WO2019025776A1

WO2019025776A1 - Nairovirus diagnostic assay

Info

Publication number: WO2019025776A1
Application number: PCT/GB2018/052167
Authority: WO
Inventors: Laura BONNEY; Robert Watson
Original assignee: The Secretary Of State For Health
Priority date: 2017-07-31
Filing date: 2018-07-30
Publication date: 2019-02-07
Also published as: GB201712284D0

Abstract

The present invention provides nucleic acid products and corresponding methods for identifying the presence or absence of Nairovirusin a sample.

Description

NAIROVIRUS DIAGNOSTIC ASSAY

The invention relates to nucleic acid products and to the detection of Nairovirus. More specifically, the invention relates to new primers and probes for detecting the presence of Nairovirus in a sample.

Tick-bourne viruses of the Nairovirus genus are the cause of a range of medically and agriculturally important diseases. Ganjam and Nairobi sheep disease are pathogens of agricultural importance, causing severe disease in sheep and goats and have a significant economic impact in affected countries. Several members of the Nairovirus genus have been reported to cause human disease including Dugbe, Nairobi Sheep disease, Erve virus and Crimean-Congo-Hemorrhagic fever (CCHF) virus (CCHFV). CCHFV has had the greatest impact. CCHFV is present in large parts of the globe, and is the cause of a virulent hemorrhagic fever in humans. There is, at present, no effective prophylaxis or treatment for CCHFV. With case numbers increasing in many countries in recent years, the WHO has described CCHF as "one of the most rapidly emerging viral hemorrhagic fevers in Africa, Asia and Europe". CCHFV has several transmission routes and is both zoonotic and can be passed from person-to-person, causing high fatality rates in the nosocomial setting.

The current Nairovirus diagnostics; RT-PCR, virus isolation and serology are slow and confined to a conventional laboratory. There is considerable interest in affected countries in simple and fieldable new technologies to allow detection of CCHF and other Nairovirus strains in remote rural settings with limited resources. This would enable surveillance of ticks and reservoir species and provide access to testing in the agricultural setting and for cut-off communities. A rapid-turnaround diagnostic for use in the hospital or clinic would also allow timely patient isolation, to prevent spread to other patients and hospital staff.

There are, at present, no FDA-approved diagnostics for identification of CCHFV or other Nairovirus strains. The only molecular assay on the market and current gold-standard CCHF PCR assay is the Altona RealStar® CCHFV RT-PCR Kit. Whilst existing RT-PCR based methods can provide high levels of sensitivity (with some detecting down to a single genome copy), these methods are hampered by significant limitations. For example, existing RT-PCR based methods are slow (typically 1.5 hour run-time on a standard RT-PCR machine), and are therefore not ideally-suited to high-throughput screening.

Moreover, existing RT-PCR based methods have a high power requirement, which is typically accommodated by a mains electrical source. This power-requirement significantly l reduces portability of these methods, and can severely limit their suitability for use at the point-of-care (POC) which, in the context of Nairovirus detection, is often in remote rural locations. Thermocyclers are also expensive, which further limits their suitability for widespread use.

Existing RT-PCR based methods also require intensive sample processing work, and are typically highly sensitive to sample quality. This increases sample preparation time and complexity, and typically requires the involvement of skilled technicians.

In view of the various limitations with existing RT-PCR based detection methods, there have been efforts to develop serological assays for detecting CCHFV. The small number of commercial products on the market include; the gold-standard serology-based Eurolmmun I FA and the Vector-Best IgG, IgM and antigen-detection ELISAs for CCHFV. These antibody-based detection assays are advantageous because they are relatively simple to perform, and provide a readout that is easy to understand. However, these serological methods are unable to detect early infection or determine viral loads, and they are less sensitive than existing RT-PCR based assays. They are also labour intensive and have a slow turnaround time, typically several hours.

There is therefore an urgent and unmet need for a rapid, simple and fieldable technology that would enable reliable identification of Nairovirus.

The present invention solves one or more of the above-identified problems by providing a flexible, accurate, rapid, portable robust and sensitive method for detecting Nairovirus, particularly CCHFV.

Specifically, the invention provides a method for detecting Nairovirus in a sample, via detection of Nairovirus nucleic acid in the sample. The terms "detecting Nairovirus in a sample" and "detecting Nairovirus nucleic acid in a sample" (and the like) are, within the context of the invention, used interchangeably. The Nairovirus detected or quantified using primers and/or probe of the invention is typically CCHFV.

According to the invention, methods for detecting Nairovirus in a sample typically comprise amplifying the amount of Nairovirus nucleic acid in the sample. In such cases, the skilled person will appreciate that "detection of Nairovirus nucleic acid in the sample" typically involves detection of Nairovirus nucleic acid amplicon and/or the corresponding amplification product. In one embodiment, said amplification product comprises a fluorescent moiety, and the detection of Nairovirus nucleic acid in the sample comprises detection of said fluorescent moiety.

The primers and probes of the invention have been developed to be highly suitable for in-field testing using a "lab-in-a-suitcase" format, and can achieve real-time detection of Nairovirus (such as CCHFV) within as little as 5-25 minutes, e.g. when used in an RPA assay. The primers and probes of the invention are highly suitable for use in the detection of Nairovirus in remote rural settings, as well as peripheral hospital sites (where there is greater access to acute samples of serum, saliva and urine). The inventors believe that the present invention will revolutionise the global response to Nairovirus.

The invention is highly suitable for use in a multiplex format, for detection of other medically and/or agriculturally important pathogens. Thus, in one embodiment, the primers and probe of the invention are for use in a multiplex format. In one embodiment, the primers and probe of the invention are for use in a multiplex format, for the detection of CCHFV as well as one or more other viruses that show haemorrhagic symptoms, such as Ebola virus, Lassa virus and/or Marburg virus. In one embodiment, the primers and probe of the invention are for use in a multiplex format, for the detection of CCHFV as well as one or more other members of the Nairovirus genus e.g. selected from Nairobi sheep disease and Dugbe. In one embodiment, the primers and probe of the invention are for use in a multiplex format, for the detection of CCHFV and other medically important viruses circulating in a CCHFV outbreak. In one embodiment, the primers and probe of the invention are for use in a multiplex format, for the detection of CCHFV and other medically important viruses circulating in a CCHFV outbreak, and vectored by ticks e.g. Ganjam, Dugbe, Nairobi Sheep disease and/or Erve virus. In one embodiment, the primers and probe of the invention are for use in a multiplex format, for the detection of CCHFV as well as one or more other arboviruses, optionally selected from the group consisting of Sinbis Virus Disease, Dengue, Chikungunya, Yellow Fever, West Nile Encephalitis, Japanese B Encephalitis, Venezuelan Equine Encephalitis, Dengue hemorrhagic fever, LaCrosse Encephalitis, Rift Valley Fever, Western Equine Encephalitis, Mayaro Fever, Eastern Equine Encephalitis, Powassan Encephalitis, California Encephalitis, Russian Spring Fever, Kyasanur Forest Disease, Sandfly fever, St. Louis Encephalitis, Colorado Tick Fever, Louping-ill, Oropouche Fever, Barmah Forest Fever and Murray Valley Encephalitis. In one embodiment, the primers and probe of the invention are for use in a multiplex format, for the detection of CCHFV as well as one or more other viral haemorrhagic fever viruses. Other viral haemorrhagic fever viruses include, for example Ebola virus, Lassa virus, Marburg virus, Dengue fever, yellow fever, Rift Valley Fever, Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Lujo virus, Junin virus, Machupo virus, Sabia virus, Guanarito virus, Garissa virus and/or llesha virus. In one embodiment, multiplex assays of the invention are performed as separate RPA assays, each directed to the respective pathogens. In one embodiment, multiplex assays of the invention are performed in the format of a PAN-genus RPA assay. Multiplex assays of the invention are well-suited for use with samples from individuals from areas of high pathogen prevalence; particularly for screening applications. Multiplex assays of the invention are also well-suited for use with samples from individuals exhibiting CCHFV-like symptoms; particularly for identifying whether said individual is infected with CCHFV and/or a different pathogen.

