EP4247981A1 - Method of detecting sars-cov2 - Google Patents
Method of detecting sars-cov2Info
- Publication number
- EP4247981A1 EP4247981A1 EP21820467.5A EP21820467A EP4247981A1 EP 4247981 A1 EP4247981 A1 EP 4247981A1 EP 21820467 A EP21820467 A EP 21820467A EP 4247981 A1 EP4247981 A1 EP 4247981A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- sequence
- seq
- primer
- nucleotides
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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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
- 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 a method for detecting a target nucleic acid sequence of a ribonucleic acid (RNA) from SARS-CoV-2 comprising strand-invasion based DNA amplification.
- RNA ribonucleic acid
- the invention also relates to oligonucleotides, compositions and kits suitable for use in this method, and their use for detection of SARS-CoV-2 in a sample.
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic that has killed over a million individuals, disrupted economies, and continues to spread widely.
- SARS-CoV-2 The disease caused by SARS-CoV-2 is known as coronavirus disease 2019 (COVID-19).
- COVID-19 coronavirus disease 2019
- Nucleic acid amplification tests are becoming the method of choice for routine diagnosis of viral pathogens such as SARS-CoV-2 within clinical laboratory settings. They offer superior sensitivity and specificity over serology, immunoassays and traditional viral culture-based detection methods (Mahony et al. 2011).
- PCR Polymerase chain reaction
- RT-PCR real-time reverse transcription polymerase chain reaction
- a limitation of RT-PCR is that it can be prone to false negative results due to sample derived inhibition, and in particular the RNA extraction step can become a limiting factor in the performance of the method.
- RT-PCR also requires the use of heavy and sophisticated thermal cyclers and skilled personnel, which consequently limits its use for in field or point-of-care applications.
- RT-PCR assays are also timeconsuming, and there remains a need for assays which are capable of detecting the presence of SARS-CoV-2 in a sample which is both rapid, specific and sensitive.
- the present invention relates to detecting the presence of an RNA from SARS- CoV-2 in a sample.
- the method of the invention for detecting the presence of an RNA from SARS-CoV-2 in a sample uses a reverse transcriptase and an upstream primer, a downstream primer, and a strand invasion oligonucleotide, wherein each of said at least one upstream primer, at least one downstream primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence.
- the reverse transcriptase synthesises complementary DNA (cDNA) from the RNA present in the sample, forming double stranded (duplex) nucleic acid molecules.
- the primers and strand invasion oligonucleotide provide for amplification of a target nucleic acid sequence from SARS-CoV-2. If the target nucleic acid sequence from SARS- CoV-2 is present in the duplex nucleic acid molecules formed from the activity of the reverse transcriptase in the method, the strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of the upstream primer and downstream primer, thereby permitting amplification. Typically, the amplification is performed under isothermal conditions, without a requirement for thermal denaturation of duplex nucleic acid molecules.
- RNA to cDNA The reverse transcription of RNA to cDNA is typically carried out simultaneously with strand-invasion based amplification and therefore the detection of the presence of an RNA from SARS-CoV-2 can be performed directly from a sample suspected to comprise SARS-CoV-2 RNA.
- the method of the invention allows for highly specific and sensitive detection of RNA from SARS-CoV-2. Furthermore, the method of the invention is advantageous compared to the conventional RT-PCR method used for detection of RNA in various respects. For example, it can provide for improved sensitivity, reduced detection time and be less susceptible to sample-derived inhibition.
- the inventors have further identified particularly effective target nucleic acid sequences for detection of SARS-CoV-2 employing the methods of the invention, and particularly effective primers and strand invasion oligonucleotides for use in amplification of these target sequences.
- the above target sequences, primers and oligonucleotides may also be used for detection of cDNA previously prepared from samples containing SARS- CoV-2 RNA.
- the invention provides a method for detecting the presence of an RNA from SARS-CoV-2 in a sample, said method comprising contacting said sample with a reverse transcriptase, at least one upstream primer, at least one downstream primer, at least one strand invasion oligonucleotide and a recombinase under conditions promoting amplification of a target nucleic acid sequence from SARS-CoV-2, wherein each of said at least one upstream primer, at least one downstream primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence; and wherein said strand invasion oligonucleotide renders at least a portion of said target nucleic acid sequence single-stranded, to allow the binding of said upstream primer and a downstream primer.
- the invention further provides a method for detecting a target nucleic acid sequence from SARS-CoV-2 in a sample, said method comprising contacting said sample with at least one upstream primer, at least one downstream primer and at least one strand invasion oligonucleotide under conditions promoting amplification of said target nucleic acid sequence, wherein each said primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence; and wherein said strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of said upstream primer and a downstream primer.
- said target sequence from SARS-CoV-2 is from the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2.
- RdRP RNA-dependent RNA polymerase
- the target nucleic acid sequence from SARS-CoV-2 is SEQ ID NO: 4 or a variant of thereof.
- detecting the amplification of said target nucleic acid sequence indicates presence of RNA from SARS- CoV-2 in said sample
- the invention additionally provides a composition or a kit, the composition or kit comprising: (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide, wherein said upstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 1 or a variant thereof, said downstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 2 or a variant thereof, and said strand invasion oligonucleotide is an oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant thereof.
- the invention also provides a method for diagnosis of an infection by SARS-CoV- 2 in a subject, comprising carrying out a method of the invention for detecting the presence of an RNA from SARS-CoV-2 in a sample from said subject.
- Figure 1 - shows multiple sequence alignment of a portion of SARS-CoV-2 genome against homologous portions of sequence SARS-CoV-2 isolates and related respiratory viruses. Highlighted portion shows the chosen amplicon region for the methods of the present invention. Positions marked with “ ” (period/full stop) shows identity.
- MT628280.1 Severe acute respiratory syndrome coronavirus 2 isolate SARS- CoV-2/human/USA/CA-CZB-1629/2020
- NC_045512.2 Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1
- MT655135.1 Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/ESP/HUD-98203801/2020
- MT370892.1 Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV- 2/human/USA/NY-PV08122/2020
- MT415322.1 Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/IND/GMCKN443/2020
- CoVl AY278741.1 SARS coronavirus Urbani
- CoVl AY291451.1 SARS coronavirus TW1
- NL63 MG428704.1 Human coronavirus
- Figures 2-22 show sensitivity of the CVD1 assay.
- Synthetic SARS-CoV-2 genomic RNA (gRNA) template for assay development was purchased from Twist Bioscience.
- Figures 2-20 show assay performance using different primer pairs in combination with the CVD1-IO1 strand invasion oligonucleotide (with reference to Table 1 of Example 2 and Table 2 of Example 3 below):
- Figures 21 and 22 shows a separate comparison of the two tested strand invasion oligonucleotides for the CVD1 assay (CVD1-IO1 and CVD 1-IO2), which were tested with the F3 and R9 primer pairing.
- Figure 23 shows the performance of an LNA containing oligonucleotide probe for the CVD1 assay.
- the sample comprises RNA. If the RNA is present in the sample in a suitable form allowing for detection according to the invention, the sample may be used directly.
- a sample e.g. a sample from a subject, in particular a subject suspected of being infected with SARS-CoV- 2
- the processing may be by heating or treatment with detergents.
- the SARS-CoV-2 RNA may also be derived, obtained or extracted from the sample.
- RNA nucleic acids
- extracting nucleic acids in particular RNA
- purifying nucleic acids in particular RNA
- Total nucleic acid may be isolated or RNA may be isolated separately.
- a sample is processed in an appropriate manner such that the RNA in the sample is provided in a convenient form for contacting with the reverse transcriptase, primers and strand invasion oligonucleotide.
- the sample is a clinical sample, for example a sample obtained from a patient suspected of having an infection by SARS-CoV-2.
- the RNA may be genomic RNA of SARS-CoV-2.
- RNA from SARS-CoV-2 is typically single-stranded.
- RNA can be obtained or derived from the sample.
- reference samples for example for particular pathogens/particular viral strains
- environmental samples may be used in the present invention.
- Suitable environmental samples may be samples from wastewater. Suitable types of clinical sample vary according to the particular type of infection that is present, or suspected of being present in a subject.
- the sample may be blood, plasma, saliva, serum, sputum, urine or a stool sample. Samples from a subject suspected of being infected with SARS-CoV-2 may be collected by nose-throat swab from nasal and pharyngeal cavity.
- samples may be an aspirate from a subject, such as an endotracheal aspirate or a nasopharyngeal aspirate.
- the sample may be stored in universal transport media (UTM).
- UDM universal transport media
- the samples are taken from animal subjects, such as mammalian subjects.
- the samples will commonly be taken from human subjects, but the present invention is also applicable in general to domestic animals, livestock, birds and fish.
- the invention may be applied in a veterinary or agricultural setting.
- the sample (e.g. from a subject) may be a sample which has been processed such that the RNA has already been reverse transcribed and/or converted to cDNA.
- the methods of the invention does not comprise a step of reverse transcription and/or contacting the sample with a reverse transcriptase.
- a suitable sample of cDNA or dsDNA amplified from the starting RNA is provided as a template.
- the sample is contacted with a reverse transcriptase or any other enzyme (such as a polymerase) having reverse transcriptase activity under conditions promoting reverse transcription of the RNA present in the sample into cDNA.
- Suitable conditions include any conditions used to provide for activity of reverse transcriptase enzymes known in the art.
- the conditions typically include the presence of one or more species of primer, dNTPs (dATP, dTTP, dUTP, dCTP and dGTP) or analogues thereof, suitable buffering agents/pH and other factors which are required for enzyme performance or stability.
- the primer may be oligo d(T).
- a mixture of random primers (such as random hexamers) may be used.
- the same primers to be used for amplification of the target nucleic acid sequence may be used also for priming reverse transcription of the RNA.
- the conditions may also include the presence of detergents and stabilising agents.
- the conditions may also include additional proteins.
- Preferably the additional proteins may be single-strand DNA binding proteins.
- the temperature used is typically isothermal, i.e constant throughout the reverse transcription process. Preferably, the temperature is identical to use for amplification of the target nucleic acid sequence, such that reverse transcription and amplification occur simultaneously in the same reaction temperature, without need for temperature cycling (as in RT-PCR), This provides significant benefits for speed of detection.
- the temperature used typically depends on the nature of the reverse transcriptase enzyme and other enzyme components, and also reflects the hybridisation temperature required for the primer(s).
- Suitable reverse transcriptases include Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and functional fragments or variants thereof. Particular suitable reverse transcriptases include Improm, GoScript, MMuLV, AMV, RevertAid transcriptases. In some embodiments, a polymerase with reverse transcriptase activity may also be used. Suitable polymerases with reverse transcriptase activity are known in the art, such as GspSSD LF DNA Polymerase.
- the target nucleic acid sequence is a region (or amplicon) suitable for detection by strand-invasion based amplification.
- the selection of suitable target nucleic acid sequences, and the consequent design of primer and strand invasion oligonucleotides for detection of those sequences is an important consideration. Examples of appropriate sequences are provided herein.
- the target nucleic acid sequences exemplified herein are presented as DNA sequences. However, it should be understood that any DNA sequence described herein also encompasses its RNA equivalent. Thus for example the target sequence of SEQ ID NO: 4 has as its RNA equivalent SEQ ID NO: 31. Accordingly, where an RNA is detected in accordance with the invention, this RNA will include the target nucleic acid sequence in RNA form. This RNA sequence will be reverse transcribed to cDNA and then subject to amplification, such that the target nucleic acid sequence which acts as the template for the primers and strand invasion oligonucleotide will typically be in DNA form.
- the target nucleic acid sequence will be unique to its genome. This allows for a highly qualitative, unambiguous determination of the presence of SARS-CoV-2 in the sample, even if closely related pathogens (such as other coronaviruses) exist.
- the target nucleic acid sequence will thus typically differ from any homologous nucleic acid sequence in a related species.
- the target nucleic acid sequence will comprise several mismatches with a homologous nucleic acid sequence in a related species.
- the target nucleic acid sequence is not present in any other type of virus, or not present in any other coronavirus or respiratory virus. More preferably, the target nucleic acid sequence is specific to SARS-CoV-2.
- the target nucleic acid sequence or amplicon is of a sufficient length to provide for hybridisation of the upstream and downstream primers and strand invasion oligonucleotide in a suitable manner to different portions of the target sequence.
- the amplicon is at least 45 nucleotides in length, more preferably at least 50, at least 55, at least 60, at least 65 or at least 70 nucleotides in length, as measured from the 5’ site of binding of the upstream primer to the 5’ site of binding of the downstream primer.
- the amplicon may be about 45 to about 85, about 55 to about 80, or about 65 to about 75 nucleotides in length.
- an amplicon may be selected which is to be invaded at two separate upstream and downstream locations by one or more strand invasion oligonucleotides, as described in WO/2015/185655, incorporated herein by reference. This permits use of longer amplicons, such as those of about 70 to about 500 nucleotides in length, about 70 to about 400, about 100 to about 400, or about 100 to about 200 nucleotides in length.
- the target nucleic acid sequence may be present in any region of the viral genome, provided it has the necessary characteristics for specific detection of the virus as discussed above.
- the target nucleic acid sequence is from the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2.
- the target nucleic acid sequence for detection of SARS-CoV-2 preferably comprises SEQ ID NO: 4 or a variant thereof. It should be understood that the target nucleic acid sequence which provides the template for amplification will be present in a duplex (for example, the duplex formed through the reverse transcription) which comprises a sense strand representing SEQ ID NO: 4 or a variant thereof and a complementary anti-sense strand. The upstream and downstream primers used to amplify the target nucleic acid sequence bind to opposing strands of this duplex.
- the target nucleic acid sequence may comprise a naturally occurring variant sequence of SEQ ID NO: 4 which is present in a different strain.
- Variants of SEQ ID NO: 4 may comprise a region which is partly or fully complementary or identical to at least 35 contiguous nucleotides, more typically at least 40 or at least 45 contiguous nucleotides, preferably at least 55, at least 60 or at least 65 contiguous nucleotides of SEQ ID NO: 4.
- the variants may comprise a region which has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 mismatches (substitutions) with respect to a region of the corresponding original target sequence of SEQ ID NO: 4.
- the variants may comprise a region of at least 35 nucleotides in length which has 1, 2, 3, 4, 5, or 6 mismatches, such as 1-3 or 1-5 mismatches, to a corresponding region of at least 35 contiguous nucleotides of the corresponding original target sequence.
- the variants may comprise a region of at least 45, 55, or 65 nucleotides in length which has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as 1-5 or 1-8 mismatches to a corresponding region of an equivalent length in the corresponding original target sequence.
- Any mismatches in the variant sequence may be at least 2, at least 4, at least 5, or at least 10 nucleotides apart.
- the variants may comprise a region of at least 35, at least 45, at least 55, or at least 65 nucleotides in length which is in full complementarity or identity with the original target sequence.
- the variants comprise only one or two mismatches to SEQ ID NO: 4 in a region corresponding to the strand-invasion oligonucleotide-complementary region of SEQ ID NO: 4. More preferably, the variants comprise a region which is in full complementarity or identity with the strand-invasion oligonucleotide-complementary region of SEQ ID NO: 4.
- the target nucleic acid sequence comprises or consists of SEQ ID NO: 4 and a complementary antisense strand.
- a target nucleic acid sequence from SARS- COV-2 to be amplified may comprise SEQ ID NO: 4 (or a variant thereof) and additional contiguous nucleotides at the 5’ and/or the 3’ termini.
- the additional contiguous nucleotides may be identical to the corresponding nucleotides from the RNA- dependent RNA polymerase (RdRP) gene of SARS-CoV-2.
