe is preferably a TaqMan® probe.
- the polynucleotide probes are preferably detectably-labelled. Suitable detectable labels are known in the art.
- the detectable labels are preferably fluorescent molecules or dyes, such as fluorescein derivatives. Suitable fluorescent molecules or dyes include, but are not limited to, 6- carboxyfluorescein (FAM), 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (VIC®) and tetrachlorofluorescein (TET).
- a suitable quencher for use with these dyes in TaqMan®, molecular beacon or scorpion probes is tetramethylrhodamine (TAMRA).
- the detectable label is most preferably 6-carboxyfluorescein (FAM).
- Polynucleotide probes are also available from commercial sources (such as Biolegio).
- target cDNA polynucleotide comprising or consisting of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. Specific hybridisation is discussed above.
- the target cDNA may be any length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
- the target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 5.
- the target cDNA polynucleotide may be double stranded or single stranded.
- the target cDNA polynucleotide is preferably single stranded.
- the target cDNA polynucleotide may be isolated, substantially isolated, purified or substantially purified as discussed above. The target cDNA polynucleotide is not naturally occurring.
- RNA virus such as SARS-CoV-2. titre
- the invention also provides methods for measuring the RNA virus, such as SARS-CoV-2, titre in a sample.
- the method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of RNA virus, such as SARS-CoV-2, in a sample as discussed above may be used.
- the quantitative method uses real time RT-PCR. If RNA virus, such as SARS-CoV-2, is present, the method comprises evaluating the cycle quantification (Cq) value and thereby measuring the RNA virus, such as SARS-CoV-2, titre.
- the method preferably comprises evaluating the Cq value against standard Cq values generated from standard dilution curves.
- the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels.
- the threshold level is preferably 10% of the end point fluorescence. Autocalling may be used to set the threshold level.
- the Cq is the PCR cycle number at which the sample reaction curve intersects the threshold line. This value indicates how many cycles it took to detect a real signal from the sample.
- Real-Time PCR machines typically calculate the Cq value for each of sample.
- the Cq value changes depending on the amount of RNA virus, such as SARS-CoV-2, present in the sample.
- RNA virus such as SARS-CoV-2
- These standard curves allow the amount of RNA virus, such as SARS-CoV-2, to be measured in sample.
- the Cq is predictive of the amount of the RNA virus, such as SARS-CoV-2, in the sample.
- a Cq value of about 16.0 to about 20.9, preferably about 18.0 to about 19.9 indicates the sample contains about 10 6 virions/pl
- a Cq value of about 21.0 to about 25.9 indicates the sample contains about 10 5 virions/pl
- a Cq value of about 26.0 to about 28.9, preferably from about 26.0 to about 27.9 indicates the sample contains about 10 4 virions/pl
- a Cq value of about 29.0 to about 31.9, preferably 30.1 indicates the sample contains about 10 3 virions/pl
- a Cq value of about 32.0 to about 33.9 preferably from about 33.0 to about 33.9
- a Cq value of above 34.0 or above indicates the sample contains 10 or fewer virions/pl.
- a Cq value of 38.0, 39.0 or 40.0 or above preferably indicates the sample contains 5 or fewer virions/pl.
- the virions are preferably SARS-CoV-2 virions.
- All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to one significant figure.
- the sample refers to the (undiluted) sample that is added to (and diluted by) the RT-PCR assay mixture. The concentrations do not relate to the final concentration of the virions in the diluted RT-PCR assay mixture.
- the dilution factor or concentration factor needs to be taken into account when calculating the the amount of RNA virus, such as SARS-CoV-2, present in the sample.
- the invention also provides a method for differentiating between high and low RNA virus, such as SARS-CoV-2, titres in a sample.
- the method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of RNA virus, such as SARS- CoV-2, in a sample as discussed above may be used.
- the method uses real time RT-PCR. If RNA virus, such as SARS-CoV-2, is present, the method comprises evaluating the cycle quantification (Cq) value and thereby determining whether the sample has a low or high RNA virus, such as SARS-CoV-2, titre. Cq measurement and evaluation is discussed above.
- Cq cycle quantification
- the Cq value changes depending on the amount of RNA virus, such as SARS-CoV-2, in a sample.
- RNA virus such as SARS-CoV-2
- These standard curves allow the amount of RNA virus, such as SARS-CoV-2, to be measured in a sample.
- the Cq is predictive of the amount of the RNA virus, such as SARS-CoV-2, in the sample.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 2 virions/pl or lower
- a high RNA virus such as SARS-CoV-2
- titre is about 10 3 virions/pl or higher.
- a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2
- titre a Cq value of 31.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 3 virions/pl or higher
- a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 31.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 4 virions/pl or higher.
- a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 28.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 4 virions/pl or higher.
- a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 28.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 5 virions/pl or higher.
- a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 25.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 5 virions/pl or higher
- a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 25.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 6 virions/pl or higher.
- a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 20.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- a low RNA virus such as SARS-CoV-2
- a high RNA virus such as SARS-CoV-2
- titre is about 10 6 virions/pl or higher.
- a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 20.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
- the virions are preferably SARS-CoV-2 virions.
- All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to one significant figure.
- the quantitative methods of the invention preferably use a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1, a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2 and a probe which comprises or consists of the sequence shown in SEQ ID NO: 3.
- the probe is preferably a TaqMan® probe.
- the method is preferably conducted under the same conditions as the Examples. These conditions are discussed above.
- the sample used in the methods of the invention may be any sample.
- the invention is typically carried out on a sample that is known to contain or suspected to contain RNA virus, such as SARS- CoV-2.
- the invention may be carried out on any sample whose RNA virus, such as SARS-CoV-2, status is unknown to confirm the presence or absence of RNA virus, such as SARS- CoV-2.
- the sample may be a biological sample.
- the sample may be obtained from or extracted from any organism or microorganism.
- the organism or microorganism may be archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista.
- the sample may be obtained from or extracted from any virus.
- the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs.
- the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton.
- the sample may be derived from human or animal food.
- the sample is preferably a fluid sample.
- the sample typically comprises a body fluid of the patient.
- the sample may be urine, lymph, saliva, mucus, amniotic fluid, blood, plasma or serum.
- the sample is preferably a nasopharyngeal sample, a saliva sample (also known an oropharyngeal sample) or a blood sample.
- the sample may be a non-biological sample. Any non-biological sample can be tested.
- the non-biological sample is preferably a fluid sample. Examples of non-biological samples include, but are not limited to, surgical fluids, water such as drinking water, sea water or river water, reagents for laboratory tests and wet swabs of surfaces or materials.
- the sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells.
- the sample may be tested immediately upon being taken.
- the sample may also be typically stored prior to assay, preferably below -70°C.
- the invention also provides a method of determining whether or not a patient is infected with an RNA virus, such as SARS-CoV-2.
- the invention therefore relates to the diagnosis of an RNA virus, such as SARS-CoV-2, infection.
- the diagnostic method of the invention may be carried out in conjunction with other assays or genetic tests.
- the method comprises conducting a method of the invention for determining the presence or absence of an RNA virus, such as SARS-CoV-2, on a sample from the patient. Any of the methods discussed above may be used.
- the presence of an RNA virus, such as SARS-CoV-2, in the sample indicates the presence of the RNA virus, such as SARS-CoV-2, in the patient.
- the presence of an RNA virus, such as SARS-CoV-2, in the sample indicates the patient is infected with the RNA virus, such as SARS-CoV-2.
- the absence of an RNA virus, such as SARS- CoV-2, from the sample typically indicates the absence of the RNA virus, such as SARS-CoV-2, in the patient.
- the absence of an RNA virus, such as SARS-CoV-2, from the sample typically indicates the patient is not infected with the RNA virus, such as SARS-CoV-2.
- the absence of an RNA virus, such as SARS-CoV-2, from the sample may indicate that the particular sample from the patient does not contain the RNA virus, such as SARS-CoV-2, and does not necessarily mean the patient is not infected.
- the diagnostic method preferably uses a nasopharyngeal sample or a saliva sample (also known as an oropharyngeal sample).
- a nasopharyngeal sample or a saliva sample also known as an oropharyngeal sample.
- the absence of an RNA virus, such as SARS-CoV-2, from these samples does typically indicate the patient is not infected with the RNA virus, such as SARS-CoV-2.
- the invention also provides a method of measuring the titre of RNA virus, such as SARS-CoV- 2, in a patient.
- the invention also provides a method for differentiating between high and low RNA virus, such as SARS-CoV-2, titres in a patient.
- These methods comprise conducting the quantitative method of the invention for measuring the titre of RNA virus, such as SARS-CoV-2, or for differentiating between high and low RNA virus, such as SARS-CoV-2, titres on a sample from the patient.
- Low RNA virus, such as SARS-CoV-2, titres, high RNA virus, such as SARS-CoV-2, titres and methods for measuring them are discussed above. Any of these may be used on a sample from the patient.
- the quantitative method preferably uses a nasopharyngeal sample or a saliva sample (also known as an oropharyngeal sample).
- the diagnostic and quantitative methods preferably comprise taking a sample from the patient before conducting the RT-PCR assay.
- the patient displays the symptoms of infection with the RNA virus, such as SARS- CoV-2, i.e. the patient is known or expected to be infected with the RNA virus, such as SARS-CoV-2.
- the patient may be asymptomatic, i.e. the patient’s RNA virus, such as SARS-CoV-2, status is unknown or the patient is expected not to be infected with the RNA virus, such as SARS-CoV-2.
- the patient may be susceptible to, or at risk from, infection with the RNA virus, such as SARS-CoV-2.
- the patient may have underlying health conditions which make infection with the RNA virus, such as SARS-CoV-2, particularly serious.
- the patient is generally a human patient.
- the patient may be a fetus, a newborn, an infant, a juvenile or an adult.
- the invention provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention.
- the method comprises administering to the patient a therapeutically effective amount of an anti-viral treatment.
- the invention also provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient.
- the method comprises (a) identifying the patient as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-viral treatment.
- the invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention.
- the invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient, wherein the method comprises (a) identifying the patient as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention and (b) administering the substance of composition to the patient.
- the substance or composition is preferably an anti- viral substance or composition.
- the invention also provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention.
- the method comprises administering to the patient a therapeutically effective amount of an anti- viral treatment.
- the invention also provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient comprising (a) identifying the patient as having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-viral treatment.
- the high RNA virus, such as SARS-CoV-2, titre is about 10 3 virions/pl or higher.
- a Cq value of 31.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
- a high RNA virus, such as SARS-CoV-2, titre is about 10 4 virions/pl or higher.
- a Cq value of 25.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
- a high RNA virus, such as SARS-CoV-2, titre is about 10 5 virions/pl or higher.
- a Cq value of 28.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
- a high RNA virus such as SARS-CoV-2, titre is about 10 6 virions/pl or higher.
- a Cq value of 20.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
- the invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as being having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention.
- the invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient, wherein the method comprises (a) identifying the patient as being having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention and (b) administering the substance or composition to the patient.
- the substance or composition is preferably an anti-viral substance or composition
- any treatment, substance or composition may be used in the invention. Suitable anti- viral treatments, substances and compositions are well known.
- the anti-SARS-CoV or anti-SARS-CoV-2 treatment, substance or composition may inhibit viral entry into cells by inhibiting ACE2 receptors, such as ACE2 receptor antibodies or arbidol, or inhibiting TMPRSS2, such as camosat mesylate.
- the anti-SARS-CoV or anti-SARS-CoV-2 treatment, substance or composition may inhibit 3- chymotrypsin-like protease, such as lopinavir or darunavir.
- the anti-viral treatment, substance or composition may inhibit viral replication by inhibiting viral RNA-dependent RNA polymerase (RdRP), such as ribavirin, remdesivir or favipiravir.
- RdRP viral RNA-dependent RNA polymerase
- the anti-viral treatment, substance or composition may be an anti-viral small interfering RNA (siRNA) designed to inhibit entry of the virus into cells and/or inhibit viral replication by targeting RNA virus, such as SARS-CoV-2, genes involved in these processes.
- RNA virus such as SARS-CoV-2
- the anti-RNA virus, such as SARS-CoV-2, treatment, substance or composition may be an anti-IL-6 therapy or antibody, such as tocilizumab or sarilumab.
- the anti-RNA virus, such as SARS-CoV-2, treatment, substance or composition may be any anti-inflammatory and inhibitor of immune responses.
- the anti-influenza treatment may be a neuraminidase inhibitors, such as laninamivir, favipiravir, peramivir, zanamivir or oseltamivir, or an inhibitor of the viral M2 protein, such as amantadine or rimantadine.
- the invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention.
- the method comprises administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody.
- the invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody.
- High SARS-CoV-2 titres and methods of measuring them are discussed above.
- the invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention.
- the invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the an anti-IL-6 therapy or antibody to the patient.
- the invention concerns administering to the patient a therapeutically effective mount of the treatment, substance or composition to the patient.
- a therapeutically effective amount is an amount which ameliorates one or more symptoms of the RNA virus, such as SARS-CoV-2, infection.
- a therapeutically effective amount is preferably a number which abolishes one or symptoms of the RNA virus, such as SARS-CoV-2, infection.
- a therapeutically effective amount may cure or abolish the RNA virus, such as SARS-CoV-2, infection. Suitable amounts are discussed in more detail below.
- composition of the invention may be administered to any suitable patient. Suitable patients are discussed above with reference to the diagnostic embodiments of the invention.
- the invention may be used in combination with other means of, and substances for, treating the disease or disorder or providing pain relief
- the treatment, substance or composition may be used in combination with existing treatments for the RNA virus, such as SARS-CoV-2, infection including intensive care treatment and the use of ventilators.