Accordingly, the invention provides a composition comprising: a nucleic acid probe comprising: (i) nucleic acid sequence of any one of SEQ ID NOs: 29-110; or (ii) nucleic acid sequence exhibiting at least 85% identity to any one of SEQ ID NOs: 29-110; or (iii) nucleic acid sequence comprising 15 or more consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 29-110; wherein group "3" = modification that functions to block polymerase extension; "5" = dT-fluorophore; "6" = an abasic nucleotide analog; and "7" = dT-quencher group (suitable for group "5"); and/or b) a forward nucleic acid primer and a reverse nucleic acid primer; the forward nucleic acid primer comprising: i) nucleic acid sequence CGTGCCGCTTACGCC (SEQ ID NO: 2); or ii) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of SEQ ID NO: 2; and the reverse nucleic acid primer comprising: i) nucleic acid sequence TGTGAAAGTGTCCAT (SEQ ID NO: 16); or ii) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of SEQ ID NO: 16.

In one embodiment, group "3" is selected from (i) C₃-spacer, (ii) a phosphate, (iii) a biotin- TEG, or (iv) an amine; group "5" is selected from (i) dT-fluorescein, (ii) TAMRA and (iii) Cy5; group "6" is D-spacer; and group "7" is selected from (i) dT-BHQ1 and (ii) dT-BHQ2. In one embodiment, group "3" is propanol; group "5" is dT-fluorescein; group "6" is D-spacer; and group "7" is dT-BHQ1 .

In one embodiment, the forward nucleic acid primer comprises or consists of (i) the nucleic acid sequence of any one of SEQ ID NOs: 1-14; or (ii) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-14; or (iii) nucleic acid sequence comprising 15 or more consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 1 or any one of SEQ ID NOs: 1-14.

In one embodiment, the reverse nucleic acid primer comprises or consists of: (i) the nucleic acid sequence of or any one of SEQ I D NOs: 15-28; or (ii) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 15-28; or (iii) nucleic acid sequence comprising 15 or more consecutive nucleic acids of the nucleic acid sequence of any one of SEQ ID NOs: 15-28.

In one embodiment, the composition comprises: (i) forward nucleic acid primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1 ; (ii) reverse nucleic acid primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15; and (iii) nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29.

The invention also provides a kit comprising a composition as described above.

The invention also provided a device comprising a composition as described above.

In one embodiment, the kit or the device comprises part or whole of a TwistAmp Basic kit, TwistAmp Basic-RT kit, TwistAmp exo kit, TwistAmp exo-RT kit, TwistAmp fpg kit, and/or TwistAmp nfo kit.

The invention also provides use of a composition, kit or device as described above, in a method of detecting Nairovirus in a sample.

The invention also provides a method for detecting the presence of Nairovirus in a sample or detecting the absence of said Nairovirus in said sample, said method comprising: A) combining said sample with a composition as described above; B) allowing nucleic acid present in the sample to contact the primers and/or probes within the composition; and C) performing a nucleic acid amplification technique; wherein amplification of nucleic acid in the sample confirms that nucleic acid from Nairovirus is present within the sample, and wherein the absence of amplification of nucleic acid in the sample confirms that nucleic acid from Nairovirus is absent from the sample. In one embodiment, the nucleic acid amplification technique is an isothermal nucleic acid amplification technique, such as Recombinase Polymerase Amplification (RPA).

In one embodiment, the sample is from an individual, typically an animal, typically a human. In such an embodiment, the sample is typically selected from blood, plasma, saliva, serum, sputum, urine, cerebral spinal fluid, semen, cells, a cellular extract, a tissue sample, a tissue biopsy, a stool sample, a swab from any body site and/or one or more organs; typically blood, serum, urine, saliva and/or organ(s). In one embodiment, the animal is a tick, typically an ixodid tick. In such an embodiment, the sample is typically homogenised tick(s).

In one embodiment, the sample is a crude sample.

In one embodiment, the method is for use in surveillance of Nairovirus (e.g. CCHFV) prevalence.

In one embodiment, the method is for use in diagnosing Nairovirus infection in an individual. In one embodiment, upon identification of Nairovirus infection in the individual, said individual is provided with an appropriate treatment or therapy.

Treatment or therapy for CCHFV infection is primarily supportive, and typically includes paying careful attention to fluid balance and correction of electrolyte abnormalities, oxygenation and hemodynamic support, and appropriate treatment of secondary infections. Separately or in addition, treatment for CCHFV infection comprises administration of an antiviral drug. In one embodiment, said antiviral drug is ribavirin.

There now follows a brief description of the Figures, which illustrate embodiments of the present invention.

Figure 1 : Design of primers and probes of the invention.

Figure 2: RPA sensitivity with a synthetic RNA template of Europe I strain AY277672. The data are represented graphically as (a.) Delta Rn against time (minutes), in the form of a table of time to positive (TTP value) vs target copy number (b.). Values shown are the mean of 3 independent experiments, each of which were performed with three replicates. The threshold was set at delta Rn 50,000.

Figure 3: Gel electrophoresis showing the products of a basic RT-RPA, (following PCR clean-up) performed with synthetic RNA fragments from a selection of CCHF strains; AY277672, DQ21 1638, NC005302 and DQ211643.

The inventors have conducted a detailed analysis of the sequences of all 7 phylogenetic clades of CCHFV. Based on the results of this analysis, the inventors have designed new primers and probes which allow detection of CCHFV sequences from current outbreak strains. Despite significant sequence variation between the various CCHFV clades, the primers and probes of the invention were surprisingly able to detect CCHFV extracts representing all 7 phylogenetic clades, and a panel of clinical and tick samples from a recent outbreak. This surprising result is highly desirable, because it drastically reduces the risk of false negatives.

Moreover, the primers and probes of the invention are highly selective, and did not show cross-reactivity, with related viral strains. This is highly desirable because it reduces the risk of false positives.

Another advantage provided by the primers and probes of the invention is that they provide high sensitivity (capable of detecting as little as 10 genome copies/μΙ) and specificity, even when used with crude sample preparations (both of human and tick origin). The invention therefore avoids the requirement for highly stringent sample preparation, reduces the requirement for skilled technicians, and greatly reduces the cost and time spent on sample preparation, as compared to conventional methods.

A further advantage provided by the invention is that the mode of detection of Nairovirus is flexible; it can be performed using real-time/quantitative PCR (qPCR), standard PCR, or isothermal techniques such as recombinase polymerase amplification (RPA), depending on the equipment available. The primers and probes of the invention allow this flexibility in the mode of detection because their annealing temperatures are compatible with the reverse transcriptase enzyme. Isothermal methods (such as RPA) are preferred, because they achieve the high sensitivity that is provided by RT-PCR (detecting down to a single genome copy), and are also faster, simpler, more portable, lower cost, and more simple-to-use (and may thus be performed by an untrained operator). These advantages are highly desirable for real-world detection of Nairovirus. In a preferred embodiment, the primers and probes of the invention are used in an RPA assay. The RPA assay requires forward and reverse primers (i.e. a "primer pair") and a probe for detection. The probe is typically a fluorescent probe. Wherein the amplification method is a "conventional" PCR method (such as RT-PCR), the method typically employs primers of the invention (typically a primer pair).

The RPA is a simple, rapid (5-25 minutes run time) isothermal molecular detection method, suitable for field-use, with no false-positives. It can be performed with crude sample preparations, with minimal hands-on time and can be detected on a low-power, lightweight and portable device. RPA methods therefore represent a significant advantage over conventional PCR-based methods, particularly for in-field use.

Thus, in one embodiment, the samples used in the detection methods of the invention undergo minimal preparation. For example, blood samples may be allowed to clot; serum and/or urine samples may be diluted in a lysis buffer; and ticks may be homogenized in a lysis buffer).

In one embodiment, reaction components (e.g. primers and probe of the invention) are provided in freeze-dried form. In such embodiments, liquid sample may be used to solubilise the reaction components.

Commercial RPA kits are readily available (e.g. from TwistDX), and contain freeze-dried reagents for long-term storage. The RPA assay is a simple-to-perform, rapid (5-20 minutes run time) isothermal molecular detection method, which shows particularly high sensitivity and speed, even compared to other isothermal methods (e.g. LAM P, SDA, RCA and NASBA). For example, the RPA assay is preferred over loop-mediated-isothermal amplification (LAMP) because it is more amenable to multiplexing and is performed at a lower temperature (37-42 degrees rather than 60-65 degrees), leading to lower power requirements. The RPA assay is preferred over NASBA because it is faster (NASBA is described as requiring 2 hours for amplification). RPA is typically carried out as a one-tube amplification reaction, involving a single low reaction temperature (typically 37-42° C). Readouts from the RPA assay are flexible and include e.g. gel-based, real-time, simple fluorescence detection and lateral-flow. The Nairovirus RPA assay has the flexibility to be used either in a lightweight portable detection device for field testing, or in a laboratory setting in a rapid, high-throughput system.

