Patent Application
20240279753
The present invention relates to a nucleic acid assay and a method for amplifying or amplifying and detecting nucleic acids. In particular embodiments, the invention relates to an assay for detecting SARS-CoV-2 nucleic acids.
The SARS-CoV-2 pandemic has led to development of a number of assays for detecting the virus. Two of the most common are antigen-based lateral flow assays, which detect presence of specific viral proteins, using conjugated antibodies to bind spike, envelope, membrane or nucleocapsid proteins; or RT-PCR assays, which amplify specific viral genome sequences to detect presence of the virus. Each of these has advantages and disadvantages; lateral flow assays are rapid, giving results in as little as 30 minutes, but relatively less sensitive and prone to false negatives. RT-PCR tests are more accurate, but are not seen as a rapid option given the need generally to return samples to a laboratory for testing. It would be beneficial to have alternative testing options available. Further, given rates of viral mutation in the genes and proteins normally assayed, there may be reductions in sensitivity of these assays. It would therefore be beneficial to adopt a different region of the viral genome in the assay.
Various isothermal amplification methods are known for amplification of nucleic acids which may be suitable for incorporating into an alternative assay for SARS-CoV-2. These include NASBA (nucleic acid sequence-based amplification); LAMP (loop-mediated isothermal amplification); HAD (helicase-dependent amplification); RCA (rolling circle amplification); MDA (multiple displacement amplification); WGA (whole genome amplification, including MALBAC, LIANTI, DOP-PCR); and RPA (recombinase polymerase amplification). The benefit of isothermal amplification is that it does not require thermal cycling to amplify the target, and so may not require specific equipment to carry out the assay.
A further isothermal technique is RT-LIDA (reverse transcription lesion-induced DNA amplification). The LIDA technique is described generally in U.S. Pat. No. 9,193,993, and is a method for isothermally amplifying a DNA sequence involving hybridizing a destabilizing DNA template to complementary nucleotide fragments to form a first nicked duplex; ligating the first nicked duplex to form a product duplex comprising the DNA sequence and the template, wherein the product duplex is capable of dissociating to release the DNA sequence and the template; and repeating these steps to generate multiple copies of the template and the DNA sequence. Where the initial template is RNA, then an initial step is also included to generate cDNA from the RNA. RT-LIDA is also described in Alladin-Mustan et al, “Reverse transcription lesion-induced DNA amplification: An instrument-free isothermal method to detect RNA”; Analytica Chimica Acta, Volume 1149, 2021, 238130, https://doi.org/10.1016/j.aca.2020.12.005.
In developing the SARS-CoV-2 assay described herein, the present inventor has further developed a modification of RT-LIDA which may provide improved amplification results. In particular, in conventional RT-LIDA, self-ligation of primers can be an issue, resulting in false positives. It is believed that the modified assay presented here reduces the occurrence of such false positives, among other advantages.
Although the specific assay described herein is an RT-LIDA amplification of a portion of the SARS-CoV-2 genome, it will be understood that a) the same portion of SARS-CoV-2 genome may be detected using other methods, in particular isothermal amplification; and that b) the modified RT-LIDA described herein is of more general applicability than for this assay. A key benefit of the assay described herein is that it allows for isothermal amplification and detection in a single reaction vessel, and can be performed with no specialised equipment beyond the assay reagents, if desired.
According to an aspect of the present invention, there is provided an assay for SARS-CoV-2 wherein a portion of a nucleic acid coding for a Leu-Thr-Asp (LTD) sequence at or near the terminus of the ORF9c protein is amplified and detected.
Also provided is a method for detecting SARS-CoV-2 in a sample, the method comprising: generating cDNA from an RNA present in the sample; amplifying a portion of the cDNA using an amplification process specific for a portion of the cDNA corresponding to the SARS-CoV-2 genome coding for ORF9c; and detecting the presence of a portion of amplified cDNA coding for a Leu-Thr-Asp (LTD) sequence at or near the terminus of the ORF9c protein.