- the treatment, substance or composition of the invention may be formulated using any suitable method.
- Formulation with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art.
- the exact nature of a formulation will depend upon several factors including the composition to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19 th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.
- the treatment, substance or composition may be administered by any route. Suitable routes include, but are not limited to, enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical and other appropriate routes. If the lungs are being treated, the treatment, substance or composition is preferably administered by inhalation.
- compositions may be prepared together with a physiologically acceptable carrier or diluent.
- the treatment, substance or composition may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient.
- excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.
- Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
- the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
- Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
- the suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
- Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
- binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
- Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions typically take the form of solutions or suspensions and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
- compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness.
- the composition preferably comprises human serum albumin.
- Plasma-Lyte A® is a sterile, nonpyrogenic isotonic solution for intravenous administration.
- Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6H1 !NaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO2»3H2O); 37 mg of Potassium Chloride, USP (KC1); and 30 mg of Magnesium Chloride, USP (MgC12*6H2O). It contains no antimicrobial agents.
- the pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
- the most preferred pharmaceutically acceptable carrier or diluent is a pharmaceutically acceptable transfection reagent.
- a pharmaceutical acceptable transfection reagent is suitable for administration to patients.
- the pharmaceutical acceptable transfection reagent may be liposomes, preferably cationic liposomes, polymers, preferably cationic polymers, and dendrimers.
- the pharmaceutical acceptable transfection reagent is more preferably a pharmaceutically acceptable PEI transfection reagent, such as a linear PEI transfection reagent.
- the most preferred pharmaceutical acceptable transfection reagent is GMP in v/vo-jetPEI®.
- composition is administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective.
- quantity to be administered depends on the subject to be treated, capacity of the patient’s immune system and the degree of treatment desired. Precise amounts required to be administered may depend on the judgement of the practitioner and may be peculiar to each patient.
- any suitable dose of the treatment, substance or composition may be administered to a patient.
- the dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
- a typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific substance, the age, weight and conditions of the subject to be treated and the frequency and route of administration.
- daily dosage levels are from 5 mg to 2 g.
- from about 0.01 to about 50mg per kg of patient of sRNA may administered, such as from about 0.05 to about 40, from about 0.
- At least about 0.01 mg per kg of patient may administered, such as at least about 0.05, at least about 0. 1, at least about 0.5, at least about 1, at least about 2, at least about 5, at least about 10, at least about 20, at least at least about 30 or at least about 40 mg per kg.
- doses may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.
- regular intervals include, but are not limited to, every day, every week, every fortnight or every month.
- the reaction mix was lOuL 2x oasig mastermix, 2uL primer/probe mix (resuspended in template preparation buffer) and 8uL sample.
- the reaction conditions were as follows: (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
- the threshold level was 10% of the end point fluorescence.
- the CFX and Lightcycler autocalling was also used.
- Example 1 was repeated with increasing dilutions of Human 2019-nCoV RNA (Purified RNA of Coronavirus strain "BetaCoV/Germany/BavPatl/2020 p. 1" grown in cell culture obtained from EVAg: https://www. European-virus-archive. com/nucleic-acid/human-2019-ncov-ma).
- the concentrations shown relate to the concentrations of RNA before it was added to the RT-PCR reaction mix.
- the results are shown in Figures 1 to 3.
- the mean Cq values are shown in Tables 3 to 5 below. Table 3 - Figure 1 Cq values
- Example 1 was repeated with a variety of different preparations of the sample. Initial experiments were assessment of PCR inhibition caused by sample preparation to optimise the method. No SARS-Cov-2 RNA was used at this point. Swab samples were prepared in Copan diagnostics universal transport medium (UTM) (366C), Sigma- virucult viral transport medium (VTM) (MW951S), phosphate buffered saline (PBS) and saline. PBS was prepared in house using PBS tablets (P32080) supplied by Melford laboratories. lOOmL of PBS contained: 137mM NaCl, 11.9mM Phosphate buffer, 2.7mM KC1. The saline used was STERETS Normasol, Ref: 99766774, Sodium chloride solution 0.9%w/v.
- UDM Copan diagnostics universal transport medium
- VTM Sigma- virucult viral transport medium
- PBS phosphate buffered saline
- PBS phosphate buffered saline
- Each of the swab samples were diluted at the following dilutions: no dilution (neat), 1:5, 1:10, 1:15 and 1 :25.
- the sample was diluted in a dilution solution shown in Table 6.
- Trizma was manufactured in house to pH8.5, IM. lOOrnL of Trizma ph8.5, IM contains: 4.42g Trizma hydrochloride (Sigma-Aldrich, Cat number: T3253) and 8.72g Trizma base, Supplier: Sigma- Aldrich, Cat number: T1503). Glycerol, bidistilled 99.5%, was used (VWR, Cat number: 24388.295). EDTA (0.5M), pH8.0 was used (Thermo Fisher Scientific, Cat number: AM9260G). Nuclease-free water was obtained from Fisher Scientific (Cat number: BP561).
- Example 3 was repeated with different optimisations of the sample for RT-PCR.
- Samples were prepared as in Example 3 and UTM and VTM samples were diluted 1:15 (6uL of sample + 84uL of dilution solution) and PBS and saline samples were diluted 1:5 (18uL sample + 72uL of dilution solution) using the dilution solution shown in Table 6.
- nuclease-free water (Fisher Scientific: Cat number: BP561) according to the formulation shown in Table 7 as in Example 3.
- the 90uL diluted samples produced above were optimised using the optimisation solution shown in Table 7. 3uL (termed 1.5x), 2uL (termed lx), luL (termed 0.5x) or 0.4uL (termed 0.2x) of the optimisation solution was added to the 90uL of the diluted sample. After this, 20uL of IEC RNA was added to each of the optimised samples. 8uL of each optimised sample containing IEC RNA was then tested in direct RT-PCR (as in Example 1). The results of the direct RT-PCR reactions are shown in Table 9.
- Example 3 Samples were prepared as in Example 4: UTM and VTM samples were diluted 1:15 (6uL of sample + 84uL of dilution solution) and PBS and saline samples were diluted 1:5 (18uL sample + 72uL of dilution solution) using the dilution solution shown in Table 6 and 2uL of the optimisation solution shown in Table 7 was added to each of the 90uL diluted samples. After this, 20uL of IEC RNA was added to each of the 92uL to provide 112uL samples. 8uL of each diluted and optimised sample containing IEC RNA was tested using direct RT-PCR (as in Example 1). The components in the 8uL sample added to the direct RT-PCR reaction and IEC Cq values are shown in Table 10.
- Swabs were prepared in ImL PBS with or without 1% or 5% (by volume) Triton® X-100 or Triton® X-100 (Sigma- Aldrich).
- Example 5 The samples were then prepared exactly as shown in Example 5. Samples were diluted 1:5 using dilution solution (18uL of sample + 72uL of dilution solution), 2uL of optimisation solution was added to the 90uL diluted sample and then 20uL IEC was added. 8uL of the sample was then tested using direct RT-PCR (as in Example 1). The results are shown in Table 12.
- the sample preparation buffer comprised Trizma, EDTA, polyoxyethylene isooctylcyclohexyl ether (Triton® X-100 reduced) and glycerol (as shown in Table 13 below).
- the PCR reaction mix and final PCR reaction volume included potassium chloride (KC1) and magnesium chloride (MgC12) (as shown in Table 14 below).
- sample preparation buffer and PCR reaction mix were tested using RNA extracted from heat inactivated SARS-CoV-2 cell culture preparations.
- Sample preparation buffer was contrived with 1000 copies of SARS-CoV-2 RNA. 5pl of the sample preparation buffer was then removed and pipetted into the PCR reaction tube with 15uL of PCR reaction mix to create a total PCR reaction volume of 20uL.
- Example 8 Version 2 PROmate 2G Example 7 was repeated but there was also an additional internal extraction control (IEC) which was freeze dried with the RNase inhibitor in a bead and which was added to the sample preparation buffer during the workflow. The results are shown in Figure 5.
- IEC internal extraction control
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Abstract
The invention relates to novel compositions and methods for extracting RNA from an RNA virus or obtaining viral RNA from a sample and for determining the presence or absence of an RNA virus, such as SARS-CoV-2.
Description
KIT AND METHOD
Field of the Invention
The invention relates to novel kits and methods for extracting RNA from an RNA virus or obtaining viral RNA from a sample and for determining the presence or absence of an RNA virus, such as SARS-CoV-2.
Background to the Invention
Coronaviruses are members of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales. A novel coronavirus, 2019-nCoV or COVID-19, capable of infecting humans was identified in Wuhan, China and then led to a worldwide pandemic. Based on its phylogenetic relationships and genomic structures, the virus belongs to genera Betacoronavirus and has close similarities to severe acute respiratory syndrome-related coronaviruses (SARS-CoV). The virus uses ACE2 as the entry receptor-like SARS-CoV. These similarities lead the Coronavirus Study Group of the International Committee on Taxonomy of Viruses to term the virus SARS-CoV-2. Since the virus is novel, new methods are required to test for the virus and distinguish it from other similar coronaviruses. The sequence of SARS-Cov-2 has been published under accession no. MN908947 (https://www.ncbi.nlm.nih.gov/nuccore/MN908947.3'). Sequence information from around the world nevertheless indicates that numerous variants of the virus exist.
Summary of the Invention
The inventors have surprisingly identified new compositions and methods for extracting ribonucleic acid (RNA) from a variety of RNA viruses and for determining the presence or absence of such RNA viruses, such as SARS-CoV-2, in a variety of samples. The invention provides: a kit for extracting RNA from an RNA virus comprising (a) an inactivation solution comprising t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced), (b) a dilution solution comprising TRIZMA® (2-Amino-2- (hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA) and (c) an optimisation solution comprising potassium chloride (KC1) and magnesium chloride (MgCl2); a sample preparation buffer for obtaining viral RNA from a sample comprising (a) t- octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced) and (b) TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA);
a kit for determining the presence or absence of an RNA virus comprising (i) a sample preparation buffer solution of the invention and (ii) a PCR reaction mix comprising potassium chloride (KC1) and magnesium chloride (MgCl2); a method of extracting RNA from an RNA virus, if present, in a sample, comprising (a) contacting the sample with an inactivation solution comprising Triton® X-100 and/or Triton® X-100 reduced, (b) diluting the inactivated sample produced in (a) with a dilution solution comprising TRIZMA®, glycerol and EDTA and (c) adding to the diluted sample produced in (b) an optimisation solution comprising KC1 and MgCl2 and thereby extracting the RNA from the RNA virus, if present; a method of obtaining viral RNA, if present, from a sample, comprising contacting the sample with a sample preparation buffer of the invention and thereby obtaining the viral RNA, if present, from the sample; a method of determining the presence or absence of an RNA virus in a sample, wherein the method comprises (i) extracting RNA from the RNA virus in the sample, if present, or obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reversetranscription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample; a method for measuring the titre of an RNA virus in a sample, if present, wherein the method comprises (i) extracting RNA from the RNA virus in the sample, if present, or obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a real time RT-PCR method on the sample produced in (i) and, if the virus is present, evaluating the cycle quantification (Cq) value and thereby measuring the viral titre; a method of determining whether or not a patient is infected with an RNA virus, wherein the method comprises conducting a method of the invention on a sample from the patient and thereby determining whether or not a patient is infected with the virus; a method of measuring the titre of an RNA virus in a patient, wherein the method comprises conducting a method of the invention on a sample from the patient and thereby measuring the viral titre in the patient; a method of treating an RNA virus infection in a patient identified as being infected with the RNA virus using a method of the invention, comprising administering to the patient a therapeutically effective amount of an anti-viral treatment and thereby treating the infection in the patient; a substance or composition for use in a method of treating an RNA virus infection in a patient identified as being infected with the RNA virus using a method of the invention;
a method of treating an RNA virus infection in a patient identified as having a high RNA virus titre using a method of the invention, comprising administering to the patient a therapeutically effective amount of an anti-viral treatment and thereby treating the RNA virus infection in the patient; and a substance or composition for use in a method of treating an RNA virus infection in a patient identified as being having a high RNA viral titre using a method of the invention.
Description of the Figures
Figure 1 shows the standard curve of SARS-Cov-2 whole genomic RNA (EvaG) using a real time RT-PCR assay (curves from left to right: 104 copies/pl, 103 copies/pl, 102 copies/pl and 10 copies/pl).
Figure 2 shows the standard curve of SARS-Cov-2 whole genomic RNA (EvaG) using a real time RT-PCR assay (curves from left to right: 104 copies/pl, 102 copies/pl, 10 copies/pl and 0.59 copies/pl).
Figure 3 shows the standard curve of SARS-Cov-2 whole genomic RNA (EvaG) using a real time RT-PCR assay (curves from left to right: 106 copies/pl, 104 copies/pl, 102 copies/pl, 10 copies/pl and 5 copies/pl).
Figure 4 shows results using the sample preparation buffer of the invention. The control is the positive control template. Other curves represent samples amplifying at the limit of detection.
Figure 5 shows results using the sample preparation buffer of the invention. The control is the positive control template. Other curves represent samples amplifying at the limit of detection.
Description of the Sequence Listing
SEQ ID NO: 1 shows the forward primer used in the Examples.
SEQ ID NO: 2 shows the reverse primer used in the Examples.
SEQ ID NO: 3 shows the sequence of the probe used in the Examples.
SEQ ID NO: 4 shows the cDNA amplified in the Examples.
SEQ ID NO: 5 shows the reverse complement of SEQ ID NO: 3.
Detailed Description of the Invention
It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.