Advantageously, the RPA assay accommodates the use of crude sample preparations when used with primers and probes of the invention. In an exemplary RPA assay of the invention, samples are collected in-field and undergo minimal processing (e.g. blood is allowed to clot, serum and/ or urine is diluted in a lysis buffer and ticks homogenized in a lysis buffer). A simple workflow involves adding sample to the RPA assay buffer and mixing with freeze-dried pellets. A magnesium start typically initiates the RPA reaction and the RPA can be followed in real-time at a single low temperature on a portable device. The RPA assay has the flexibility to be used in a rapid, high-throughput test e.g. during an outbreak, and avoids the above-mentioned limitations of conventional PCR and serological detection methods.

The invention is highly-suited to a number of important applications in the detection of Nairovirus. For example, the invention can help support local surveillance of Nairovirus, in the animal reservoir, in Nairovirus-positive tick populations, and in humans. This enables rapid targeting of vector control measures including the prediction of emerging Nairovirus epizootics before they enter the human population.

The invention also permits faster and more widespread screening of suspect cases than existing methods. The invention is therefore ideally suited to surveillance of Nairovirus prevalence (e.g. using human samples). Moreover, the improved detection of Nairovirus, provided by the invention, would significantly improve the identification of suitable patients, who would benefit from treatment or therapy.

The invention allows the screening of a variety of samples, such as blood, urine, saliva and organs, and can therefore be used to test for suspected cases, and help avoid transfusion and transplant-mediated transmission of Nairovirus. The invention is well-suited to rapid turnaround screening, and is highly applicable to applications where a rapid clinical decision is required e.g. for organ transplant. The invention can also be used in screening travellers returning from a Nairovirus affected area (or an area suspected of being affected). The invention can also be used in testing symptomatic individuals and pregnant women, sexual health and family planning screening in affected countries.

The invention is also suitable for diagnosing a Nairovirus infection in an individual e.g. for confirming whether an individual suspected of Nairovirus infection is infected with Nairovirus. In one embodiment, the individual is a tick or an animal that forms part of the animal reservoir for the virus. An such instances, "infection" refers to a host tick or a reservoir animal that is carryng Nairovirus.

In one embodiment, the primers and probes of the invention are used in a multiplex method, as described above. The invention also provides compositions and kits for use in said multiplex methods.

The invention provides nucleic acid primers and probes for the detection of Nairovirus nucleic acid in a sample.

In one embodiment, detection of Nairovirus nucleic acid in a sample comprises the use of a primer pair of the invention (i.e. a forward and reverse primer).

In one embodiment, detection of Nairovirus nucleic acid in a sample comprises the use of probe of the invention. In one embodiment, detection of Nairovirus nucleic acid in a sample comprises the use of primers and probes of the invention. A primer pair of the invention and a probe of the invention are typically used in the RPA method.

In one embodiment, the invention provides a composition comprising primers and/or probes of the invention. Said composition may be provided in any form, typically lyophilised, liquid, or frozen. Compositions of the invention typically comprise e.g. salts and/or stabilisers and/or components required for DNA amplification.

Primers and probes of the invention may be referred to as "oligonucleotide" probes and primers.

Primers and probes of the invention are isolated nucleic acids. "CCHV RPA Fw"

A preferred primer of the invention is "CCHV RPA Fw" (SEQ ID NO: 1):

AGAAACACGTGCCGCTTACGCCCACAGTGTT

"CCHV RPA Fw" is a "forward" primer that binds to the sense strand of the CCHFV S- segment gene, as shown at Figure 1.

The invention also provides fragments of "CCHV RPA Fw" that comprise 15 or more consecutive nucleic acids of SEQ ID NO:1 , e.g. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or at least 31 consecutive nucleic acids of SEQ ID NO: 1. Preferred fragments of SEQ ID NO: 1 comprise the sequence CGTGCCGCTTACGCC (SEQ ID NO: 2).

The inventors believe that sensitivity of the amplification technique (typically the RPA assay), when using the primers and probe of the invention, may be further improved by slightly modifying the primers and probe described in the Examples, by "frame-shifting" the primers and/or probe by up to 6bp in the 5' or 3' direction or altering the size by +/- 15 bp, preferably +/- 8bp (relative to the AY277672 DNA sequence shown in Figure 1).

Thus, the invention also provides the following forward primers: aAGAAACACGTGCCGCTTACGCCCACAGTGT (SEQ ID NO: 3)

aaAGAAACACGTGCCGCTTACGCCCACAGTG (SEQ ID NO: 4)

caaAGAAACACGTGCCGCTTACGCCCACAGT (SEQ ID NO: 5)

tcaaAGAAACACGTGCCGCTTACGCCCACAG (SEQ ID NO: 6)

ctcaaAGAAACACGTGCCGCTTACGCCCACA (SEQ ID NO: 7)

tctcaaAGAAACACGTGCCGCTTACGCCCAC (SEQ ID NO: 8)

GAAACACGTGCCGCTTACGCCCACAGTGTTc (SEQ ID NO: 9)

AAACACGTGCCGCTTACGCCCACAGTGTTct (SEQ ID NO: 10)

AACACGTGCCGCTTACGCCCACAGTGTTctc (SEQ ID NO: 11)

ACACGTGCCGCTTACGCCCACAGTGTTctct (SEQ ID NO: 12)

CACGTGCCGCTTACGCCCACAGTGTTctctt (SEQ ID NO: 13)

ACGTGCCGCTTACGCCCACAGTGTTctcttg (SEQ ID NO: 14)

The invention also provides fragments of any one of SEQ ID NOs: 1-14 that comprise 15 or more consecutive nucleic acids of any one of SEQ ID NOs: 1-14, e.g. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30 or at least 31 consecutive nucleic acids of any one of SEQ ID NOs: 1-14. Preferred fragments of SEQ ID NOs: 1-14 comprise the sequence CGTGCCGCTTACGCC (SEQ ID NO: 2).

Other preferred fragments lack one or more nucleic acids e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 5' terminus, and/or one or more nucleic acids e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 3' terminus of SEQ ID NO: 1 or of SEQ ID NOs: 1-14, while retaining the ability to bind specifically to CCHFV nucleic acid.

Primers of the invention also include variants of any one of SEQ ID NOs: 1-14. Such variants typically consist of a nucleic acid sequence having 85% or more identity, e.g. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs: 1-14.

In some embodiments, the above-disclosed forward primers of the invention comprise additional nucleic acids at the 5' end of the above-disclosed primer sequences. Preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (shown in Figure 1 and in SEQ ID NO: 11 1) that are directly upstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed forward primers. In some embodiments, the above-disclosed primers of the invention comprise up to 15 additional nucleic acids {e.g. 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 5' end. Such primers also fall within the scope of the invention.

In some embodiments, the above-disclosed forward primers of the invention comprise additional nucleic acids at the 3' end of the above-disclosed primer sequences. Preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (e.g. shown in Figure 1) that are directly downstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed forward primers. In some embodiments, the above-disclosed primers of the invention comprise up to 15 additional nucleic acids {e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 3' end. Such primers also fall within the scope of the invention.

In some embodiments, the above-disclosed forward primers of the invention comprise additional nucleic acids at the 5' and 3' ends of the above-disclosed primer sequences. For example, in some embodiments, the above-disclosed primers of the invention comprise up to 15 additional nucleic acids {e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 5' end, and up to 15 additional nucleic acids {e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 3' end; e.g. 1 additional nucleic acid at their 5' end and at their 3' end; 2 additional nucleic acids at their 5' end and at their 3' end; 3 additional nucleic acids at their 5' end and at their 3' end; 4 additional nucleic acids at their 5' end and at their 3' end; 5 additional nucleic acids at their 5' end and at their 3' end; 6 additional nucleic acids at their 5' end and at their 3' end; 7 additional nucleic acids at their 5' end and at their 3' end; 8 additional nucleic acids at their 5' end and at their 3' end; 9 additional nucleic acids at their 5' end and at their 3' end; or 10 additional nucleic acids at their 5' end and at their 3' end. Such primers also fall within the scope of the invention.