- a target nucleic acid sequence from SARS-CoV-2 may be SEQ ID NO: 30. In some embodiments, a target nucleic acid sequence from SARS-CoV-2 may be the RNA equivalent of the sequences described herein, such as SEQ ID NO: 31 or SEQ ID NO: 32.
- More than one target nucleic acid sequence may be detected in a method of the invention, by providing two or more sets of upstream primer, downstream primer and strand invasion oligonucleotide, each set adapted for detection of a different target nucleic acid sequence.
- Suitable upstream and downstream primers are selected based on the target nucleic acid sequence of interest, and having regard to the site of binding of the strand invasion oligonucleotide that renders at least a portion of the target nucleic acid sequence singlestranded to allow the binding of the upstream primer and downstream primer.
- the upstream and downstream primers comprise a sequence that is partly or fully complementary or identical to the target and optionally a 5’ and/or 3’ flanking non- complementary sequence.
- the upstream and downstream primers may consist entirely of partly or fully complementary or identical sequence to the target.
- the length of the primer sequence that is complementary or identical to the target is sufficient to provide specific hybridisation to the target nucleic acid sequence.
- the length of complementary or identical sequence is typically at least 10 nucleotides, more preferably at least 15, at least 16, or at least 17 nucleotides.
- the length of complementary or identical sequence may be 10-25, 15-25, 10-30 or 15-30 nucleotides.
- the length of complementary or identical sequence may most preferably be 15-20 nucleotides.
- sequence lengths refer to portions of the primers which may be partly or fully complementary or identical to the target nucleic acid sequence. Mismatches may be present between the primers and the target sequence at particular positions while still allowing for specific amplification and detection of the target sequence, in particular having regard to the combined use of upstream and downstream primers and a strand invasion oligonucleotide to achieve amplification. There may be 1, 2, 3, 4 or 5 mismatches between the complementary or identical region of the primer and the corresponding region of the target sequence.
- the primer is designed to allow for specific detection of SARS-CoV-2.
- the primer typically specifically or selectively hybridises to a complementary sequence found only in the pathogen of interest.
- the primer may also hybridise to other sequences, such as sequences found in other pathogens, provided that when used in combination with the second primer and strand invasion oligonucleotide, specific amplification of a sequence found only in the pathogen of interest is obtained.
- Specific or selective hybridisation refers to the binding of a primer only to a particular nucleotide sequence under given conditions, when that sequence is present in a nucleic acid in a sample, such as a complex biological mixture including total cellular and foreign DNA or RNA.
- Appropriate hybridisation conditions are known in the art. See for example, Sambrook, Fritsche and Maniatis “Molecular Cloning: A Laboratory Manual”, 2nd Ed. Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety. Appropriate hybridisation conditions are also provided in the Examples below. As is known to the skilled person, appropriate hybridisation conditions may vary depending on the length of a primer and its base composition. Hybridisation is typically performed at the same temperature as amplification, and thus also depends on the activity profile of the polymerase and recombinase enzymes employed.
- the upstream and downstream primer will be less than 30 nucleotides in total in length, more preferably less than 25 nucleotides in length, such as 15 to 25, or 15 to 23 nucleotides in length. It is particularly preferred that primers of less than 30 nucleotides in length are used where a recombinase is used for strand invasion. The primers are not capable of acting as substrates for recombinases.
- the upstream (or forward) primer binds to the 5’ region of one strand of the duplex target nucleic acid sequence, at a position proximal or overlapping with the 5’ binding site of the strand invasion oligonucleotide.
- the downstream (or reverse) primer binds to the 5’ region of the opposing strand of the duplex target nucleic acid sequence to the upstream primer, at a position proximal or overlapping with the 3’ binding site of the strand invasion oligonucleotide.
- the 5’ binding sites of the upstream and downstream primers are typically at least 45 nucleotides, more preferably at least 50, at least 55, at least 60, or at least 65 nucleotides apart on the duplex target sequence.
- the upstream and/or downstream primer may have a region of sequence overlap with the sequence of the strand invasion oligonucleotide.
- the region of sequence overlap is typically 1-8 nucleotides in length, preferably 4-6 nucleotides in length, and may be at least 3, at least 4, at least 5 or at least 6 nucleotides in length.
- the downstream primer may also have correspondingly defined regions of sequence overlap with the sequence of the strand invasion oligonucleotide.
- the primer binding instead at a position that is proximal in the target sequence to the binding site of the strand invasion oligonucleotide.
- a primer binds proximal to the strand invasion oligonucleotide
- upstream and downstream primers for binding to a target nucleic acid sequence from SARS-CoV-2 are provided herein.
- Preferred upstream and downstream primers for detection and amplification of the target nucleic acid sequence of SEQ ID NO: 4 are the upstream (forward) primer of SEQ ID NO: 1 (or variants thereof) and the downstream primer of SEQ ID NO: 2 (or variants thereof).
- Variants of SEQ ID NO: 1 or 2 may be oligonucleotides of up to 30 nucleotides in length comprising a region which is partly or fully complementary or identical to at least 10 contiguous nucleotides of the corresponding original primer sequence of SEQ ID NO: 1 or 2 respectively.
- said variants of SEQ ID NO: 1 or 2 will comprise a region which is partly or fully complementary or identical to at least 11, 12, 13, 14 or 15 contiguous nucleotides of the corresponding original primer sequence of SEQ ID NO: 1 or 2 respectively.
- the above variants may comprise a region which has 1, 2, 3, 4, or 5 mismatches (substitutions) with respect to the corresponding region of the original primer sequence (and thus the target sequence) and thus is partly complementary or identical thereto.
- the variants may comprise a region of at least 10 nucleotides in length which has 1, 2, or 3 mismatches, such as 1 or 2 mismatches to a corresponding region of at least ten contiguous nucleotides of the corresponding original primer sequence.
- the variants may comprise a region of at least 13, 14 or 15 nucleotides in length which has 1, 2, 3, 4 or 5 mismatches, such as 1-3 mismatches to a corresponding region of an equivalent length in the corresponding original primer sequence.
- Any mismatches in the variant primer sequence may be at least 2, at least 4, at least 5, or at least 10 nucleotides apart.
- the variants may comprise a region of at least 10, 11, 12, 13, 14 or 15 nucleotides in length which is in full complementarity or identity with the original primer sequence.
- Variants of SEQ ID NO: 1 or 2 may also be oligonucleotides of up to 30 nucleotides in length which have at least 70% sequence identity to the sequence of the corresponding original primer sequence, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, at least 95% sequence identity.
- upstream and downstream primers for detection and amplification of the target nucleic acid sequence of SEQ ID NO: 4 are the upstream (forward) primers of SEQ ID NOs: 7, 11, 12 and 14 and the downstream primer of SEQ ID NO: 23.
- upstream primers of SEQ ID NOs: 7, 11, 12 and 14
- downstream primer of SEQ ID NO: 23 are the upstream primers of SEQ ID NOs: 23, 12, 13, 12 and 14 and the downstream primer of SEQ ID NO:
- the upstream primer comprises any one of the sequence of SEQ ID Nos: 7, 11, 12 and 14;
- the downstream primer comprises the sequence of SEQ ID NO: 23.
- the upstream primer consists of any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14;
- the downstream primer consists of the sequence of SEQ ID NO: 23.
- the upstream primer comprises SEQ ID NO: 7;
- the downstream primer comprises the sequence of SEQ ID NO: 23.
- the upstream primer consists of SEQ ID NO: 7;
- the downstream primer consists of SEQ ID NO: 23.
- variant primers may comprise region(s) complementary or identical to the 5’ or 3’ flanking nucleotide sequence(s) to the binding region of the original primers in the gene comprising the target nucleic acid sequence, such as 5-10 nucleotides from the 5’ flanking region and/or 3-region.
- the variant primers may additionally comprise sequence unrelated to the target sequence.
- Any upstream or downstream primer used in the invention may comprise one or more modified nucleotides and/or a detectable label, for example a fluorescent dye.
- a suitable strand invasion oligonucleotide is selected based on the target nucleic acid sequence of interest, and having regard to the site of binding of the upstream and downstream primers and the requirement for the strand invasion oligonucleotide to render the target nucleic acid sequence single-stranded in the relevant regions to allow for the binding of the upstream primer and downstream primer.
- the strand invasion oligonucleotide comprises a sequence that is complementary or identical to the target and optionally additional flanking non-complementary or nonidentical sequence(s).
- the length of the sequence that is complementary or identical to the target may be determined by the skilled person empirically and is sufficient to provide for efficient strand invasion of the target nucleic acid sequence, optionally under isothermal conditions.
- the complementary sequence may comprise RNA-DNA complementary base pairing and modified nucleotides.
- the length of complementary or identical sequence is at least 25 or at least 27 nucleotides, typically at least 30 nucleotides, such as least 32, at least 33 or at least 35 nucleotides, more preferably at least 36, 37, 38, 39 or 40 nucleotides in length or greater.
- the length of complementary or identical sequence may be 30-50, 32-50, 35-50, 40-50, 35 to 48, 35 to 46, 38 to 45 or 40 to 45 nucleotides in length.
- sequence lengths refer to a portion of the strand invasion oligonucleotide which may be partly or fully complementary or identical to the target nucleic acid sequence. Mismatches may be present between the strand invasion oligonucleotide and the target sequence at particular positions while still allowing for specific amplification and detection of the target sequence, in particular having regard to the combined use of upstream and downstream primers and a strand invasion oligonucleotide to achieve amplification. There may be 1, 2, 3, 4, 5, 6, 7, or 8 mismatches between the complementary or identical region of the strand invasion oligonucleotide and the corresponding region of the target sequence, depending on the total length of complementary or identical sequence. Preferably though there is only 1 or 2 mismatches between the strand invasion oligonucleotide sequence and the corresponding region of the target sequence.
- the complementary or identical sequence of the strand invasion oligonucleotide is designed to allow for specific detection of SARS-CoV-2.
- the strand invasion oligonucleotide preferably specifically or selectively hybridises to a complementary sequence found only in SARS-CoV-2.
- the strand invasion oligonucleotide may also hybridise to other sequences, such as sequences found in other pathogens, provided that when used in combination with the primers, specific amplification of a sequence found only in SARS-CoV-2 is obtained.
- the complementary or identical sequence of the strand invasion oligonucleotide hybridises to a portion of the target sequence intervening the binding regions for the upstream and downstream primers (and typically overlapping with one or more thereof).
- the strand invasion oligonucleotide may have a region of overlap of 1-8 nucleotides, preferably 4-6 nucleotides in length, such as a region of at least 3, at least 4, at least 5 or at least 6 nucleotides in length, with the upstream and/or downstream primers.
- the 5’ portion of the complementary or identical sequence of the strand invasion oligonucleotide typically binds within 25 nucleotides or less, more preferably 20 nucleotides or less from the 5’ boundary of the duplex target nucleotide sequence to be melted (the amplicon).
- the strand invasion oligonucleotide optionally further comprises non- complementary or non-identical sequence region(s) to the target that flank the complementary or identical sequence region.
- the strand invasion oligonucleotide may comprise a non-complementary or non-identical 5’ region which may be of any nucleotide sequence.
- the 5’ non-complementary region may assist binding of recombinase and is also referred to herein as the “seeding” region.
- the 5’ non-complementary region is typically at least 3 nucleotides in length, more typically at least 6, at least 8, at least 10 nucleotides in length. More preferably, the 5’ non-complementary region is of at least 12, most preferably at least 14 nucleotides in length. The 5 ’non-complementary region may be greater than 14 nucleotides in length and may be up to 20 nucleotides in length. The 5’ non-complementary region may be 10-20, 10-17, 10-14, 12-20, 12-17 or 12-15 nucleotides in length, more preferably about 12 to about 20 or about 14 to about 17 nucleotides in length.
- the 5’ non-complementary region may be of any nucleotide composition but preferably is selected to have particular nucleotide compositions described herein as being advantageous in the invention.
- the 5’ non-complementary region comprises a greater number of pyrimidine nucleotides (cytosine or thymidine) compared to purine nucleotides (adenine and guanine).
- the 5’ non-complementary region may comprise at least 60%, at least 70%, at least 80%, or at least 90% pyrimidine nucleotides (cytosine or thymidine) compared to purine nucleotides (adenine and guanine).
- the 5’ non-complementary region comprises or consists essentially of pyrimidine (cytosine or thymidine) nucleotides.
- the non-complementary region may comprise, consist or consist essentially of a mixture of cytosine and thymidine nucleotides.
- a mixture of pyrimidine nucleotides is present, a greater number of cytosine residues than thymidine residues is present.
- the non-complementary region consists essentially of, or consists of, cytosine nucleotides.
- the 5 ’non-complementary region is of about 12 to about 20 nucleotides in length and comprises, consists or consists essentially of a mixture of cytosine and guanine nucleotides. In particularly preferred embodiments, the 5’non-complementary region is of about 14 to about 20 nucleotides in length and comprises, consists or consists essentially of cytosine nucleotides. In some embodiments, the 5’non-complementary region is about 14 nucleotides in length and consists or consists essentially of cytosine residues.
- the strand invasion oligonucleotide may be designed with a seeding region which is shorter than 14 nucleotides in length if the 5’ portion of the complementary or identical sequence of the strand invasion oligonucleotide has a high proportion of pyrimidine nucleotides.
- the strand invasion oligonucleotide may be designed with a seeding region which is shorter than 14 nucleotides (such as a seeding region of 13, 12, 11, 10, 9 or 8 nucleotides) if the first 10 nucleotides at the 5’ portion of the complementary or identical sequence of the strand invasion oligonucleotide comprises at least 6 pyrimidine nucleotides.
- the strand invasion oligonucleotide is typically at least 30 nucleotides in length where a recombinase is used in conjunction with the oligonucleotide.
- the strand invasion oligonucleotide is preferably at least 35, at least 40 or at least 45 nucleotides in length, more preferably at least 50, and may be at least 55 nucleotides in length or greater.
- the strand invasion oligonucleotide may be 40-70, 45-70, 45-70, 50-70, 55-70, 45-65, 50-65, 50-60 or 55-65 nucleotides in length.
- the strand invasion oligonucleotide may comprise a 3’ non-compl ementary region typically of 1-3 nucleotides in length which comprises nucleotides which block polymerase extension such as invdT.
- typically the strand invasion oligonucleotide has a non-extendible 3 ’terminus, such that it cannot serve as a substrate for DNA amplification, and the target sequence is then only amplified on the further binding of the specific upstream and downstream primers. This avoids formation of non-specific amplification products.
- the strand invasion oligonucleotide may comprise one, two, three, four, five, six, seven, eight or more modified nucleotides in its 3’region, such as in the 10- 15 or 10-20 nucleotides from the 3 ’terminus.
- the strand- invasion oligonucleotide may comprise a 3’ modification of the 3 ’terminal nucleotide, and may be a dideoxynucleotide, or comprise a 3 ’amino-allyl group, a 3 ’carbon spacer, 3 ’phosphate, 3 ’biotin, 3 ’sialyl, or 3 ’thiol.
- the 3’ nucleotide may be a nucleotide incorporated in a reversed orientation by a 3 ’-3’ linkage.
- the 3’ region of the strand-invasion oligonucleotide may comprise nucleotides with poor substrate capability for DNA polymerases, such as PNA (peptide nucleic acid) nucleotides, LNA (locked nucleic acid), 2’-5’ linked DNA or 2’-O-methyl RNA, or combinations thereof.
- the strand-invasion oligonucleotide is a PNA oligomer comprised wholly of PNA
- such an oligonucleotide can destabilise and invade duplex DNA in the absence of a recombinase enzyme.
- the methods of the invention may be performed without presence of a recombinase enzyme.