In addition, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a solution” includes two or more solutions, reference to “a virus” includes two or more viruses, reference to “a sample” includes two or more such samples, reference to “a patient” includes two or more such patients, and the like.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
Terminology
In all instances herein, SARS-CoV-2 is interchangeable with 2019-nCoV or COVID-19.
In all instances herein, extracting RNA from an RNA virus is interchangeable with inactivating an RNA virus. The new kits and methods of the invention may be for inactivating an RNA virus.
In all instances herein, extracting RNA from an RNA virus is interchangeable with obtaining viral RNA from a sample. The new kits and methods of the invention may be for obtaining viral RNA from a sample.
In all instances herein, extracting RNA from an RNA virus is interchangeable with preparing the RNA in an RNA virus for reverse-transcription polymerase chain reaction (RT-PCR). The new kits and methods of the invention for preparing the RNA in an RNA virus for reverse-transcription polymerase chain reaction (RT-PCR).
In all instances herein, determining is interchangeable with detecting. The new kits and methods of the invention may be for detecting the presence or absence of an RNA virus, such as SARS- CoV-2, in a sample or patient. Also, in all instances, the new kits and methods of the invention may be for determining/detecting/identifying whether or not a sample contains or comprises an RNA virus, such as SARS-CoV-2, or whether or not a patient is infected with an RNA virus, such as SARS-CoV-2.
Advantages of the invention
The inventors have surprisingly identified new kits and methods for extracting RNA from a variety of RNA viruses in a variety of samples. The kits and methods have several advantages.
First, the new kits and methods are capable of preparing the RNA from an RNA virus for RT- PCR in a safe manner. The inactivation solution inactivates the virus by disrupting the viral envelope and reducing its ability to infect the person using the kit or conducting the method.
Second, the new kits and methods do not require complicated inactivation steps (for instance using heat) and/or RNA extraction steps (for instance using magnetic beads). As will be clear from the
discussion below, the kits and methods of the invention are not only safe, but also straightforward and quick to use. This further increases the safety of the kits and the methods of the invention by minimising exposure of the user to infective virus. It also means the presence or absence of the virus can be detected/determined more quickly.
Third, the new kits and methods are optimised to allow the RNA in an RNA virus to be detected using RT-PCR with a high degree of sensitivity without complicated RNA extraction steps, such as using magnetic beads. This facilitates the speed and ease with which the kits and methods can be used to detect low viral titres. As shown in the Examples, the method is capable of detecting 10 of fewer copies of the SARs-CoV-2 RNA genome per uL of sample.
Products of the invention
The invention provides a kit for extracting RNA from an RNA virus. The kit comprises an inactivation solution comprising t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced). The kit also comprises a dilution solution comprising TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA). The kit further comprises an optimisation solution comprising potassium chloride (KC1) and magnesium chloride (MgCl2). The three different solutions are typically provided in separate containers so they may be used in the method of the invention. The containers are typically sealed. The containers are typically labelled to indicate their contents.
The kit may be for extracting RNA from one or more RNA viruses as discussed below with reference to the method of the invention.
The invention also provides a sample preparation buffer for obtaining viral RNA from a sample. The viral RNA is typically from an RNA virus. The sample preparation buffer may be for extracting RNA from an RNA virus. The sample preparation buffer comprises (a) t- octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced) and (b) TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA). In this instance, the sample preparation buffer comprises the components of the inactivation solution and the dilution solution discussed above. The sample preparation buffer may be for obtaining viral RNA from one or more RNA viruses from a sample or extracting RNA from one or more RNA viruses as discussed below with reference to the method of the invention.
The invention also provides a kit for determining the presence or absence of an RNA virus. The kit comprises (i) a sample preparation buffer solution of the invention and (ii) a PCR reaction mix
comprising potassium chloride (KC1) and magnesium chloride (MgCT). Parts (i) and (ii) are typically provided in separate containers so they may be used in the method of the invention. The containers are typically sealed. The containers are typically labelled to indicate their contents.
Inactivation solution
The inactivation solution comprises t-octylphenoxypoly ethoxy ethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced). The inactivation solution may comprise Triton® X-100, Triton® X-100 reduced or a mixture of both. Triton® X-100 and Triton® X-100 reduced are detergents which disrupt viral envelopes and inactivate RNA viruses.
Triton® X-100 is also known as t-octylphenoxypoly ethoxy ethanol and oly ethylene glycol tertoctylphenyl ether. These terms are interchangeable.
Triton® X-100 reduced is also known as polyoxyethylene (10) isooctylcyclohexyl ether.
The inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 and/or Triton® X-100 reduced by volume. For instance, the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 and/or Triton® X- 100 reduced by volume. The inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 and/or Triton® X-100 reduced.
The inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 by volume. For instance, the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 by volume. The inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 by volume.
The inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 reduced by volume. For instance, the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 reduced by volume. The inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 reduced by volume.
The inactivation solution preferably comprises from about 0.1% to about 10% of Triton® X- 100 and Triton® X-100 reduced by volume. For instance, the inactivation solution may comprise from about 0.5% to about 8.0% or from about 1.0% to about 5.0% Triton® X-100 and Triton® X-100 reduced by volume. The inactivation solution preferably comprises about 1.0% or about 5.0% of Triton® X-100 and Triton® X-100 reduced. In these embodiments, the amounts by volume of each add together to give the relevant values. For instance, 2.5% of Triton® X-100 and 2.5% Triton® X- 100 reduced give 5% in total. If a mixture of Triton® X-100 and Triton® X-100 reduced is used, they
may be present in any ratio, such as 1: 10, 1 :5, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 5:1 or 10: 1. The inactivation solution preferably comprises only Triton® X-100.
The inactivation solution preferably comprises universal transport medium (UTM), virus transport medium (VTM), PBS or saline. The Triton® X-100 and/or Triton® X-100 reduced are preferably present in UTM, VTM, PBS or saline. UTM is commercially available from a variety of sources, such as Copan diagnostics (catalogue number UTM - 366C). VTM is commercially available from a variety of sources, such as Sigma- virucult VTM (catalogue number MW951S). PBS can be prepared using standard methods. For instance, PBS can be prepared using PBS tablets (P32080) supplied by Melford laboratories. In this instance, lOOmL of PBS contains 137mM NaCl, 11.9mM Phosphate buffer and 2.7mM KC1. Saline can be prepared using standard methods. For instance, the saline could be STERETS Normasol (Ref: 99766774), which is sodium chloride solution 0.9%w/v. The PBS and saline are typically prepared using nuclease-free water as discussed below.
The inactivation solution preferably comprises or consists of PBS or saline with from about 1% to about 5.0% of Triton® X-100 and/or Triton® X-100 reduced by volume. This solution may comprise any of the amounts of Triton® X-100 and/or Triton® X-100 reduced discussed above. The inactivation solution more preferably comprises or consists of PBS or saline with from about 1.0% to about 5.0% of Triton® X-100 by volume. This solution may comprise any of the amounts of Triton® X-100 discussed above.
The inactivation solution preferably comprises or consists of PBS or saline with about 1% or about 5.0% of Triton® X-100 and/or Triton® X-100 reduced by volume. This solution may comprise any of the amounts of Triton® X-100 and/or Triton® X-100 reduced discussed above. The inactivation solution more preferably comprises or consists of PBS or saline with about 1.0% or about 5.0% of Triton® X-100 by volume. This solution may comprise any of the amounts of Triton® X-100 discussed above.
The kit may comprise any volume of inactivation solution, such as at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2 mL, at least about 5mL, at least about lOmL, at least about 20mL, or at least about 50mL. The kit may comprise higher volumes of the inactivation solution, such as at least about lOOmL, at least about 500mL, at least about IL or more. The kit typically comprises sufficient inactivation solution to allow an RNA virus in any of the samples discussed below, such as a saliva swab (also known as an oropharyngeal swab) or a nasopharyngeal swab, to be inactivated.
The inactivation solution is typically nuclease-free. This can be achieved by obtaining nuclease-free components or preparing nuclease free components using nuclease-free water. Nuclease-
free water is commercially available, such as from Fisher Scientific (Cat number: BP561) and Sigma Aldrich (Cat number W4502). Nuclease-free water in the context of the invention typically means DNAase-free and RNAase-free water.
Dilution solution
The dilution solution comprises TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA).
TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol) is well known in the art and can be prepared using standard methods. For instance, it can be prepared to pH8.5, IM using 4.42g Trizma hydrochloride (Sigma- Aldrich, Cat number: T3253) and 8.72g Trizma base (Sigma- Aldrich, Cat number: T1503). Glycerol is commercially available, such as bidistilled 99.5% (Supplier: VWR (Cat number: 24388.295). EDTA is also commercially available, such as 0.5M, pH8.0 from Thermo Fisher Scientific (Cat number: AM9260G). The dilution solution typically comprises or consists of the three components in nuclease-free water.
The dilution solution preferably comprises from about 5.0mM to about 20.0mM TRIZMA®. The dilution solution more preferably comprises from about 8.0mM to about 15.0mM TRIZMA®. The dilution solution more preferably comprises about lO.OmM TRIZMA®.
The dilution solution preferably comprises from about 0.5mM to about 3mM glycerol. The dilution solution more preferably comprises from about l.OmM to about 2.0mM glycerol. The dilution solution more preferably comprises about 1.4mM, such as about 1.368mM, glycerol.
The dilution solution preferably comprises from about 0.1 to about l.OmM ethylenediaminetetraacetic acid (EDTA). The dilution solution more preferably comprises from about 0.2 to about 0.8mM ethylenediaminetetraacetic acid (EDTA). The dilution solution more preferably comprises about 0.5mM ethylenediaminetetraacetic acid (EDTA).
The dilution solution preferably comprises from about 5.0mM to about 20.0mM TRIZMA®, from about 0.5mM to about 3mM glycerol and from about 0.1 to about l.OmM ethylenediaminetetraacetic acid (EDTA). The dilution solution more preferably comprises from about 8.0mM to about 15.0mM TRIZMA®, from about l.OmM to about 2.0mM glycerol and from about 0.2 to about 0.8mM ethylenediaminetetraacetic acid (EDTA). The dilution solution more preferably comprises about lO.OmM TRIZMA®, about 1.4mM, such as about 1.368mM, glycerol and about 0.5mM ethylenediaminetetraacetic acid (EDTA).
The dilution solution preferably comprises or consists of nuclease-free water with from about 5.0mM to about 20.0mM TRIZMA®, from about 0.5mM to about 3.0mM glycerol and from about 0.1
to about l.OmM ethylenediaminetetraacetic acid (EDTA). The dilution solution more preferably comprises or consists of nuclease-free water with from about 8.0mM to about 15.0mM TRIZMA®, from about l.OmM to about 2.0mM glycerol and from about 0.2 to about 0.8mM ethylenediaminetetraacetic acid (EDTA). The dilution solution more preferably comprises or consists of nuclease-free water with about lOrnM TRIZMA®, about 1.4mM, such as about 1.368mM, glycerol and about 0.5mM ethylenediaminetetraacetic acid (EDTA).
The dilution solution most preferably has the composition shown in Table 6.
The kit may comprise any volume of dilution solution, such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL. The kit may comprise higher volumes of the dilution solution, such as at least about lOOmL, at least about 500mL, at least about IL or more. The kit typically comprises sufficient dilution solution to dilute the inactivated sample as discussed below with reference to the method of the invention.
The dilution solution is typically nuclease-free. This can be achieved by obtaining or preparing nuclease-free components. Nuclease-free water is discussed above.
Optimisation solution
The optimisation solution comprises potassium chloride (KC1) and magnesium chloride. KC1 is commercially available, for instance from Sigma-Aldrich (Cat number: P9541). MgCl2, such as MgC12.6H2O, is commercially available, such as from VWR (Cat number: 25108.925). The optimisation solution typically comprises or consists of KC1 and MgCl2 in nuclease-free water.
The optimisation solution preferably comprises from about 0.1M to about 1.0M KC1. The optimisation solution more preferably comprises from about 0.2M to about 0.8M KC1 or from about 0.3M to about 0.6M KC1. The optimisation solution more preferably comprises about 0.5M, about 0.58M or about 0.5749M KC1.
The optimisation solution preferably comprises from about 0.5M to about 10M MgCl2. The optimisation solution more preferably comprises from about 1.0 to about 7.0M MgCl2 or from about 1.5M to about 5.0M MgCl2. The optimisation solution more preferably comprises about 3M, about 2.9M or about 2.91622M MgCl2.
The optimisation solution preferably comprises from about 0.1M to about 1.0M KC1 and from about 0.5M to about 10M MgCl2. The optimisation solution more preferably comprises from about 0.2M to about 0.8M KC1 and from about 1.0M to about 7.0M MgCl2. The optimisation solution more preferably comprises from about 0.3M to about 0.6M KC1 and from about 1.5M to about 5.0M MgCl2.
The optimisation solution more preferably comprises about 0.5M, about 0.58M or about 0.5749M KC1 and about 3M, about 2.9M or about 2.91622M MgCl2.
The optimisation solution preferably comprises or consists of nuclease-free water with from about 0. IM to about 1.0M KC1 and from about 0.5M to about 10M MgCl2. The optimisation solution more preferably comprises or consists of nuclease-free water with from about 0.2M to about 0.8M KC1 and from about 1.0M to about 7.0M MgCl2. The optimisation solution more preferably comprises or consists of nuclease-free water with from about 0.3M to about 0.6M KC1 and from about 1.5M to about 5.0M MgCl2. The optimisation solution more preferably comprises or consists of nuclease-free water with about 0.5M, about 0.58M or about 0.5749M KC1 and about 3M, about 2.9M or about 2.91622M MgCl2.
The optimisation solution most preferably has the composition shown in Table 7.