"CCHF RPA Rev"

A preferred primer of the invention is "CCHF RPA Rev" (SEQ ID NO: 15):

TAGGAGTTTGTGAAAGTGTCCATAAGTCCATT

"CCHF RPA Rev" is a "reverse" primer that binds to the reverse strand of the CCHFV S-segment gene, as shown at Figure 1. The invention also provides fragments of "CCHF RPA Rev" that comprise 15 or more consecutive nucleic acids of SEQ ID NO: 15, e.g. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , or at least 32consecutive nucleic acids of SEQ ID NO: 15. Preferred fragments of SEQ ID NO: 15 comprise the sequence TGTGAAAGTGTCCAT (SEQ ID NO: 16).

The inventors believe that sensitivity of the RPA assay, when using the primers and probe of the invention, may be further improved by slightly modifying the primers and probe described in the Examples, by "frame-shifting" the primers and/or probe by up to 6bp in the 5' or 3' direction or altering the size by +/- 15 bp, preferably +/- 8bp (relative to the AY277672 DNA sequence shown in Figure 1).

Thus, the invention also provides the following reverse primers:

gTAGGAGTTTGTGAAAGTGTCCATAAGTCCAT (SEQ ID NO: 17)

agTAGGAGTTTGTGAAAGTGTCCATAAGTCCA (SEQ ID NO: 18)

gagTAGGAGTTTGTGAAAGTGTCCATAAGTCC (SEQ ID NO: 19)

ggagTAGGAGTTTGTGAAAGTGTCCATAAGTC (SEQ ID NO: 20)

aggagTAGGAGTTTGTGAAAGTGTCCATAAGT (SEQ ID NO: 21)

aaggagTAGGAGTTTGTGAAAGTGTCCATAAG (SEQ ID NO: 22)

AGGAGTTTGTGAAAGTGTCCATAAGTCCATTt (SEQ ID NO: 23)

GGAGTTTGTGAAAGTGTCCATAAGTCCATTtc (SEQ ID NO: 24)

GAGTTTGTGAAAGTGTCCATAAGTCCATTtcc (SEQ ID NO: 25)

AGTTTGTGAAAGTGTCCATAAGTCCATTtccc (SEQ ID NO: 26)

GTTTGTGAAAGTGTCCATAAGTCCATTtccct (SEQ ID NO: 27)

TTTGTGAAAGTGTCCATAAGTCCATTtccctt (SEQ ID NO: 28)

The invention also provides fragments of any one of SEQ ID NOs: 15-28 that comprise 15 or more consecutive nucleic acids of any one of SEQ ID NOs: 15-28, e.g. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , or at least 32 consecutive nucleic acids of any one of SEQ ID NOs: 15-28. Preferred fragments of SEQ ID NOs: 15-28 comprise the sequence TGTGAAAGTGTCCAT (SEQ ID NO: 16).

Other preferred fragments lack one or more nucleic acids e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 5' terminus, and/or one or more nucleic acids e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 3' terminus of SEQ ID NO: 15 or of SEQ ID NOs: 15-28, while retaining the ability to bind specifically to CCHFV nucleic acid.

Primers of the invention also include variants of any one of SEQ ID NOs: 15-28. Such variants typically consist of an amino acid sequence having 85% or more identity, e.g. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs: 15-28.

In some embodiments, the above-disclosed reverse primers of the invention comprise additional nucleic acids at the 5' end of the above-disclosed primer sequences. Preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (corresponding to the reverse complement of the sequence shown in e.g. Figure 1 and SEQ ID NO: 11 1) that are directly upstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed reverse primers. In some embodiments, the above-disclosed primers of the invention comprise up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 5' end. Such primers also fall within the scope of the invention.

In some embodiments, the above-disclosed reverse primers of the invention comprise additional nucleic acids at the 3' end of the above-disclosed primer sequences. Preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (corresponding to the reverse complement of the sequence shown in Figure 1) that are directly downstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed reverse primers. In some embodiments, the above-disclosed primers of the invention comprise up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 3' end. Such primers also fall within the scope of the invention.

In some embodiments, the above-disclosed reverse primers of the invention comprise additional nucleic acids at the 5' and 3' ends of the above-disclosed primer sequences. For example, in some embodiments, the above-disclosed reverse primers of the invention comprise up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 5' end, and up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 3' end; e.g. 1 additional nucleic acid at their 5' end and at their 3' end; 2 additional nucleic acids at their 5' end and at their 3' end; 3 additional nucleic acids at their 5' end and at their 3' end; 4 additional nucleic acids at their 5' end and at their 3' end; 5 additional nucleic acids at their 5' end and at their 3' end; 6 additional nucleic acids at their 5' end and at their 3' end; 7 additional nucleic acids at their 5' end and at their 3' end; 8 additional nucleic acids at their 5' end and at their 3' end; 9 additional nucleic acids at their 5' end and at their 3' end; or 10 additional nucleic acids at their 5' end and at their 3' end. Such primers also fall within the scope of the invention.

"CCHF RPA Probe 1"

A preferred probe of the invention is "CCHF RPA Probe 1" (SEQ ID NO: 29):

CCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCG3

Wherein : 3= propanol,

5= dT-fluorescein,

6= D-spacer,

7= dT-BHQ1

"CCHF RPA Probe 1" is an "EXO" probe, which has been designed for use in an RPA assay.

Other modifications may be present at groups 3, 5, 6 and 7. According to the invention:

3 = modification that functions to block polymerase extension

5 = dT-fluorophore

6 = an abasic nucleotide analog

7 = dT-quencher group (suitable for group "5")

In one embodiment:

3 = selected from (i) C₃-spacer, (ii) a phosphate, (iii) a biotin-TEG, or (iv) an amine

5= selected from (i) dT-fluorescein, (ii) TAMRA, and Cy5

6= a tetrahydrofuran residue ("THF", sometimes referred to as a "D-spacer"), 7= selected from (i) dT-BHQ1 (preferably wherein group "5" is dT-fluorescein) and (ii) dT-BHQ2 (preferably wherein group "5" is TAMRA or Cy5)

The probe of the invention is typically 46-52 nucleic acids in length. Whilst the probes of the invention described herein typically include group "5" at the 5' of group "7", the skilled person will appreciate that the relative position of groups "5" and "7", may be swapped. Said probes fall within the scope of the invention.

Preferred fragments of SEQ ID NO: 29 comprise the sequence AGTGTTCTCTTGAGTG567G (SEQ ID NO: 30).

As shown in Figure 1 , the CCHF RPA Probe 1 corresponds to the "CCGCTTACGCCCACAGTGTTCTCTTGAGTGTCTGCAAAATGGAAAACAAGATCG" (SEQ ID NO: 136) region of the AY277672 sequence, except that the underlined "TCT" residues have been substituted with dT-fluorescein, D-spacer, and dTHQ1 , respectively.

Probes of the invention comprise groups "5", "6" and "7". Groups "5" and "7" are preferably separated by 2 to 6 bases. In a preferred embodiment, groups "5" and "7" are separated by group "6" only (i.e. said groups are preferably positioned directly adjacent to each other).

In another embodiment, groups "5" and "7" are separated by group "6" and 1 or more (e.g. 1 , 2, 3, 4, or 5) additional nucleic acid residues. Said one or more additional nucleic acid residues typically correspond to the corresponding residues in the AY277672 DNA sequence provided in SEQ ID NO: 11 1. In one embodiment, said 1 or more (e.g. 1 , 2, 3, 4, or 5) additional nucleic acid residues are not present in a probe of the invention.

Whilst SEQ ID NO: 29 includes groups "5", "6" and "7" at the positions corresponding to nucleic acids "TCT" (as shown above), other nucleic acids may instead be substituted for groups "5", "6" and "7" (i.e. groups "5", "6" and "7" may be present at different position within probes of the invention). In a preferred embodiment, groups "5", "6" and "7" are substituted for nucleic acids "TCT", as shown above.

In a preferred embodiment, primers of the invention that oppose the direction of the probe do not overlap with the probe, to avoid the occurrence of primer-probe dimers. In a preferred embodiment, secondary structures that could cause probes to fold back on themselves are avoided.