- a strand invasion oligonucleotide suitable for detection of the target nucleic acid sequence of SEQ ID NO: 4 comprises SEQ ID NO: 3.
- a strand invasion oligonucleotide suitable for detection of the target nucleic acid sequence of SEQ ID NO: 4 is SEQ ID NO: 24.
- a particularly preferred strand invasion oligonucleotide is a modified derivative of SEQ ID NO: 24, most preferably SEQ ID NO: 25.
- a strand invasion oligonucleotide used in the invention comprises one or more modified oligonucleotides in its 3 ’region to block its use as a polymerase substrate.
- a modified derivative of SEQ ID NO: 3 or 24 may comprise one, two, three, four, five, six, seven, eight or more modified nucleotides in its 3’region, typically in the 10-15 or 10-20 nucleotides from the 3’terminus. The modifications may be selected from any of those discussed above.
- the modified derivative may be a PNA oligomer of corresponding sequence to SEQ ID NO: 3 or 24.
- variant strand invasion oligonucleotides may be used.
- Variants of SEQ ID NOs: 3, 24 and 25 are typically oligonucleotides of greater than 30 nucleotides, more preferably at least 35, at least 40, or at least 45 nucleotides in length, comprising a region which is partly or fully complementary or identical to at least 30 contiguous nucleotides of the corresponding original target-binding sequence present in SEQ ID NOs: 3, 24 and 25.
- the target-binding sequence of SEQ ID NOs: 3, 24 and 25 is the region which has complementarity or identity to the target.
- said variants will comprise a region which is partly or fully complementary or identical to at least 32, 35, 37, 40, 42 or 45 contiguous nucleotides of the target-binding sequence present in SEQ ID NOs: 3, 24 and 25.
- the above variants may comprise a region which has 1, 2, 3, 4, 5, 6, 7 or 8 mismatches (substitutions) with respect to the corresponding target-binding region of the original strand invasion oligonucleotide of SEQ ID NOs: 3, 24 or 25 (and thus the target sequence) and thus is partly complementary or identical thereto.
- the variants may comprise a region of at least 30 nucleotides in length which has 1, 2, 3, or 4, such as 1-4 or 1-3 mismatches to a corresponding region of at least 40 contiguous nucleotides of the corresponding original strand invasion oligonucleotide.
- the variants may comprise a region of at least 35, 40, 42, or 45 nucleotides in length which has 1, 2, 3, 4, 5 or 6, such as 1-5, or 1-3 mismatches to a corresponding region of an equivalent length in the corresponding original strand invasion oligonucleotide.
- Any mismatches in the variant strand invasion oligonucleotide sequence may be at least 2, at least 4, at least 5, or at least 10 nucleotides apart.
- the variants may comprise a region of at least 32, 35, 37, 40, 42 or 45 nucleotides in length which is in full complementarity or identity with the target-binding region of the original strand invasion oligonucleotide.
- Variants of SEQ ID NOs: 3, 24 and 25 may also be oligonucleotides of greater than 30 nucleotides in length comprising a target-binding region which has at least 70% sequence identity to the target-binding sequence of the corresponding original strand invasion oligonucleotide, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, at least 95% sequence identity.
- variant strand invasion oligonucleotides may comprise additional sequence(s) complementary or identical to the 5’ and/or 3’ flanking nucleotide sequence(s) to the binding region for the original strand invasion oligonucleotide in the gene from which the target nucleic acid sequence is derived, such as 5-10 or 5-15 nucleotides from the 5’ flanking region and/or 3-region.
- the remaining sequence of the variant strand invasion oligonucleotides is typically unrelated to the target sequence, and also typically unrelated to the original strand invasion oligonucleotide.
- the variant strand invasion oligonucleotides further comprise one or more modified oligonucleotides in their 3 ’region such as, two, three, four, five, six, seven, eight or more modified nucleotides, which may be in the 10-15 or 10-20 nucleotides from the 3’terminus
- modified oligonucleotides such as, two, three, four, five, six, seven, eight or more modified nucleotides, which may be in the 10-15 or 10-20 nucleotides from the 3’terminus
- the modifications may be selected from any of those discussed above.
- a strand invasion oligonucleotide of the invention may further comprise a detectable label, for example a fluorescent dye.
- the nucleic acid derived from the sample is contacted with the upstream and downstream primers and the strand invasion oligonucleotide for detection purposes, under conditions promoting amplification of the target nucleic acid sequence.
- Such conditions typically comprise the presence of a DNA polymerase enzyme.
- Suitable conditions include any conditions used to provide for activity of polymerase enzymes known in the art.
- the conditions typically include the presence of all dNTPs (dATP, dTTP, dCTP, dUTP and dGTP or analogues thereof), suitable buffering agents/pH and other factors which are required for enzyme performance or stability.
- the conditions may include the presence of detergents and stabilising agents.
- the temperature used is typically isothermal, i.e constant throughout the method of detection and/or amplification process of the invention. The temperature used typically depends on the nature of the polymerase enzyme and other enzyme components, and also reflects the hybridisation temperature required for the primers and strand invasion oligonucleotides.
- a suitable temperature is from around 41 °C to around 45 °C, preferably from around 42 °C to around 44 °C.
- the polymerase used typically has strand-displacement activity.
- strand displacement is used herein to describe the ability of a DNA polymerase, optionally in conjunction with accessory proteins, to displace complementary strands on encountering a region of double stranded DNA during DNA synthesis.
- Suitable DNA polymerases include poll from E. coli. B. siibliHs. or B. stearothermophilus, and functional fragments or variants thereof, and T4 and T7 DNA polymerases and functional fragments or variants thereof.
- a preferred polymerase is Bsu DNA polymerase or a functional fragment or variant thereof.
- the conditions may further comprise the presence of a recombinase.
- a recombinase system may be used in the method of the invention.
- the recombinase system may be of prokaryotic or eukaryotic origin, and may be bacterial, yeast, phage, or mammalian.
- the recombinase may polymerise onto a single-stranded oligonucleotide in the 5 ’-3’ or 3 ’-5; direction.
- the recombinase may be derived from a myoviridae phage, such as T4, T2, T6, Rb69, Aehl, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rbl4, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rbl6, Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3, or phage LZ2.
- a myoviridae phage such as T4, T2, T6, Rb69, Aehl, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rbl4, Rb32, Aeromonas phage 25, Vibrio phage
- the T4 recombinase UvsX (Accession number: P04529) or a functional variant or fragment thereof is used.
- the Rad systems of eukaryotes or the recA-Reco system of E. coli or other prokaryotic systems may also be used.
- the conditions may further comprise the presence of recombinase accessory proteins, such as single-stranded binding protein (e.g. gp32, accession number P03695) and recombinase loading agent (e.g. UvsY, accession number NP 049799.2).
- recombinase accessory proteins such as single-stranded binding protein (e.g. gp32, accession number P03695) and recombinase loading agent (e.g. UvsY, accession number NP 049799.2).
- the conditions comprise the presence of the T4 gp32, UvsX and UvsY proteins.
- the recombinase (such as UvsX), and where used the recombinase loading agent (such as UvsY) and single stranded DNA binding protein (such as gp32), can each be native, hybrid or mutant proteins from the same or different myoviridae phage sources.
- a native protein may be a wild type or natural variant of a protein.
- the conditions may further comprise other factors used to enhance the efficiency of the recombinase such as compounds used to control DNA interactions, for example proline, DMSO or crowding agents which are known to enhance loading of recombinases onto DNA (Lavery P et. Al JBC 1992, 26713, 9307-9314; W02008/035205).
- other factors used to enhance the efficiency of the recombinase such as compounds used to control DNA interactions, for example proline, DMSO or crowding agents which are known to enhance loading of recombinases onto DNA (Lavery P et. Al JBC 1992, 26713, 9307-9314; W02008/035205).
- the conditions may also comprise the presence of an ATP regeneration system.
- ATP regeneration systems are known to the person skilled in the art, and include glycolytic enzymes.
- Suitable components of an ATP regeneration system may include one or more of phosphocreatine, creatine kinase, myokinase, pyrophosphatase, sucrose and sucrose phosphorylase.
- the conditions may further comprise the presence of ATP.
- magnesium ions such as magnesium ions, DTT or other reducing agents, salts, BSA/PEG or other crowding agents may also be included.
- the various components described above, inclusive of the primers and strand invasion oligonucleotide, may be provided in varying concentrations to provide for DNA amplification.
- the skilled person can select suitable working concentrations of the various components in practice.
- detecting the amplification of said target nucleic acid sequence indicates presence of RNA from SARS-CoV-2 in said sample.
- a method of the present invention may comprise additional steps of monitoring and/or detecting the amplification of the target nucleic acid sequence.
- One or both of the primers, or the strand invasion oligonucleotide may incorporate a label or other detectable moiety. Any label or detectable moiety may be used. Examples of suitable labels include radioisotopes or fluorescent moieties, and FRET pairs of a fluorophore and acceptor moiety. Alternatively, or additionally one or more probes that detect the amplified DNA may be used, again incorporating a label or other detectable moiety. The probes may bind at any suitable location in the amplicon. Probes detecting different amplified target sequences may signal at different fluorescent wavelengths to provide for multiplex detection.
- An oligonucleotide probe suitable for detecting the amplification of SEQ ID NO: 4 in accordance with the methods of the present invention is a probe having the sequence of SEQ ID NO: 27 or a variant thereof.
- Variants of SEQ ID NO: 27 are typically oligonucleotides of 12 to 25 or 12 to 20 nucleotides, comprising a region which is partly or fully complementary or identical to at least 8, 9 or 10 contiguous nucleotides of the corresponding original target-binding sequence present in SEQ ID NO: 27.
- the target-binding sequence of SEQ ID NO: 29 is the region which has complementarity or identity to the target.
- Variants of SEQ ID NO: 27 may comprise a region which has 1, 2, 3, 4 or 5 mismatches (substitutions) with respect to the corresponding target-binding region of the original strand invasion oligonucleotide of SEQ ID NO: 27 (and thus the target sequence) and thus is partly complementary or identical thereto.
- the variants may comprise a region of at least 10 nucleotides in length which has 1, 2 or 3 such as 1-3 or 1-2 mismatches to a corresponding region of the corresponding original oligonucleotide probe sequence.
- Any mismatches in the variant strand invasion oligonucleotide sequence may be at least 2, at least 4 or at least 5 nucleotides apart.
- Variants of SEQ ID NO: 27 may also be oligonucleotides of greater than 10 nucleotides in length comprising a target-binding region which has at least 70% sequence identity to the target-binding sequence of the corresponding original strand invasion oligonucleotide, preferably at least 80% or at least 90% sequence identity.
- the probe may be as described in WO 2015/075198, incorporated herein by reference.
- the probe may be a variant oligonucleotide of SEQ ID NO: 27 comprising a fluorophore, a quencher and a region complementary to said target nucleic acid sequence, wherein the sequence of said oligonucleotide probe comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides.
- the sequence of said oligonucleotide probe comprises at least 50% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides.
- the nucleotide sequence of the oligonucleotide probe is composed solely of ribonucleotides (which may be natural ribonucleotides or modified ribonucleotides) or solely of PNA nucleotides.
- the nucleotide sequence of the probe may be composed solely of natural ribonucleotides, solely of modified ribonucleotides, or of a mixture of natural and modified ribonucleotides.
- the oligonucleotide probe may have a mixed backbone of RNA nucleotides and PNA nucleotides or modified RNA nucleotides and PNA nucleotides.
- Preferred modified ribonucleotides include 2’-fluoro ribonucleotides, 2’-O- methyl ribonucleotides, and LNA (locked nucleic acid) nucleotides, and combinations thereof.
- a particularly preferred modified ribonucleotide is LNA. Any of the above percentage contents for modified RNA nucleotides may apply specifically to the proportion of 2’-fluoro ribonucleotides, 2’-O-methyl ribonucleotides, or LNA nucleotides in the probe.
- the probe may be composed solely of 2’-fluoro ribonucleotides, 2’-O-methyl ribonucleotides, or LNA nucleotides.
- Other suitable modified ribonucleotides include 2'-O-methoxy-ethyl and other 2'-substitutions.
- SEQ ID NO: 27 which comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotide is SEQ ID NO: 28.
- Dyes which intercalate with amplified DNA may also be used to detect the amplified DNA, such as SYBR green and thiazole orange.
- the detection of the signal from the amplified DNA may be made by any suitable system, including real-time PCR.
- the invention further provides each of the: upstream (forward) primers of SEQ ID NOs: 1 and 5-14; downstream (reverse) primers of SEQ ID NOs: 2 and 15-23 and strand invasion oligonucleotides of SEQ ID NOs: 3 and 24-26 and variants and modified derivatives of each thereof as products per se, and compositions and formulations comprising said primers and strand invasion oligonucleotides.
- the primers and optionally the strand invasion oligonucleotide may be used in any method for detection a target nucleic acid from SARS-CoV-2. Typically, the method is a strand-invasion based DNA amplification method.
- any suitable DNA amplification method that allows for specific detection of a target nucleic acid from SARS-CoV-2 may be used.
- the upstream and downstream primers may be used in a DNA amplification method that does not require use of a strand invasion oligonucleotide, such as PCR. Combinations of primers and strand invasion oligonucleotides
- primers and strand invasion oligonucleotides (and variants/modified derivatives thereof) described for use in connection with particular target sequences herein are also described together for use in combination for amplification of that target sequence.
- an upstream primer comprising the sequence of SEQ ID NO: 1 or a variant thereof
- a downstream primer comprising the sequence of SEQ ID NO: 2 or a variant thereof
- a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant or modified derivative thereof
- the same principles apply to the other primer/strand invasion oligonucleotides described for each target sequence herein.
- upstream and downstream primers for detection and amplification of the target nucleic acid sequence of SEQ ID NO: 4 are the upstream (forward) primers of SEQ ID NOs: 7, 11, 12 and 14 and the downstream primer of SEQ ID NO: 23. As such, in some embodiments:
- the upstream primer comprises any one of the sequence of SEQ ID Nos: 7, 11, 12 and 14;
- the downstream primer comprises the sequence of SEQ ID NO: 23.
- the upstream primer consists of any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14;
- the downstream primer consists of the sequence of SEQ ID NO: 23.
- the upstream primer comprises SEQ ID NO: 7;
- the downstream primer comprises the sequence of SEQ ID NO: 23.
- the upstream primer consists of SEQ ID NO: 7;
- the downstream primer consists of SEQ ID NO: 23.
- the invention also provides compositions and kits comprising (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide for the detection of a target nucleic acid from SARS-CoV-2.
- the upstream primer, downstream primer and strand invasion oligonucleotide are as described above.
- the composition or kit may be suitable for detection of SARS-CoV-2, in accordance with the method of the invention, or an alternative DNA amplification method.
- composition or kit is suitable for use for detecting the target nucleic acid sequence of SEQ ID NO: 4, typically the upstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 1 or a variant thereof, the downstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 2 or a variant thereof, and the strand invasion oligonucleotide is an oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant thereof.
- the oligonucleotide primers are typically less than 30 nucleotides in length.
- the strand invasion oligonucleotide may have the sequence of any one of SEQ ID NOs: 24-26.
- the strand invasion oligonucleotide has the sequence of SEQ ID NO: 25.
- the strand invasion oligonucleotide is typically at least 30 nucleotides in length.
- composition may be for example a solution, lyophilisate, suspension, or an emulsion in an oily or aqueous vehicle.
- the at least two oligonucleotides may be provided as a mixture, or in separate containers.
- the kit optionally further comprises instructions for use in a method of the invention.
- the kit may comprise a means for detection of amplified DNA.
