WO2018077891A1

WO2018077891A1 - Method for detecting rsv by strand-invasion based dna amplification

Info

Publication number: WO2018077891A1
Application number: PCT/EP2017/077190
Authority: WO
Inventors: Kevin EBOIGBODIN; Kirsi MOILANEN; Sonja ELF
Original assignee: Orion Diagnostica Oy
Priority date: 2016-10-25
Filing date: 2017-10-24
Publication date: 2018-05-03
Also published as: GB201618035D0

Abstract

A method for detecting a ribonucleic acid (RNA) comprising a target nucleic acid sequence by strand-invasion based DNA amplification is provided, together with oligonucleotides, compositions and kits suitable for use in this method.

Description

METHOD FOR DETECTING RSV BY STRAND-INVASION BASED DNA

AMPLIFICATION

Field of the Invention

The invention relates to a method for detecting a target nucleic acid sequence of a ribonucleic acid (RNA) 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 pathogens, particularly Respiratory Syncytial Virus (RSV). Background to the Invention

Nucleic acid amplification tests are becoming the method of choice for routine diagnosis of pathogens such as viruses within clinical laboratory settings. They offer superior sensitivity and specificity over serology, immunoassays and traditional viral culture-based detection methods [Mahony et al. 2011].

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays have been developed for example for the diagnosis of Respiratory Syncytial Virus (RSV) 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 [Drosten et al. 2002]. Hence 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.

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 2014/173963A1.

Summary of the Invention

The present invention relates to detection of a target nucleic acid sequence of an

RNA in a sample, in particular a target nucleic acid sequence of a Respiratory Syncytial Virus (RSV). The method of the invention for detecting a target nucleic acid sequence present in an RNA uses a reverse transcriptase and at least one upstream primer, at least one downstream primer, and at least one strand invasion oligonucleotide, each comprising a region complementary to said target nucleic acid sequence. The reverse transcriptase synthesises a complementary DNA from the RNA. In combination, the primers and strand invasion oligonucleotide provide for amplification of the target nucleic acid sequence from duplex nucleic acids formed 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 acids. The reverse transcription of RNA to cDNA is typically carried out simultaneously with strand-invasion based amplification and detection of duplex nucleic acids comprising the target nucleic acid sequence derived from the starting RNA template.

In preferred embodiments, more than one species of upstream primer or downstream primer and/or more than one species of strand invasion oligonucleotide may be used for detection. This advantageously allows for improved sensitivity of detection of multiple subtypes of RSV in a single reaction.

RSV is a member of the Paramyxoviridae family which harbors a non-segmented negative sense single-stranded RNA as its genetic material. Two major serotypes of RSV exist; RSV A and RSV B which differ in their genetic makeup and surface glycoproteins. The inventors have shown that the method of the invention allows for highly specific and sensitive detection of RNA targets, such as genomic RNA of (RSV). 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 RSV A and RSV B 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 RSV A and RSV B RNA.

The invention provides a method for detecting a ribonucleic acid (RNA) comprising a target nucleic acid sequence in a sample, said method comprising contacting said sample with a reverse transcriptase, 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 a duplex nucleic acid comprising said target nucleic acid sequence single-stranded, to allow the binding of said upstream primer and a downstream primer.

The NA comprising the target nucleic acid sequence is typically from an

RSV virus.

The invention further provides a method for detecting a target nucleic acid sequence from an RSV A and/or RSV B gene 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.

The method may be for detecting a target nucleic acid sequence from an RSV A gene or a target nucleic acid sequence from an RSV B gene. Preferably, the method is for detecting a target nucleic acid sequence from an RSV A gene and a target nucleic acid sequence from an RSV B gene. Preferably said target sequence is in the N-gene area of RSV A or RSV B Preferably the amplified target nucleic acid sequence is SEQ ID NO: 1 or a variant thereof.

The invention additionally provides a composition or a kit, the composition or kit comprising at least two oligonucleotides selected from (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide,

wherein said upstream primer is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2 or a variant thereof, said downstream primer is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3 or a variant thereof, and said strand invasion oligonucleotide is an oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 5 or a variant thereof.

In a preferred embodiment, the composition or kit comprises an upstream primer which is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2, a first downstream primer which is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3, a second downstream primer which is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 4, a first strand invasion oligonucleotide which is an

oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 7, and a second strand invasion oligonucleotide which is an

oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 8.

The invention also provides a method for diagnosis of an infection by a pathogen in a subject, comprising carrying out a method of the invention for detecting a ribonucleic acid (RNA) comprising a target nucleic acid sequence of a pathogen in a sample from said subject. Preferably, the pathogen is an RSV. The invention also provides a

method for diagnosis of an infection by an RSV A and/or RSV B virus in a subject, comprising carrying out a method of the invention for detecting a target nucleic acid sequence from anRSV A and/or RSV B gene in a sample from said subject. Brief Description of the Figures

Figure 1 shows alignment of oligonucleotides used for SIBA in NPl assay described in Example 1 with sequences for RSV A and B obtained from various sources. Italic sequence denotes 2'-0-methyl RNA sequence present in invasion oligonucleotides. Bold poly-cytosine denotes the non-homologous seeding area present in invasion oligonucleotides. Underlined sequence denotes sequence overlap between the forward or reverse primers and the respective invasion oligonucleotide. Mismatches to RSV A sequences are shaded.

Figure 2 shows sensitivity of (a) RSV A and (b) RSV B RT-SIBA assays detecting the N gene area of RSV (NP1 assay). Serial dilutions of RNA from RSV A or RSV B were prepared and used as template in RT-SIBA assays. Figure 2 (c) shows RSV A detection with RSV A or RSV B detecting oligonucleotides and their combination. Figure 2 (d) shows RSV B detection with RSV A or RSV B detecting oligonucleotides and their combination. Specificity for RSV A or RSV B was also confirmed with assays from RNA for other viruses (e).

Figure 3 shows sensitivity of further RSV A and RSV B RT-SIBA assays for the N gene of RSV: (a) RSV A detection with NP- 1.1 assay, (b) RSV B detection with NP- 1.1 assay, (c) RSV A and RSV B detection with NP-2 assay, (d) RSV A detection with NP-3 assay.