In one embodiment, probe of the invention comprises a sequence selected from:

567GTTCTCTTGAGTGTCTG3 (SEQ ID NO: 31)

A567TTCTCTTGAGTGTCTG3 (SEQ ID NO: 32) AG567TCTCTTGAGTGTCTG3 (SEQ ID NO: 33)

AGT567CTCTTGAGTGTCTG3 (SEQ ID NO: 34)

AGTG567TCTTGAGTGTCTG3 (SEQ ID NO: 35)

AGTGT567CTTGAGTGTCTG3 (SEQ ID NO: 36)

AGTGTT567TTGAGTGTCTG3 (SEQ ID NO: 37)

AGTGTTC567TGAGTGTCTG3 (SEQ ID NO: 38)

AGTGTTCT567GAGTGTCTG3 (SEQ ID NO: 39)

AGTGTTCTC567AGTGTCTG3 (SEQ ID NO: 40)

AGTGTTCTCT567GTGTCTG3 (SEQ ID NO: 41)

AGTGTTCTCTT567TGTCTG3 (SEQ ID NO: 42)

AGTGTTCTCTTG567GTCTG3 (SEQ ID NO: 43)

AGTGTTCTCTTGA567TCTG3 (SEQ ID NO: 44)

AGTGTTCTCTTGAG567CTG3 (SEQ ID NO: 45)

AGTGTTCTCTTGAGT567TG3 (SEQ ID NO: 46)

AGTGTTCTCTTGAGTG567G3 (SEQ ID NO: 47)

AGTGTTCTCTTGAGTGT5673 (SEQ ID NO: 48)

In one embodiment, probe of the invention comprises a sequence selected from: cacagtgttc567tgagtgtctg (SEQ ID NO: 61), preferably:

aacacgtgccgcttacgcccacagtgttc567tgagtgtctgcaaa (SEQ ID NO: 62) cacagtgttc56t7gagtgtctg (SEQ ID NO: 63), preferably:

aacacgtgccgcttacgcccacagtgttc56t7gagtgtctgcaaa (SEQ ID NO: 64) cacagtgttc5c67gagtgtctg (SEQ ID NO: 65), preferably:

acacgtgccgcttacgcccacagtgttc5c67gagtgtctgcaaaa (SEQ ID NO: 66) cagtgttctc56gag7gtctgc (SEQ ID NO: 67), preferably:

cacgtgccgcttacgcccacagtgttctc56gag7gtctgcaaaat (SEQ ID NO: 68) cagtgttctc5t6ag7gtctgc (SEQ ID NO: 69), preferably:

acgtgccgcttacgcccacagtgttctc5t6ag7gtctgcaaaatg (SEQ ID NO: 70) cagtgttctc5tg6g7gtctgc (SEQ ID NO: 71), preferably:

cgtgccgcttacgcccacagtgttctc5tg6g7gtctgcaaaatgg (SEQ ID NO: 72) cagtgttctc5tga67gtctgc (SEQ ID NO: 73), preferably: gtgccgcttacgcccacagtgttctc5tga67gtctgcaaaatgga (SEQ ID NO: 74) agtgttctct56ag7gtctgcaa (SEQ ID NO: 75), preferably:

acgtgccgcttacgcccacagtgttctct56ag7gtctgcaaaatg (SEQ ID NO: 76) agtgttctct5g6g7gtctgcaa (SEQ ID NO: 77), preferably:

cgtgccgcttacgcccacagtgttctct5g6g7gtctgcaaaatgg (SEQ ID NO: 78) agtgttctct5ga67gtctgcaa (SEQ ID NO: 79), preferably:

gtgccgcttacgcccacagtgttctct5ga67gtctgcaaaatgga (SEQ ID NO: 80) agtgttctct56agtg7ctgcaa (SEQ ID NO: 81), preferably:

acgtgccgcttacgcccacagtgttctct56agtg7ctgcaaaatg (SEQ ID NO: 82) agtgttctct5g6gtg7ctgcaa (SEQ ID NO: 83), preferably:

cgtgccgcttacgcccacagtgttctct5g6gtg7ctgcaaaatgg (SEQ ID NO: 84) agtgttctct5ga6tg7ctgcaa (SEQ ID NO: 85), preferably:

gtgccgcttacgcccacagtgttctct5ga6tg7ctgcaaaatgga (SEQ ID NO: 86) agtgttctct5gag6g7ctgcaa (SEQ ID NO: 87), preferably:

tgccgcttacgcccacagtgttctct5gag6g7ctgcaaaatggaa (SEQ ID NO: 88) agtgttctct5gagt67ctgcaa (SEQ ID NO: 89), preferably:

gccgcttacgcccacagtgttctct5gagt67ctgcaaaatggaaa (SEQ ID NO: 90) ttctcttgag567ctgcaaaatg (SEQ ID NO: 91), preferably:

gccgcttacgcccacagtgttctcttgag567ctgcaaaatggaaa (SEQ ID NO: 92) ttctcttgag56tc7gcaaaat (SEQ ID NO: 93), preferably:

gccgcttacgcccacagtgttctcttgag56tc7gcaaaatggaaa (SEQ ID NO: 94) ttctcttgag5g6c7gcaaaat (SEQ ID NO: 95), preferably:

ccgcttacgcccacagtgttctcttgag5g6c7gcaaaatggaaaa (SEQ ID NO: 96) ttctcttgag5gt67gcaaaat (SEQ ID NO: 97), preferably: cgcttacgcccacagtgttctcttgag5gt67gcaaaatggaaaac (SEQ ID NO: 98) acagcaaaga56aga7gaacaaa (SEQ ID NO: 99), preferably:

gaaaacaagatcgaggtgaacagcaaaga56aga7gaacaaatggt (SEQ ID NO: 100) acagcaaaga5g6ga7gaacaaa (SEQ ID NO: 101), preferably:

aaaacaagatcgaggtgaacagcaaaga5g6ga7gaacaaatggtt (SEQ ID NO: 102) acagcaaaga5ga6a7gaacaaa (SEQ ID NO: 103), preferably:

aaacaagatcgaggtgaacagcaaaga5ga6a7gaacaaatggttt (SEQ ID NO: 104) agcaaaga5gag67gaacaaa (SEQ ID NO: 105), preferably:

aacaagatcgaggtgaacagcaaaga5gag67gaacaaatggtttg (SEQ ID NO: 106) gaacaaatgg567gaggagttta (SEQ ID NO: 107), preferably:

tgaacagcaaagatgagatgaacaaatgg567gaggagtttaaaaa (SEQ ID NO: 108) gtttgaggag567aaaaaggga (SEQ ID NO: 109), preferably:

aagatgagatgaacaaatggtttgaggag567aaaaagggaaatgg (SEQ ID NO: 110)

Preferably, probes of the invention comprise 30 or more nucleic acids at the 5' of group "6". Said 30 or more nucleic acids typically correspond to the corresponding nucleic acids in the AY277672 sequence.

Preferably, probes of the invention comprise 15 or more nucleic acids at the 3' of group "6". Said 15 or more nucleic acids typically correspond to the corresponding nucleic acids in the AY277672 sequence.

Preferably, probes of the invention comprise 30 or more nucleic acids at the 5' of group "6", and 15 or more nucleic acids at the 3' of group "6". Said 30 or more nucleic acids and said 15 or more nucleic acids typically correspond to the corresponding nucleic acids in the AY277672 sequence.

Within the present disclosure, groups "5", "6" and "7" are not treated as sequence variants when discussing "consecutive nucleic acids", "sequence identity" and the like. In such cases, groups "5", "6" and "7" are assessed as though they are the corresponding nucleic acids in the AY277672 sequence. Independent of the nucleic acid sequence in the probe, group "3" is present at the 3' end of the probe. Group "3" is present at the 3' end of the probe to block the polymerase extension. In a preferred embodiment, group "3" is propanol.

Thus, the invention also provides the following probes: gCCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATC3 (SEQ ID NO: 49) tgCCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGAT3 (SEQ ID NO: 50) gtgCCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGA3 (SEQ ID NO: 51) cgtgCCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAG3 (SEQ ID NO: 52) acgtgCCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAA3 (SEQ ID NO: 53) cacgtgCCGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACA3 (SEQ ID NO: 54)

CGCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCGa3 (SEQ ID NO: 55)

GCTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCGag3 (SEQ ID NO: 56)

CTTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCGagg3 (SEQ ID NO: 57)

TTACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCGaggt3 (SEQ ID NO: 58)

TACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCGaggtg3 (SEQ ID NO: 59)

ACGCCCACAGTGTTCTCTTGAGTG567GCAAAATGGAAAACAAGATCGaggtga3 (SEQ ID NO: 60)

The invention also provides fragments of any one of SEQ ID NOs: 29-110 that comprise 15 or more consecutive nucleic acids of any one of SEQ ID NOs: 29-110, e.g. at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 consecutive nucleic acids of any one of SEQ ID NOs: 31-48. Preferred fragments of SEQ ID NOs: 29-110 comprise the sequence AGTGTTCTCTTGAGTG567G (SEQ ID NO: 30).