- the kit or composition optionally comprises one or more probes that detect amplified DNA (such as the oligonucleotide probe of SEQ ID NO: 27 or a variant thereof, preferably an oligonucleotide probe of SEQ ID NO: 28).
- the kit or composition optionally comprises one or more of a DNA polymerase, a reverse transcriptase, a recombinase, and recombinase accessory proteins.
- the kit may comprise both a reverse transcriptase and a DNA polymerase, or both a reverse transcriptase and a recombinase.
- the DNA polymerase is Bsu polymerase.
- the recombinase is bacteriophage T4 UvsX, optionally in combination with the recombinase accessory proteins UvsY and gp32.
- the kit or composition may further comprise dNTPs, suitable buffers and other factors which are required for DNA amplification in the method of the invention as described above.
- the present invention is particularly advantageous in the medical setting.
- the detection methods of the invention provide a highly specific test to allow for determination of whether a clinical sample contains a target nucleic acid sequence from SARS-CoV-2. Additionally, the method may be applied for screening of carriers of SARS-CoV-2.
- SARS-CoV-2 The determination of whether or not SARS-CoV-2 is present may be in the context of any disease or illness present or suspected of being present in a patient. Such diseases may include those caused by, linked to, or exacerbated by the presence of SARS- CoV-2, and in particular COVID-19. Thus, a patient may display symptoms indicating the presence of SARS-CoV-2, such as a respiratory illness, and a sample may be obtained from the patient in order to determine the presence of SARS-CoV-2 by the method described above.
- the invention thus provides a method of diagnosing an infection caused by SARS- CoV-2 in a subject, comprising determining the presence of a target nucleic acid sequence from SARS-CoV-2 according to the invention in a sample from said subject.
- a particularly preferred embodiment of the invention is the identification of SARS- CoV-2 present in patients having symptoms of a respiratory illness, and in particular COVID-19.
- the invention thus provides a diagnostic method for respiratory illnesses that are caused by SARS-CoV-2.
- the method provides for a dramatic improvement in the patient management of respiratory illnesses because it allows for the optimal therapeutic treatment for a given patient. Thereby the test would reduce the length of hospital stays, the frequency of re-admission and reduce costs.
- the amplification method of the invention has particular benefits over other nucleic acid amplification methods in the clinical setting. RT-PCR requires the use of heavy and sophisticated thermal cyclers and skilled personnel, which consequently limits its use for in field or point-of-care applications.
- the amplification method of the invention can be carried out isothermally only requiring small instruments, enabling it to be performed within point-of-care settings. This provides benefits for prompt treatment of patients and limitation of the misuse of antibiotics or antiviral drugs by early identification of the infecting pathogen associated with a respiratory illness.
- the diagnostic method may conveniently be performed based on nucleic acid derived from a sample of a patient, providing an indication to clinicians whether the respiratory illness or the symptoms of the respiratory illness is due to an infection by SARS-CoV-2.
- the medical treatment can then be optimised, for example by use of appropriate antiviral compounds and/or other appropriate therapies.
- the present invention also provides a method of treating a subject infected with SARS-CoV-2, wherein a sample from the subject has been determined to comprise an RNA from SARS-CoV-2 and/or a target nucleic acid sequence from SARS-CoV-2 according to the methods described herein.
- treating a subject infected with SARS-CoV-2 also includes alleviating the symptoms of SARS-CoV-2 in said subject. Treatment of a subject infected with SARS-CoV-2 may be with any suitable methods known in the art.
- Suitable treatment of SARS-CoV-2 infection include administration of antiviral compounds such as remdesivir, administration of one or more antibodies (such as antibodies which are capable of binding viral particles, proteins and/or nucleic acids from SARS-CoV-2), and/or administration of one or more steroids such as dexamethasone.
- antiviral compounds such as remdesivir
- antibodies such as antibodies which are capable of binding viral particles, proteins and/or nucleic acids from SARS-CoV-2
- steroids such as dexamethasone
- Example 1 Selection of target sequences and oligonucleotides for detection of SARS-CoV-2 using RT-SIBA
- the isothermal nucleic acid amplification method known as Strand Invasion Based Amplification (SIBA®) with high analytical sensitivity and specificity has previously been described in Hoser et al.
- RT- SIBA reverse transcription SIBA
- CVD1 is a specific reverse transcription SIBA (RT-SBA) assay developed for detection of SARS-CoV-2 genomic RNA.
- Nextstrain (nextstrain.org) and NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) were utilized in the design of the RT-SIBA CVD1 assay.
- RNA-dependent RNA polymerase (RdRP) gene showing high sequence conservation among sequenced SARS-CoV-2 isolates was selected for CVD1 assay target area.
- RdRP was recently shown to be a relevant target sequence in the literature (Chan et al. 2020) and recognized both by WHO (Laboratory testing for coronavirus diease (COVID-19) in suspected human cases, interim guidance dated 19 March 2020; WHO Reference: WHO/COVID-19/laboratory/2020.5) and FDA (Ward et al. 2020).
- the RdRP gene sequence was analysed for target nucleic acid sequence which would be suitable for amplification by RT-SIBA.
- a region corresponding to SEQ ID NO: 4 was found to be particularly suitable due to its conservation in SARS-CoV-2 in combination with low homology to related respiratory pathogens and human genomic DNA.
- This particular amplicon was also shown to provide a region which could be used to design a strand invasion oligonucleotide such that it would have a pyrimidine-rich 5’ end, which would benefit designs of a seeding region.
- RT-SIBA reactions described herein were performed using a commercial SIBA reagent kit (Aidian, Finland) with the addition of 16U of GoScriptTM Reverse Transcriptase (Promega).
- UvsX and gp32 enzymes were each used at 0.25 mg/ml and the reaction were started using 10 mM magnesium acetate.
- the forward and reverse primers and the IO were each used at 200 nM final concentrations.
- the oligonucleotides used were purchased from Eurofins Genomics (Ebersberg, Germany) or Integrated DNA Technologies (Leuven, Belgium) and purified by the manufacturer using either reverse-phase HPLC (for primers and probes) or PAGE (for the invasion oligonucleotides).
- SIBA amplification was detected using SYBR Green 1 (1 : 100,000 dilution).
- RT- SIBA reactions were incubated at 41 °C for 60 minutes, and fluorescence readings were taken at 60-second intervals on an Agilent MX Pro 3005P (Agilent Technologies, Inc., CA, United States). After incubation for 60 minutes, the instrument was set to run a melt curve from 40°C to 95°C in order to further assess the specificity of SIBA reactions.
- RT-SIBA CVD1 assay amplification primers were screened to determine optimal combination for rapid amplification and acceptable specificity. The primers tested are shown in the tables below.
- Amplification occurs before 20 min (so that amplification provides a fast result)
- CVD1-IO1 and CVD 1-IO2 were tested with CVD1-F3 + CVD1-R9 amplification primer combination using 200 cp of synthetic SARS-CoV-2 gRNA as a template. No template control was used to evaluate the specificity of the invasion oligonucleotides.
- Figures 21 and 22 shows the results of the comparison.
- the results showed that the CVD 1 -IO1 and CVD 1-102 invasion oligonucleotides were both functional and specific for detection of SARS-Cov-2 RNA with SIB A CVD1 assay.
- CVD 1 -101 was further shown to be the better performing oligonucleotide due to the faster generation of signal.
- a locked nucleic acid (LNA) probe specific for the assay of the invention was developed.
- a functional assay specific probe can be particularly useful if the assay is multiplexed with internal amplification and/or extraction control assay or another analyte assay.
- CVD1-LNA-R0X probe (CA+GT+C+CA+GT+C+C+A; SEQ ID NO: 28, where a preceding “+” indicates an LNA modification) was designed to have ROX fluorophore at the 5’ end and an Iowa Black quencher at the 3’ end (/56- ROXN/CA+GT+C+CA+GT+C+C+A/3IABkFQ/; SEQ ID NO: 29). A total of 7 LNA bases was included in the design.
- CVD1 assay sensitivity and limit of detection was evaluated with 1000, 200, 100, 25, 10 and 0 copies of synthetic SARS-CoV-2 gRNA in final reaction mixture.
- the CVD1 assay was performed as described above and incubated for 60 min in 42 °C using CVD1- F3, CVD1-R9 and CVD1-IO1 assay oligonucleotides. Data was analyzed using 20 minutes cut-off time. The reactions were continued for an additional 40 minutes to evaluate late amplification events especially in no template control (NTC - 0 copies) reactions.
- Hoser M, Mansukoski HK, Morrical SW and Eboigbodin KE Strand Invasion Based Amplification (SIBA): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte.
- SIBA Strand Invasion Based Amplification
- Mahony JB, Petrich A, Smieja M Molecular diagnosis of respiratory virus infections.
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Abstract
A method for detecting a ribonucleic acid (RNA) from SARS-CoV-2 by strand-invasion based DNA amplification is provided, together with oligonucleotides, compositions and kits suitable for use in this method.
Description
METHOD OF DETECTING SARS-COV2
Field of the Invention
The invention relates to a method for detecting a target nucleic acid sequence of a ribonucleic acid (RNA) from SARS-CoV-2 comprising strand-invasion based DNA amplification. The invention also relates to oligonucleotides, compositions and kits suitable for use in this method, and their use for detection of SARS-CoV-2 in a sample.
Background to the Invention
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic that has killed over a million individuals, disrupted economies, and continues to spread widely. The disease caused by SARS-CoV-2 is known as coronavirus disease 2019 (COVID-19). The ability to quickly diagnose the presence of an infection in a subject remains vital in helping governments control the spread of COVID-19.
Nucleic acid amplification tests are becoming the method of choice for routine diagnosis of viral pathogens such as SARS-CoV-2 within clinical laboratory settings. They offer superior sensitivity and specificity over serology, immunoassays and traditional viral culture-based detection methods (Mahony et al. 2011).
Polymerase chain reaction (PCR) based assays, and in particular real-time reverse transcription polymerase chain reaction (RT-PCR) assays, have been developed for detecting the presence of SARS-CoV-2 and the diagnosis of COVID-19, and remain the most common platform used. A limitation of RT-PCR is that it can be prone to false negative results due to sample derived inhibition, and in particular the RNA extraction step can become a limiting factor in the performance of the method. RT-PCR also requires the use of heavy and sophisticated thermal cyclers and skilled personnel, which consequently limits its use for in field or point-of-care applications. RT-PCR assays are also timeconsuming, and there remains a need for assays which are capable of detecting the presence of SARS-CoV-2 in a sample which is both rapid, specific and sensitive.
An isothermal DNA amplification process relying on an upstream primer, a downstream primer, and a strand invasion oligonucleotide is described in WO
2009/150467 Al and Hoser et al. 2014. Detection of C. difficile bacterial DNA has been described in WO 2014/173963 Al.
Summary of the Invention
The present invention relates to detecting the presence of an RNA from SARS- CoV-2 in a sample. The method of the invention for detecting the presence of an RNA from SARS-CoV-2 in a sample uses a reverse transcriptase and an upstream primer, a downstream primer, and a strand invasion oligonucleotide, wherein each of said at least one upstream primer, at least one downstream primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence. The reverse transcriptase synthesises complementary DNA (cDNA) from the RNA present in the sample, forming double stranded (duplex) nucleic acid molecules. In combination, the primers and strand invasion oligonucleotide provide for amplification of a target nucleic acid sequence from SARS-CoV-2. If the target nucleic acid sequence from SARS- CoV-2 is present in the duplex nucleic acid molecules formed from the activity of the reverse transcriptase in the method, the strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of the upstream primer and downstream primer, thereby permitting amplification. Typically, the amplification is performed under isothermal conditions, without a requirement for thermal denaturation of duplex nucleic acid molecules. The reverse transcription of RNA to cDNA is typically carried out simultaneously with strand-invasion based amplification and therefore the detection of the presence of an RNA from SARS-CoV-2 can be performed directly from a sample suspected to comprise SARS-CoV-2 RNA.
The inventors have shown that the method of the invention allows for highly specific and sensitive detection of RNA from SARS-CoV-2. Furthermore, the method of the invention is advantageous compared to the conventional RT-PCR method used for detection of RNA in various respects. For example, it can provide for improved sensitivity, reduced detection time and be less susceptible to sample-derived inhibition.
The inventors have further identified particularly effective target nucleic acid sequences for detection of SARS-CoV-2 employing the methods of the invention, and
particularly effective primers and strand invasion oligonucleotides for use in amplification of these target sequences. The above target sequences, primers and oligonucleotides may also be used for detection of cDNA previously prepared from samples containing SARS- CoV-2 RNA.
The invention provides a method for detecting the presence of an RNA from SARS-CoV-2 in a sample, said method comprising contacting said sample with a reverse transcriptase, at least one upstream primer, at least one downstream primer, at least one strand invasion oligonucleotide and a recombinase under conditions promoting amplification of a target nucleic acid sequence from SARS-CoV-2, wherein each of said at least one upstream primer, at least one downstream primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence; and wherein said strand invasion oligonucleotide renders at least a portion of said target nucleic acid sequence single-stranded, to allow the binding of said upstream primer and a downstream primer.
The invention further provides a method for detecting a target nucleic acid sequence from SARS-CoV-2 in a sample, said method comprising contacting said sample with at least one upstream primer, at least one downstream primer and at least one strand invasion oligonucleotide under conditions promoting amplification of said target nucleic acid sequence, wherein each said primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence; and wherein said strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of said upstream primer and a downstream primer.
Typically said target sequence from SARS-CoV-2 is from the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2. Preferably the target nucleic acid sequence from SARS-CoV-2 is SEQ ID NO: 4 or a variant of thereof.
In accordance with the method of the invention disclosed herein, detecting the amplification of said target nucleic acid sequence indicates presence of RNA from SARS- CoV-2 in said sample
The invention additionally provides a composition or a kit, the composition or kit comprising: (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide, wherein said upstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 1 or a variant thereof, said downstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 2 or a variant thereof, and said strand invasion oligonucleotide is an oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant thereof.
The invention also provides a method for diagnosis of an infection by SARS-CoV- 2 in a subject, comprising carrying out a method of the invention for detecting the presence of an RNA from SARS-CoV-2 in a sample from said subject.
Figure 1 - shows multiple sequence alignment of a portion of SARS-CoV-2 genome against homologous portions of sequence SARS-CoV-2 isolates and related respiratory viruses. Highlighted portion shows the chosen amplicon region for the methods of the present invention. Positions marked with “ ” (period/full stop) shows identity. The following are the identity of the strains recited in the multiple sequence alignment: MT628280.1 : Severe acute respiratory syndrome coronavirus 2 isolate SARS- CoV-2/human/USA/CA-CZB-1629/2020; NC_045512.2: Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1; MT655135.1 : Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/ESP/HUD-98203801/2020; MT370892.1 : Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV- 2/human/USA/NY-PV08122/2020; MT415322.1 : Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/IND/GMCKN443/2020; CoVl AY278741.1: SARS coronavirus Urbani; CoVl AY291451.1: SARS coronavirus TW1; NL63
MG428704.1: Human coronavirus NL63 isolate Kilifi_HH_5402_20-May-2010; 229E MT438696.1: Human coronavirus 229E isolate HCoV-229E/USA/UNM_0186/2016; HKU1 KF686343.1: Human coronavirus HKU1 strain HKUl/human/USA/HKUl- 13/2010; OC43 MN306053.1: Human coronavirus OC43 strain HCoV_OC43/Seattle/US A/SC9430/2018
Figures 2-22 show sensitivity of the CVD1 assay. Synthetic SARS-CoV-2 genomic RNA (gRNA) template for assay development was purchased from Twist Bioscience. Figures 2-20 show assay performance using different primer pairs in combination with the CVD1-IO1 strand invasion oligonucleotide (with reference to Table 1 of Example 2 and Table 2 of Example 3 below):
Figure 2 - Primer Fl with Primer R8
Figure 3 - Primer F2 with Primer R1
Figure 4 - Primer F2 with Primer R4
Figure 5 - Primer F2 with Primer R5
Figure 6 - Primer F2 with Primer R7
Figure 7 - Primer F2 with Primer R8
Figure 8 - Primer F3 with Primer R5
Figure 9 - Primer F3 with Primer R6
Figure 10 - Primer F3 with Primer R7
Figure 11 - Primer F3 with Primer R8
Figure 12 - Primer Fl with Primer R9
Figure 13 - Primer F3 with Primer R9
Figure 14 - Primer F4 with Primer R9
Figure 15 - Primer F5 with Primer R9
Figure 16 - Primer F6 with Primer R9
Figure 17 - Primer F9 with Primer R9
Figure 18 - Primer F10 with Primer R9
Figure 19 - Primer Fl 1 with Primer R9
Figure 20 - Primer Fl 2 with Primer R9
Figures 21 and 22 shows a separate comparison of the two tested strand invasion oligonucleotides for the CVD1 assay (CVD1-IO1 and CVD 1-IO2), which were tested with the F3 and R9 primer pairing.