Figure 4 shows sensitivity of an RSV B RT-SIBA assay for the M2-1 gene of RSV.

Figure 5 shows sensitivity of RSV A and RSV B RT-SIBA assays for the polymerase gene area of RSV: (a) RSV A and RSV B detection with POL-1 assay, (b) RSV A detection with AB4 assay. Total copy number of template RNA per reaction was 10 - 1000 cp/reaction. Y- axis: SybrGreen 1 fluoresence intensity (arbitrary units); X-axis: time (minutes). The results indicated that RT-SIBA could be used to reproducibly detect as low as 10 copies of RSV RNA per reaction. NTC = no template control. Figure 6 shows sequences of the invention and the assays in which they are preferably used.

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.

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, the sample may be processed only by heating or treatment with detergents. However, the nucleic acid may also be derived, obtained or extracted from the sample. Methods for processing samples containing nucleic acids, extracting nucleic acids and/or purifying nucleic acids for use in detection 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 nucleic acid 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, or having an infection by a pathogen, in particular a viral pathogen. The viral pathogen is preferably an RSV virus. The RNA may be genomic RNA of a virus. The RNA may also be bacterial mRNA. RNA may also be useful for bacterial detection, owing to the very large number of ribosomes present in bacterial cells which effectively amplify the concentration of target sequences. The RNA is typically single- stranded RNA but double-stranded RNA may also be used.

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 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. RSV samples may preferably be collected by nasal or nasopharyngeal (NP) swabs from nasal or nasopharyngeal cavity. The swab sample may be stored in universal transport media (UTM) or viral transport media (VTM).

Nasopharyngeal aspirates or nasopharyngeal washes may also be used as a sample material for detection of RSV.

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.

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 RNA from 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 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, all four dNTPs, dATP, dTTP, 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.

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: 1 has as its RNA equivalent SEQ ID NO: 41. 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 a pathogen 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 the pathogen in the sample, even if closely related organisms 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 RSV viruses. Preferably, the target nucleic acid sequence is not present in any other type of virus, or not present in any other respiratory virus. The target nucleic acid sequence may be specific to a particular subtype of RSV. The known RSV subtypes include RSV A and RSV B. The target nucleic acid sequence may be specific for RSV A or RSV B. Alternatively, the target nucleic acid sequence may be present in both RSV A and RSV B. The target nucleic acid sequence may be specific for a particular genotype of RSV. Alternatively, the target nucleic acid sequence may be inclusive for multiple different genotypes of RSV A and/or RSV B, and thus is typically present and can be detected in more than one genotypes of RSV A and/or RSV B. Preferably, the target nucleic acid sequence is inclusive for at least two or three, more preferably at least five, at least seven, at least ten different genotypes, typically pathogenic genotypes. Typically, the target nucleic acid sequence is inclusive for the most clinically relevant genotypes, in respiratory illnesses caused by RSVA or RSV B.

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, or at least 65 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 70, about 50 to about 70, about 55 to about 70 or about 60 to about 70 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 RSV virus, 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 present in the N-gene area of RSV. The target nucleic acid sequence from the N-gene area may comprise a nucleotide sequence within nucleotides 1500-1600 of an RSV genome, typically comprising nucleotides 1511-1578 of RSV A deposited as ATCC VR-1540 or nucleotides corresponding thereto. The target nucleic acid may comprise nucleotides 1509-1576 of RSV B deposited as ATCC VR-955 or nucleotides corresponding thereto.

The target nucleic acid sequence for the N-gene area of RSV preferably comprises

SEQ ID NO: 1 or a variant thereof (for detection of the N-gene of RSV). It should be understood that the target nucleic acid sequence which provides the template for amplification will be present in a duplex which comprises a sense strand representing SEQ ID NO: 1 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 upstream primer binds upstream of the site of invasion of the duplex by the strand invasion oligonucleotide and the downstream primer binds downstream of the invasion site. This binding is shown in Figure 1. The strand invasion oligonucleotide may bind to the opposing strand to that bound by the upstream or downstream primer (invasion may be initiated based on binding to either strand of the duplex). Accordingly the terms "upstream" and "downstream" refer to the position of binding of the primers on the duplex with respect to the invasion site. Where the primer binds on the same strand as the invasion oligonucleotide, an upstream primer will bind 5' to the invasion oligonucleotide and a downstream primer will bind 3 ' to the invasion oligonucleotide.

The target nucleic acid sequence may comprise a naturally occurring variant sequence of SEQ ID NO: 1 which is present in a different subtype or genotype of RSV . SEQ ID NO: 1 represents a conserved sequence from the RSV A subtype. Other related target sequences to SEQ ID NO: 1 from the N-gene area of use in the invention include SEQ ID NOs 34 and 35. SEQ ID NOs 42 and 43 represent naturally occurring variant sequences from the RSV B subtype.

Variants of SEQ ID NO: 1 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 50 or at least 55 contiguous nucleotides of SEQ ID NO: 1. The variants may comprise a region which has 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 or more mismatches (substitutions) with respect to a region of the corresponding original target sequence of SEQ ID NO: 1.

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 40, 45, or 50 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, 40, or 45 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: 1 in a region corresponding to the strand-invasion oligonucleotide-complementary region of SEQ ID NO: 1. 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: 1.

Corresponding variants to those described above for SEQ ID NO: 1 are also described herein for SEQ ID NOs 34 and 35 and for the RSV B target sequences of SEQ ID NOs 42 and 43.

Most preferably, the target nucleic acid sequence comprises SEQ ID NO: 1, 34, 35, 42 or 43 or consists of SEQ ID NO: 1 , 34, 35, 42, or 43 and a complementary antisense strand.

An additional target nucleic acid sequence from the N-gene area that may be used for detection of RSV is SEQ ID NO: 19 or a variant thereof. Variants of SEQ ID NO: 19 may be selected according to the same criteria set out above in relation to SEQ ID NO: 1.