The kit may comprise any volume of optimisation solution, such as at least about 2uL, at least about 5uL, lOuL, at least about 20uL, at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2 mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL. The kit may comprise higher volumes of the dilution solution, such as at least about lOOmL, at least about 500mL, at least about IL or more. The kit typically comprises sufficient optimisation solution to optimise the diluted sample as discussed below with reference to the method of the invention.
The optimisation solution is typically nuclease-free. This can be achieved by obtaining or preparing nuclease-free components.
Sample preparation buffer and associated kit of the invention
The sample preparation buffer of the invention comprises (a) t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced). The sample preparation buffer preferably comprises polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced). Any of the embodiments discussed above with reference to the inactivation solution, especially the concentrations and ratios of the components, equally apply to (a) of the sample preparation buffer. The sample preparation buffer more preferably comprises about 0.5% t- octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced). The sample preparation buffer most preferably comprises about 0.5% polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced).
The sample preparation buffer of the invention also comprises (b) TRIZMA® (2-Amino-2- (hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA). Any of the
embodiments discussed above with reference to the dilution solution, especially the concentrations of the components, equally apply to (a) of the sample preparation buffer. The sample preparation buffer is effectively a TRIZMA®-EDTA buffer comprising t-octylphenoxypoly ethoxy ethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced) and glycerol. The sample preparation buffer is typically nuclease-free. This can be achieved by obtaining or preparing nuclease-free components. The sample preparation buffer typically comprises or consists of the four or five components in nuclease-free water.
The sample preparation buffer preferably comprises about 1% IM or 0.01M TRIZMA® (2- Amino-2-(hydroxymethyl)-l,3-propanediol). The sample preparation buffer preferably comprises about 0.1% 0.5M or 0.5mM EDTA. The sample preparation buffer preferably comprises about 10% glycerol. The sample preparation buffer preferably comprises about 1% IM or O.OlmM TRIZMA® (2-Amino-2- (hydroxymethyl)-l,3-propanediol), about 0.1% 0.5M or 0.5mM EDTA and about 10% glycerol.
The sample preparation buffer may be present as any volume, such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL. The sample preparation buffer may be present in higher volumes, such as at least about lOOmL, at least about 500mL, at least about IL or more. The sample preparation buffer is preferably present in about ImL.
The invention also provides a kit for determining the presence or absence of an RNA virus comprising (i) a sample preparation buffer of the invention and (ii) a PCR reaction mix comprising potassium chloride (KC1) and magnesium chloride (MgCl2). PCR reaction mixes are known in the art and are discussed below and in the Examples. The PCR reaction mix may be any of those. Any of the embodiments discussed above with reference to the optimisation solution, especially the concentrations of the components, equally apply to (ii) of the kit comprising the sample preparation buffer. The PCR reaction mix preferably comprises from about 5mM to about 25mM potassium chloride (KC1) and from about 5mM to about 25mM magnesium chloride (MgCl2), such as from about 6mM to about 24mM, from about 7mM to about 23mM, from about 8mM to about 22mM, from about 9mM to about 21mM or from about lOmM to about 20mM potassium chloride (KC1) and from about 6mM to about 24mM, from about 7mM to about 23mM, from about 8mM to about 22mM, from about 9mM to about 21mM or from about lOmM to about 20mM magnesium chloride (MgCl2). The PCR reaction mix preferably comprises about 18.8mM potassium chloride (KC1) and about 18.73mM magnesium chloride (MgCl2). In a preferred embodiment, 5uL of sample preparation buffer is mixed with 15uL of the PCR reaction mix giving a final potassium chloride (KC1) concentration of 14. ImM and a final magnesium chloride (MgCl2) concentration of 14.05mM in the 20uL RT-PCR reaction.
The kit may comprise any volume of sample preparation buffer, such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL. The kit may comprise higher volumes of the sample preparation buffer, such as at least about lOOmL, at least about 500mL, at least about IL or more. The kit typically comprises about ImL of sample preparation buffer. The kit may comprise any volume of PCR reaction mix, such as such as at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL.
Internal extraction control (IEC)
The kit of the invention preferably further comprises an internal extraction control (IEC) RNA. When performing RNA extraction and RT-PCR, it is advantageous to have an exogenous source of RNA template that is spiked into the extraction. This control RNA is then co-purified with the sample RNA and can be detected as a positive control for the extraction process (internal extraction control). Successful co-purification and RT- PCR amplification of the control RNA also indicates that PCR inhibitors are not present at a high concentration. IEC RNA can be prepared or is commercially available.
The kit may comprise any volume of IEC, such as at least about 20uL, at least about 50uL, at least about lOOuL, at least about 200uL, at least about 500uL, at least about ImL, at least about 2mL, at least about 5mL, at least about lOmL, at least about 20mL or at least about 50mL. The kit may comprise higher volumes of the dilution solution, such as at least about lOOmL, at least about 500mL, at least about IL or more. The kit typically comprises sufficient IEC to conduct the method of the invention.
Other components
The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the methods below to be carried out. Such reagents or instruments include, but are not limited to, one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a patient (such as a vessel or an instrument comprising a needle or a swab) or tubes in which quantitative reactions can be done. The kit may, optionally, comprise instructions to enable the kit to be used in the methods of the invention or details regarding which patients may be tested. The kit may further comprise any of the reagents required to conduct RT-PCR discussed below, including
primers, probes, a reverse transcriptase, heat-stable DNA polymerase and negative control, such as nuclease-free water.
Method of extracting/ obtaining RNA of the invention
The invention provides a method of extracting RNA from an RNA virus, if present, in a sample. The method can only extract the RNA from the RNA virus if the virus is present and, as explained in more detail below, the sample may not contain the virus.
The method comprises using a kit of the invention. First (a), the method comprises contacting the sample with an inactivation solution comprising Triton® X-100 and/or Triton® X-100 reduced. Second (b), the method comprises diluting the inactivated sample with a dilution solution comprising TRIZMA®, glycerol and EDTA. Third (c), the method comprises adding to the diluted sample an optimisation solution comprising KC1 and MgCl2.
The inactivation solution may be any of those discussed above with reference to the kits of the invention.
The dilution solution may be any of those discussed above with reference to the kits of the invention.
The optimisation solution may be any of those discussed above with reference to the kits of the invention.
Any amount of the inactivation solution may be contacted with the sample, such as about ImL, about 2mL, about 5mL or more. A saliva swab (also known as an oropharyngeal swab) or a nasopharyngeal swab is preferably contacted with about ImL inactivation solution. A saliva swab (also known as an oropharyngeal swab) or a nasopharyngeal swab is more preferably contacted with about ImL inactivation solution comprising PBS or saline.
The inactivated sample in (a) may be diluted in step (b) by about 1:5, about 1:10, about 1 : 15 or about 1:25. Dilution of 1:5 means 1 part (in terms of volume) of the inactivated sample is combined with 4 parts dilution sample (to form 5 parts in total). If the inactivation solution comprises UTM or VTM, the inactivated sample in (a) is preferably diluted in step (b) by about 1:15. For instance, 6uL of inactivated sample is combined with 84uL of dilution solution. If the inactivation solution comprises PBS or saline, the inactivated sample in (a) is preferably diluted in step (b) by about 1:5. For instance, 18uL of inactivated sample is preferably combined with 72uL of dilution solution. If the inactivation solution comprises PBS or saline, the inactivated sample in (a) may be diluted in step (b) by about 1:10. For instance, 9uL of inactivated sample is preferably combined with 81uL of dilution solution.
Step (c) preferably comprises adding 1 part (by volume) of the optimisation solution to 30 parts or 45 parts of the diluted sample produced in (b). For instance, step (c) preferably comprises adding 3uL of the optimisation solution to the 90uL of the diluted sample produced in (b) or 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b). If the inactivation solution comprises UTM, VTM or PBS, step (c) preferably comprises adding 1 part (by volume) of the optimisation solution to 45 parts of the diluted sample produced in (b), such as adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b). If the inactivation solution comprises saline, step (c) preferably comprises adding 1 part (by volume) of the optimisation solution to 30 parts or 45 parts of the diluted sample produced in (b), such as adding 2uL or 3uL of the optimisation solution to the 90uL of the diluted sample produced in (b). Step (c) most preferably comprises adding 1 part (by volume) of the optimisation solution to 45 parts of the diluted sample produced in (b), such as adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
The method preferably further comprises step (d) further diluting the optimised sample produced in step (c).
In a preferred embodiment, the inactivation solution in step (a) comprises PBS or saline, step (b) comprises diluting the inactivated sample produced in (a) 1:5 with the dilution solution and step (c) comprises adding 1 part of the optimisation solution to 45 parts of the diluted sample produced in (b). Preferably, the inactivation solution in step (a) comprises PBS or saline, step (b) comprises diluting 18uL of inactivated sample produced in (a) with 72uL of the dilution solution and step (c) comprises adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
In another preferred embodiment, the inactivation solution in step (a) comprises UTM or VTM, step (b) comprises diluting the inactivated sample produced in (a) 1:15 with the dilution solution and step (c) comprises adding 1 part of the optimisation solution to 45 parts of the diluted sample produced in (b). Preferably, the inactivation solution in step (a) comprises UTM or VTM, step (b) comprises diluting 6uL of the inactivated sample produced in (a) with 84uL dilution solution and step (c) comprises adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b).
In both preferred embodiments, the method preferably further comprises (d) adding 5 parts of nuclease free water or IEC to 23 parts of the optimised sample produced in (c). For instance, the method preferably further comprises (d) adding 20uL of nuclease free water or IEC RNA to the 92uL of the optimised sample produced in (c).
The method preferably comprises (a) contacting the sample with an inactivation solution comprising or consisting of PBS or saline with Triton® X-100 and/or Triton® X-100 reduced, (b)
diluting 18uL of inactivated sample produced in (a) with 72uL of the dilution solution, (c) adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b) and (d) adding 20uL of nuclease free water or IEC RNA to 92uL of the optimised sample produced in (c).
The method preferably comprises (a) contacting the sample with an inactivation solution comprising or consisting of UTM or VTM with Triton® X-100 and/or Triton® X-100 reduced, (b) diluting 6uL of inactivated sample produced in (a) with 84uL of the dilution solution, (c) adding 2uL of the optimisation solution to the 90uL of the diluted sample produced in (b) and (d) adding 20uL of nuclease free water or IEC RNA to 92uL of the optimised sample produced in (c).
The invention also provides a method of obtaining viral RNA, if present, from a sample, comprising contacting the sample with a sample preparation buffer of the invention and thereby obtaining the viral RNA, if present, from the sample. The method can only obtain the viral RNA if the virus is present and, as explained in more detail below, the sample may not contain the virus. The sample preparation buffer may be any of those discussed above. Any amount of the sample preparation buffer may be contacted with the sample, such as about ImL, about 2mL, about 5mL or more. An oropharyngeal swab or a nasopharyngeal swab is preferably contacted with about ImL of the sample preparation buffer. The method may involve the use of the kit of the invention comprising the sample preparation buffer of the invention.
The method of the invention preferably does not comprise any additional methods of inactivating the virus and/or extracting RNA from the virus. The method of the invention is sufficient to inactivate the virus and extract the RNA from the virus. The method of the invention is sufficient to obtain viral RNA from the sample. The method of the invention preferably does not comprise heat inactivation or using a viral inactivation kit. The method preferably does not comprise using an RNA extraction kit. Such kits are well known in the art, such as QIAamp® virus RNA mini kit (Qiagen). The method of the invention preferably does not comprise using magnetic beads to extract the RNA. Magnetic bead kits are well known in the art, such as Automated extraction system GenoXtract® from HAIN Lifescience GmbH (Brucker) using GXT DNA/RNA Extraction kit (Catalogue no: 12.01.02, 96 samples).
RNA virus
The invention may be carried out with any RNA virus. The RNA virus is typically from the realm Riboviria. The RNA virus may be from the kingdom of Orthornavirae or Pararnavirae. The RNA virus is preferably from the kingdoms of Orthornavirae.
The RNA virus is preferably from the family of Coronaviridae . The virus may be the severe acute respiratory syndrome (SARS) coronavirus (also known as SARS-CoV or SARS-CoV-1). The terms severe acute respiratory syndrome (SARS) coronavirus, SARS-CoV and SARS-CoV-1 are interchangeable. The virus may be the Middle East respiratory syndrome (MERS) coronavirus, also known as camel flu. The virus is preferably SARS-CoV-2.
The RNA virus is preferably from the family of Orthomyxoviridae . The virus may an influenza virus. The influenza virus is preferably an influenza A virus, such as H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1,H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 or H10N7. Influenza A viruses infect humans, pigs, birds, horses and bats. The influenza virus is preferably an influenza B virus, which infects humans and seals. The influenza virus is preferably an influenza D virus, which infects pigs and cattle. The influenza virus is preferably an influenza C virus, which infects humans, pigs and dogs. The influenza virus is preferably an infectious salmon anemia virus, which infects Atlantic salmon. The influenza virus is preferably Thogotovirus or Dhori virus, which infects ticks, mosquitos and mammals, includeing humans.
The invention may extract the RNA from multiple RNA viruses or two or more RNA viruses, such as 3 or more, 4 or more, 5 or more or 10 or more viruses. For instance, the invention may extract RNA from one or more of (i) SARS-CoV, (ii) MERS coronavirus and (iii) SARS- CoV-2, such as (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii). The invention may extract RNA from a coronavirus and an influenza virus. For instance, the invention may extract RNA from one or more of (i) SARS-CoV, (ii) MERS coronavirus and (iii) SARS- CoV-2 (including as defined above) and an influenza virus. The invention may extract RNA from one or more of (i) SARS-CoV, (ii) MERS coronavirus and (iii) SARS-CoV-2 (including as defined above) and one or more of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1,H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2 and H10N7.