Other preferred fragments lack one or more nucleic acids e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 5' terminus, and/or one or more nucleic acids e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the 3' terminus of SEQ ID NO: 29 or SEQ ID NOs: 29-110, while retaining the ability to bind specifically to CCHFV nucleic acid.

Probes of the invention also include variants of any one of SEQ ID NOs: 29-110. Such variants typically consist of an amino acid sequence having 85% or more identity, e.g. at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ I D NOs: 29-1 10. In one embodiment, probes of the invention consist of any one of SEQ I D NOs: 29-1 10.

In some embodiments, the above-disclosed probes of the invention comprise additional nucleic acids at the 5' end of the above-disclosed probe sequences. Preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (e.g. shown in Figure 1 ) that are directly upstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed probes. In some embodiments, the above- disclosed probes of the invention comprise up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 5' end. Such probes also fall within the scope of the invention.

In some embodiments, the above-disclosed probes of the invention comprise additional nucleic acids at the 3' end of the above-disclosed probe sequences. Preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (shown in Figure 1 ) that are directly downstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed probes. In some embodiments, the above- disclosed probes of the invention comprise up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 3' end. Such probes also fall within the scope of the invention.

In some embodiments, the above-disclosed probes of the invention comprise additional nucleic acids at the 5' and 3' ends of the above-disclosed probe sequences. For example, in some embodiments, the above-disclosed probes of the invention comprise up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 5' end, and up to 15 additional nucleic acids (e.g. 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 additional nucleic acids) at their 3' end (preferably, said additional nucleic acids correspond to the nucleic acids in the AY277672 DNA sequence (shown in Figure 1 ) that are directly downstream to nucleic acids in the AY277672 DNA sequence that bind to the above-disclosed probes); e.g. 1 additional nucleic acid at their 5' end and at their 3' end; 2 additional nucleic acids at their 5' end and at their 3' end; 3 additional nucleic acids at their 5' end and at their 3' end; 4 additional nucleic acids at their 5' end and at their 3' end; 5 additional nucleic acids at their 5' end and at their 3' end; 6 additional nucleic acids at their 5' end and at their 3' end; 7 additional nucleic acids at their 5' end and at their 3' end; 8 additional nucleic acids at their 5' end and at their 3' end; 9 additional nucleic acids at their 5' end and at their 3' end; or 10 additional nucleic acids at their 5' end and at their 3' end. Such primers also fall within the scope of the invention.

All nucleic acid sequences presented herein are presented in a 5'-to-3' (left-to-right) orientation.

Primers and probes of the invention are capable of binding to the CCHFV S-segment gene, as shown at Figure 1 . Thus, identification of CCHFV (e.g. in a sample) is based on the detection of nucleic acid sequence(s) that correspond to the CCHFV S-segment gene. The AY277672 DNA sequence in Figure 1 and in SEQ I D NO: 1 1 1 provides a CCHFV reference sequence.

Probes of the invention are typically "Exo" probes (typically from TwistDX), for use in an RPA assay.

The invention also relates to other types of probe, for use in combination with primers of the invention. Since the preferred method of the invention involves an RPA assay, said other types of probe are preferably also compatible with the RPA assay, for example "nfo" probes and "fpg" probes.

Thus, in one embodiment, the invention provides primers of the invention and an nfo probe. Nfo probes are typically for use in lateral flow detection, which may involve use of e.g. biotin or digoxigenin tags.

Thus, in one embodiment, the invention provides primers of the invention and a fpg probe, typically from TwistDX. As explained in TwistDX product information sheets, Fpg probes are typically oligonucleotides that are modified at the 5' end with a quencher group and that contain a fluorophore label on an abasic nucleotide analogue 4 to 5 nucleotides downstream of the quencher (i.e. at position 5 or 6). The fluorophore is attached to the ribose of the abasic site via a C-O-C linker (a so-called dR-group). In addition, TwistAmpR fpg probes are blocked from polymerase extension by a suitable 3 ' modification (such as a C3- spacer, a phosphate, a Biotin-TEG or an amine). The fluorescent signal generated by the fluorophore (typically Carboxy-fluorescein) will normally be quenched by the 5 ' quencher group (typically a Black Hole Quencher (BHQ)). In a double stranded context the dR-fluorophore residue, the 'gap ' in the probe, presents a substrate for a number of DNA repair enzymes, including the enzyme fpg present in the TwistAmpR fpg kit. Typically the % sequence identity is determined over a length of contiguous nucleic acid residues. A primer or probe specific for the CCHFV nucleic acid may, for example, have at least 80% sequence identity to CCHFV nucleic acid, measured over at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 14, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80 or more nucleic acid residues, up to the entire length of the primer or probe specific for the CCHFV nucleic acid.

Sequence identity may be determined with respect to any primer or probe disclosed herein.

Primers and probes of the invention may be complementary to the CCHFV nucleic acid. Typically the primer or probe specific for CCHFV nucleic acid is complementary over a length of contiguous nucleic acid residues. Typically the primer or probe specific for CCHFV nucleic acid is complementary over a length of at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 14, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31 , at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 70, at least 80, at least 90, or more nucleic acid residues, up to the full length of the primer or probe.

A primer or probe of the invention may be complementary to the reverse sequence of the CCHFV nucleic acid. Typically the primer or probe specific for CCHFV nucleic acid is complementary over a length of contiguous nucleic acid residues of the reverse sequence. Typically, the primer or probe specific for CCHFV nucleic acid is complementary over a length of at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 14, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 45, at least 50, at least 55, at least 60, at least 70, at least 80, at least 90, or more nucleic acid residues, up to the full length of the primer or probe.

A primer or probe of the invention may be complementary to a variant of a CCHFV nucleic acid. Typically the primer or probe of the invention is complementary to a variant having at least 80% sequence identity to the CCHFV nucleic acid. A sequence identity of at least 80% includes at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% sequence identity.

Variants of the specific sequences provided above may be defined by reciting the number of nucleotides that differ between the variant sequences and the specific reference sequence, preferably with reference to the AY277672 DNA sequence in Figure 1 and in SEQ ID NO: 11 1. These differences may result from the addition, deletion and/or substitution of one or more nucleotide position within the variant sequence compared with the reference sequence. Thus, in one embodiment, the sequence may comprise (or consist of) a nucleotide sequence that differs from the specific sequences provided at no more than ten nucleotide positions, no more than nine nucleotide positions, no more than eight nucleotide positions, no more than seven nucleotide positions, no more than six nucleotide positions, no more than five nucleotide positions, no more than four nucleotide positions, no more than three nucleotide positions, no more than two nucleotide positions or no more than one nucleotide position. Conservative substitutions are preferred. The term variants as defined herein also encompasses splice variants. As noted above, when specifically discussing probes of the invention, groups "5", "6" and "7" are not treated as sequence variant, and are assessed as though they are the corresponding nucleic acids in the AY277672 sequence.

Typically, for RPA-based methods, the probe is typically 10 to 80 nucleotides in length, preferably 20 to 80 nucleotides in length, more preferably 30 to 70 nucleotides in length, even more preferably 40 to 60 nucleotides in length, and even more preferably 46 to 54 nucleotides in length.

The forward primer of the invention is typically 15-50 nucleotides in length. For RPA-based methods, the forward primer is preferably 15-45 nucleotides in length, more preferably 30-40 nucleotides in length, most preferably 33-37 nucleotides in length.

The reverse primer of the invention is typically 15-50 nucleotides in length. For RPA-based methods, the reverse primer is preferably 15-45 nucleotides in length, more preferably 30-40 nucleotides in length, most preferably 33-37 nucleotides in length.

The primers and probes of the invention are specially designed to hybridise to nucleic acid from all 7 clades of CCHFV. In the context of the present invention, the term "hybridises" includes hybridising to the sense strand of a target sequence, the reverse of a target sequence, the complement of a target sequence or the reverse complement of a target sequence. Wherein the amplification method is PCR, it is preferred that the binding conditions for primers and probes of the invention are such that a high level of specificity is provided - i.e. hybridisation of the primers and/or probes occurs under "stringent conditions". In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target (or complement) sequence hybridises to a perfectly matched probe or primer. In this regard, the Tm of oligonucleotides, probes or primers of the present invention, at a salt concentration of about 0.02M or less at pH 7, is for example above 60°C, such as about 70°C.