Other target nucleic acid sequences that may be detected in accordance with the invention are in the M2-1 gene area of an RSV or in the Polymerase gene area of an RSV.

A preferred target nucleic acid sequences for the M2-1 gene area of an RSV is SEQ ID

NO: 29. Preferred target nucleic acid sequences for the Polymerase gene area of an RSV are SEQ ID NOs 14 and 24. Thus, the target nucleic acid sequence to be detected may comprise any of SEQ ID NOs 14, , 24 and 29 or comprise a variant of any thereof. Variants of SEQ ID NOs 14, 24 and 29 may be selected according to the same criteria set out above in relation to SEQ ID NO: 1.

Additional variant target nucleic acid sequences provided herein are the longer consensus sequences from which the above amplicons are derived, as described below. Thus, the consensus sequences of any of SEQ ID NOs 36-40 or fragments thereof may be amplified in accordance with the invention.

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. For example, a method of the invention may detect both SV A and RSV B.

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 single- stranded 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 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 sequence to the target. The length of the primer sequence that is complementary to the target is sufficient to provide specific hybridisation to the target nucleic acid sequence. The length of complementary sequence is typically at least 10 nucleotides, more preferably at least 15, at least 16, or at least 17 nucleotides. The length of complementary sequence may be 10-25, 15-25, 10-30 or 15-30 nucleotides.

It should be understood that the above sequence lengths refer to portions of the primers which may be partly or fully complementary 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 region of the primer and the

corresponding region of the target sequence. Preferably, the primer is designed to allow for specific detection of a pathogen of interest (typically an RSV virus). 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 one strand of the duplex target nucleic acid sequence, at a position upstream of the site of invasion by the strand invasion oligonucleotide. Where the upstream primer binds on the same strand as the strand invasion oligonucleotide (in the 5' region of this strand), it typically binds proximal or overlapping with the 5' binding site of the strand invasion oligonucleotide. The

downstream (or reverse) primer binds to the opposing strand of the duplex target nucleic acid sequence to the upstream primer, at a position upstream of the site of invasion by the strand invasion oligonucleotide. Where the downstream primer binds on the same strand as the strand invasion oligonucleotide (in the 3 ' region of this strand), it typically binds 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 of target nucleotide sequences in RSV genes are provided herein. Preferred upstream and downstream primers for detection of the target sequence of SEQ ID NO: lare the primers of SEQ ID NOs 2 and 3, or variants thereof.

Variants of SEQ ID NOs 2 and 3 may be oligonucleotides of up to 30 nucleotides in length comprising a region which is partly or fully complementary to at least 10 contiguous nucleotides of the corresponding original primer sequence of SEQ ID NO: 2 or

3. Preferably, said variants will comprise a region which is partly or fully identical to at least 11, 12, 13, 14 or 15 contiguous nucleotides of the corresponding original primer sequence of SEQ ID NO: 2 or 3. Where the original primer sequence is longer than 16 nucleotides in length, such as up to 21 nucleotides in length, the variants may

correspondingly comprise a region which is partly or fully identical to 16, 17, 18, 19 or 20 contiguous nucleotides thereof.

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 identical to the original primer sequence. 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 identity with the original primer sequence.

Variants of SEQ ID NOs 2 and 3 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.

Additionally, the variant primers may comprise region(s) complementary 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.

Variants of the upstream and downstream primers of the invention further include variants which are specific for RSV A, or specific for RSV B. It should be understood that the term "specific" in the context of the invention defines an ability to more sensitively and/or rapidly detect a particular subtype of RSV. A variant specific for RSV A may thus also be capable of detecting RSVB, but for example with a slower detection time. Variants may have improved specificity for RSV A or RSV B through incorporating sequence regions which bind and/or amplify a target sequence from RSV A or RSV B with greater efficiency than the corresponding target sequence from the other RSV subtype. For example, an RSV A specific primer may be fully complementary to a target sequence from RSV A, but have a mismatch with the corresponding target sequence from RSV B, causing it to bind to and/or amplify the RSV B sequence with lower efficiency. An RSV A-specific primer may be preferred for amplification of SEQ ID NO: 1, 34 or 35 or a variant of any thereof. An RSV B-specific primer may be preferred for amplification of SEQ ID NO: 42 or 43 or a variant thereof.

A preferred example of an RSV A specific primer is provided by the downstream primer of SEQ ID NO: 3. A preferred example of an RSV B-specific primer is provided by the downstream primer of SEQ ID NO: 4, which is a variant of SEQ ID NO: 3 having a single mismatch to SEQ ID NO: 3 (at nucleotide position 7, C instead of T, as shown in Figure 1). A variant of SEQ ID NO: 3 which is specific for RSV B more generally is any variant as described above (such as an oligonucleotide of up to 30 nucleotides in length which has a region partly identical to at least 10 contiguous nucleotides of the

corresponding original primer sequence of SEQ ID NO: 3) wherein the partly identical region comprises the mismatch present in SEQ ID NO: 4. The only mismatch between the partly identical region and the corresponding region of the original primer sequence of SEQ ID NO: 3 may be that present in SEQ ID NO: 4. An RSV B specific variant of SEQ ID NO: 3 may comprise a sequence which is identical to SEQ ID NO: 3 but for the mismatch present in SEQ ID NO: 4. An RSV-B specific primer which comprises the sequence of SEQ ID NO: 4 may also be used.

RSV A or RSV B specific variants with an analogous definition may also be provided for other primers described herein wherein a mismatch is present between their corresponding target sequences in RSV A and RSV B, and this influences binding and/or amplification efficiency with the primer.

Where an RSVA or RSV B specific primer (upstream or downstream) is used, it may be used as the only upstream or downstream primer, to thereby provide for improved detection of RSV A or RSV B where this is desired. Alternatively, a primer specific for RSV A (upstream or downstream) and a primer specific for RSV B (upstream or downstream) may be included together to provide for improved detection of both RSV A and RSV B, in the context of desired detection of any RSV / both the A and B subtypes of RSV. In preferred embodiments, an assay for detection of the N-gene area of RSV includes RSV A and RSV B specific primers for the target nucleic acid sequence of SEQ ID NO: 1 or a variant thereof. The RSV A and RSV B specific primers may be the downstream primers of SEQ ID NOs 3 and 4, or RSV A/RSV B specific variants thereof as applicable.