Method of determining the presence or absence of an RNA virus in a sample
The invention provides various methods of determining the presence or absence of an RNA virus, such as SARS-CoV-2, in a sample. Any number of RNA viruses may be detected as discussed above. All of the methods comprise extracting RNA from the RNA virus in the sample, if present, using a method of the invention. Any of the methods and kits discussed above may be used.
The method comprises (i) extracting RNA from the RNA virus in the sample, if present, or obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample. The method may comprise (i) extracting RNA from the RNA virus in the sample, if present, using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample. The method may comprise (i) obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
In one embodiment, the method comprises conducting a RT-PCR assay on the sample that has been subjected to the extraction method of the invention. After conducting the extraction methods discussed above, one 14th of the sample is preferably used in the RT-PCR. Various embodiments discussed above result in final volume of 112uL. In such embodiments, 8uL of the extracted sample is used in RT-PCR. The RT-PCR is preferably direct RT-PCR. In direct RT-PCR, the sample, preferably 8uL, is directly added to the RT-PCR assay after conducting the extraction method of the invention without any intervening steps.
In another embodiment, the method comprises conducting a RT-PCR assay on the viral RNA obtained from the sample using the sample preparation buffer of the invention. In a preferred embodiment, the method preferably comprises contacting an oropharyngeal swab or a nasopharyngeal swab with about ImL sample preparation buffer and then adding about 5uL of the sample preparation buffer to about 15uL of the PCR reaction mix to form a total RT-PCR reaction volume of about 20uL. If the method of the invention uses a kit of the invention comprising the sample preparation buffer of the invention, the PCR reaction mix comprises potassium chloride (KC1) and magnesium chloride (MgCl2).
Methods for conducting RT-PCR are well known in the art and any suitable conditions may be used. Reverse transcription involves the use of the enzyme reverse transcriptase to convert viral RNA into cDNA. In the context of the invention, PCR involves using the primers to amplify a specific portion of the cDNA, namely the cDNA amplicon, which typically contains the target cDNA of interest.
Methods for reverse transcription are well known in the art and reverse transcriptases are commercially available (e.g. Superscript® II reverse transcriptase (Invitrogen) and Affinity script
(Agilent)). Methods typically construct cDNA from random cDNA hexamers included in the reaction mixture.
PCR involves amplifying a cDNA amplicon using a heat-stable DNA polymerase and a pair of forward and reverse polynucleotide primers. Because the newly synthesized cDNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. Many polymerase chain methods are known to those of skill in the art and may be used in the methods of the invention. For example, cDNA can be subjected to 20 to 40 cycles of amplification in a thermocycler as follows: 95°C for 30 sec, 52° to 60°C for 1 min, and 72°C for 1 min, with a final extension step of 72°C for 5 min. For another example, cDNA can be subjected to 20 to 40 polymerase chain reaction cycles in a thermocycler at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54°C to 58°C for 1 min, an extension step at 70°C for 1 min, with a final extension step at 70°C for 5 min. Heat stable DNA polymerases are commercially available (such as GoTaq® G2 (Promega)).
The RT-PCR assay may be conducted using the conditions set out in the Examples. The reverse transcriptase is preferably Affinity script (Agilent). The DNA polymerase is preferably GoTaq® G2 (Promega). Both enzymes are preferably present in a 2x mastermix. The reaction mix is preferably lOuL 2x oasig mastermix, 2uL primer/probe mix (resuspended in template preparation buffer) and 8uL sample. The reaction conditions are preferably (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
The RT-PCR method is preferably real time RT-PCR. This method is known in the art.
The RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one-step reactions are routine in the art.
Step (ii) of the method preferably comprises reverse transcribing any RNA in the sample, contacting any cDNA produced with a pair of primers under conditions which allow the primers to amplify their cDNA amplicon and determining the presence or absence of the cDNA amplicon. Detection of the cDNA amplicon indicates that the sample contains the RNA virus (i.e. indicates the presence of RNA virus in the sample). A lack of detection of the cDNA amplicon indicates that the sample does not contain the RNA virus (i.e. indicates the absence of RNA virus from the sample).
Suitable probes and primers for use in the method of the invention are known in the art. Specific types of probes and primers that may be used in the invention are discussed below with
reference to the preferred probes and primers for use in the invention. Probes and primers for detecting SARS-CoV, MERS coronavirus and influenza viruses are known in the art. As a result, the skilled person can detect any RNA virus or any combination of RNA viruses.
Method of determining the presence or absence of SARS-CoV -2 in a sample
In various preferred embodiments, the method of the invention is for determining the presence or absence of SARS-CoV -2 in a sample.
In particular, the invention provides a method of determining the presence or absence of SARS- CoV-2 in a sample, wherein the method comprises (i) extracting RNA from the SARS-CoV -2 in the sample, if present, or obtaining SARS-CoV -2 RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample. The method may comprise (i) extracting RNA from the SARS-CoV-2 in the sample, if present, using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample. The method may comprise (i) obtaining viral RNA, if present, from the sample using a method of the invention and (ii) conducting a reverse-transcription polymerase chain reaction (RT- PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
In one embodiment, step (ii) comprises using a preferred pair of primers discussed below and detecting the cDNA amplicon amplified by the primers if present. Any method may be used to detect the cDNA amplicon. For instance, the method may use SYBR Green to detect the cDNA amplicon. When the SYBR Green binds to the cDNA, it emits light and the intensity of the fluorescence increases as the cDNA amplicons accumulate. This technique is easy to use since designing of probes is not necessary given lack of specificity of its binding. The method preferably uses a preferred polynucleotide probe discussed below which specifically hybridises to the cDNA amplicon. The probe may be any of those discussed below. The use of the probe allows the specific cDNA amplicon to be identified if present. The probe is preferably a TaqMan® probe.
In another embodiment, step (ii) comprises using a pair of primers that amplifies a cDNA amplicon comprising (or consisting of) a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3 if present. The cDNA amplicon may be any of those discussed below. The cDNA amplicon preferably comprises or consists of SEQ ID NO: 4. The cDNA amplicon preferably comprises or consists of SEQ
ID NO: 5. The method also comprises using a preferred polynucleotide probe discussed below which comprises the sequence shown in SEQ ID NO: 3 or a variant thereof as defined above to detect the cDNA amplicon if present. The polynucleotide probe may be any of those discussed below. The probe is preferably a TaqMan® probe. The polynucleotide probe specifically hybridises to cDNA amplicon if present.
In another embodiment, step (ii) comprises using a preferred a pair of primers discussed below and a preferred polynucleotide probe discussed below. Any of the primers and probes discussed below may be used. The pair of primers preferably comprises a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1 and a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2. The polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3. The pair of primers amplifies a cDNA amplicon as discussed below and then this is specifically detected using the polynucleotide probe discussed below.
In all embodiments, detection of the cDNA amplicon (i.e. the presence of the cDNA amplicon) indicates that the sample contain SARS-CoV-2 (i.e. indicates the presence of SARS-CoV-2 in the sample). A lack of detection of the cDNA amplicon (i.e. the absence of the cDNA amplicon) indicates that the sample does not contain SARS-CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample). Detection of multiple copies of the cDNA amplicon (i.e. the presence of multiple copies of the cDNA amplicon) indicates that the sample contain SARS-CoV-2 (i.e. indicates the presence of SARS-CoV-2 in the sample). A lack of detection of multiple copies of the cDNA amplicon (i.e. the absence of multiple copies of the cDNA amplicon) indicates that the sample does not contain SARS- CoV-2 (i.e. indicates the absence of SARS-CoV-2 from the sample).
In all embodiments, the method preferably comprises (a) reverse transcribing any RNA in the sample, (b) contacting any cDNA produced in (a) with the pair of primers under conditions which allow the primers to amplify their cDNA amplicon and (c) determining the presence or absence of the cDNA amplicon. Step (c) preferably uses a polynucleotide probe as described below.
The RT-PCR assay is preferably a one-step RT-PCR assay conducted in one tube or vessel. Such one-step reactions are routine in the art.
Preferred primers for use in detecting SARS-CoV-2
Disclosed herein are a preferred pair of primers for determining the presence or absence of SARS-CoV-2 in a sample.
The forward primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 1 or a variant thereof having at least about 80% homology to SEQ ID NO: 1 based on sequence identity over its entire length.
The reverse primer comprises a polynucleotide having the sequence shown in SEQ ID NO: 2 or a variant thereof having at least about 80% homology to SEQ ID NO: 2 based on sequence identity over its entire length.
A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial.
A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2’0-methyl, 2’ methoxy-ethyl, phosphorami date, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides are most preferably adenosine, guanosine, uridine and cytidine
The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides are preferably deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5’ or 3’ side of a nucleotide.
Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5- methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxy adenosine monophosphate (dAMP), deoxy adenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate
(dUTP), deoxy cytidine monophosphate (dCMP), deoxy cytidine diphosphate (dCDP) and deoxy cytidine triphosphate (dCTP), 5-methyl-2’ -deoxy cytidine monophosphate, 5-methyl-2’ -deoxy cytidine diphosphate, 5-methyl-2’ -deoxy cytidine triphosphate, 5-hydroxymethyl-2’ -deoxy cytidine monophosphate, 5-hydroxymethyl-2’ -deoxy cytidine diphosphate and 5-hydroxymethyl-2’- deoxy cytidine triphosphate. The nucleotides are preferably selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. The nucleotides are preferably selected from dAMP, dTMP, dGMP or dCMP.
The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2’ amino pyrimidines (such as 2’-amino cytidine and 2’- amino uridine), 2’-hyrdroxyl purines (such as , 2’ -fluoro pyrimidines (such as 2’ -fluorocytidine and 2’fluoro uridine), hydroxyl pyrimidines (such as 5’-a-P-borano uridine), 2’-O-methyl nucleotides (such as 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine and 2’-O-methyl uridine), 4’- thio pyrimidines (such as 4’ -thio uridine and 4’ -thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2’ -deoxy uridine, 5-(3-aminopropyl)-uridine and 1,6-diaminohexyl-N- 5-carbamoylmethyl uridine).
One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag. The label may be any suitable label which allows the polynucleotide to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 1251, 35S, enzymes, antibodies, antigens, other polynucleotides and ligands such as biotin.
The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2’0-methyl, 2’ methoxy-ethyl, phosphorami date, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.
The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LN A), morpholino nucleic acid or other synthetic polymers with nucleotide side chains. The polynucleotide may comprise any of the nucleotides discussed above, including the modified nucleotides.
The polynucleotide may be DNA or RNA. In the context of the invention, a primer polynucleotide that is RNA has the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 if all of the instances of dAMP, dTMP, dGMP and dCMP are replaced with AMP, UMP, GMP and CMP
respectively. In other words, the DNA nucleotides are replaced with their corresponding RNA nucleotides and T is replaced with U.
The polynucleotide is preferably DNA.
The polynucleotide may be single stranded or double stranded. The primer polynucleotide is preferably single stranded.
Any of the polynucleotides discussed herein, such as the primers, probes, cDNA amplicons and target cDNA sequences, may be isolated, substantially isolated, purified or substantially purified. The polynucleotide is isolated or purified if it is completely free of any other components, such as buffer, other polynucleotides, virus material or cells. The polynucleotide is substantially isolated or substantially purified if it is only mixed with carriers or diluents, such as buffers or excipients, which will not interfere with its intended use, such as in a RT-PCR assay. The primer polynucleotides are not naturally occurring.
The primer may comprise a variant sequence based on SEQ ID NO: 1 or 2. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 1 or 2. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2 and which retains its ability to specifically hybridise to a sequence that is complementary to SEQ ID NO: 1 or 2. A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 1 or 2 and which retains its ability to amplify the complementary DNA (cDNA) amplicon amplified from SARS-CoV-2 cDNA by SEQ ID NOs: 1 and 2 (i.e. SEQ ID NO: 4). The term "amplified" refers to the process of making multiple copies of the polynucleotide, such as cDNA, from a single polynucleotide or fewer polynucleotides.
A variant “specifically hybridises” to its target sequence when it hybridises with preferential or high affinity to the partner but does not substantially hybridise, does not hybridise or hybridises with only low affinity to other polynucleotides, especially other sequences in SARS-CoV-2. Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al. , 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley- Interscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCl and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M
NaCl, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCl, 1% SDS at 37 °C, followed by a wash in 0. IX (0.0165 M Na+) SSC at 60 °C. A variant “specifically hybridises” if it hybridises to its partner with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotides. More preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotides. Preferably, the variant hybridises to its partner with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a polynucleotide which differs from its partner by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The variant typically hybridises to its target sequence with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.
The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 1 or 2, but may be longer or shorter. A variant of SEQ ID NO: 1 is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. A variant of SEQ ID NO; 2 is preferably at least 20 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is at least about 80% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence has at least about 80% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 84% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 84% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 88% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 88% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 92% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 92% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence is preferably at least about 96% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 1, a variant sequence preferably has at least about 96% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 1.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 79% or at least about 79. 17% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 79% or at least about 79. 17% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 80% or at least about 83.3% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 80% or at least about 83.3% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 85% or at least about 87.5% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 85% or at least about 87.5% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 90% or at least about 91.67% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least
about 90% or at least about 91.67% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 2.
Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence is preferably at least about 95% or at least about 95.8% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 2, a variant sequence preferably has at least about 95% or at least about 95.8% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 2.
Methods of measuring homology based on sequence identity or identity are known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity (e.g. used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
The PILEUP and BLAST algorithms can also be used to calculate identity, homology or line up sequences (typically on their default settings), for example as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum
probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
A variant is typically identical to SEQ ID NO: 1 over at least about 20, at least about 21, at least about 22, at least about 23, at least about 24 or at least about 25 consecutive nucleotides. A variant is typically identical to SEQ ID NO: 2 over at least about 20, at least about 21, at least about 22, at least about 23 or at least about 24 consecutive nucleotides.