The SARS-CoV-2 genome includes a gene coding for ORF9c (previously known as ORF14). This is a 70 amino acid protein which was previously of unknown function and present in Human SARS and Bat CoV. In SARS-CoV-2 the ORF9c protein is 73 amino acids long and has a 9 bp insert coding 3 additional amino acids (LTD) at the terminus of the transcript. A comparison of the ORF9c amino acid sequences from SARS-CoV-2 (SEQ ID NO: 12), Human SARS (SEQ ID NO: 13), and Bat CoV (SEQ ID NO: 14) is given in
The cDNA sequence encoding the LTD insertion is AAC TGT CTA (SEQ ID NO: 1). The genomic sequence will of course be the corresponding RNA sequence (UUG ACA GAU, SEQ ID NO: 2). Amplification may be of a DNA sequence spanning this insertion, and detection via a probe which binds a sequence comprising this insertion.
In preferred embodiments, amplification is via RT-LIDA. LIDA is a simple-to-implement amplification technique based on a modification of the Ligase Chain Reaction (LCR). It operates at room-temperatures between 18° C. to 37° C. to provide rapid (≤20 minutes) amplification of a selected target. It uses four oligonucleotide primers and a single enzyme making it significantly less complex compared to other isothermal chemistries.
Also provided is a method of amplifying a target RNA molecule in a sample, the method comprising:
As discussed herein, the inventor has determined that use of a DNA ligase having no single base overhang or blunt end ligating ability provides for enhanced accuracy and reduced background from the method. In preferred embodiments, the ligase is PBCV-1 DNA ligase, although others may be used, including engineered ligases which have been modified from the natural form to reduce or remove a single base overhang or blunt end ligating ability. The reduction of background amplification from use of such a ligase means that components can be included in the reaction mix which accelerate the ligase reaction (for example, crowding agents, such as PEG). With conventional ligases having single base overhang ligating ability, such as T4 ligase, these have to be omitted because they promote the co-amplification of this background.
The destabilising primers may include one or more features selected from the presence of an abasic site or a mismatch with the corresponding complementary sequence. In embodiments, one primer includes a mismatch, and one primer includes an abasic site. Preferably the P1c* primer includes a mismatch; this may be an A:T mismatch (that is, a perfectly complementary sequence may include a G or C, whereas the mismatch includes an A or a T). Preferably the mismatch is internal to the primer; that is, at least 2, 3, 4, or more nucleotides distant from the 5′ and the 3′ end. In embodiments, the P1(csp) primer includes an abasic site. The abasic site is preferably at an end of the primer, preferably the 5′ end.
The primers are designed so as to hybridise to contiguous portions of the target. To allow for ligation, the upstream primer of each pair (that is, the primer which hybridises to a region on the target 5′-wards of the other primer) includes a phosphate group at the 5′ end. For example, the P2p and P1(csp) primers may include this phosphate group.
The method may further comprise the step of detecting at least one of the cDNA strands. Preferably at least one of the primers includes a label. For example, the P2* primer may include a label. In embodiments, one primer from each pair includes a detectable label (eg, the P2* primer and the P1c* primer). This allows detection of both cDNA strands. The label may be a fluorescent label, for example, a fluorescein group.
The detection step may comprise capturing at least one of the cDNA strands via a complementary oligonucleotide (Ro, a reporter oligonucleotide) immobilised on a solid support. Where the cDNA strand is labelled, this can be used to localise the label to a specific location; for example, in order to develop a line or other indicator to show detection of the target sequence.
In an embodiment, the immobilised complementary oligonucleotide Ro may initially be hybridised to a partially-complementary oligonucleotide (for example, an oligonucleotide having one or more mismatches, or an oligonucleotide which is shorter than the immobilised oligonucleotide); and capturing the cDNA strand comprises 35 allowing the cDNA strand to displace the partially-complementary oligonucleotide. It will be apparent that the partially-complementary oligonucleotide is partially identical to the cDNA. In a preferred embodiment the partially-complementary oligonucleotide is shorter than the immobilised oligonucleotide and is shorter than the cDNA. The relative lengths are preferably selected such that, in the event that displaced oligonucleotide hybridises to free cDNA or primers, the ligase is unable to activate. This reduces false positives and further allows the detection step to take place in the same environment as the amplification step (that is, in the presence of the primers). In embodiments, the immobilised complementary oligonucleotide Ro and partially-complementary oligonucleotide (Qo) include a reporter-quencher pair (for example, Ro may include a reporter and Qo includes a quencher). Displacement of the Qo oligonucleotide by the cDNA strand separates the reporter-quencher pair and allows the reporter to be detected. This approach avoids the need to include a label in the initial primers. Any compatible reporter-quencher pair may be used; for example FAM or VIC for the reporter dye and TAMRA for the quencher dye. In other embodiments, the oligonucleotides Ro and Qo may be linked as a single molecule, either through an intervening section of nucleic acid, or via a non-nucleic acid linker. In further embodiments, where a reporter-quencher pair is used, the Ro oligonucleotide need not be immobilised on a solid support, but may be in solution. In an example, the Ro and Qo oligonucleotides in solution may be joined by a linker (nucleic acid or non-nucleic acid).