An alternative downstream primer which may be used in combination with upstream primer comprising SEQ ID NO: 2 or a variant thereof for detection of the target sequence of SEQ ID NO: 1 is the downstream primer comprising SEQ ID NO: 9 or a variant thereof.

An additional upstream and downstream primer pair which may be used for detection of the target sequence of SEQ ID NO: 1 is the primer pair comprising the sequences of SEQ ID NO: 10 and 11 or variants thereof. An additional target nucleic acid sequence from the N-gene area that may be used for detection of RSV is SEQ ID NO: 19 or a variant thereof. For detection of the target nucleic acid sequence of SEQ ID NO: 19 or a variant thereof, upstream and downstream primers comprising respectively the sequences of SEQ ID NOs 20 and 21 or variants thereof are provided herein. For the target nucleic acid sequence of SEQ ID NO: 29 or a variant thereof, from the M2-1 gene area, primers comprising the sequences of SEQ ID NOs 30 and 31 or variants thereof are provided. Additionally, for the target nucleic acid sequence of SEQ ID NO: 14 or a variant thereof, from the Polymerase gene area of RSV, primers comprising the sequences of SEQ ID NOs 15 and 16 or variants thereof are provided. Also, for the additional target nucleic acid sequence from the Polymerase gene area of SEQ ID NO: 24 or a variant thereof, primers comprising the sequences of SEQ ID NOs 25 and 26 or variants thereof are provided.

All of the above primers are typically oligonucleotides of less than 30 nucleotides in length. Suitable variant sequences for all primers described above may be selected in accordance with the detailed criteria described above, including those described in relation to variants of the primers of SEQ ID NOs 2 and 3.

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 (since it may invade on either strand) and optionally additional flanking non-complementary or non-identical 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 a pathogen of interest (typically an RSV virus). Thus, the strand invasion oligonucleotide preferably specifically or selectively hybridises to a complementary sequence found only in the pathogen of interest. 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 the pathogen of interest 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, or 12-17 nucleotides in length, more preferably about 14 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 or consists essentially of pyrimidine (cytosine or guanine) nucleotides. The non-complementary region may comprise, consist or consist essentially of a mixture of cytosine and guanine nucleotides. Preferably, where a mixture of pyrimidine nucleotides is present, a greater number of cytosine residues than guanine 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 14 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 especially preferred 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 comprise a 3 ' non-complementary region typically of 1 -3 nucleotides in length which comprises nucleotides which block polymerase extension such as invdT.

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.

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'-0-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 in RSV genes are provided herein. A preferred strand invasion oligonucleotide for detection of the target sequence of SEQ ID NO: 1 is or comprises SEQ ID NO: 5 or a variant thereof. RSV A and RSV B specific variants of SEQ ID NO: 5 are provided. A preferred RSV B specific variant of SEQ ID NO: 5 is or comprises SEQ ID NO: 6. A particularly preferred strand invasion oligonucleotide is or comprises a modified derivative of SEQ ID NO: 5 or 6, most preferably SEQ ID NO: 7 or 8.

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: 5 or 6 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: 5 or 6. In addition or alternatively to modified derivatives, variant strand invasion oligonucleotide sequences may be used.

A variant of SEQ ID NO 5 is typically an oligonucleotide 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 NO: 5. The target-binding sequence of SEQ ID NO: 5 is the region which has complementarity or identity to the target duplex comprising SEQ ID NO: 1 or a variant thereof. The target-binding sequence may bind to the duplex on either strand to initiate invasion of the duplex, and thus with reference to SEQ ID NO: 1 may comprise a region having complementarity to SEQ ID NO: 1 if binding on the strand comprising SEQ ID NO: 1. Alternatively, the target-binding sequence may bind to the opposing strand of the duplex to that comprising SEQ ID NO: 1, and thus comprise a region having identity to SEQ ID NO: 1 (and thus complementary to the sequence on the opposing strand). A variant of SEQ ID NO: 5 may thus bind on either strand of a target duplex comprising SEQ ID NO: 1 and comprise a target-binding sequence which is partly or fully identical to the target-binding sequence present in SEQ ID NO: 5, or a target-binding sequence which is partly or fully complementary to the target-binding sequence present in SEQ ID NO: 5.

Preferably, a said variant 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 NO: 5.

The above variant 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 NO: 5 (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.

A variant of SEQ ID NO: 5 may also be an oligonucleotide 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, a variant strand invasion oligonucleotide 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 oligonucleotide is typically unrelated to the target sequence, and also typically unrelated to the original strand invasion oligonucleotide.

A variant strand invasion oligonucleotide may 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.

Variants of the strand invasion oligonucleotides of the invention further include variants which are specific for RSV A or RSV B. The term "specific" is to be understood as defined above in relation to RSV A or RSV B specific primers. An RSV A specific strand invasion oligonucleotide may for example be fully complementary or fully identical to a target sequence from RSV A, but have a mismatch/lack full complementarity with a corresponding target sequence from RSV B such that it binds to and/or amplifies the RSV B sequence with lower efficiency. An RSV A or RSV B specific strand invasion oligonucleotide may be used in combination with an RSV A or RSV B specific upstream and/or downstream primer. An RSV A-specific strand invasion oligonucleotide may be preferred for amplification of SEQ ID NO: 1, 34 or 35 or a variant of any thereof. An RSV

B-specific strand invasion oligonucleotide may be preferred for amplification of SEQ ID

NO: 42 or 43 or a variant thereof.