Figure 23 shows the performance of an LNA containing oligonucleotide probe for the CVD1 assay.
Detailed Description of the Invention
It is to be understood that different applications of the disclosed 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 polypeptide” includes two or more such polypeptides, and the like. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
6
RECTIFIED SHEET (RULE 91) ISA/EP
Methods of detection
Sample
In embodiments where reverse transcription is carried out, the sample comprises RNA. If the RNA is present in the sample in a suitable form allowing for detection according to the invention, the sample may be used directly. Alternatively, a sample (e.g. a sample from a subject, in particular a subject suspected of being infected with SARS-CoV- 2) may be initially processed before being used in a method according to the present invention. In some cases, the processing may be by heating or treatment with detergents. In some cases, the SARS-CoV-2 RNA may also be derived, obtained or extracted from the sample. Methods for processing samples containing nucleic acids (in particular RNA), extracting nucleic acids (in particular RNA) and/or purifying nucleic acids (in particular RNA) for use in detection and/or amplification methods are well-known in the art. Total nucleic acid may be isolated or RNA may be isolated separately.
Typically, a sample is processed in an appropriate manner such that the RNA in the sample is provided in a convenient form for contacting with the reverse transcriptase, primers and strand invasion oligonucleotide.
Commonly, the sample is a clinical sample, for example a sample obtained from a patient suspected of having an infection by SARS-CoV-2. The RNA may be genomic RNA of SARS-CoV-2. RNA from SARS-CoV-2 is typically single-stranded.
However, any sample can be used, provided that RNA can be obtained or derived from the sample. Thus, reference samples (for example for particular pathogens/particular viral strains), or environmental samples may be used in the present invention. Suitable environmental samples may be samples from wastewater. Suitable types of clinical sample vary according to the particular type of infection that is present, or suspected of being present in a subject. The sample may be blood, plasma, saliva, serum, sputum, urine or a stool sample. Samples from a subject suspected of being infected with SARS-CoV-2 may be collected by nose-throat swab from nasal and pharyngeal cavity. Other suitable samples may be an aspirate from a subject, such as an endotracheal aspirate or a nasopharyngeal aspirate. The sample may be stored in universal transport media (UTM).
In preferred embodiments, the samples are taken from animal subjects, such as mammalian subjects. The samples will commonly be taken from human subjects, but the present invention is also applicable in general to domestic animals, livestock, birds and fish. For example, the invention may be applied in a veterinary or agricultural setting.
Therefore, in some embodiments of the methods of the present invention, the sample (e.g. from a subject) may be a sample which has been processed such that the RNA has already been reverse transcribed and/or converted to cDNA. In such embodiments, the methods of the invention does not comprise a step of reverse transcription and/or contacting the sample with a reverse transcriptase. In embodiments where reverse transcription of RNA has previously been carried out, a suitable sample of cDNA or dsDNA amplified from the starting RNA is provided as a template.
Reverse transcription
The sample is contacted with a reverse transcriptase or any other enzyme (such as a polymerase) having reverse transcriptase activity under conditions promoting reverse transcription of the RNA present in the sample into cDNA. Suitable conditions include any conditions used to provide for activity of reverse transcriptase enzymes known in the art. The conditions typically include the presence of one or more species of primer, dNTPs (dATP, dTTP, dUTP, dCTP and dGTP) or analogues thereof, suitable buffering agents/pH and other factors which are required for enzyme performance or stability. The primer may be oligo d(T). A mixture of random primers (such as random hexamers) may be used. Alternatively, the same primers to be used for amplification of the target nucleic acid sequence may be used also for priming reverse transcription of the RNA.
The conditions may also include the presence of detergents and stabilising agents. The conditions may also include additional proteins. Preferably the additional proteins may be single-strand DNA binding proteins. The temperature used is typically isothermal, i.e constant throughout the reverse transcription process. Preferably, the temperature is identical to use for amplification of the target nucleic acid sequence, such that reverse transcription and amplification occur simultaneously in the same reaction temperature, without need for temperature cycling (as in RT-PCR), This provides significant benefits
for speed of detection. The temperature used typically depends on the nature of the reverse transcriptase enzyme and other enzyme components, and also reflects the hybridisation temperature required for the primer(s).
Suitable reverse transcriptases include Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and functional fragments or variants thereof. Particular suitable reverse transcriptases include Improm, GoScript, MMuLV, AMV, RevertAid transcriptases. In some embodiments, a polymerase with reverse transcriptase activity may also be used. Suitable polymerases with reverse transcriptase activity are known in the art, such as GspSSD LF DNA Polymerase.
Target nucleic acid sequence
The target nucleic acid sequence is a region (or amplicon) suitable for detection by strand-invasion based amplification. The selection of suitable target nucleic acid sequences, and the consequent design of primer and strand invasion oligonucleotides for detection of those sequences is an important consideration. Examples of appropriate sequences are provided herein. The target nucleic acid sequences exemplified herein are presented as DNA sequences. However, it should be understood that any DNA sequence described herein also encompasses its RNA equivalent. Thus for example the target sequence of SEQ ID NO: 4 has as its RNA equivalent SEQ ID NO: 31. Accordingly, where an RNA is detected in accordance with the invention, this RNA will include the target nucleic acid sequence in RNA form. This RNA sequence will be reverse transcribed to cDNA and then subject to amplification, such that the target nucleic acid sequence which acts as the template for the primers and strand invasion oligonucleotide will typically be in DNA form.
Typically, where SARS-CoV-2 is to be detected, the target nucleic acid sequence will be unique to its genome. This allows for a highly qualitative, unambiguous determination of the presence of SARS-CoV-2 in the sample, even if closely related pathogens (such as other coronaviruses) exist. The target nucleic acid sequence will thus typically differ from any homologous nucleic acid sequence in a related species. Typically,
the target nucleic acid sequence will comprise several mismatches with a homologous nucleic acid sequence in a related species.
Specific aspects of the invention relate to detection of SARS-CoV-2. Preferably, the target nucleic acid sequence is not present in any other type of virus, or not present in any other coronavirus or respiratory virus. More preferably, the target nucleic acid sequence is specific to SARS-CoV-2.
The target nucleic acid sequence or amplicon is of a sufficient length to provide for hybridisation of the upstream and downstream primers and strand invasion oligonucleotide in a suitable manner to different portions of the target sequence. Preferably, the amplicon is at least 45 nucleotides in length, more preferably at least 50, at least 55, at least 60, at least 65 or at least 70 nucleotides in length, as measured from the 5’ site of binding of the upstream primer to the 5’ site of binding of the downstream primer. The amplicon may be about 45 to about 85, about 55 to about 80, or about 65 to about 75 nucleotides in length. In some instances, an amplicon may be selected which is to be invaded at two separate upstream and downstream locations by one or more strand invasion oligonucleotides, as described in WO/2015/185655, incorporated herein by reference. This permits use of longer amplicons, such as those of about 70 to about 500 nucleotides in length, about 70 to about 400, about 100 to about 400, or about 100 to about 200 nucleotides in length.
In the preferred aspects related to detection of SARS-CoV-2, the target nucleic acid sequence may be present in any region of the viral genome, provided it has the necessary characteristics for specific detection of the virus as discussed above. Preferably, the target nucleic acid sequence is from the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2.
The target nucleic acid sequence for detection of SARS-CoV-2 preferably comprises SEQ ID NO: 4 or a variant thereof. It should be understood that the target nucleic acid sequence which provides the template for amplification will be present in a duplex (for example, the duplex formed through the reverse transcription) which comprises a sense strand representing SEQ ID NO: 4 or a variant thereof and a complementary anti-sense strand. The upstream and downstream primers used to amplify the target nucleic acid sequence bind to opposing strands of this duplex.
The target nucleic acid sequence may comprise a naturally occurring variant sequence of SEQ ID NO: 4 which is present in a different strain.
Variants of SEQ ID NO: 4 may comprise a region which is partly or fully complementary or identical to at least 35 contiguous nucleotides, more typically at least 40 or at least 45 contiguous nucleotides, preferably at least 55, at least 60 or at least 65 contiguous nucleotides of SEQ ID NO: 4. The variants may comprise a region which has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 mismatches (substitutions) with respect to a region of the corresponding original target sequence of SEQ ID NO: 4.
Thus, for instance the variants may comprise a region of at least 35 nucleotides in length which has 1, 2, 3, 4, 5, or 6 mismatches, such as 1-3 or 1-5 mismatches, to a corresponding region of at least 35 contiguous nucleotides of the corresponding original target sequence. The variants may comprise a region of at least 45, 55, or 65 nucleotides in length which has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, such as 1-5 or 1-8 mismatches to a corresponding region of an equivalent length in the corresponding original target sequence. Any mismatches in the variant sequence may be at least 2, at least 4, at least 5, or at least 10 nucleotides apart.
Alternatively, the variants may comprise a region of at least 35, at least 45, at least 55, or at least 65 nucleotides in length which is in full complementarity or identity with the original target sequence.
Preferably, the variants comprise only one or two mismatches to SEQ ID NO: 4 in a region corresponding to the strand-invasion oligonucleotide-complementary region of SEQ ID NO: 4. More preferably, the variants comprise a region which is in full complementarity or identity with the strand-invasion oligonucleotide-complementary region of SEQ ID NO: 4.
Most preferably, the target nucleic acid sequence comprises or consists of SEQ ID NO: 4 and a complementary antisense strand.
Other variant target nucleic acid sequences that may be amplified and detected in accordance with the invention include nucleic acid sequences which are longer than SEQ ID NO: 4, provided that the sequence derives from the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2. In other words, a target nucleic acid sequence from SARS-
COV-2 to be amplified may comprise SEQ ID NO: 4 (or a variant thereof) and additional contiguous nucleotides at the 5’ and/or the 3’ termini. Preferably, the additional contiguous nucleotides may be identical to the corresponding nucleotides from the RNA- dependent RNA polymerase (RdRP) gene of SARS-CoV-2. In some embodiments, a target nucleic acid sequence from SARS-CoV-2 may be SEQ ID NO: 30. In some embodiments, a target nucleic acid sequence from SARS-CoV-2 may be the RNA equivalent of the sequences described herein, such as SEQ ID NO: 31 or SEQ ID NO: 32.
More than one target nucleic acid sequence may be detected in a method of the invention, by providing two or more sets of upstream primer, downstream primer and strand invasion oligonucleotide, each set adapted for detection of a different target nucleic acid sequence.
Upstream and downstream primers
Suitable upstream and downstream primers are selected based on the target nucleic acid sequence of interest, and having regard to the site of binding of the strand invasion oligonucleotide that renders at least a portion of the target nucleic acid sequence singlestranded to allow the binding of the upstream primer and downstream primer.
The upstream and downstream primers comprise a sequence that is partly or fully complementary or identical to the target and optionally a 5’ and/or 3’ flanking non- complementary sequence. Alternatively, the upstream and downstream primers may consist entirely of partly or fully complementary or identical sequence to the target. The length of the primer sequence that is complementary or identical to the target is sufficient to provide specific hybridisation to the target nucleic acid sequence. The length of complementary or identical sequence is typically at least 10 nucleotides, more preferably at least 15, at least 16, or at least 17 nucleotides. The length of complementary or identical sequence may be 10-25, 15-25, 10-30 or 15-30 nucleotides. The length of complementary or identical sequence may most preferably be 15-20 nucleotides.
It should be understood that the above sequence lengths refer to portions of the primers which may be partly or fully complementary or identical to the target nucleic acid sequence. Mismatches may be present between the primers and the target sequence at
particular positions while still allowing for specific amplification and detection of the target sequence, in particular having regard to the combined use of upstream and downstream primers and a strand invasion oligonucleotide to achieve amplification. There may be 1, 2, 3, 4 or 5 mismatches between the complementary or identical region of the primer and the corresponding region of the target sequence.
Preferably, the primer is designed to allow for specific detection of SARS-CoV-2. Thus, the primer typically specifically or selectively hybridises to a complementary sequence found only in the pathogen of interest. However, the primer may also hybridise to other sequences, such as sequences found in other pathogens, provided that when used in combination with the second primer and strand invasion oligonucleotide, specific amplification of a sequence found only in the pathogen of interest is obtained.
Specific or selective hybridisation refers to the binding of a primer only to a particular nucleotide sequence under given conditions, when that sequence is present in a nucleic acid in a sample, such as a complex biological mixture including total cellular and foreign DNA or RNA. Appropriate hybridisation conditions are known in the art. See for example, Sambrook, Fritsche and Maniatis “Molecular Cloning: A Laboratory Manual”, 2nd Ed. Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety. Appropriate hybridisation conditions are also provided in the Examples below. As is known to the skilled person, appropriate hybridisation conditions may vary depending on the length of a primer and its base composition. Hybridisation is typically performed at the same temperature as amplification, and thus also depends on the activity profile of the polymerase and recombinase enzymes employed.
Typically the upstream and downstream primer will be less than 30 nucleotides in total in length, more preferably less than 25 nucleotides in length, such as 15 to 25, or 15 to 23 nucleotides in length. It is particularly preferred that primers of less than 30 nucleotides in length are used where a recombinase is used for strand invasion. The primers are not capable of acting as substrates for recombinases.
The upstream (or forward) primer binds to the 5’ region of one strand of the duplex target nucleic acid sequence, at a position proximal or overlapping with the 5’ binding site of the strand invasion oligonucleotide. The downstream (or reverse) primer binds to the 5’
region of the opposing strand of the duplex target nucleic acid sequence to the upstream primer, at a position proximal or overlapping with the 3’ binding site of the strand invasion oligonucleotide. The 5’ binding sites of the upstream and downstream primers are typically at least 45 nucleotides, more preferably at least 50, at least 55, at least 60, or at least 65 nucleotides apart on the duplex target sequence.
The upstream and/or downstream primer may have a region of sequence overlap with the sequence of the strand invasion oligonucleotide. The region of sequence overlap is typically 1-8 nucleotides in length, preferably 4-6 nucleotides in length, and may be at least 3, at least 4, at least 5 or at least 6 nucleotides in length. The downstream primer may also have correspondingly defined regions of sequence overlap with the sequence of the strand invasion oligonucleotide.
Alternatively, there may be no sequence overlap between the upstream and/or downstream primer and the strand invasion oligonucleotide, with the primer binding instead at a position that is proximal in the target sequence to the binding site of the strand invasion oligonucleotide.