Each polynucleotide in the primer may be any length. The forward primer is preferably at least 20 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The reverse primer is at least 20 nucleotides in length, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length.
The forward primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably comprises or consists of the exact sequence shown in SEQ ID NO: 2.
The forward primer preferably is the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably is the exact sequence shown in SEQ ID NO: 2.
The forward primer preferably is DNA and comprises/consist of the exact sequence shown in SEQ ID NO: 1. The reverse primer preferably is DNA and comprises/consists of the exact sequence shown in SEQ ID NO: 2.
The pair of primers may comprise any combination of primers discussed above. The pair of primers may comprise a forward primer that comprises/consist of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above. The pair of primers may comprise a forward primer that comprises/consists of a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of the exact sequence of SEQ ID NO: 2. The pair of primers may comprise a forward primer that comprises/consists a variant of SEQ ID NO: 1 as discussed above and a reverse primer that comprises/consists of a variant of SEQ ID NO: 2 as discussed above.
The pair of primers more preferably comprises a forward primer which comprises or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer which comprises or consists of the exact sequence shown in SEQ ID NO: 2. The pair of primers more preferably comprises a forward primer that is the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is the exact sequence shown in SEQ ID NO: 2.
The pair of primers most preferably comprises a forward primer that is DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 1 and a reverse primer that is preferably DNA and comprises/ or consists of the exact sequence shown in SEQ ID NO: 2.
Primers having any of the sequences above may be manufactured using standard techniques. Custom primers having specific sequences are commercially available from various suppliers (such as Biolegio).
Preferred cDNA amplicons for use in detecting SARS-CoV-2
Also described herein is a complementary DNA (cDNA) amplicon amplified from SARS-CoV- 2 cDNA by the pair of primers discussed above. The cDNA amplicon is amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. Specific hybridisation is defined above with reference to the preferred primers. The cDNA amplicon is preferably amplified from SARS-CoV-2 cDNA and comprises or consists of a sequence which is complementary to the sequence shown in SEQ ID NO: 3.
SARS-CoV-2 cDNA is cDNA produced by the reverse transcription of SARS-CoV-2 RNA. The sequence of SARS-CoV-2 cDNA is published (see above) and methods for reverse transcribing it are known in the art and discussed in the Example. Once the SARS-CoV-2 cDNA has been reversed transcribed, the pair of primers discussed above may be used to amplify the cDNA amplicon using polymerase chain reaction (PCR). Methods for conducting PCR using primers are known in the art and discussed below and in the Examples. A cDNA amplicon is amplified from SARS-CoV-2 cDNA if it comprises a portion/part of the SARS-CoV-2 cDNA. In other words, it comprises a portion/part of the sequence of the SARS-CoV-2 cDNA.
The term "amplified" refers to the process of making multiple copies of the cDNA from a single polynucleotide or fewer polynucleotides. An amplicon is the cDNA that results from amplification. In the context of the invention, primers, such as the primers discussed above, may be used to amplify a part of the SARS-CoV-2 cDNA resulting in the shorter cDNA amplicons. These amplicons typically contain the cDNA target sequence being used in the context of the invention.
The cDNA amplicon is typically a polynucleotide comprising nucleotides selected from dAMP, dTMP, dGMP or dCMP as discussed above. The cDNA may be double stranded or single stranded. The cDNA is preferably single stranded.
The cDNA amplicon may be any length, such as from about 20 to about 100 nucleotides in the length, such as from about 25 to about 90, from about 30 to about 95 or from about 40 to about 90 nucleotides in length. The cDNA amplicon is preferably 82 nucleotides in length.
The cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 4. The cDNA amplicon preferably comprises or consists of the sequence shown in SEQ ID NO: 5.
The cDNA amplicon may be isolated, substantially isolated, purified or substantially purified as discussed above. The cDNA amplicon is not naturally occurring.
Preferred polynucleotide probes for use in detecting SARS-CoV-2
Also described herein are preferred polynucleotide probes for determining the presence or absence of SARS-CoV-2 in a sample.
In one embodiment, the polynucleotide probe specifically hybridises to the cDNA amplicon described above. The polynucleotide probe comprises or consists of a sequence which specifically hybridises to the cDNA amplicon described above. The polynucleotide probe preferably comprises or consists of a sequence which specifically hybridises to a target sequence on the cDNA amplicon described above. The target sequence may be any length, such as from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
Polynucleotides and specific hybridisation are defined above with reference to the preferred primers. The probe polynucleotide may be any of the polynucleotides discussed above. The polynucleotide probe is preferably DNA. The polynucleotide probe is preferably single stranded DNA. The polynucleotide probe may be any length, such as from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The polynucleotide probe is preferably the same length as the target sequence it detects. The target sequence is preferably specific for SARS- CoV-2. The target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus. The target sequence is preferably found/present in SARS-Cov-2 cDNA but is not found in the RNA of any other virus or in any human RNA. The target sequence is preferably found/present in the cDNA amplicon described above. The target sequence is preferably part of SEQ ID NO: 4. The target sequence is preferably the sequence shown in SEQ ID NO: 5.
In another embodiment, the polynucleotide probe comprises or consists of the sequence shown in SEQ ID NO: 3 or a variant thereof having at least about 80% homology to SEQ ID NO: 3 based on sequence identity over its entire length. Polynucleotides are defined above with reference to the preferred primers. The probe polynucleotide may be any of the polynucleotides discussed above. The polynucleotide probe is preferably DNA. The polynucleotide probe is preferably single stranded DNA.
The polynucleotide probe may be any length, such as from 20 to 30 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
A variant sequence is a polynucleotide that has a nucleotide sequence which varies from that of SEQ ID NO: 3 and which retains its ability to specifically hybridise to the target sequence of SEQ ID NO: 5. Specific hybridisation is discussed above. The variant sequence must not recognise or hybridise to any cDNA sequence from any other virus.
The variant sequence may comprise any of the nucleotides discussed above, including the modified nucleotides. The variant sequence is typically the same length as SEQ ID NO: 3, but may be longer or shorter. A variant of SEQ ID NO: 3 is preferably at least 20 nucleotides in length, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence is at least about 80% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence has at least about 80% identity to that sequence. Homology based on sequence identity and identity are interchangeable herein. This allows for variation, deletion, addition or a combination thereof of five nucleotides within the sequence of SEQ ID NO: 3.
Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence is preferably at least about 84% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 84% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of four nucleotides within the sequence of SEQ ID NO: 3.
Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence is preferably at least about 88% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 88% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of three nucleotides within the sequence of SEQ ID NO: 3.
Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence is preferably at least about 92% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 92% identity to that sequence. This allows for variation, deletion, addition or a combination thereof of two nucleotides within the sequence of SEQ ID NO: 3.
Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence is preferably at least about 96% homologous to that sequence based on nucleotide identity. Over the entire length of the sequence of SEQ ID NO: 3, a variant sequence preferably has at least about 96% identity to that
sequence. This allows for variation, deletion, addition or a combination thereof of one nucleotide within the sequence of SEQ ID NO: 3.
The polynucleotide probe preferably comprises or consists of the sequence shown in SEQ ID NO: 3.
The polynucleotide probe may be isolated, substantially isolated, purified or substantially purified as discussed above. The polynucleotide probe is not naturally occurring.
The polynucleotide probe is preferably DNA probe, a TaqMan® probe, a molecular beacon or a scorpion probe. DNA probes hybridise to the, typically complementary, target sequence and then can be detected. TaqMan® probes are known in the art and are polynucleotides that have a fluorescent dye attached to the 5' end and a quencher to the 3' end. The polymerase used in PCR cleaves hybridised probes freeing the fluorescent dye from quenching such it can be detected. Molecular beacon probes are known in the art and are similar to TaqMan® probes except (rather than using cleavage to separate the dye from the quencher) hybridisation to the target sequence separates the dye from the quencher. Scorpion probes are known the art and are similar to molecular beacons except the 3' end also contains a sequence that is complementary to the extension product of the primer on the 5' end which opens the probe on hybridisation and allows the dye to be detected. The polynucleotide probe is preferably a TaqMan® probe.
The polynucleotide probes are preferably detectably-labelled. Suitable detectable labels are known in the art. The detectable labels are preferably fluorescent molecules or dyes, such as fluorescein derivatives. Suitable fluorescent molecules or dyes include, but are not limited to, 6- carboxyfluorescein (FAM), 2'-chloro-7'phenyl-l,4-dichloro-6-carboxy-fluorescein (VIC®) and tetrachlorofluorescein (TET). A suitable quencher for use with these dyes in TaqMan®, molecular beacon or scorpion probes is tetramethylrhodamine (TAMRA).
The detectable label is most preferably 6-carboxyfluorescein (FAM).
Polynucleotide probes are also available from commercial sources (such as Biolegio).
Preferred target cDNA sequence for use in detecting SARS-CoV-2
Also described herein is a preferred target cDNA polynucleotide comprising or consisting of a sequence (a) to which the sequence shown in SEQ ID NO: 3 specifically hybridises and/or (b) which is complementary to the sequence shown in SEQ ID NO: 3. Specific hybridisation is discussed above. The target cDNA may be any length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The target cDNA polynucleotide preferably comprises or consists of the sequence shown in SEQ ID NO: 5. The target cDNA polynucleotide may be double stranded or single stranded. The
target cDNA polynucleotide is preferably single stranded. The target cDNA polynucleotide may be isolated, substantially isolated, purified or substantially purified as discussed above. The target cDNA polynucleotide is not naturally occurring.
Methods of measuring the RNA virus, such as SARS-CoV-2. titre
The invention also provides methods for measuring the RNA virus, such as SARS-CoV-2, titre in a sample. The method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of RNA virus, such as SARS-CoV-2, in a sample as discussed above may be used. The quantitative method uses real time RT-PCR. If RNA virus, such as SARS-CoV-2, is present, the method comprises evaluating the cycle quantification (Cq) value and thereby measuring the RNA virus, such as SARS-CoV-2, titre. The method preferably comprises evaluating the Cq value against standard Cq values generated from standard dilution curves.
In real time RT-PCR, the threshold line is the level of detection or the point at which a reaction reaches a fluorescent intensity above background levels. The threshold level is preferably 10% of the end point fluorescence. Autocalling may be used to set the threshold level.
The Cq is the PCR cycle number at which the sample reaction curve intersects the threshold line. This value indicates how many cycles it took to detect a real signal from the sample. Real-Time PCR machines typically calculate the Cq value for each of sample.
As is clear from Example 2 (Figures 1 to 3), the Cq value changes depending on the amount of RNA virus, such as SARS-CoV-2, present in the sample. These standard curves allow the amount of RNA virus, such as SARS-CoV-2, to be measured in sample. In other words, the Cq is predictive of the amount of the RNA virus, such as SARS-CoV-2, in the sample.
In a preferred method, a Cq value of about 16.0 to about 20.9, preferably about 18.0 to about 19.9, indicates the sample contains about 106 virions/pl, a Cq value of about 21.0 to about 25.9 indicates the sample contains about 105 virions/pl, a Cq value of about 26.0 to about 28.9, preferably from about 26.0 to about 27.9, indicates the sample contains about 104 virions/pl, a Cq value of about 29.0 to about 31.9, preferably 30.1, indicates the sample contains about 103 virions/pl, a Cq value of about 32.0 to about 33.9, preferably from about 33.0 to about 33.9, indicates the sample contains about 102 virions/pl, a Cq value of above 34.0 or above indicates the sample contains 10 or fewer virions/pl. A Cq value of 38.0, 39.0 or 40.0 or above preferably indicates the sample contains 5 or fewer virions/pl. In all cases, the virions are preferably SARS-CoV-2 virions. All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to one significant figure. In all concentration cases in this paragraph, the sample refers to the
(undiluted) sample that is added to (and diluted by) the RT-PCR assay mixture. The concentrations do not relate to the final concentration of the virions in the diluted RT-PCR assay mixture. If the sample, for instance from a patient, is diluted or concentrated before it is used in the RT-PCR assay, the dilution factor or concentration factor needs to be taken into account when calculating the the amount of RNA virus, such as SARS-CoV-2, present in the sample.
The invention also provides a method for differentiating between high and low RNA virus, such as SARS-CoV-2, titres in a sample. The method comprises conducting any of the methods discussed above. Any of the methods for determining the presence or absence of RNA virus, such as SARS- CoV-2, in a sample as discussed above may be used. The method uses real time RT-PCR. If RNA virus, such as SARS-CoV-2, is present, the method comprises evaluating the cycle quantification (Cq) value and thereby determining whether the sample has a low or high RNA virus, such as SARS-CoV-2, titre. Cq measurement and evaluation is discussed above.
As is clear from Example 2 (Figures 1 to 3), the Cq value changes depending on the amount of RNA virus, such as SARS-CoV-2, in a sample. These standard curves allow the amount of RNA virus, such as SARS-CoV-2, to be measured in a sample. In other words, the Cq is predictive of the amount of the RNA virus, such as SARS-CoV-2, in the sample.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 102 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 103 virions/pl or higher. In this method, a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 31.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 10 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 103 virions/pl or higher. In this method, a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 31.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 102 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 104 virions/pl or higher. In this method, a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 28.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 10 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 104 virions/pl or higher. In this
method, a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 28.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 102 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 105 virions/pl or higher. In this method, a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 25.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 10 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 105 virions/pl or higher. In this method, a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 25.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 102 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 106 virions/pl or higher. In this method, a Cq value of about 32.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 20.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In a preferred method, a low RNA virus, such as SARS-CoV-2, titre is about 10 virions/pl or lower and a high RNA virus, such as SARS-CoV-2, titre is about 106 virions/pl or higher. In this method, a Cq value of about 34.0 or above indicates the sample has a low RNA virus, such as SARS- CoV-2, titre and a Cq value of 20.9 or lower indicates the sample has a high RNA virus, such as SARS- CoV-2, titre.