In some embodiments, at least one of the primer sequences includes a tag (for example, a nucleic acid tag) which is not part of the target sequence to be amplified. This tag may be used in the detection or other steps of the method, as described herein.
In embodiments, the primer and other sequences are as follows:
In further embodiments, the Ro and Qo oligonucleotide sequences are:
It is also possible to use a similar method to amplify DNA, with the addition of a DNA unfolding enzyme (eg, a recombinase or helicase) to unwind an initial double stranded DNA molecule. Thus, also provided by the present invention is a method of amplifying a target DNA molecule in a sample, the method comprising:
The SSB may be a bacterial SSB. The DNA unfolding enzyme may be a DNA helicase, or may be a DNA recombinase, preferably RecA.
The DNA ligase is preferably PBCV-1 DNA ligase. Other features of this aspect of the invention may be the same as for the RNA amplification method described herein. It will be noted that no displacement DNA is required in this method, as the ligated DNA will spontaneously dissociate from the template once formed. Further, we believe that this method—using SSB and helicase or recombinase—can also serve to replace the disDNA from the RT-LIDA method described herein. As such, a further aspect of the invention provides a method of amplifying a target RNA molecule in a sample, the method comprising:
Also provided is a kit comprising oligonucleotides having the sequences of SEQ ID NOs: 3-7, and optionally also SEQ ID NOs: 8 and 9. The oligonucleotide having SEQ ID NO: 8 may be provided immobilised on a solid support, and with the oligonucleotide of SEQ ID NO: 9 hybridised thereto.
The present inventor has also found that the reaction mix can be separated into a liquid master mix and a lyophilised reagent component is possible. The invention therefore further provides a kit for amplification of a target RNA sequence, the kit comprising:
The kit may further comprise a solid support having a reporter oligonucleotide immobilised thereon, and a quencher oligonucleotide hybridised to the reporter oligonucleotide.
The master mix may comprise 1.05 UM DNA ligase, 50 mM Tris, 10 mM MgCl2, 1 mM ATP and 10 mM DTT. In embodiments, the master mix and/or the lyophilised components may comprise a crowding agent, preferably PEG.
In embodiments, the solid support is in the form of a reaction vessel, where the reaction may take place. The lyophilised reagents may be provided in the reaction vessel. In this way, a sample containing RNA to be detected and the master mix may be added to the reaction vessel containing the lyophilised reagents, and the whole amplification and detection process take place in the vessel.
A yet further aspect of the invention relates to an alternative liquid master mix. In this embodiment, the master mix comprises Tris, manganese cations, DTT, and below 1 mM ATP, at pH greater than 8. A particularly preferred master mix comprises 50 mM Tris-HCl, 5 mM MnCl2, 10 μM ATP, 10 mM DTT, at pH 8.5. This differs from the conventional master mix for PBCV-1, which contains higher ATP concentrations, has a lower pH, and contains MgCl2 rather than MnCl2. See for example the product data sheet for SplintR ligase available from New England Biolabs. As explained further herein, this modified master mix reduces the production of adenylylated DNA, a byproduct which can reduce production of the correct ligated product. The mix may further comprise PBCV-1 DNA ligase, preferably at 1.05 UM, and/or may further comprise a crowding agent, preferably PEG. This master mix may also be provided together with lyophilised reagents as described.
Coronaviruses (CoVs) (order Nidovirales, family Coronaviridae, subfamily Coronavirinae) are enveloped viruses with a positive sense, single-stranded RNA genome. With genome sizes ranging from 26 to 32 kilobases (kb) in length. They infect humans and cause disease to varying degrees, from upper respiratory tract infections (URTIs) resembling the common cold, to lower respiratory tract infections (LRTIs) such as bronchitis, pneumonia, and even severe acute respiratory syndrome (SARS). SARS-CoV-2, SARS-COV and MERS-COV cause severe infections that lead to high mortality rates.