Where a primer binds proximal to the strand invasion oligonucleotide, typically there is 25 nucleotides or less, more preferably 20 nucleotides or less, 15 nucleotides or less, or 10 nucleotides or less between the relevant binding site of the strand invasion oligonucleotide and the 5’ end of the primer. This ensures that the primer is able to hybridise to the single-stranded region created by binding of the strand invasion oligonucleotide.
Specific examples of suitable upstream and downstream primers for binding to a target nucleic acid sequence from SARS-CoV-2 are provided herein. Preferred upstream and downstream primers for detection and amplification of the target nucleic acid sequence of SEQ ID NO: 4 are the upstream (forward) primer of SEQ ID NO: 1 (or variants thereof) and the downstream primer of SEQ ID NO: 2 (or variants thereof).
Variants of SEQ ID NO: 1 or 2 may be oligonucleotides of up to 30 nucleotides in length comprising a region which is partly or fully complementary or identical to at least 10 contiguous nucleotides of the corresponding original primer sequence of SEQ ID NO: 1 or 2 respectively. Preferably, said variants of SEQ ID NO: 1 or 2 will comprise a region
which is partly or fully complementary or identical to at least 11, 12, 13, 14 or 15 contiguous nucleotides of the corresponding original primer sequence of SEQ ID NO: 1 or 2 respectively.
The above variants may comprise a region which has 1, 2, 3, 4, or 5 mismatches (substitutions) with respect to the corresponding region of the original primer sequence (and thus the target sequence) and thus is partly complementary or identical thereto. Thus, for instance, the variants may comprise a region of at least 10 nucleotides in length which has 1, 2, or 3 mismatches, such as 1 or 2 mismatches to a corresponding region of at least ten contiguous nucleotides of the corresponding original primer sequence. The variants may comprise a region of at least 13, 14 or 15 nucleotides in length which has 1, 2, 3, 4 or 5 mismatches, such as 1-3 mismatches to a corresponding region of an equivalent length in the corresponding original primer sequence. Any mismatches in the variant primer sequence may be at least 2, at least 4, at least 5, or at least 10 nucleotides apart.
Alternatively, the variants may comprise a region of at least 10, 11, 12, 13, 14 or 15 nucleotides in length which is in full complementarity or identity with the original primer sequence.
Variants of SEQ ID NO: 1 or 2 may also be oligonucleotides of up to 30 nucleotides in length which have at least 70% sequence identity to the sequence of the corresponding original primer sequence, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, at least 95% sequence identity.
Particularly preferred combinations of upstream and downstream primers for detection and amplification of the target nucleic acid sequence of SEQ ID NO: 4 are the upstream (forward) primers of SEQ ID NOs: 7, 11, 12 and 14 and the downstream primer of SEQ ID NO: 23. As such, in some embodiments of the methods disclosed herein:
(a) the upstream primer comprises any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14;
(b) the downstream primer comprises the sequence of SEQ ID NO: 23.
In some embodiments of the methods disclosed herein:
(a) the upstream primer consists of any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14;
(b) the downstream primer consists of the sequence of SEQ ID NO: 23.
In some embodiments of the methods disclosed herein:
(a) the upstream primer comprises SEQ ID NO: 7;
(b) the downstream primer comprises the sequence of SEQ ID NO: 23.
In some embodiments of the methods disclosed herein:
(a) the upstream primer consists of SEQ ID NO: 7;
(b) the downstream primer consists of SEQ ID NO: 23.
Additionally, the variant primers may comprise region(s) complementary or identical to the 5’ or 3’ flanking nucleotide sequence(s) to the binding region of the original primers in the gene comprising the target nucleic acid sequence, such as 5-10 nucleotides from the 5’ flanking region and/or 3-region. The variant primers may additionally comprise sequence unrelated to the target sequence.
Any upstream or downstream primer used in the invention may comprise one or more modified nucleotides and/or a detectable label, for example a fluorescent dye.
Strand invasion oligonucleotide
A suitable strand invasion oligonucleotide is selected based on the target nucleic acid sequence of interest, and having regard to the site of binding of the upstream and downstream primers and the requirement for the strand invasion oligonucleotide to render the target nucleic acid sequence single-stranded in the relevant regions to allow for the binding of the upstream primer and downstream primer.
The strand invasion oligonucleotide comprises a sequence that is complementary or identical to the target and optionally additional flanking non-complementary or nonidentical sequence(s). The length of the sequence that is complementary or identical to the target may be determined by the skilled person empirically and is sufficient to provide for efficient strand invasion of the target nucleic acid sequence, optionally under isothermal conditions. The complementary sequence may comprise RNA-DNA complementary base pairing and modified nucleotides. Typically, the length of complementary or identical sequence is at least 25 or at least 27 nucleotides, typically at least 30 nucleotides, such as
least 32, at least 33 or at least 35 nucleotides, more preferably at least 36, 37, 38, 39 or 40 nucleotides in length or greater. The length of complementary or identical sequence may be 30-50, 32-50, 35-50, 40-50, 35 to 48, 35 to 46, 38 to 45 or 40 to 45 nucleotides in length.
It should be understood that the above sequence lengths refer to a portion of the strand invasion oligonucleotide which may be partly or fully complementary or identical to the target nucleic acid sequence. Mismatches may be present between the strand invasion oligonucleotide and the target sequence at particular positions while still allowing for specific amplification and detection of the target sequence, in particular having regard to the combined use of upstream and downstream primers and a strand invasion oligonucleotide to achieve amplification. There may be 1, 2, 3, 4, 5, 6, 7, or 8 mismatches between the complementary or identical region of the strand invasion oligonucleotide and the corresponding region of the target sequence, depending on the total length of complementary or identical sequence. Preferably though there is only 1 or 2 mismatches between the strand invasion oligonucleotide sequence and the corresponding region of the target sequence.
Preferably, the complementary or identical sequence of the strand invasion oligonucleotide is designed to allow for specific detection of SARS-CoV-2. Thus, the strand invasion oligonucleotide preferably specifically or selectively hybridises to a complementary sequence found only in SARS-CoV-2. However, the strand invasion oligonucleotide may also hybridise to other sequences, such as sequences found in other pathogens, provided that when used in combination with the primers, specific amplification of a sequence found only in SARS-CoV-2 is obtained.
The complementary or identical sequence of the strand invasion oligonucleotide hybridises to a portion of the target sequence intervening the binding regions for the upstream and downstream primers (and typically overlapping with one or more thereof). The strand invasion oligonucleotide may have a region of overlap of 1-8 nucleotides, preferably 4-6 nucleotides in length, such as a region of at least 3, at least 4, at least 5 or at least 6 nucleotides in length, with the upstream and/or downstream primers.
The 5’ portion of the complementary or identical sequence of the strand invasion oligonucleotide typically binds within 25 nucleotides or less, more preferably 20 nucleotides or less from the 5’ boundary of the duplex target nucleotide sequence to be melted (the amplicon).
The strand invasion oligonucleotide optionally further comprises non- complementary or non-identical sequence region(s) to the target that flank the complementary or identical sequence region. The strand invasion oligonucleotide may comprise a non-complementary or non-identical 5’ region which may be of any nucleotide sequence. The 5’ non-complementary region may assist binding of recombinase and is also referred to herein as the “seeding” region.
The 5’ non-complementary region is typically at least 3 nucleotides in length, more typically at least 6, at least 8, at least 10 nucleotides in length. More preferably, the 5’ non-complementary region is of at least 12, most preferably at least 14 nucleotides in length. The 5 ’non-complementary region may be greater than 14 nucleotides in length and may be up to 20 nucleotides in length. The 5’ non-complementary region may be 10-20, 10-17, 10-14, 12-20, 12-17 or 12-15 nucleotides in length, more preferably about 12 to about 20 or about 14 to about 17 nucleotides in length. The 5’ non-complementary region may be of any nucleotide composition but preferably is selected to have particular nucleotide compositions described herein as being advantageous in the invention. Preferably, the 5’ non-complementary region comprises a greater number of pyrimidine nucleotides (cytosine or thymidine) compared to purine nucleotides (adenine and guanine). For example, the 5’ non-complementary region may comprise at least 60%, at least 70%, at least 80%, or at least 90% pyrimidine nucleotides (cytosine or thymidine) compared to purine nucleotides (adenine and guanine). More preferably, the 5’ non-complementary region comprises or consists essentially of pyrimidine (cytosine or thymidine) nucleotides. The non-complementary region may comprise, consist or consist essentially of a mixture of cytosine and thymidine nucleotides. Preferably, where a mixture of pyrimidine nucleotides is present, a greater number of cytosine residues than thymidine residues is present. Most preferably, the non-complementary region consists essentially of, or consists of, cytosine nucleotides. In preferred embodiments, the 5 ’non-complementary region is of
about 12 to about 20 nucleotides in length and comprises, consists or consists essentially of a mixture of cytosine and guanine nucleotides. In particularly preferred embodiments, the 5’non-complementary region is of about 14 to about 20 nucleotides in length and comprises, consists or consists essentially of cytosine nucleotides. In some embodiments, the 5’non-complementary region is about 14 nucleotides in length and consists or consists essentially of cytosine residues.
Alternatively, the strand invasion oligonucleotide may be designed with a seeding region which is shorter than 14 nucleotides in length if the 5’ portion of the complementary or identical sequence of the strand invasion oligonucleotide has a high proportion of pyrimidine nucleotides. For example, the strand invasion oligonucleotide may be designed with a seeding region which is shorter than 14 nucleotides (such as a seeding region of 13, 12, 11, 10, 9 or 8 nucleotides) if the first 10 nucleotides at the 5’ portion of the complementary or identical sequence of the strand invasion oligonucleotide comprises at least 6 pyrimidine nucleotides.
The strand invasion oligonucleotide is typically at least 30 nucleotides in length where a recombinase is used in conjunction with the oligonucleotide. The strand invasion oligonucleotide is preferably at least 35, at least 40 or at least 45 nucleotides in length, more preferably at least 50, and may be at least 55 nucleotides in length or greater. The strand invasion oligonucleotide may be 40-70, 45-70, 45-70, 50-70, 55-70, 45-65, 50-65, 50-60 or 55-65 nucleotides in length.
The strand invasion oligonucleotide may comprise a 3’ non-compl ementary region typically of 1-3 nucleotides in length which comprises nucleotides which block polymerase extension such as invdT. In this respect, typically the strand invasion oligonucleotide has a non-extendible 3 ’terminus, such that it cannot serve as a substrate for DNA amplification, and the target sequence is then only amplified on the further binding of the specific upstream and downstream primers. This avoids formation of non-specific amplification products. The strand invasion oligonucleotide may comprise one, two, three, four, five, six, seven, eight or more modified nucleotides in its 3’region, such as in the 10- 15 or 10-20 nucleotides from the 3 ’terminus. The strand- invasion oligonucleotide may comprise a 3’ modification of the 3 ’terminal nucleotide, and may be a dideoxynucleotide,
or comprise a 3 ’amino-allyl group, a 3 ’carbon spacer, 3 ’phosphate, 3 ’biotin, 3 ’sialyl, or 3 ’thiol. The 3’ nucleotide may be a nucleotide incorporated in a reversed orientation by a 3 ’-3’ linkage. Alternatively or additionally, the 3’ region of the strand-invasion oligonucleotide may comprise nucleotides with poor substrate capability for DNA polymerases, such as PNA (peptide nucleic acid) nucleotides, LNA (locked nucleic acid), 2’-5’ linked DNA or 2’-O-methyl RNA, or combinations thereof.
Where the strand-invasion oligonucleotide is a PNA oligomer comprised wholly of PNA, such an oligonucleotide can destabilise and invade duplex DNA in the absence of a recombinase enzyme. Thus, where a PNA oligonucleotide is used, the methods of the invention may be performed without presence of a recombinase enzyme.
Specific examples of suitable strand invasion oligonucleotides for target nucleotide sequences from SARS-CoV-2 are provided herein. A strand invasion oligonucleotide suitable for detection of the target nucleic acid sequence of SEQ ID NO: 4 comprises SEQ ID NO: 3. A strand invasion oligonucleotide suitable for detection of the target nucleic acid sequence of SEQ ID NO: 4 is SEQ ID NO: 24. A particularly preferred strand invasion oligonucleotide is a modified derivative of SEQ ID NO: 24, most preferably SEQ ID NO: 25.
As discussed above, it is preferred that a strand invasion oligonucleotide used in the invention comprises one or more modified oligonucleotides in its 3 ’region to block its use as a polymerase substrate. Thus, a modified derivative of SEQ ID NO: 3 or 24 may comprise one, two, three, four, five, six, seven, eight or more modified nucleotides in its 3’region, typically in the 10-15 or 10-20 nucleotides from the 3’terminus. The modifications may be selected from any of those discussed above. The modified derivative may be a PNA oligomer of corresponding sequence to SEQ ID NO: 3 or 24.
In addition to modified derivatives of SEQ ID NOs: 3 and 24, variant strand invasion oligonucleotides may be used.
Variants of SEQ ID NOs: 3, 24 and 25 are typically oligonucleotides of greater than 30 nucleotides, more preferably at least 35, at least 40, or at least 45 nucleotides in length, comprising a region which is partly or fully complementary or identical to at least 30 contiguous nucleotides of the corresponding original target-binding sequence present in
SEQ ID NOs: 3, 24 and 25. The target-binding sequence of SEQ ID NOs: 3, 24 and 25 is the region which has complementarity or identity to the target.
Preferably, said variants will comprise a region which is partly or fully complementary or identical to at least 32, 35, 37, 40, 42 or 45 contiguous nucleotides of the target-binding sequence present in SEQ ID NOs: 3, 24 and 25.
The above variants may comprise a region which has 1, 2, 3, 4, 5, 6, 7 or 8 mismatches (substitutions) with respect to the corresponding target-binding region of the original strand invasion oligonucleotide of SEQ ID NOs: 3, 24 or 25 (and thus the target sequence) and thus is partly complementary or identical thereto. Thus, for instance, the variants may comprise a region of at least 30 nucleotides in length which has 1, 2, 3, or 4, such as 1-4 or 1-3 mismatches to a corresponding region of at least 40 contiguous nucleotides of the corresponding original strand invasion oligonucleotide. The variants may comprise a region of at least 35, 40, 42, or 45 nucleotides in length which has 1, 2, 3, 4, 5 or 6, such as 1-5, or 1-3 mismatches to a corresponding region of an equivalent length in the corresponding original strand invasion oligonucleotide. Any mismatches in the variant strand invasion oligonucleotide sequence may be at least 2, at least 4, at least 5, or at least 10 nucleotides apart.
Alternatively, the variants may comprise a region of at least 32, 35, 37, 40, 42 or 45 nucleotides in length which is in full complementarity or identity with the target-binding region of the original strand invasion oligonucleotide.
Variants of SEQ ID NOs: 3, 24 and 25 may also be oligonucleotides of greater than 30 nucleotides in length comprising a target-binding region which has at least 70% sequence identity to the target-binding sequence of the corresponding original strand invasion oligonucleotide, preferably at least 75%, at least 80%, more preferably at least 85%, at least 90%, at least 95% sequence identity.
Additionally, the variant strand invasion oligonucleotides may comprise additional sequence(s) complementary or identical to the 5’ and/or 3’ flanking nucleotide sequence(s) to the binding region for the original strand invasion oligonucleotide in the gene from which the target nucleic acid sequence is derived, such as 5-10 or 5-15 nucleotides from the 5’ flanking region and/or 3-region.
The remaining sequence of the variant strand invasion oligonucleotides is typically unrelated to the target sequence, and also typically unrelated to the original strand invasion oligonucleotide.