In all cases, the virions are preferably SARS-CoV-2 virions. All of the Cq values above are preferably mean Cq values derived from multiple experiments. The Cq values are typically expressed to one significant figure.
The quantitative methods of the invention preferably use a forward primer which comprises or consists of the sequence shown in SEQ ID NO: 1, a reverse primer which comprises of consists of the sequence shown in SEQ ID NO: 2 and a probe which comprises or consists of the sequence shown in SEQ ID NO: 3. The probe is preferably a TaqMan® probe. The method is preferably conducted under the same conditions as the Examples. These conditions are discussed above.
Sample
The sample used in the methods of the invention may be any sample. The invention is typically carried out on a sample that is known to contain or suspected to contain RNA virus, such as SARS- CoV-2. Alternatively, the invention may be carried out on any sample whose RNA virus, such as SARS-CoV-2, status is unknown to confirm the presence or absence of RNA virus, such as SARS- CoV-2. The sample may be a biological sample. The sample may be obtained from or extracted from any organism or microorganism. The organism or microorganism may be archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista. The sample may be obtained from or extracted from any virus. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep, fish, chickens or pigs or may alternatively be pets such as cats or dogs. Alternatively, the sample may be of plant origin, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton. The sample may be derived from human or animal food.
The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus, amniotic fluid, blood, plasma or serum. The sample is preferably a nasopharyngeal sample, a saliva sample (also known an oropharyngeal sample) or a blood sample.
The sample may be a non-biological sample. Any non-biological sample can be tested. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include, but are not limited to, surgical fluids, water such as drinking water, sea water or river water, reagents for laboratory tests and wet swabs of surfaces or materials.
The sample is typically processed prior to being used in the invention, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be tested immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C.
Method of diagnosis of the invention
The invention also provides a method of determining whether or not a patient is infected with an RNA virus, such as SARS-CoV-2. The invention therefore relates to the diagnosis of an RNA virus, such as SARS-CoV-2, infection. The diagnostic method of the invention may be carried out in conjunction with other assays or genetic tests. The method comprises conducting a method of the
invention for determining the presence or absence of an RNA virus, such as SARS-CoV-2, on a sample from the patient. Any of the methods discussed above may be used. The presence of an RNA virus, such as SARS-CoV-2, in the sample indicates the presence of the RNA virus, such as SARS-CoV-2, in the patient. The presence of an RNA virus, such as SARS-CoV-2, in the sample indicates the patient is infected with the RNA virus, such as SARS-CoV-2. The absence of an RNA virus, such as SARS- CoV-2, from the sample typically indicates the absence of the RNA virus, such as SARS-CoV-2, in the patient. The absence of an RNA virus, such as SARS-CoV-2, from the sample typically indicates the patient is not infected with the RNA virus, such as SARS-CoV-2. The absence of an RNA virus, such as SARS-CoV-2, from the sample may indicate that the particular sample from the patient does not contain the RNA virus, such as SARS-CoV-2, and does not necessarily mean the patient is not infected. The diagnostic method preferably uses a nasopharyngeal sample or a saliva sample (also known as an oropharyngeal sample). The absence of an RNA virus, such as SARS-CoV-2, from these samples does typically indicate the patient is not infected with the RNA virus, such as SARS-CoV-2.
The invention also provides a method of measuring the titre of RNA virus, such as SARS-CoV- 2, in a patient. The invention also provides a method for differentiating between high and low RNA virus, such as SARS-CoV-2, titres in a patient. These methods comprise conducting the quantitative method of the invention for measuring the titre of RNA virus, such as SARS-CoV-2, or for differentiating between high and low RNA virus, such as SARS-CoV-2, titres on a sample from the patient. Low RNA virus, such as SARS-CoV-2, titres, high RNA virus, such as SARS-CoV-2, titres and methods for measuring them are discussed above. Any of these may be used on a sample from the patient.
The quantitative method preferably uses a nasopharyngeal sample or a saliva sample (also known as an oropharyngeal sample).
The diagnostic and quantitative methods preferably comprise taking a sample from the patient before conducting the RT-PCR assay.
Typically, the patient displays the symptoms of infection with the RNA virus, such as SARS- CoV-2, i.e. the patient is known or expected to be infected with the RNA virus, such as SARS-CoV-2. The patient may be asymptomatic, i.e. the patient’s RNA virus, such as SARS-CoV-2, status is unknown or the patient is expected not to be infected with the RNA virus, such as SARS-CoV-2. The patient may be susceptible to, or at risk from, infection with the RNA virus, such as SARS-CoV-2. The patient may have underlying health conditions which make infection with the RNA virus, such as SARS-CoV-2, particularly serious.
The patient is generally a human patient. The patient may be a fetus, a newborn, an infant, a juvenile or an adult.
Therapeutic methods of the invention
The invention provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti-viral treatment.
The invention also provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient. The method comprises (a) identifying the patient as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-viral treatment.
The invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention. The invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient, wherein the method comprises (a) identifying the patient as being infected with the RNA virus, such as SARS-CoV-2, using a method of the invention and (b) administering the substance of composition to the patient. The substance or composition is preferably an anti- viral substance or composition.
The invention also provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention. The method comprises administering to the patient a therapeutically effective amount of an anti- viral treatment. The invention also provides a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient comprising (a) identifying the patient as having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-viral treatment. In a preferred method, the high RNA virus, such as SARS-CoV-2, titre is about 103 virions/pl or higher. In this method, a Cq value of 31.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
In a preferred method, a high RNA virus, such as SARS-CoV-2, titre is about 104 virions/pl or higher. In this method, a Cq value of 25.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
In a preferred method, a high RNA virus, such as SARS-CoV-2, titre is about 105 virions/pl or higher. In this method, a Cq value of 28.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
In a preferred method, a high RNA virus, such as SARS-CoV-2, titre is about 106 virions/pl or higher. In this method, a Cq value of 20.9 or lower indicates the sample has a high RNA virus, such as SARS-CoV-2, titre.
The invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient identified as being having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention. The invention also provides a substance or composition for use in a method of treating an RNA virus, such as SARS-CoV-2, infection in a patient, wherein the method comprises (a) identifying the patient as being having a high RNA virus, such as SARS-CoV-2, titre using a method of the invention and (b) administering the substance or composition to the patient. The substance or composition is preferably an anti-viral substance or composition
Any treatment, substance or composition may be used in the invention. Suitable anti- viral treatments, substances and compositions are well known. The anti-SARS-CoV or anti-SARS-CoV-2 treatment, substance or composition may inhibit viral entry into cells by inhibiting ACE2 receptors, such as ACE2 receptor antibodies or arbidol, or inhibiting TMPRSS2, such as camosat mesylate. The anti-SARS-CoV or anti-SARS-CoV-2 treatment, substance or composition may inhibit 3- chymotrypsin-like protease, such as lopinavir or darunavir. The anti-viral treatment, substance or composition may inhibit viral replication by inhibiting viral RNA-dependent RNA polymerase (RdRP), such as ribavirin, remdesivir or favipiravir. The anti-viral treatment, substance or composition may be an anti-viral small interfering RNA (siRNA) designed to inhibit entry of the virus into cells and/or inhibit viral replication by targeting RNA virus, such as SARS-CoV-2, genes involved in these processes. Some patients infected with RNA virus, such as SARS-CoV-2, especially patients with a high RNA virus, such as SARS-CoV-2, titre, suffer serious immune responses, including cytokine storms. IL-6 has been shown to be partly responsible for such cytokine storms. The anti-RNA virus, such as SARS-CoV-2, treatment, substance or composition may be an anti-IL-6 therapy or antibody, such as tocilizumab or sarilumab. The anti-RNA virus, such as SARS-CoV-2, treatment, substance or composition may be any anti-inflammatory and inhibitor of immune responses. The anti-influenza treatment may be a neuraminidase inhibitors, such as laninamivir, favipiravir, peramivir, zanamivir or oseltamivir, or an inhibitor of the viral M2 protein, such as amantadine or rimantadine.
The invention also provides a method of treating SARS-CoV-2 in a patient identified as having a high SARS-CoV-2 titre using a method of the invention. The method comprises administering to the
patient a therapeutically effective amount of an anti-IL-6 therapy or antibody. The invention also provides a method of treating SARS-CoV-2 in a patient comprising (a) identifying the patient as having a high SARS-CoV-2 titre using a method of the invention and (b) administering to the patient a therapeutically effective amount of an anti-IL-6 therapy or antibody. High SARS-CoV-2 titres and methods of measuring them are discussed above.
The invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient identified as being having a high SARS-CoV-2 titre using a method of the invention. The invention also provides an anti-IL-6 therapy or antibody for use in a method of treating SARS-CoV-2 in a patient, wherein the method comprises (a) identifying the patient as being having a high SARS-CoV-2 titre using a method of the invention and (b) administering the an anti-IL-6 therapy or antibody to the patient.
The invention concerns administering to the patient a therapeutically effective mount of the treatment, substance or composition to the patient. A therapeutically effective amount is an amount which ameliorates one or more symptoms of the RNA virus, such as SARS-CoV-2, infection. A therapeutically effective amount is preferably a number which abolishes one or symptoms of the RNA virus, such as SARS-CoV-2, infection. A therapeutically effective amount may cure or abolish the RNA virus, such as SARS-CoV-2, infection. Suitable amounts are discussed in more detail below.
The composition of the invention may be administered to any suitable patient. Suitable patients are discussed above with reference to the diagnostic embodiments of the invention.
The invention may be used in combination with other means of, and substances for, treating the disease or disorder or providing pain relief In some cases, the treatment, substance or composition may be used in combination with existing treatments for the RNA virus, such as SARS-CoV-2, infection including intensive care treatment and the use of ventilators.
The treatment, substance or composition of the invention may be formulated using any suitable method. Formulation with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. The exact nature of a formulation will depend upon several factors including the composition to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.
The treatment, substance or composition may be administered by any route. Suitable routes include, but are not limited to, enteral or parenteral routes such as via buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical and other appropriate
routes. If the lungs are being treated, the treatment, substance or composition is preferably administered by inhalation.
Pharmaceutical compositions may be prepared together with a physiologically acceptable carrier or diluent. The treatment, substance or composition may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, of the like and combinations thereof.
Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active substance, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.
Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions typically take the form of solutions or suspensions and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.
In addition, if desired, the pharmaceutical compositions of the invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. The composition preferably comprises human serum albumin.
One suitable carrier or diluents is Plasma-Lyte A®. This is a sterile, nonpyrogenic isotonic solution for intravenous administration. Each 100 mL contains 526 mg of Sodium Chloride, USP (NaCl); 502 mg of Sodium Gluconate (C6H1 !NaO7); 368 mg of Sodium Acetate Trihydrate, USP (C2H3NaO2»3H2O); 37 mg of Potassium Chloride, USP (KC1); and 30 mg of Magnesium Chloride,
USP (MgC12*6H2O). It contains no antimicrobial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
If the treatment, substance or composition is a siRNA, the most preferred pharmaceutically acceptable carrier or diluent is a pharmaceutically acceptable transfection reagent. A pharmaceutical acceptable transfection reagent is suitable for administration to patients. The pharmaceutical acceptable transfection reagent may be liposomes, preferably cationic liposomes, polymers, preferably cationic polymers, and dendrimers. The pharmaceutical acceptable transfection reagent is more preferably a pharmaceutically acceptable PEI transfection reagent, such as a linear PEI transfection reagent. The most preferred pharmaceutical acceptable transfection reagent is GMP in v/vo-jetPEI®.
The composition is administered in a manner compatible with the dosage formulation and in such amount will be therapeutically effective. The quantity to be administered depends on the subject to be treated, capacity of the patient’s immune system and the degree of treatment desired. Precise amounts required to be administered may depend on the judgement of the practitioner and may be peculiar to each patient.
Any suitable dose of the treatment, substance or composition may be administered to a patient. The dose may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.01 to 50 mg per kg of body weight, according to the activity of the specific substance, the age, weight and conditions of the subject to be treated and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. For example, from about 0.01 to about 50mg per kg of patient of sRNA may administered, such as from about 0.05 to about 40, from about 0. 1 to about 30, from about 0.5 to about 20, from about 1 to about 10 or from about 2 to about 5 mg per kg. At least about 0.01 mg per kg of patient may administered, such as at least about 0.05, at least about 0. 1, at least about 0.5, at least about 1, at least about 2, at least about 5, at least about 10, at least about 20, at least at least about 30 or at least about 40 mg per kg.
These doses may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily. Other suitable regular intervals include, but are not limited to, every day, every week, every fortnight or every month.
The following Examples illustrate the invention.
Examples
Example 1 - RT-PCR
Ten RT-PCR assays were conducted on Human 2019-nCoV RNA (Purified RNA of Coronavirus strain "BetaCoV/Germany/BavPatl/2020 p.l" grown in cell culture obtained from EVAg: https ://www. european-virus-archive. com/nucleic-acid/human-2019-ncov-ma). In all instances, the primers had the sequences shown in SEQ ID NOs: 1 and 2 and the TaqMan® probe had the sequence shown in SEQ ID NO: 3. The probes and primers were sourced from Biolegio. Reverse transcriptase (Affinity script) and DNA polymerase (GoTaq® G2 by Promega) were used in a 2x mastermix. The reaction mix was lOuL 2x oasig mastermix, 2uL primer/probe mix (resuspended in template preparation buffer) and 8uL sample. The reaction conditions were as follows: (1) one cycle of reverse transcription for 10 minutes at 55°C, (2) one cycle of initial denaturation and Taq activation for 2 minutes at 95°C and (3) 45 cycles of denaturation for 10 seconds at 95°C and annealing and extension for 60 seconds at 60°C.