A preferred example of a sequence for inclusion in an RSV A specific strand invasion oligonucleotide is provided by SEQ ID NO: 5 itself, and more preferably the modified derivative of SEQ ID NO:7. A preferred example of a sequence for inclusion in an RSV B specific strand invasion oligonucleotide is provided by SEQ ID NO: 6 , which is a variant of SEQ ID NO: 5 having a single mismatch thereto in its target-complementary region (at nucleotide position 31, C instead of T, as shown in Figure 1), and more preferably the modified derivative of SEQ ID NO: 8. A variant of SEQ ID NO: 5 which is specific for RSV B more generally is any variant described above (such as an

oligonucleotide of greater than 30 nucleotides in length comprising a region which is partly complementary or identical to at least 30 contiguous nucleotides of the corresponding original target-binding sequence present in SEQ ID NO: 5) wherein the region partly complementary or identical to said target-binding sequence represents the mismatch specifically bound by SEQ ID NO: 6. The said region of the variant may comprise a partly identical region to the target-binding sequence of SEQ ID NO: 5 comprising said mismatch. Alternatively, the said region of the variant may comprise a partly

complementary region to the target-binding region of SEQ ID NO: 5, including a sequence complementary to said mismatch bound by SEQ ID NO: 6.

The only mismatch between the partly identical or partly complementary region of the variant and the target-binding sequence of SEQ ID NO: 5 may be the above mismatch. An RSV B specific variant of SEQ ID NO: 5 may comprise a target-binding region identical or complementary to that of SEQ ID NO: 5 but for the above mismatch.

Where an RSV A or RSV B specific strand invasion oligonucleotide is used, it may be used as the only strand invasion oligonucleotide, to thereby provide for improved detection of RSV A or RSV B where this is desired. Alternatively, a strand invasion oligonucleotide specific for RSV A and a strand invasion oligonucleotide specific for RSV B may be included together for improved detection of both said subtypes of RSV.

In preferred embodiments, an assay for the detection of the N-gene area of RSV includes RSV A and RSV B specific strand invasion oligonucleotides for the target nucleic acid sequence of SEQ ID NO: 1 or a variant thereof. The RSV A and RSV B specific strand invasion oligonucleotides preferably comprise SEQ ID NOs 5 and 6, or RSV A/RSV B specific variants thereof or modified derivatives of any thereof, preferably SEQ ID NOs 7 and 8. Further preferably, said RSV A and RSV B specific strand invasion oligonucleotides are used in combination with RSV A and RSV B specific upstream and/or downstream primers for SEQ ID NO: 1 or a variant thereof, optimally downstream primers comprising SEQ ID NOs 3 and 4 or RSV A/RSV B specific variants thereof. A strand invasion oligonucleotide comprising SEQ ID NO: 5 or a variant thereof may alternatively be provided together with an upstream primer comprising SEQ ID NO: 2 or a variant thereof, and a downstream primer comprising SEQ ID NO: 9 or a variant thereof, for detection of the N-gene area target nucleic acid sequence of SEQ ID NO: 34 or a variant thereof.

In an additional embodiment for detection of the N-gene area target nucleic acid sequence of SEQ ID NO: 35 or a variant thereof, the strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 12 or a variant and/or modified derivative thereof is provided. This strand invasion oligonucleotide is preferably used in combination with upstream and downstream primers comprising SEQ ID NOs 10 and 11 or variants thereof. A preferred such strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 13.

For detection of the additional N-gene area target nucleic acid sequence of SEQ ID NO: 19 or a variant thereof, a strand invasion oligonucleotide comprising the sequence of SEQ ID NOs 22 or a variant and/or modified derivative thereof is provided herein. This strand invasion oligonucleotide is preferably used in combination with upstream and downstream primers comprising SEQ ID NOs 20 and 21 or variants thereof. A preferred such strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 23.

For the target nucleic acid sequence of SEQ ID NO: 29 or a variant thereof, from the M2-1 gene area, a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 32 or a variant and/or modified derivative thereof is provided herein. This strand invasion oligonucleotide is preferably used in combination with upstream and downstream primers comprising SEQ ID NOs 30 and 31 or variants thereof. A preferred such strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 33.

Additionally, for the target nucleic acid sequence of SEQ ID NO: 14 or a variant thereof, from the Polymerase gene area of SV, a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 17 or a variant and/or modified derivative thereof is provided herein. This strand invasion oligonucleotide is preferably used in combination with upstream and downstream primers comprising SEQ ID NOs 15 and 16 or variants thereof. A preferred such strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 18.

Also, for the additional target nucleic acid sequence from the Polymerase gene area of SEQ ID NO: 24 or a variant thereof, a strand invasion oligonucleotide comprising the sequence of SEQ ID NO: 27 or a variant and/or modified derivative thereof is provided herein. This strand invasion oligonucleotide is preferably used in combination with upstream and downstream primers comprising SEQ ID NOs 25 and 26 or variants thereof. A preferred such strand invasion oligonucleotide comprises the sequence of SEQ ID NO: 28.

All of the above strand invasion oligonucleotides are typically oligonucleotides of greater than 30 nucleotides in length. It will further be understood that suitable modified derivatives and variants of all strand invasion oligonucleotides described above may be selected in accordance with the same detailed criteria described above in relation to variants and modified derivatives of strand invasion oligonucleotides including those described with reference to SEQ ID NO: 5.

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 four dNTPs, dATP, dTTP, dCTP 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 amplification process. 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. Where Bsu polymerase is used, a suitable temperature is 40 degrees centigrade.

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. subtilis, 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; WO2008/035205).

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.

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. The probe may be as described in WO 2015/075198, incorporated herein by reference. Thus, the probe may be an oligonucleotide 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. 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 primers and strand invasion

oligonucleotides of or comprising SEQ ID NOs 2 to 13, 15 to 18, 20 to 23, 25 to 28 and 30 to 33 and variants and modified derivatives of each thereof (preferably SEQ ID NOs 7, 8,

13, 23, 33, 18, 28) as applicable as products per se, and compositions and formulations comprising said primers and strand invasion oligonucleotides. The primers and optionally the strand invasion oligonucleotide(s) may be used in any method for detection of an RSV. Typically, the method is a strand-invasion based DNA amplification method. However, any suitable DNA amplification method that allows for specific detection of an RSV 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 of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2 or a variant thereof, a downstream primer of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3 or a variant thereof, and a strand invasion oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 5 or a variant or modified derivative thereof (typically comprising one or more modified nucleotides in its 3' region), are described for use in methods for amplification of the target sequence of SEQ ID NO: 1 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.