The variant strand invasion oligonucleotides further comprise one or more modified oligonucleotides in their 3 ’region such as, two, three, four, five, six, seven, eight or more modified nucleotides, which may be in the 10-15 or 10-20 nucleotides from the 3’terminus The modifications may be selected from any of those discussed above.
A strand invasion oligonucleotide of the invention may further comprise a detectable label, for example a fluorescent dye.
Amplification of the target nucleic acid sequence
The nucleic acid derived from the sample is contacted with the upstream and downstream primers and the strand invasion oligonucleotide for detection purposes, under conditions promoting amplification of the target nucleic acid sequence. Such conditions typically comprise the presence of a DNA polymerase enzyme. Suitable conditions include any conditions used to provide for activity of polymerase enzymes known in the art.
The conditions typically include the presence of all dNTPs (dATP, dTTP, dCTP, dUTP and dGTP or analogues thereof), suitable buffering agents/pH and other factors which are required for enzyme performance or stability. The conditions may include the presence of detergents and stabilising agents. The temperature used is typically isothermal, i.e constant throughout the method of detection and/or amplification process of the invention. The temperature used typically depends on the nature of the polymerase enzyme and other enzyme components, and also reflects the hybridisation temperature required for the primers and strand invasion oligonucleotides. In some embodiments, where a polymerase suitable for isothermal amplification (such as Bsu polymerase) is used, a suitable temperature is from around 41 °C to around 45 °C, preferably from around 42 °C to around 44 °C.
The polymerase used typically has strand-displacement activity. The term “strand displacement” is used herein to describe the ability of a DNA polymerase, optionally in
conjunction with accessory proteins, to displace complementary strands on encountering a region of double stranded DNA during DNA synthesis. Suitable DNA polymerases include poll from E. coli. B. siibliHs. or B. stearothermophilus, and functional fragments or variants thereof, and T4 and T7 DNA polymerases and functional fragments or variants thereof. A preferred polymerase is Bsu DNA polymerase or a functional fragment or variant thereof.
The conditions may further comprise the presence of a recombinase. Any recombinase system may be used in the method of the invention. The recombinase system may be of prokaryotic or eukaryotic origin, and may be bacterial, yeast, phage, or mammalian. The recombinase may polymerise onto a single-stranded oligonucleotide in the 5 ’-3’ or 3 ’-5; direction. The recombinase may be derived from a myoviridae phage, such as T4, T2, T6, Rb69, Aehl, KVP40, Acinetobacter phage 133, Aeromonas phage 65, cyanophage P-SSM2, cyanophage PSSM4, cyanophage S-PM2, Rbl4, Rb32, Aeromonas phage 25, Vibrio phage nt-1, phi-1, Rbl6, Rb43, Phage 31, phage 44RR2.8t, Rb49, phage Rb3, or phage LZ2. In a preferred embodiment, the T4 recombinase UvsX (Accession number: P04529) or a functional variant or fragment thereof is used. The Rad systems of eukaryotes or the recA-Reco system of E. coli or other prokaryotic systems may also be used.
The conditions may further comprise the presence of recombinase accessory proteins, such as single-stranded binding protein (e.g. gp32, accession number P03695) and recombinase loading agent (e.g. UvsY, accession number NP 049799.2). In a preferred embodiment, the conditions comprise the presence of the T4 gp32, UvsX and UvsY proteins.
The recombinase (such as UvsX), and where used the recombinase loading agent (such as UvsY) and single stranded DNA binding protein (such as gp32), can each be native, hybrid or mutant proteins from the same or different myoviridae phage sources. A native protein may be a wild type or natural variant of a protein.
The conditions may further comprise other factors used to enhance the efficiency of the recombinase such as compounds used to control DNA interactions, for example
proline, DMSO or crowding agents which are known to enhance loading of recombinases onto DNA (Lavery P et. Al JBC 1992, 26713, 9307-9314; W02008/035205).
The conditions may also comprise the presence of an ATP regeneration system. Various ATP regeneration systems are known to the person skilled in the art, and include glycolytic enzymes. Suitable components of an ATP regeneration system may include one or more of phosphocreatine, creatine kinase, myokinase, pyrophosphatase, sucrose and sucrose phosphorylase. The conditions may further comprise the presence of ATP.
Additional components such as magnesium ions, DTT or other reducing agents, salts, BSA/PEG or other crowding agents may also be included.
The various components described above, inclusive of the primers and strand invasion oligonucleotide, may be provided in varying concentrations to provide for DNA amplification. The skilled person can select suitable working concentrations of the various components in practice.
Detection of presence of amplified DNA
The presence of amplified DNA resulting from the contacting of the target nucleic acid sequence with the primers and strand invasion oligonucleotide under conditions promoting DNA amplification may be monitored by any suitable means. Accordingly, in methods of the present invention, detecting the amplification of said target nucleic acid sequence indicates presence of RNA from SARS-CoV-2 in said sample. In some embodiments, a method of the present invention may comprise additional steps of monitoring and/or detecting the amplification of the target nucleic acid sequence.
One or both of the primers, or the strand invasion oligonucleotide may incorporate a label or other detectable moiety. Any label or detectable moiety may be used. Examples of suitable labels include radioisotopes or fluorescent moieties, and FRET pairs of a fluorophore and acceptor moiety. Alternatively, or additionally one or more probes that detect the amplified DNA may be used, again incorporating a label or other detectable moiety. The probes may bind at any suitable location in the amplicon. Probes detecting different amplified target sequences may signal at different fluorescent wavelengths to provide for multiplex detection. An oligonucleotide probe suitable for detecting the
amplification of SEQ ID NO: 4 in accordance with the methods of the present invention is a probe having the sequence of SEQ ID NO: 27 or a variant thereof.
Variants of SEQ ID NO: 27 are typically oligonucleotides of 12 to 25 or 12 to 20 nucleotides, comprising a region which is partly or fully complementary or identical to at least 8, 9 or 10 contiguous nucleotides of the corresponding original target-binding sequence present in SEQ ID NO: 27. The target-binding sequence of SEQ ID NO: 29 is the region which has complementarity or identity to the target.
Variants of SEQ ID NO: 27 may comprise a region which has 1, 2, 3, 4 or 5 mismatches (substitutions) with respect to the corresponding target-binding region of the original strand invasion oligonucleotide of SEQ ID NO: 27 (and thus the target sequence) and thus is partly complementary or identical thereto. Thus, for instance, the variants may comprise a region of at least 10 nucleotides in length which has 1, 2 or 3 such as 1-3 or 1-2 mismatches to a corresponding region of the corresponding original oligonucleotide probe sequence. Any mismatches in the variant strand invasion oligonucleotide sequence may be at least 2, at least 4 or at least 5 nucleotides apart.
Variants of SEQ ID NO: 27 may also be oligonucleotides of greater than 10 nucleotides in length comprising a target-binding region which has at least 70% sequence identity to the target-binding sequence of the corresponding original strand invasion oligonucleotide, preferably at least 80% or at least 90% sequence identity.
The probe may be as described in WO 2015/075198, incorporated herein by reference. Thus, the probe may be a variant oligonucleotide of SEQ ID NO: 27 comprising a fluorophore, a quencher and a region complementary to said target nucleic acid sequence, wherein the sequence of said oligonucleotide probe comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides. In some embodiments, the sequence of said oligonucleotide probe comprises at least 50% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides. In particularly preferred embodiments, the nucleotide sequence of the oligonucleotide probe is composed solely of ribonucleotides (which may be natural ribonucleotides or modified ribonucleotides) or solely of PNA nucleotides. The nucleotide sequence of the probe may be composed solely of natural ribonucleotides, solely of modified ribonucleotides, or of a mixture of natural
and modified ribonucleotides. The oligonucleotide probe may have a mixed backbone of RNA nucleotides and PNA nucleotides or modified RNA nucleotides and PNA nucleotides. Preferred modified ribonucleotides include 2’-fluoro ribonucleotides, 2’-O- methyl ribonucleotides, and LNA (locked nucleic acid) nucleotides, and combinations thereof. A particularly preferred modified ribonucleotide is LNA. Any of the above percentage contents for modified RNA nucleotides may apply specifically to the proportion of 2’-fluoro ribonucleotides, 2’-O-methyl ribonucleotides, or LNA nucleotides in the probe. Alternatively, the probe may be composed solely of 2’-fluoro ribonucleotides, 2’-O-methyl ribonucleotides, or LNA nucleotides. Other suitable modified ribonucleotides include 2'-O-methoxy-ethyl and other 2'-substitutions. A particularly preferred variant of SEQ ID NO: 27 which comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotide is SEQ ID NO: 28.
Dyes which intercalate with amplified DNA may also be used to detect the amplified DNA, such as SYBR green and thiazole orange.
The detection of the signal from the amplified DNA may be made by any suitable system, including real-time PCR.
Primers and oligonucleotides
The invention further provides each of the: upstream (forward) primers of SEQ ID NOs: 1 and 5-14; downstream (reverse) primers of SEQ ID NOs: 2 and 15-23 and strand invasion oligonucleotides of SEQ ID NOs: 3 and 24-26 and variants and modified derivatives of each thereof as products per se, and compositions and formulations comprising said primers and strand invasion oligonucleotides. The primers and optionally the strand invasion oligonucleotide may be used in any method for detection a target nucleic acid from SARS-CoV-2. Typically, the method is a strand-invasion based DNA amplification method. However, any suitable DNA amplification method that allows for specific detection of a target nucleic acid from SARS-CoV-2 may be used. The upstream and downstream primers may be used in a DNA amplification method that does not require use of a strand invasion oligonucleotide, such as PCR.
Combinations of primers and strand invasion oligonucleotides
It will be understood that the particular primers and strand invasion oligonucleotides (and variants/modified derivatives thereof) described for use in connection with particular target sequences herein are also described together for use in combination for amplification of that target sequence. Thus, for example, an upstream primer comprising the sequence of SEQ ID NO: 1 or a variant thereof, a downstream primer comprising the sequence of SEQ ID NO: 2 or a variant thereof, and a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant or modified derivative thereof, are described for use in methods for amplification of the target sequence of SEQ ID NO: 4 or a variant thereof, and also for inclusion in combination in kits and compositions. The same principles apply to the other primer/strand invasion oligonucleotides described for each target sequence herein.
Particularly preferred combinations of upstream and downstream primers for detection and amplification of the target nucleic acid sequence of SEQ ID NO: 4 are the upstream (forward) primers of SEQ ID NOs: 7, 11, 12 and 14 and the downstream primer of SEQ ID NO: 23. As such, in some embodiments:
(a) the upstream primer comprises any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14;
(b) the downstream primer comprises the sequence of SEQ ID NO: 23.
In some embodiments:
(a) the upstream primer consists of any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14;
(b) the downstream primer consists of the sequence of SEQ ID NO: 23.
In some embodiments:
(a) the upstream primer comprises SEQ ID NO: 7;
(b) the downstream primer comprises the sequence of SEQ ID NO: 23.
In some embodiments:
(a) the upstream primer consists of SEQ ID NO: 7;
(b) the downstream primer consists of SEQ ID NO: 23.
Compositions and kits
The invention also provides compositions and kits comprising (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide for the detection of a target nucleic acid from SARS-CoV-2. The upstream primer, downstream primer and strand invasion oligonucleotide are as described above. The composition or kit may be suitable for detection of SARS-CoV-2, in accordance with the method of the invention, or an alternative DNA amplification method.
Where the composition or kit is suitable for use for detecting the target nucleic acid sequence of SEQ ID NO: 4, typically the upstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 1 or a variant thereof, the downstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 2 or a variant thereof, and the strand invasion oligonucleotide is an oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant thereof. The oligonucleotide primers are typically less than 30 nucleotides in length. The strand invasion oligonucleotide may have the sequence of any one of SEQ ID NOs: 24-26. Preferably, the strand invasion oligonucleotide has the sequence of SEQ ID NO: 25. The strand invasion oligonucleotide is typically at least 30 nucleotides in length.
The above composition may be for example a solution, lyophilisate, suspension, or an emulsion in an oily or aqueous vehicle.
In the above kit, the at least two oligonucleotides may be provided as a mixture, or in separate containers. The kit optionally further comprises instructions for use in a method of the invention. The kit may comprise a means for detection of amplified DNA.
The kit or composition optionally comprises one or more probes that detect amplified DNA (such as the oligonucleotide probe of SEQ ID NO: 27 or a variant thereof, preferably an oligonucleotide probe of SEQ ID NO: 28). The kit or composition optionally comprises one or more of a DNA polymerase, a reverse transcriptase, a recombinase, and recombinase accessory proteins. The kit may comprise both a reverse transcriptase and a DNA polymerase, or both a reverse transcriptase and a recombinase. Preferably, the DNA polymerase is Bsu polymerase. Preferably, the recombinase is bacteriophage T4 UvsX,
optionally in combination with the recombinase accessory proteins UvsY and gp32. The kit or composition may further comprise dNTPs, suitable buffers and other factors which are required for DNA amplification in the method of the invention as described above.
Diagnosis of an infection by a pathogen and medical applications
The present invention is particularly advantageous in the medical setting. The detection methods of the invention provide a highly specific test to allow for determination of whether a clinical sample contains a target nucleic acid sequence from SARS-CoV-2. Additionally, the method may be applied for screening of carriers of SARS-CoV-2.
The determination of whether or not SARS-CoV-2 is present may be in the context of any disease or illness present or suspected of being present in a patient. Such diseases may include those caused by, linked to, or exacerbated by the presence of SARS- CoV-2, and in particular COVID-19. Thus, a patient may display symptoms indicating the presence of SARS-CoV-2, such as a respiratory illness, and a sample may be obtained from the patient in order to determine the presence of SARS-CoV-2 by the method described above.
The invention thus provides a method of diagnosing an infection caused by SARS- CoV-2 in a subject, comprising determining the presence of a target nucleic acid sequence from SARS-CoV-2 according to the invention in a sample from said subject.
A particularly preferred embodiment of the invention is the identification of SARS- CoV-2 present in patients having symptoms of a respiratory illness, and in particular COVID-19.
The invention thus provides a diagnostic method for respiratory illnesses that are caused by SARS-CoV-2. The method provides for a dramatic improvement in the patient management of respiratory illnesses because it allows for the optimal therapeutic treatment for a given patient. Thereby the test would reduce the length of hospital stays, the frequency of re-admission and reduce costs. The amplification method of the invention has particular benefits over other nucleic acid amplification methods in the clinical setting. RT-PCR requires the use of heavy and sophisticated thermal cyclers and skilled personnel, which consequently limits its use for in field or point-of-care applications. The
amplification method of the invention can be carried out isothermally only requiring small instruments, enabling it to be performed within point-of-care settings. This provides benefits for prompt treatment of patients and limitation of the misuse of antibiotics or antiviral drugs by early identification of the infecting pathogen associated with a respiratory illness.
The diagnostic method may conveniently be performed based on nucleic acid derived from a sample of a patient, providing an indication to clinicians whether the respiratory illness or the symptoms of the respiratory illness is due to an infection by SARS-CoV-2. Depending on the outcome of the test the medical treatment can then be optimised, for example by use of appropriate antiviral compounds and/or other appropriate therapies.
As such, the present invention also provides a method of treating a subject infected with SARS-CoV-2, wherein a sample from the subject has been determined to comprise an RNA from SARS-CoV-2 and/or a target nucleic acid sequence from SARS-CoV-2 according to the methods described herein. As used herein, treating a subject infected with SARS-CoV-2 also includes alleviating the symptoms of SARS-CoV-2 in said subject. Treatment of a subject infected with SARS-CoV-2 may be with any suitable methods known in the art. Examples of suitable treatment of SARS-CoV-2 infection include administration of antiviral compounds such as remdesivir, administration of one or more antibodies (such as antibodies which are capable of binding viral particles, proteins and/or nucleic acids from SARS-CoV-2), and/or administration of one or more steroids such as dexamethasone.