The coronaviral genome encodes four major structural proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein, all of which are required to produce a structurally complete viral particle. The E protein is the smallest of the major structural proteins. During the replication cycle, E is abundantly expressed inside the infected cell, but only a small portion is incorporated into the virion envelope. E participates in viral assembly, release of virions and pathogenesis of the virus. Much of the protein is localised at the site of intracellular trafficking, the ER, Golgi, and ERGIC, where it participates in CoV assembly and budding.
Sequenced genomes of SARS-CoV-2 strains combined with comparative analysis of the SARS-COV genome organization and transcription has been used to construct a tentative list of gene products. It was suggested that SARS-CoV-2 had 16 predicted non-structural proteins constituting a polyprotein (wORF1ab), followed by (at least) 13 downstream open reading frames (ORFs): Surface glycoprotein (or Spike), ORF3a, ORF3b, Envelope, Membrane, ORF6, ORF7a, ORF7b, ORF8, Nucleocapsid, ORF9a, ORF9b and ORF10. The three viral species whose proteins shared the highest similarity were consistently the same: human SARS coronavirus (SARS-COV), bat coronavirus (BtCoV), as well as another bat betacoronavirus (BtRf-BetaCoV). A genome map of SARS-CoV-2 is shown in
A growing number of mutations have already been identified in clinical samples of SARS-CoV-2. It is therefore important to design an assay to regions that are impacted least by mutations, or by selecting a chemistry that can report mutations back to the clinician or researcher.
A number of these mutations have been shown to span sites used in commercially available assays which have altered the performance of these tests. In some, multiple mutations across the primer, probe, or primer and probe sites have been observed.
Rather than design a set of primers and probes to a variable region in the spike protein, we selected a region that is not present in other SARS or coronaviruses and is relatively conserved in SARS-CoV-2. We have designed a test that spans an insertion unique to SARS-CoV-2 making it highly specific. An advantage of selecting this target is that testing for SARS-CoV-2 can be carried out using a single target; other assays for SARS-CoV-2 typically require use of at least two separate genomic targets due to the potential for cross reactivity with other coronaviruses from non-unique targets.
Specifically, we have designed a RT-LIDA assay targeted to ORF9c (previously known as ORF14). This is a 70 amino acid protein which was previously of unknown function and present in Human SARS and Bat CoV. In SARS-CoV-2 the ORF9c protein is 73 amino acid long and has a 9 bp insert coding 3 additional amino acids at the terminus of the transcript (
The overall process of RT-LIDA is shown in
The reaction then moves into the exponential phase, in which the DNA primers P2p and P2* and the destabilising DNA primers P1c* and P1(csp) alternately hybridise to the c-DNA-II or F-DNA-I strands, are ligated, and then spontaneously dissociate from the hybridised strand due to the presence of the destabilising features of an abasic site and an internal mismatch in the destabilising primers. Each cycle thus doubles the number of cDNA strands. In this illustration, the destabilising primers include a fluorescent label, allowing detection of the F-DNA-I strand after formation.
The conventional RT-LIDA process uses T4 ligase as the ligation enzyme. However, as shown in
We therefore investigated the use of alternative ligases lacking single base overhang ligation ability. PBCV-1 ligase is described in Nucleic Acids Research, 2003, Vol. 31, No. 17 DOI: 10.1093/nar/gkg665; and ligation of RNA-splinted DNA by PBCV-1 ligase is described in G Lohman et al, “Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase”; Nucleic acids research, 42(3), 1831-1844. https://doi.org/10.1093/nar/gkt1032.
The results of using PBCV-1 ligase in positive and negative control samples are shown in
Under recommended conditions, PBCV-1 reaction buffer contains 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, and the reaction is carried out at pH 7.5 at 25° C. However, this can result in generation of adenylylated DNA primers which reduce yield of the correct ligation product. PBCV-1 DNA ligase binds to a nicked DNA duplex containing reactive 3′-OH and 5′-PO4 termini. It does not bind to a continuous DNA duplex, to a tailed duplex or even to a nicked ligand containing non-ligatable 3′-OH and 5′-OH termini.