Compositions and kits

The invention also provides compositions and kits comprising at least two oligonucleotides selected from (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide. The upstream primer, downstream primer and strand invasion oligonucleotide are as described above. The composition or kit may comprise an upstream and a downstream primer, an upstream primer and a strand invasion

oligonucleotide, or a downstream primer and a strand invasion oligonucleotide. Preferably, the composition or kit comprises an upstream primer, a downstream primer and a strand invasion oligonucleotide. The composition or kit may be suitable for detection of an RSV, 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: 1, typically the upstream primer is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2 or a variant thereof, the downstream primer is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3 or a variant thereof, and the strand invasion oligonucleotide is an oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 5 or a variant thereof. The strand invasion oligonucleotide may comprise the sequence of SEQ ID NO: 7. The composition or kit may further comprise an additional strand invasion oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 8 or an SV B specific variant thereof. The composition or kit may further comprise an additional downstream primer of less than 30 nucleotides in length comprising or consisting of a sequence which is an an RSV B specific variant of SEQ ID NO: 3, such as SEQ ID NO: 4. The composition or kit may comprise both an additional downstream primer and an additional strand invasion oligonucleotide as described above.

The additional combinations of upstream primer(s), downstream primer(s) and strand invasion oligonucleotide(s) described above for the target sequences of SEQ ID NO 1 and described for the target sequences of SEQ ID NOs 14, 19, 24 and 29 may also be selected to provide compositions and kits in a similar manner to that described above.

The composition or kit may provide a first set of oligonucleotides allowing for detection of the target nucleic acid sequence of SEQ ID NO: 1 and additionally a second set of oligonucleotides allowing for detection of the target nucleic acid sequence of any of SEQ ID NO:s 14,19, 24 and 29, .

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. 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 a pathogen, particularly an RSV. The method may be applied to a range of disease settings associated with RSV. Additionally, the method may be applied for screening of carriers of RSV.

The determination of whether or not RSV 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 RSV . Thus, a patient may display symptoms indicating the presence of RSV, such as a respiratory illness, and a sample may be obtained from the patient in order to determine the presence of RSV and optionally also the serotype, subtype or strain thereof by the method described above.

The invention thus provides a method of diagnosing an infection caused by RSV in a subject, comprising determining the presence of a target nucleic acid sequence from an RSV according to the invention in a sample from said subject. The method may further comprise other steps of identifying the subtype and/or genotype of RSV , such as by serology, immunoassay or viral culture from a sample provided by the subject.

A particularly preferred embodiment of the invention is the identification of RSV present in patients having a respiratory illness.

The invention thus provides a diagnostic method for respiratory illnesses that are caused by RSV.

Respiratory Syncytial Virus (RSV) is one of the most common cause of respiratory infections among young children and the elderly, and is alsoassociated with high morbidity and mortality rate among children as well as in elderly and immunocompromised patients (Hall et al. 2001, Falsey A R et al. 2005) RSV accounts for over 60% of acute respiratory infection cases amongst young children. For children below the age of one, RSV can account for approximately 80% of lower respiratory infections (Piedimonte et al. 2014). Almost all children before the age of 3 will have been infected by RSV. Accurate diagnosis of RSV plays an important role in the management and treatment of RSV infection since its often clinically challenging to distinguish RSV from other viral or bacterial respiratory infections.

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 detection methods in the clinical setting. RT-PCR is often more sensitive than immunoassay methods but slower to provide results (Mahony et al. 2011) and 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. Although immunoassay tests are less sensitive compared with RT-PCR, the time to result for immunoassay tests can be between 15-30 mins. This is significantly faster than RT-PCR which can take over 2 hours. Furthermore immunoassay tests can be performed with relatively small footprint without the need for specialized training. This makes an immunoassay test ideal for near patient or at the point of care testing.

The amplification method of the invention surprising allows for rapid and highly sensitive detection of RSV with a detection time similar to an immunoassay method. The amplification method of the invention can also 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 is due to an infection by RSV virus. The diagnostic method may also provide an indication as to the serotype, strain or subtype of RSV and it can then be evaluated whether this is known to be resistant to any antiviral compounds. Depending on the outcome of the test the medical treatment can then be optimised, for example by use of appropriate antiviral compounds.

The following Examples illustrate the invention.

Example 1 - Selection of target sequences and oligonucleotides for detection of RSV A and B virus 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. (2014).

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.

Virus pathogen resource (ViPR) (Pickett B E et al 2012) and nucleotide database of National Center for Biotechnology Information were utilized in the design of RSV RT- SIBA assays for detection of RSV A and RSV B.

After being established, the assays were then subjected to several rounds of optimization, as described subsequently. The preferred target region (amplicon) for detecting RSV was established as SEQ ID NO: 1 from the N-gene area or naturally occurring variants thereof such as SEQ ID NOs 42 and 43, and preferred primers and strand invasion oligonucleotides were designed for amplification of these targets.

Additional amplicons from the N-gene area and other genes were also selected and preferred primers and strand invasion oligonucleotides were designed.

Example 2 - RT-SIBA method for detection of RSV

Two RSV strains, RSV-A (ATCC VR-1540) and RSV-B (ATCC VR-1400) were purchased from ATCC. RNA was extracted from these viruses using Qiamp Viral mini kit (Qiagen). RNA extraction was performed according to manufacturer's instructions. The extracted RNA was quantified using Genesig® RSV RT-PCR kit (Primerdesign™ Ltd, United Kingdom). The kit include control RSV RNA with known amount. For the quantification of the extracted RSV-A and RSV-B RNA were performed according to manufacturer's instructions using BioRad qPCR equipment (Bio-Rad Laboratories, CA, United States). The quantified RNA was subsequently used for establishing the analytical sensitivity of RT-SIBA RSV assay.