The threshold level was 10% of the end point fluorescence. The CFX and Lightcycler autocalling was also used.
The results are shown in Tables 1 and 2.
Example 2 - RT-PCR with dilutions
Example 1 was repeated with increasing dilutions of Human 2019-nCoV RNA (Purified RNA of Coronavirus strain "BetaCoV/Germany/BavPatl/2020 p. 1" grown in cell culture obtained from EVAg: https://www. european-virus-archive. com/nucleic-acid/human-2019-ncov-ma). The concentrations shown relate to the concentrations of RNA before it was added to the RT-PCR reaction mix. The results are shown in Figures 1 to 3. The mean Cq values are shown in Tables 3 to 5 below. Table 3 - Figure 1 Cq values
Example 3 - Sample dilution
Example 1 was repeated with a variety of different preparations of the sample. Initial experiments were assessment of PCR inhibition caused by sample preparation to optimise the method. No SARS-Cov-2 RNA was used at this point. Swab samples were prepared in Copan diagnostics universal transport medium (UTM) (366C), Sigma- virucult viral transport medium (VTM) (MW951S), phosphate buffered saline (PBS) and saline. PBS was prepared in house using PBS tablets (P32080) supplied by Melford laboratories. lOOmL of PBS contained: 137mM NaCl, 11.9mM Phosphate buffer, 2.7mM KC1. The saline used was STERETS Normasol, Ref: 99766774, Sodium chloride solution 0.9%w/v.
Each of the swab samples were diluted at the following dilutions: no dilution (neat), 1:5, 1:10, 1:15 and 1 :25. The sample was diluted in a dilution solution shown in Table 6.
Trizma was manufactured in house to pH8.5, IM. lOOrnL of Trizma ph8.5, IM contains: 4.42g Trizma hydrochloride (Sigma-Aldrich, Cat number: T3253) and 8.72g Trizma base, Supplier: Sigma- Aldrich, Cat number: T1503). Glycerol, bidistilled 99.5%, was used (VWR, Cat number: 24388.295). EDTA (0.5M), pH8.0 was used (Thermo Fisher Scientific, Cat number: AM9260G). Nuclease-free water was obtained from Fisher Scientific (Cat number: BP561).
An optimisation solution was prepared in nuclease-free water (Fisher Scientific: Cat number: BP561) according to the formulation shown in Table 7. Table 7 - Formulation of optimisation solution
90uL of the diluted samples produced above were optimised using the 2uL of the optimisation solution shown in Table 7. After this, 20uL of IEC RNA was added to each of the 92uL to provide 112uL samples. 8uL of each diluted and optimised sample containing IEC RNA was tested using direct RT-PCR (as in Example 1). The results of the direct RT-PCR reactions are shown in Table 8.
On the basis of the results in Table 8, additional experiments were conducted with 1:15 dilutions of UTM and VTM samples and 1:5 dilutions of PBS and saline samples.
Example 4 - Sample optimisation
Example 3 was repeated with different optimisations of the sample for RT-PCR. Samples were prepared as in Example 3 and UTM and VTM samples were diluted 1:15 (6uL of sample + 84uL of dilution solution) and PBS and saline samples were diluted 1:5 (18uL sample + 72uL of dilution solution) using the dilution solution shown in Table 6.
An optimisation solution was prepared in nuclease-free water (Fisher Scientific: Cat number: BP561) according to the formulation shown in Table 7 as in Example 3.
The 90uL diluted samples produced above were optimised using the optimisation solution shown in Table 7. 3uL (termed 1.5x), 2uL (termed lx), luL (termed 0.5x) or 0.4uL (termed 0.2x) of the optimisation solution was added to the 90uL of the diluted sample. After this, 20uL of IEC RNA was added to each of the optimised samples. 8uL of each optimised sample containing IEC RNA was then tested in direct RT-PCR (as in Example 1). The results of the direct RT-PCR reactions are shown in Table 9.
On the basis of these results, lx optimisation solution (2uL in 90uL diluted sample) was chosen for further experiments.
Example 5 - Dilution and optimisation
Examples 3 and 4 were repeated. Samples were prepared as in Example 4: UTM and VTM samples were diluted 1:15 (6uL of sample + 84uL of dilution solution) and PBS and saline samples were diluted 1:5 (18uL sample + 72uL of dilution solution) using the dilution solution shown in Table 6 and 2uL of the optimisation solution shown in Table 7 was added to each of the 90uL diluted samples. After this, 20uL of IEC RNA was added to each of the 92uL to provide 112uL samples. 8uL of each diluted and optimised sample containing IEC RNA was tested using direct RT-PCR (as in Example 1). The components in the 8uL sample added to the direct RT-PCR reaction and IEC Cq values are shown in Table 10.
This sample preparation was compared with magnetic bead extraction of RNA using Automated extraction system GenoXtract® from HAIN Lifescience GmbH (Brucker) using GXT DNA/RNA Extraction kit (Catalogue no: 12.01.02, 96 samples) . The IEC was added to the magnetic bead extraction at the same concentration as the direct method to represent a sample at a given concentration going through both workflows. Sample was taken through both methods to directly compare Cq values. The results from RT-PCR testing of magnetic bead extracted RNA is shown in Table 11.
These results confirmed that the direct dilution and optimisation steps in accordance with the invention were comparable to using magnetic bead extraction methods.
Example 6 - Inactivation of the sample
Swabs were prepared in ImL PBS with or without 1% or 5% (by volume) Triton® X-100 or Triton® X-100 (Sigma- Aldrich).
The samples were then prepared exactly as shown in Example 5. Samples were diluted 1:5 using dilution solution (18uL of sample + 72uL of dilution solution), 2uL of optimisation solution was added to the 90uL diluted sample and then 20uL IEC was added. 8uL of the sample was then tested using direct RT-PCR (as in Example 1). The results are shown in Table 12.
These results show that the Triton® X-100 and Triton® X-100 not only inactivate the virus but also improve the RT-PCR reaction.
Example 7 - Version 1 PROmate 1G v2
The sample preparation buffer comprised Trizma, EDTA, polyoxyethylene isooctylcyclohexyl ether (Triton® X-100 reduced) and glycerol (as shown in Table 13 below). The PCR reaction mix and final PCR reaction volume included potassium chloride (KC1) and magnesium chloride (MgC12) (as shown in Table 14 below).
The sample preparation buffer and PCR reaction mix were tested using RNA extracted from heat inactivated SARS-CoV-2 cell culture preparations. Sample preparation buffer was contrived with 1000
copies of SARS-CoV-2 RNA. 5pl of the sample preparation buffer was then removed and pipetted into the PCR reaction tube with 15uL of PCR reaction mix to create a total PCR reaction volume of 20uL.
Internal extraction control (IEC) was present in the PCR reaction mix. The results are shown in Figure 4. Table 13 - Sample preparation buffer composition in 1ml
Example 8 - Version 2 PROmate 2G Example 7 was repeated but there was also an additional internal extraction control (IEC) which was freeze dried with the RNase inhibitor in a bead and which was added to the sample preparation buffer during the workflow. The results are shown in Figure 5.
Claims
1. A kit for extracting RNA from an RNA virus comprising (a) an inactivation solution comprising t-octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced), (b) a dilution solution comprising TRIZMA® (2- Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA) and (c) an optimisation solution comprising potassium chloride (KC1) and magnesium chloride (MgCL).
2. A kit according to claim 1, wherein the inactivation solution comprises from about 0.1% to about 10.0% of Triton® X-100 and/or Triton® X-100 reduced by volume.
3. A kit according to claim 1 or 2, wherein the inactivation solution comprises virus transport medium, phosphate buffered saline (PBS) or saline.
4. A kit according to any one of the preceding claims, wherein the dilution solution comprises from about 5.0mM to about 20.0mM TRIZMA®, from about 0.5mM to about 3.0mM glycerol and from about 0.1 to about l.OmM ethylenediaminetetraacetic acid (EDTA).
5. A kit according to any one of the preceding claims, wherein the optimisation solution comprises from about 0. IM to about 1.0M KC1.
6. A kit according to any one of the preceding claims, wherein the optimisation solution comprises from about 0.5M to about 10M MgCl2.
7. A kit according to any one of the preceding claims, wherein the kit further comprises an internal extraction control (IEC) RNA.
8. A sample preparation buffer for obtaining viral RNA from a sample comprising (a) t- octylphenoxypolyethoxyethanol (Triton® X-100) and/or polyoxyethylene (10) isooctylcyclohexyl ether (Triton® X-100 reduced) and (b) TRIZMA® (2-Amino-2-(hydroxymethyl)-l,3-propanediol), glycerol and ethylenediaminetetraacetic acid (EDTA).
9. A kit for determining the presence or absence of an RNA virus comprising (i) a sample preparation buffer solution according to claim 8 and (ii) a PCR reaction mix comprising potassium chloride (KC1) and magnesium chloride (MgCL).
10. A method of extracting RNA from an RNA virus, if present, in a sample, comprising (a) contacting the sample with an inactivation solution comprising Triton® X-100 and/or Triton® X-100 reduced, (b) diluting the inactivated sample produced in (a) with a dilution solution comprising TRIZMA®, glycerol and EDTA and (c) adding to the diluted sample produced in (b) an optimisation solution comprising KC1 and MgCL and thereby extracting the RNA from the RNA virus, if present.
11. A method according to claim 10, wherein the inactivation solution is as defined in any one of claims 1 to 3.
12. A method according to claim 10 or 11, wherein the dilution solution is as defined in claim 4.
13. A method according to any one of claims 10 to 12, wherein the optimisation solution is as defined in claim 5 or 6.
14. A method according to any one of claims 10 to 13, wherein the inactivation solution in step (a) comprises PBS or saline, step (b) comprises diluting the inactivated sample produced in (a) 1:5 with the dilution solution and step (c) comprises adding 1 part of the optimisation solution to 45 parts of the diluted sample produced in (b).
15. A method according to claim 14, wherein the method further comprises adding 5 parts of nuclease free water or IEC RNA to 23 parts of the optimised sample produced in (c).
16. A method of obtaining viral RNA, if present, from a sample, comprising contacting the sample with a sample preparation buffer according to claim 8 and thereby obtaining the viral RNA, if present, from the sample.
17. A method according to any one of claims 10 to 16, wherein the method does not comprise any additional methods of inactivating the virus and/or extracting RNA from the virus.
18. A method of determining the presence or absence of an RNA virus in a sample, wherein the method comprises (i) extracting RNA from the RNA virus in the sample, if present, or obtaining viral RNA, if present, from the sample using a method according to any one of claims 10 to 17 and (ii) conducting a reverse-transcription polymerase chain reaction (RT-PCR) assay on the sample produced in (i) and thereby determining the presence or absence of the virus in the sample.
19. A method according to claim 18, wherein the method comprises using a kit according to claim 9.
20. A method according to claim 18 or 19, wherein the method further comprises reverse transcribing any RNA in the sample, contacting any cDNA produced with a pair of primers under conditions which allow the primers to amplify their cDNA amplicon and determining the presence or absence of the cDNA amplicon.
21. A method according to any one of claims 18 to 20, wherein the RT-PCR assay is a one-step RT-PCR assay conducted in one tube or vessel.
22. A method according to any one of claims 18 to 21, wherein the sample is a human sample.
23. A method according to any one of claims 18 to 22, wherein the sample is a nasopharyngeal sample, a saliva (or oropharyngeal) sample or a blood sample.
24. A method according to any one of claims 10 to 23, wherein the virus is SARS-CoV-2.
25. A method for measuring the titre of an RNA virus in a sample, if present, wherein the method comprises (i) extracting RNA from the RNA virus in the sample, if present, or obtaining viral RNA, if
present, from the sample using a method according to any one of claims 10 to 17 and (ii) conducting a real time RT-PCR method on the sample produced in (i) and, if the virus is present, evaluating the cycle quantification (Cq) value and thereby measuring the viral titre.
26. A method of determining whether or not a patient is infected with an RNA virus, wherein the method comprises conducting a method according to any one of claims 18 to 24 on a sample from the patient and thereby determining whether or not a patient is infected with the virus.
27. A method of measuring the titre of an RNA virus in a patient, wherein the method comprises conducting a method according to claim 25 on a sample from the patient and thereby measuring the viral titre in the patient.
28. A method according to claim 26 or 27, wherein the method comprises taking a sample from the patient before conducting the RT-PCR assay.
29. A method of treating an RNA virus infection in a patient identified as being infected with the RNA virus using a method according to claim 26, comprising administering to the patient a therapeutically effective amount of an anti-viral treatment and thereby treating the infection in the patient.
30. A substance or composition for use in a method of treating an RNA virus infection in a patient identified as being infected with the RNA virus using a method according to claim 26.
31. A method of treating an RNA virus infection in a patient identified as having a high RNA virus titre using a method according to claim 27, comprising administering to the patient a therapeutically effective amount of an anti-viral treatment and thereby treating the RNA virus infection in the patient.
32. A substance or composition for use in a method of treating an RNA virus infection in a patient identified as being having a high RNA viral titre using a method according to claim 27.
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2021
- 2021-10-20 EP EP21802597.1A patent/EP4232596A1/en active Pending
- 2021-10-20 WO PCT/EP2021/079071 patent/WO2022084381A1/en unknown
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