The ATP-dependent DNA ligases catalyse the joining of 5′-phosphate-terminated strands to 3′-hydroxyl-terminated strands via three sequential nucleotidyl transfer reactions. In the first step, attack on the α-phosphate of ATP by DNA ligase results in displacement of pyrophosphate and formation of a covalent ligase-adenylate intermediate in which AMP is linked to the ε-amino group of a lysine. The active site lysine residue is located within a conserved motif, KxDGxR. The AMP is then transferred to the 5′-monophosphate terminus of a nicked DNA duplex to form the DNA-adenylate intermediate, which consists of an inverted (5′)-(5′) pyrophosphate bridge structure, AppDNA. Attack by the 3′-OH-terminated strand of the nicked duplex on DNA-adenylate seals the nick and releases AMP.
If AppDNA is released in solution, it can become a ‘dead end’ product under conditions of mM ATP concentrations, as free ligase rapidly reacts with ATP to adenylylate the active site of the enzyme. The adenylylated enzyme cannot bind AppDNA, as the adenylyl group on the enzyme occupies the same binding pocket as the adenylyl group on the AppDNA intermediate. UM ATP concentrations result in a higher steady state concentration of deadenylylated ligase, which can bind and react AppDNA substrates to ligated DNA effectively.
As well as using μM ATP (e.g. 10 μM), enzyme concentration and selection of Mn2+ (5 mM) instead of Mg2+ significantly reduces the formation of AppDNA and the potential for dead-end substrates. pH 8.5 also eliminates AppDNA, normally pH between 7 and 8 is used for most ligase master mixes. Hence, the invention further provides a ligase buffer comprising manganese cations, a reduced (less than 1 mM) amount of ATP, and at a pH above 8. A preferred ligase buffer for use with the methods of the invention includes 50 mM Tris-HCl, 5 mM MnCl2, 10 UM ATP, 10 mM DTT, at pH 8.5.
Further investigation demonstrated that PBCV-1 ligase additionally ligates DNA primers on an RNA template more rapidly than T4 ligase. See
An illustration of the reporting strategy is shown in
In other embodiments, the primers may also include an additional sequence tag which is not part of the target region to be amplified; this allows use of a reporter sequence which is in part complementary to the sequence tag, and does not require any sequence homology to the priming regions as such. This reduces the risk of sequences binding to the reporter or released sequences. As an example, if the sequence to be detected is the P2p-P2* ligated oligonucleotide, then the P2* primer may include an additional sequence tag: P2p-P2*-T. The reporter oligonucleotide Ro is complementary to the P2p-P2*-T sequence as a whole, whereas the quencher oligonucleotide Qo omits the tag, so would have the P2p-P2* sequence. In this embodiment, the released Qo oligonucleotide is able to serve as a template for the P1 primers; however, release only takes place as the P2p-P2*-T product accumulates, such that signal is only detected when there is genuine amplification and release. This modified displacement reporting strategy is illustrated in
A particular advantage of this combination with the described removal of false positive product formation, is that the amplification always goes to completion (100%) after a certain time regardless of input target RNA concentration, so that there is minimal requirement to evaluate fluorescence strength as an endpoint determination, it provides a yes/no result which is particularly suited to POC and OTC applications. However, monitoring of fluorescence signal as a function of time can be used to provide a quantitative measurement for professional use where determination of the quantity of RNA in the sample is important. This method can be used in both modalities.
Finally, an illustration of how the reporting strategy may appear is shown in
In summary, then, we have developed an assay for SARS-CoV-2 which identifies a highly conserved region which is distinct from closely related viruses. Further, in the process of development we have determined that use of PBCV-1 ligase in an RT-LIDA reaction has a number of benefits:
Further, adoption of the displacement reporting method would be particularly useful for LIDA and in fact is based on the same displacement mechanics of oligonucleotide association disassociation kinetics within the LIDA assay. This too can be carried out as a single step process.
We have additionally developed an improved ligation master mix, the use of which may reduce production of AppDNA during ligation, which can reduce yield of the correct ligation product.
Further, use of SSB and DNA unwinding proteins, such as RecA or helicase, can allow a single step amplification procedure to be carried out on DNA, as well as RNA.
These properties mean that the assays described herein may be carried out rapidly, in a single reaction vessel, and in a single step.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2108853.9 | Jun 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/066736 | 6/20/2022 | WO |