Premixed buffer solutions are commercially available (eg. EXPRESSHYB Hybridisation Solution from CLONTECH Laboratories, Inc.), and hybridisation can be performed according to the manufacturer's instructions.

Examples of suitable labels include detectable labels such as radiolabels or fluorescent or coloured molecules, enzymatic markers or chromogenic markers - e.g. dyes that produce a visible colour change upon hybridisation of the probe or primer. By way of example, the label may be digoxygenin, fluorescein-isothiocyanate (FITC), R-phycoerythrin, Alexa 532, carboxy-X-rhodamine (ROX), carboxytetramethylrhodamine (TAMRA), 4,5-dichloro- dimethoxy-fluorescein (JOE), BHQ-1/2/3, Cy5, Cy5.5 or Cy3. The probes or primer preferably contain a Fam label (e.g. a 5' Fam label), and/ or a minor groove binder (MGB). The label may be a reporter molecule, which is detected directly, such as by exposure to photographic or X-ray film. Alternatively, the label is not directly detectable, but may be detected indirectly, for example, in a two-phase system. An example of indirect label detection is binding of an antibody to the label.

Examples of suitable tags include "complement/ anti-complement pairs". The term "complement/ anti-complement pair" denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. Examples of suitable tags include biotin and streptavidin (or avidin). By way of example, a biotin tag may be captured using streptavidin, which may be coated onto a substrate or support such as a bead (for example a magnetic bead) or membrane. Likewise, a streptavidin tag may be captured using biotin, which may be coated onto a substrate or support such as a bead (for example a magnetic bead) or membrane. Other exemplary complement/ anti-complement pairs include receptor/ ligand pairs, antibody/ antigen (or hapten or epitope) pairs, and the like. Another example is a nucleic acid sequence tag that binds to a complementary sequence. The latter may itself be pre-labelled, or may be attached to a surface (e.g. a bead) which is separately labelled. An example of the latter embodiment is the well-known LuminexR bead system. Other exemplary pairs of tags and capture molecules include receptor/ ligand pairs and antibody/ antigen (or hapten or epitope) pairs. Where subsequent dissociation of the complement/ anti-complement pair is desirable, the complement/ anti- complement pair has a binding affinity of, for example, less than 10⁹ M^"1. One exemplary tagged oligonucleotide, probe or primer is a biotin-labelled oligonucleotide, probe or primer, which may be detected using horse-radish peroxidase conjugated streptavidin.

The probes or primers of the invention may be labelled with different labels or tags, thereby allowing separate identification of each, probe or primer when used in a method of the present invention.

Any conventional method may be employed to attach nucleic acid tags to a probe or primer of the present invention (e.g. to the 5' end of the defined binding region of the oligonucleotide, probe or primer). Alternatively, oligonucleotides, probes or primers of the invention (with pre-attached nucleic acid tags) may be constructed by commercial providers.

Detection of the Nairovirus (typically CCHFV) nucleic acid may be carried out by any known means. In this regard, the probe or amplification product may be tagged and/ or labelled, and the detection method may therefore comprise detecting said tag and/ or label. Nairovirus detection is preferably performed using an isothermal technique, most preferably RPA.

In one embodiment, the probe(s) or primer(s) of the invention comprise a tag and/ or label. Thus, in one embodiment, following hybridisation of tagged/ labelled probe/primer to target Nairovirus nucleic acid, the tag/ label becomes associated with the target nucleic acid. Thus, in one embodiment, the assay may comprise detecting the tag/ label and correlating presence of tag/ label with presence of Nairovirus nucleic acid.

In one embodiment, tag and/ or label may be incorporated during extension of the probe(s) or primer(s) of the invention. In doing so, the amplification product(s) become tagged/ labelled, and the assay may therefore comprise detecting the tag/ label and correlating presence of tag/ label with presence of amplification product, and hence the presence of Nairovirus nucleic acid.

By way of example, in one embodiment, the amplification product may incorporate a tag/ label (e.g. via a tagged/ labelled dNTP such as biotin-dNTP) as part of the amplification process, and the assay may further comprise the use of a binding partner complementary to said tag (e.g. streptavidin) that includes a detectable tag/ label (e.g. a fluorescent label, such as R-phycoerythrin). In this way, the amplified product incorporates a detectable tag/ label (e.g. a fluorescent label, such as R-phycoerythrin).

In one embodiment, the probe(s) or primer(s) and/ or the amplification product(s) may include a further tag/ label (as the complement component) to allow capture of the amplification product(s).

By way of example, a "complement/ anti-complement' pairing may be employed in which an anti-complement capture component binds to said further tag/ label (complement component) and thereby permits capture of the probe(s) and/ or amplification product(s). Examples of suitable "complement/ anti-complement" partners have been described earlier in this specification, such as a complementary pair of nucleic acid sequences, a complementary antibody-antigen pair, etc. The anti-complement capture component may be attached (e.g. coated) on to a substrate or solid support - examples of suitable substrates/ supports include membranes and/ or beads (e.g. a magnetic or fluorescent bead). Capture methods are well known in the art. For example, LuminexR beads may be employed. Alternatively, the use of magnetic beads may be advantageous because the beads (plus captured, tagged/ labelled amplification product) can easily be concentrated and separated from the sample, using conventional techniques known in the art.

Immobilisation provides a physical location for the probes, primers and/or anti-complement capture component of the invention, and may serve to fix the capture component/ probe/primer at a desired location and/ or facilitate recovery or separation of probe/primer. For example, this may be employed as part of a nfo lateral flow assay of the invention, which may employ the use of digoxigenin and biotin labels. The support may be a rigid solid support made from, for example, glass, plastic or silica, such as a bead (for example a fluorescent or magnetic bead). Alternatively, the support may be a membrane, such as nylon or nitrocellulose membrane. 3D matrices are also suitable supports for use with the present invention - e.g. polyacrylamide or PEG gels. Immobilisation to a support/ platform may be achieved by a variety of conventional means. By way of example, immobilisation onto a support such as a nylon membrane may be achieved by UV cross-linking. Alternatively, biotin-labelled molecules may be bound to streptavidin-coated substrates (and vice-versa), and molecules prepared with amino linkers may be immobilised on to silanised surfaces. Another means of immobilisation is via a poly-T tail or a poly-C tail, for example at the 3' or 5' end. Said immobilisation techniques apply equally to the probe component (and primer/primer pair component, if present) of the present invention.

In one embodiment, the probes and/or primers of the invention comprise a nucleic acid sequence tag/ label (e.g. attached to each probe at the 5' end of the defined sequence of the probe/primer that binds to target/ complement nucleic acid). In more detail, each of the probes/primers is provided with a different nucleic acid sequence tag/ label, wherein each of said tags/ labels (specifically) binds to a complementary nucleic acid sequence present on the surface of a bead. Each of the different tags/ labels binds to its complementary sequence counterpart (and not to any of the complementary sequence counterparts of the other tags), which is located on a uniquely identifiable bead. In this regard, the beads are uniquely identifiable, for example by means of fluorescence at a specific wavelength. Thus, in use, probes/primers of the invention bind to Nairovirus (e.g. CCHFV) virus nucleic acid (if present in the sample). Thereafter, (only) the bound probes may be extended (in the 3' direction) in the presence of one or more labelled dNTP (e.g. biotin labelled dNTPs, such as biotin- dCTPs).

The extended primers may be contacted with a binding partner counterpart to the labelled dNTPs (e.g. a streptavidin labelled fluorophore, such as streptavidin labelled R- phycoerythrin), which binds to those labelled dNTPs that have become incorporated into the extended primers. Thereafter, the labelled extended primers may be identified by allowing them to bind to their nucleic acid counterparts present on the uniquely identifiable beads. The latter may then be "called" (e.g. to determine the type of bead present by wavelength emission) and the nature of the primer extension (and thus the type of target/ complement nucleic acid present) may be determined.

Typically, probes/primers of the invention are oligonucleotides having sequence identity or complementarity with CCHFV nucleic acid (either the sense strand, the complementary strand or the reverse of either strand) as disclosed herein. One or more probe may be immobilised on a solid support, and used to interrogate mRNA or DNA obtained from a test sample. If the mRNA or DNA from the test sample contains the CCHFV nucleic acid targeted by the immobilised probe, it will bind to the probe, and may then be detected. The probes/primers of the invention may also be detected using PCR, such as real time PCR.