The following Examples illustrate the invention.
Example 1 - Selection of target sequences and oligonucleotides for detection of SARS-CoV-2 using RT-SIBA
The isothermal nucleic acid amplification method known as Strand Invasion Based Amplification (SIBA®) with high analytical sensitivity and specificity has previously been described in Hoser et al.
An assay incorporating the SIBA method, namely reverse transcription SIBA (RT- SIBA) was developed for rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows a one-step reverse transcription of RNA to cDNA and simultaneous amplification and detection of cDNA with SIBA. CVD1 is a specific reverse transcription SIBA (RT-SBA) assay developed for detection of SARS-CoV-2 genomic RNA. Nextstrain (nextstrain.org) and NCBI BLAST (blast.ncbi.nlm.nih.gov/Blast.cgi) were utilized in the design of the RT-SIBA CVD1 assay.
Published sequence data for SARS-CoV-2 was relatively limited during the early stages of the CO VID-19 pandemic. The RNA-dependent RNA polymerase (RdRP) gene showing high sequence conservation among sequenced SARS-CoV-2 isolates was selected for CVD1 assay target area. RdRP was recently shown to be a relevant target sequence in the literature (Chan et al. 2020) and recognized both by WHO (Laboratory testing for coronavirus diease (COVID-19) in suspected human cases, interim guidance dated 19 March 2020; WHO Reference: WHO/COVID-19/laboratory/2020.5) and FDA (Ward et al. 2020).
The RdRP gene sequence was analysed for target nucleic acid sequence which would be suitable for amplification by RT-SIBA. A region corresponding to SEQ ID NO: 4 was found to be particularly suitable due to its conservation in SARS-CoV-2 in combination with low homology to related respiratory pathogens and human genomic DNA. This particular amplicon was also shown to provide a region which could be used to design a strand invasion oligonucleotide such that it would have a pyrimidine-rich 5’ end, which would benefit designs of a seeding region.
SARS-CoV-2 sequence alignment analysis for determination of homology to related sequences (Figure 1) was performed with BioEdit version 7.2.5 (https://bioedit.software.informer.eom/7.2/). The data confirms that the selected sequence (highlighted sequence in Figure 1) is conserved among all of the sequenced SARS-CoV-2
isolates. While there is a high degree of homology to SARS-CoV-1, experiments confirmed that SARS-CoV-1 would not be detected by the methods of the present invention (data not shown).
Example 2 - RT-SIBA method and optimisation of oligonucleotide design
RT-SIBA reactions described herein were performed using a commercial SIBA reagent kit (Aidian, Finland) with the addition of 16U of GoScript™ Reverse Transcriptase (Promega).
UvsX and gp32 enzymes were each used at 0.25 mg/ml and the reaction were started using 10 mM magnesium acetate. The forward and reverse primers and the IO were each used at 200 nM final concentrations. The oligonucleotides used were purchased from Eurofins Genomics (Ebersberg, Germany) or Integrated DNA Technologies (Leuven, Belgium) and purified by the manufacturer using either reverse-phase HPLC (for primers and probes) or PAGE (for the invasion oligonucleotides).
SIBA amplification was detected using SYBR Green 1 (1 : 100,000 dilution). RT- SIBA reactions were incubated at 41 °C for 60 minutes, and fluorescence readings were taken at 60-second intervals on an Agilent MX Pro 3005P (Agilent Technologies, Inc., CA, United States). After incubation for 60 minutes, the instrument was set to run a melt curve from 40°C to 95°C in order to further assess the specificity of SIBA reactions.
Several RT-SIBA CVD1 assay amplification primers were screened to determine optimal combination for rapid amplification and acceptable specificity. The primers tested are shown in the tables below.
Table 1 - Primers used for the CVD1 RT-SIBA assay
The aim was to find primer combinations that detected 200 copies of synthetic SARS-CoV-2 RNA in < 30 minutes without false positive no template control (NTC = 0 copies) reactions. Several primer combinations were shown to be functional (Figures 2- 20).
Some primers combinations were shown to produce unspecific amplification signals late in the RT-SIBA reaction. This could be due to primer dimers and other oligonucleotide interactions for instance.
It was preferable for primer combinations to provide the following amplification characteristics:
Amplification occurs before 20 min (so that amplification provides a fast result)
Amplification does not occur in “no template” controls (as this would lead to false positive signals).
In view of the above, the results presented in Figure 13 (F3 + R9), Figure 17 (F9 + R9), Figure 18 (F10 + R9) and Figure 20 (F12 + R9) would present preferable primer combinations for the CVD1 assay described herein.
In conclusion, the most optimal combination was determined to be CVD1-F3 + CVD1-R9 + CVD1-IO1 (Figure 13).
Example 3 - Comparison of invasion oligonucleotides
Two invasion oligonucleotides were tested with the most optimal primer combination determined in the previous screening steps. CVD1-IO1 and CVD 1-IO2 were tested with CVD1-F3 + CVD1-R9 amplification primer combination using 200 cp of synthetic SARS-CoV-2 gRNA as a template. No template control was used to evaluate the specificity of the invasion oligonucleotides.
Table 2 - Strand invasion oligonucleotides for CVD1 RT-SIBA assay
*mX indicates 2’-O-Methyl modified nucleotide
Figures 21 and 22 shows the results of the comparison. The results showed that the CVD 1 -IO1 and CVD 1-102 invasion oligonucleotides were both functional and specific for detection of SARS-Cov-2 RNA with SIB A CVD1 assay. CVD 1 -101 was further shown to be the better performing oligonucleotide due to the faster generation of signal.
Example 4 - LNA Probe for CVD1 RT-SIBA assay
In order to detect amplification and presence of the selected amplicon (i.e. an amplicon comprising SEQ ID NO: 4) with a fluorescent probe, a locked nucleic acid (LNA) probe specific for the assay of the invention was developed. A functional assay specific probe can be particularly useful if the assay is multiplexed with internal amplification and/or extraction control assay or another analyte assay.
CVD1-LNA-R0X probe (CA+GT+C+CA+GT+C+C+A; SEQ ID NO: 28, where a preceding “+” indicates an LNA modification) was designed to have ROX fluorophore at the 5’ end and an Iowa Black quencher at the 3’ end (/56-
ROXN/CA+GT+C+CA+GT+C+C+A/3IABkFQ/; SEQ ID NO: 29). A total of 7 LNA bases was included in the design.
Using similar sequence design with the reverse primer, the probe competes with the primer in the reaction by binding the reverse primer annealing area. This results to fluorescence emission due to reduced quenching of the fluorophore. CVD1-LNA-ROX was concluded to be specific and functional with SARS-CoV-2 detection assay of the present invention (see Figure 23).
Example 5 - CVD1 Assay sensitivity
CVD1 assay sensitivity and limit of detection was evaluated with 1000, 200, 100, 25, 10 and 0 copies of synthetic SARS-CoV-2 gRNA in final reaction mixture. The CVD1 assay was performed as described above and incubated for 60 min in 42 °C using CVD1- F3, CVD1-R9 and CVD1-IO1 assay oligonucleotides. Data was analyzed using 20 minutes cut-off time. The reactions were continued for an additional 40 minutes to evaluate late amplification events especially in no template control (NTC - 0 copies) reactions.
The results of the reactions performed are shown below. These show that the CVD1 assay limit of detection in a 20 minutes run time was determined to be 25 copies of gRNA in reaction. No false positive reactions occurred in 20 minutes run time. 3 out of 12 NTC reactions gave false positive signal after 20 minutes.
*Sensitivity experiment was run for 60 minutes. 3/12 false positive (average Cq 30,3min) NTC reactions occurred after 20 min
References
Chan JF, Yip CC, To KK, et al. Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens. J Clin Microbiol. 2020;58(5):e00310-20 doi: 10.1128/JCM.00310-20
Hoser M, Mansukoski HK, Morrical SW and Eboigbodin KE: Strand Invasion Based Amplification (SIBA): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte. PLoS One 2014, 9(11): el 12656.
Mahony JB, Petrich A, Smieja M: Molecular diagnosis of respiratory virus infections.
Critical Reviews in Clinical Laboratory Sciences 2011, 48(5-6):217-249.
Ward S, Lindsley A, Courier J, Assa'ad A. Clinical testing for COVID-19. J Allergy Clin
Immunol. 2020;146(l):23-34. doi: 10.1016/j.jaci.2020.05.012
Claims
1. A method for detecting the presence of an RNA from SARS-CoV-2 in a sample, said method comprising contacting said sample with a reverse transcriptase, at least one upstream primer, at least one downstream primer, at least one strand invasion oligonucleotide and a recombinase under conditions promoting amplification of a target nucleic acid sequence from SARS-CoV-2, wherein each of said at least one upstream primer, at least one downstream primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence; wherein said strand invasion oligonucleotide renders at least a portion of said target nucleic acid sequence single-stranded, to allow the binding of said upstream primer and a downstream primer; and wherein said target nucleic acid sequence comprises SEQ ID NO: 4 or a variant thereof.
2. A method for detecting a target nucleic acid sequence from SARS-CoV-2 in a sample, said method comprising contacting said sample with at least one upstream primer, at least one downstream primer and at least one strand invasion oligonucleotide under conditions promoting amplification of said target nucleic acid sequence, wherein each said primer and said strand invasion oligonucleotide comprises a region complementary to said target nucleic acid sequence; wherein said strand invasion oligonucleotide renders at least a portion of the target nucleic acid sequence single-stranded to allow the binding of said upstream primer and a downstream primer; and wherein said target nucleic acid sequence comprises SEQ ID NO: 4 or a variant thereof.
3. A method according to claim 1 or 2, wherein said upstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 1 or a variant thereof, and/or
37
wherein said downstream primer is an oligonucleotide comprising the sequence of SEQ ID NO: 2 or a variant thereof; and/or wherein said strand invasion oligonucleotide is an oligonucleotide comprising the sequence of SEQ ID NO: 3 or a variant thereof.
4. A method according to any one of the preceding claims, wherein said strand invasion oligonucleotide further comprises one or more modified nucleotides in its 3 ’region.
5. A method according to any one of the preceding claims, wherein said at least one upstream primer, at least one downstream primer and/or said strand invasion oligonucleotide have sequences which are fully complementary to said target nucleic acid.
6. A method according to any one of the preceding claims, wherein each of said at least upstream primer and said at least one downstream primer is an oligonucleotide of less than 30 nucleotides in length.
7. A method according to any one of the preceding claims, wherein said strand invasion oligonucleotide is an oligonucleotide of greater than 30 nucleotides in length.
8. A method according to any one of the preceding claims, wherein said strand invasion oligonucleotide has a 5’ terminal region non-complementary to said target nucleic acid sequence.
9. A method according to claim 8, wherein said 5’ terminal region non- complementary to the target nucleic acid sequence is:
(a) at least about 10 nucleotides in length; and/or
(b) is comprised of a greater number of pyrimidine nucleotides compared to purine nucleotides.
10. The method of any one of the preceding claims, wherein detecting the
38
amplification of said target nucleic acid sequence indicates presence of RNA from SARS- CoV-2 in said sample.
11. The method of any one of the preceding claims, further comprising contacting said sample with an oligonucleotide probe comprising a detectable moiety and a region complementary to said target nucleic acid sequence.
12. The method of claim 11, wherein the probe comprises a fluorophore and a quencher.
13. The method of claim 11 or 12, wherein the probe comprises a sequence of SEQ ID NO: 27 or a variant thereof.
14. The method of any one of claims 11-13, wherein the probe comprises at least 20% RNA nucleotides, modified RNA nucleotides and/or PNA nucleotides, preferably wherein the probe comprises at least 20% LNA nucleotides.
15. The method of any one of claims 11-14, wherein the probe comprises the sequence of SEQ ID NO: 28.
16. The method of any one of the preceding claims, wherein:
(a) the upstream primer comprises any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14, or a variant thereof;
(b) the downstream primer comprises the sequence of SEQ ID NO: 23 or a variant thereof.
17. The method of any one of the preceding claims, wherein:
(a) said upstream primer comprises the sequence of SEQ ID NO: 7 or variant thereof;
(b) said downstream primer comprises the sequence of SEQ ID NO: 23 or variant thereof; and
(c) said strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 25 or variant thereof.
18. A composition comprising:
(a) an upstream primer comprising the sequence of SEQ ID NO: 1 or variant thereof;
(b) a downstream primer comprising the sequence of SEQ ID NO: 2 or variant thereof; and
(c) a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 3 or variant thereof.
19. A kit comprising:
(a) an upstream primer comprising the sequence of SEQ ID NO: 1 or variant thereof;
(b) a downstream primer comprising the sequence of SEQ ID NO: 2 or variant thereof; and
(c) a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 3 or variant thereof.
20. A composition according to claim 18, or a kit according to claim 19, wherein:
(a) the upstream primer comprises any one of the sequence of SEQ ID NOs: 7, 11, 12 and 14, or a variant thereof;
(b) the downstream primer comprises the sequence of SEQ ID NO: 23, or a variant thereof.
21. A composition according to claim 18, or a kit according to claim 19, wherein:
(a) said upstream primer comprises the sequence of SEQ ID NO: 7;
(b) said downstream primer comprises the sequence of SEQ ID NO: 23; and
(c) said strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 25.
22. A composition or kit according to any one of claims 18 to 21, further comprising
(i) a reverse transcriptase; and/or
(ii) a DNA polymerase; and/or
(iii) a recombinase.
23. A method for diagnosis of an infection by SARS-CoV-2 in a subject, comprising carrying out a method as defined in any one of claims 1 to 17 in a sample from said subject.
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GBGB2018125.1A GB202018125D0 (en) | 2020-11-18 | 2020-11-18 | Method |
PCT/EP2021/082192 WO2022106562A1 (en) | 2020-11-18 | 2021-11-18 | Method of detecting sars-cov2 |
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EP4247981A1 true EP4247981A1 (en) | 2023-09-27 |
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EP21820467.5A Pending EP4247981A1 (en) | 2020-11-18 | 2021-11-18 | Method of detecting sars-cov2 |
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EP (1) | EP4247981A1 (en) |
GB (1) | GB202018125D0 (en) |
WO (1) | WO2022106562A1 (en) |
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Publication number | Priority date | Publication date | Assignee | Title |
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EP2426221B1 (en) | 2006-05-04 | 2016-06-22 | Alere San Diego, Inc. | Recombinase polymerase amplification |
ES2638853T3 (en) | 2008-06-11 | 2017-10-24 | Orion Pharma (Uk) Limited | Isothermal nucleic acid amplification |
WO2014173963A1 (en) | 2013-04-25 | 2014-10-30 | Orion Diagnostica Oy | Strand-invasion based dna amplification method |
WO2015075198A1 (en) | 2013-11-22 | 2015-05-28 | Orion Diagnostica Oy | Detection of nucleic acids by strand invasion based amplification |
GB201410022D0 (en) | 2014-06-05 | 2014-07-16 | Orion Diagnostica Oy | Method |
DK180348B1 (en) * | 2019-12-31 | 2021-01-20 | Qlife Aps | Method and device for analysis of liquid samples |
CN111647683B (en) * | 2020-01-19 | 2023-07-21 | 上海市公共卫生临床中心 | New coronavirus 2019-nCoV nucleic acid detection kit and application thereof |
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2020
- 2020-11-18 GB GBGB2018125.1A patent/GB202018125D0/en not_active Ceased
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2021
- 2021-11-18 WO PCT/EP2021/082192 patent/WO2022106562A1/en unknown
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GB202018125D0 (en) | 2020-12-30 |
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