Primers and invasion oligonucleotides (IO) used were purchased from Integrated DNA Technologies (Leuven, Belgium). The primers and invasion IOs were purified by reverse-phase HPLC and PAGE, respectively. RT-SIBA reactions described herein were performed using a commercial SIBA reagent kit (Orion Diagnostica Oy, 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 10 for each assay were each used at final concentrations as shown below in Table 2. Two microliters of samples or RNA templates were used in total of 20 ul reactions. T

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) or a BioRad (BioRad Laboratories, CA, United States) instrument. 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.

Example 3 - Preferred assay for detection of RSV and comparison with RT-

PCR

The performance of RT-SIBA for the detection of RSV A and RSV B were compared with a previously published RT-PCR assay for the detection of RSVA and RSVB (Do et al. 2012). The concentration of probes and primer used for RT-PCR were as follows: MTH1 (forward) 400 nM, MTH2B (reverse): 400 nM, probe A0108 LNA (RSV A): 200 nM, probe B0108 LNA (RSV B): 200 M. . The RT-PCR reactions were performed using the EXPRESS qPCR SuperMix Universal and EXPRESS Superscript® Mix (Thermo Fisher Scientific). The following thermal cycling protocol was used: 50°C for 15 min (cDNA synthesis), 95°C for 2 min (reverse transcriptase and UDG

inactivation), and 45 cycles of 95°C for 30 s and 45°C for 1 min (PCR amplification).

The sensitivities of the RT-SIBA and RT-PCR assays for RSV were evaluated in at least three independent experiments by adding serial dilutions of RSV extracted RNA from 10⁵ copies to 10 copies per reactions. Five replicates of each dilution were used. RT-SIBA reactions were detected using SYBR green I dye while RT-PCR reactions incorporated a target specific probe that enabled specific detection of RSV A and RSV B subtypes. The results are shown in Figure 2 (a,b) and Table 1. Both RT-SIBA and RT-PCR reliably detected as low as 10 copies of RSV RNA. The average time to achievement of positive reactions in RT-SIBA was compared to the corresponding ct values for RT-PCR (Table 1). RT-SIBA reactions were performed at a constant temperature and consequently did not require thermal cycling of the reaction. Hence RT-SIBA data were collected at one minute intervals. SIBA RSV assays detected 100 copies of RSV A and RSV B at approximately 13 and 19 minutes, respectively. The corresponding ct values for 100 copies of RSV A and 5 RSV B RNA were approximately 27 and 30, respectively. The corresponding average time to positive reactions for RT-PCR can take around two hours due to the initial reverse transcription step as well as the instrument ramp time. RT-SIBA displayed a faster detection time compared to RT-PCR for the detection of RSV. Figure 2 (c) shows RSV A detection with RSV A or RSV B detecting oligonucleotides and their combination. Figure 10 2 (d) shows RSV B detection with RSV A or RSV B detecting oligonucleotides and their combination.

Furthermore, subsequent melt curve analysis also revealed the presence of a single specific amplicon in reactions containing RSV RNA. The RT-SIBA RSV assay did not produce any detectable amplification signal in the absence of RSV RNA (no template 15 controls). This was also the case, when related nucleic acids from other common

respiratory pathogens were added to the RT-SIBA RSV reactions (Figure 2e). These findings confirmed that the RT-SIBA RSV assay was specific.

Table 1

20

Example 4 - Alternative assay for the N-gene target sequence of SEQ ID NO: 1

25 Alternative oligonucleotides for detection of related RSV N-gene target sequences to SEQ ID NO: 1 were selected and used for RT-SIBA detection of RSV A or RSV B in assays as described in Example 2. In summary:

• The primers of SEQ ID NOs 2, 9 and strand invasion oligonucleotides of SEQ ID NOs 7, 8 were used for amplification of SEQ ID NO: 34. Results are shown in

Figure 3 (a,b).

• The primers of SEQ ID NOs 10, 11 and strand invasion oligonucleotide of SEQ ID NO: 13 were used for amplification of SEQ ID NO: 35. Results are shown in Figure 3 (c).

Example 5 - Detection of other RSV target regions with RT-SIBA

Four further target sequences for detection of RSV were identified in various genes based on the approach outlined in Example 1. Preferred amplicon regions and

primers/strand invasion oligonucleotides for their amplification were then selected, and used for RT-SIBA detection of RSV A or RSV B in assays as described in Example 2. In summary:

• An additional amplicon (SEQ ID NO: 19) was identified in the N gene area of RSV. The primers of SEQ ID NOs 20-21 and strand invasion oligonucleotide of SEQ ID NO: 23 were used for amplification of SEQ ID NO: 19. Results are shown in Figure 3 (d).

• A preferred amplicon (SEQ ID NO: 29) was identified in the M2-1 gene area of RSV. The primers of SEQ ID NOs 30-31 and strand invasion oligonucleotide of SEQ ID NO: 33 were used for amplification of SEQ ID NO: 29. Results are shown in Figure 4.

· A first amplicon (SEQ ID NO: 14) was identified within the polymerase gene area of RSV. The primers of SEQ ID NOs 15-16 and strand invasion oligonucleotide of SEQ ID NO: 18 were used for amplification of SEQ ID NO: 14. Results are shown in Figure 5 (b).

• A second amplicon (SEQ ID NO: 24) was identified within the polymerase gene area of RSV. The primers of SEQ ID NOs 25-26 and strand invasion

oligonucleotide of SEQ ID NO: 28 were used for amplification of SEQ ID NO: 24. Results are shown in Figure 5 (a). Concentrations of primers and strand invasion oligonucleotides used in the above assays were as follows, as shown in Table 2:

Table 2

References

Do L A H, van Doom H R, Bryant J E, Nghiem M N, Nguyen Van V C, Vo C K, Nguyen M D, Tran T H, Farrar J, de Jong M D (2012) A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial vims. Journal of Virological Methods 179, 250-255

Drosten C, Panning M, Guenther S, Schmitz H (2002) False-Negative Results of PCR Assay with Plasma of Patients with Severe Viral Hemorrhagic Fever. Journal of Clinical Microbiology 40(11), 4394-4395. Falsey A R, Hennessey P A, Formica M A, Cox C, Walsh E E (2005) Respiratory Syncytial Vims Infection in Elderly and High-Risk Adults. New England Journal of Medicine 352, 1749-1759

Hall C B (2001) Respiratory Syncytial Vims and Parainfluenza Vims. New England Journal of Medicine 344, 1917- 1928

Hoser M J, Mansukoski H K, Morrical S W, Eboigbodin K E (2014) 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 9, el 12656.

Mahony J B, Petrich A, Smieja M (2011) Molecular diagnosis of respiratory vims infections. Critical Reviews in Clinical Laboratory Sciences 48, 217-249. Pickett B E, Sadat E L, Zhang Y, Noronha J M, Squires R B, Hunt V, Liu M, Kumar S, Zaremba S, Gu Z, Zhou L, Larson C N, Dietrich J, Klem E B, Scheuermann R H (2012) ViPR: an open bioinformatics database and analysis resource for virology research.

Nucleic Acids Research 40, D593-D598 Piedimonte G, Perez M K (2014) Respiratory Syncytial Vims Infection and Bronchiolitis. Pediatrics in Review 35, 519-530

Claims

1. A method for detecting a ribonucleic acid (RNA) comprising a target nucleic acid sequence from a Respiratory Syncytial Virus (RSV) in a sample, said method comprising contacting said sample with a reverse transcriptase, 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 said strand invasion oligonucleotide renders at least a portion of a duplex nucleic acid comprising said target nucleic acid sequence single-stranded, to allow the binding of said upstream primer and a downstream primer.

2. A method according to claim 1, wherein said target nucleic acid sequence is from RSV A and/or RSV B.

3. A method according to claim 2, wherein said RSV target sequence is in the N-gene area of an RSV.

4. A method according to claim 2, wherein said RSV target sequence is in the M2-1 gene area of an RSV.

5. A method according to claim 2, wherein said RSV target sequence is in the Polymerase gene area of an RSV.

6. A method according to claim 3, wherein the RSV target sequence is in the N-gene and the amplified target nucleic acid sequence comprises SEQ ID NO: 1 or a variant thereof, or SEQ ID NO: 19 or a variant thereof.

7. A method according to claim 4, wherein the amplified target nucleic acid sequence comprises SEQ ID NO 29 or a variant thereof.

8. A method according to claim 5, wherein the amplified target nucleic acid sequence comprises SEQ ID NO 14 or 24 or a variant of either thereof.

9. A method for detecting a target nucleic acid sequence from an RSV A and/or RSV B gene 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 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 RSV A and/or RSV B gene and/or the amplified target nucleic acid sequence therefrom are as defined in any one of claims 3 to 8.

10. A method according to claim 9, wherein the amplified target nucleic acid sequence comprises SEQ ID NO: 1 or a variant thereof.

11. A method according to claim 6 or 10, wherein said at least one upstream primer is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2 or a variant thereof, and/or wherein said at least one downstream primer is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3 or a variant thereof, and/or wherein said at least one strand invasion oligonucleotide is an oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 5 or a variant thereof, or a modified derivative of either thereof.

12. A method according to claim 11, which is for detection of target nucleic acid sequences from RSV-A and RSV-B, comprising contacting said sample with (i) at least two downstream primers, one being an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3 or a variant thereof which is specific for a said target nucleic acid sequence from RSV-A and one being an oligonucleotide of less than 30 nucleotides in length comprising a variant of the sequence of SEQ ID NO: 3r which is specific for a said target nucleic acid sequence from RSV-B; and (ii) at least two strand invasion oligonucleotides, one being an oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 5 or a variant thereof which is specific for a said target nucleic acid sequence from SV-A, or a modified derivative of either thereof and one being an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 6 or a variant thereof which is specific for a said target nucleic acid sequence from RSV-B, or a modified derivative of either thereof.

13. A method according to claim 12, wherein said upstream primer is an

oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2 or a variant thereof, wherein said method comprises use of two downstream primers which are oligonucleotides of less than 30 nucleotides in length comprising respectively the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and wherein said method comprises use of two strand invasion oligonucleotides which are oligonucleotides of greater than 30 nucleotides in length comprising respectively the sequence of SEQ ID NO: 7 and SEQ ID NO:8.

14. A method according to any one of the preceding claims, which further comprises contacting of said sample with a recombinase.

15. A method according to any one of the preceding claims, wherein a said strand invasion oligonucleotide has a 5 ' terminal region non-complementary to the target nucleic acid sequence which is at least about 14 nucleotides in length and/or is comprised of purine nucleotides preferably consisting essentially of cytosine nucleotides.

16. A composition comprising at least two oligonucleotides selected from (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide,

wherein each said oligonucleotide is as defined in one of claims 6-13.

17. A kit comprising at least two oligonucleotides selected from (a) an upstream primer, (b) a downstream primer and (c) a strand invasion oligonucleotide,

wherein each said oligonucleotide is as defined in one of claims 6 to 13.

18. A composition according to claim 16, or a kit according to claim 17, comprising at least one oligonucleotide of (a), at least one oligonucleotide of (b), and at least one oligonucleotide of (c), as defined in claim 11.

19. A composition or kit according to claim 18, comprising at least one additional oligonucleotide of (b) and/or at least one additional oligonucleotide of (c), as defined claim 12 or 13.

20. A composition or kit according to claim 19, comprising an upstream primer which is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 2 or a variant thereof, a first downstream primer which is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 3, a second downstream primer which is an oligonucleotide of less than 30 nucleotides in length comprising the sequence of SEQ ID NO: 4, a first strand invasion oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO: 7 and a second strand invasion oligonucleotide of greater than 30 nucleotides in length comprising the sequence of SEQ ID NO:8.

21. A composition or kit according to any one of claims 16 to 20, further comprising

(i) a reverse transcriptase; and/or

(ii) a DNA polymerase; and/or

(iii) a recombinase.

22. A method for diagnosis of an infection by a Respiratory Syncytial Virus (RSV) in a subject, comprising carrying out a method as defined in any one of claims 1 to 15 in a sample from said subject.