Diagnosing a Nairovirus infection in an individual means to identify or detect the presence and/or amount of one Nairovirus in the individual. This is achieved by determining the presence and/or amount of Nairovirus nucleic acid in a sample, as described herein. Because of the sensitivity of the present invention to detect a Nairovirus infection before an overtly observable clinical manifestation, the diagnosis, identification or detection of a Nairovirus infection includes the detection of the onset of a Nairovirus infection, as defined above.

According to the present invention, Nairovirus infection may be diagnosed or detected, by determining the presence and/or amount of Nairovirus nucleic acid in a sample obtained from an individual. As used herein, "obtain" means "to come into possession of. The present invention is particularly useful in predicting and diagnosing a Nairovirus infection in an individual, who is suspected of having a Nairovirus infection, or who is at risk of a Nairovirus infection. The present invention may be used to confirm a clinical suspicion of a Nairovirus infection.

The inventors believe that the claimed primers and probes, particularly when used in an RPA assay, could replace the current PCR assays, by providing a more rapid-turnaround and portable diagnostic for testing of symptomatic humans or animals, and particularly for high- risk groups such as agricultural workers, abattoir workers, veterinarians and healthcare workers. The invention is also well-suited to in-field monitoring of CCHFV prevalence in the animal reservoir species (both livestock and wild animals and birds) and the tick vector.

The presence and/or amount of Nairovirus in a sample may be measured relative to a control or reference population, for example relative to the corresponding Nairovirus of a control or reference population. Herein the terms "controf and "reference population" are used interchangeably.

The control or reference population can be generated from one individual or a population of two or more individuals. The control or reference population, for example, may comprise three, four, five, ten, 15, 20, 30, 40, 50 or more individuals. Furthermore, the control or reference population and the individual's (test) sample that are compared in the methods of the present invention may be generated from the same individual, provided that the test and reference samples are taken at different time points and compared to one another. For example, a sample may be obtained from an individual at the start of a study period. A control or reference taken from that sample may then be compared to subsequent samples from the same individual. Such a comparison may be used, for example, to determine the progression of a Nairovirus infection in the individual by repeated classifications over time. The control or reference may be obtained, for example, from a population of Nairovirus negative individuals (i.e. individuals negative for infection by Nairovirus) or Nairovirus positive individuals (i.e. individuals positive for infection by Nairovirus).

Typically the control or reference population does not comprise Nairovirus and/or is not infected with Nairovirus (i.e. is negative for Nairovirus infection). Alternatively, the control or reference population may comprise Nairovirus and/or be infected with Nairovirus (i.e. is positive for Nairovirus infection) and may be subsequently diagnosed with a Nairovirus infection using conventional techniques. For example, a population of Nairovirus infection-positive individuals used to generate the reference or control may be diagnosed with Nairovirus infection about 24, 48, 72, 96 or more hours after biological samples were taken from them for the purposes of generating a reference or control. In one embodiment, the population of Nairovirus -positive individuals is diagnosed with Nairovirus infection using conventional techniques about 0-36 hours, about 36-60 hours, about 60-84 hours, or about 84-108 hours after the biological samples were taken.

As described herein, the present invention relates to a method for determining the presence and/or amount of Nairovirus and/or diagnosing a Nairovirus infection. Thus, in some instances it is sufficient to detect the presence of Nairovirus in a sample. In such cases, the control or reference population may be positive or negative for Nairovirus and/or Nairovirus infection. As noted herein, the Nairovirus detected or quantified using primers and/or probe of the invention is typically CCHFV.

In other instances, the amount of Nairovirus is determined relative to a control or reference population. In such cases, the amount of Nairovirus is typically increased compared with a control or reference population, the amount may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200% compared with the control or reference population.

Alternatively, if the control or reference population is positive for Nairovirus, the amount of the Nairovirus may be decreased compared with the control or reference population. For example, the amount may be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, up to total elimination of the Nairovirus. Such a Nairovirus positive control or reference population may, for example, be used to monitor an individual's response to a treatment directed to the Nairovirus (once said treatment has been established), such that if the treatment is successful, the amount of Nairovirus will decrease relative to the control or reference population over time.

The presence and/or amount of Nairovirus according to the present invention is determined by determining the presence and/or amount of Nairovirus nucleic acid in a sample.

Measurements of the Nairovirus nucleic acid may include, for example, measurements that indicate the presence, concentration, expression level, or any other value associated with the Nairovirus nucleic acid.

The presence and/or amount of said Nairovirus nucleic acid may be determined by quantitative and/or semi-quantitative and/or qualitative analysis. Thus, the presence of Nairovirus may be determined simply by identifying the presence of Nairovirus nucleic acid in a sample (qualitative analysis), with no need to determine the amount of the nucleic acid. Alternatively, the amount of the Nairovirus nucleic acid may be approximated (semiquantitative analysis). Alternatively, the amount of the Nairovirus nucleic acid may be determined (quantitative analysis). RPA is typically a semi-quantitative method.

The amount of the Nairovirus nucleic acid encompasses, but is not limited to, the mass of the nucleic acid, the molar amount of the nucleic acid, the concentration of the nucleic acid, the molarity of the nucleic acid and the copy number of the nucleic acid. This amount may be given in any appropriate units. For example, the concentration of the nucleic acid may be given in pg/ml, ng/ml or μg/ml.

The presence and/or amount of the Nairovirus nucleic acid may be measured directly or indirectly. For example, the copy number of the Nairovirus nucleic acid may be determined using recombinase polymerase amplification (RPA), PCR, qPCR or qRT-PCR. Wherein the Nairovirus nucleic acid is RNA, the expression level may be determined, for example using RPA reverse transcription RPA (RT-RPA). In a preferred embodiment RPA is used. The relative presence and/or amount of the Nairovirus nucleic acid relative to a control or reference population may be determined using any appropriate technique. Suitable standard techniques are known in the art.

As used herein, "comparison" includes any means to discern at least one difference in the presence and/or amount of the Nairovirus nucleic acid in the individual and the control or reference population. Thus, a comparison may include a visual inspection of chromatographic spectra or numerical data, and a comparison may include arithmetical or statistical comparisons of values assigned to expression of the Nairovirus nucleic acid in the individual's sample and the control or reference. Such statistical comparisons include, but are not limited to, applying a decision rule or decision tree. If at least one internal standard is used, the comparison to discern a difference between the individual and the reference or control may also include features of these internal standards, such that the presence and/or amount of the Nairovirus nucleic acid in the individual's sample is correlated to the internal standards. The comparison can confirm the presence or absence of Nairovirus, and thus to detect or diagnose a Nairovirus infection.

The presence and/or amount level of Nairovirus nucleic acid (and hence the Nairovirus) may be alternatively compared with a control or reference population for at least 12 hours, at least 24 hours, at least 30 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 1 1 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks or more.

Although the invention does not require a monitoring period to diagnose a Nairovirus infection, it will be understood that repeated classifications of the individual, i.e., repeated snapshots, may be taken over time until the individual is no longer at risk. Alternatively, the presence and/or amount of Nairovirus in a sample obtained from the individual may be compared to presence and/or amount of Nairovirus in samples obtained from the same individual at different points in time.

As used herein, an "individuaP' is an animal. The animal may be a mammal, preferably a human or non-human primate. The animal may be a tick. The individual can be normal, suspected of having a Nairovirus infection or at risk of a Nairovirus infection. It will be understood that the term "individual" embraces one or more animals, particularly ticks e.g. pooled tick samples.

As used herein, a "patient' refers to mammal, preferably a human. The patient can be normal, suspected of having a Nairovirus infection or at risk of a Nairovirus infection.

The present invention enables the rapid detection of Nairovirus. By way of example, the method of the invention is typically completed within 30 minutes to 1 hour, 15 minutes to 45 minutes, 10 to 30 minutes, 5 to 25 minutes, or 1 to 20 minutes. The method of the invention is typically completed in less than 45 minutes, less than 30 minutes, less than 20 minutes, or less than 10 minutes.

The presence and/or amount of Nairovirus, as determined by determining the presence and/or amount of Nairovirus nucleic acid may be detected, quantified or determined by any appropriate means.

The presence and/or amount of the Nairovirus nucleic acid may be determined in a sample obtained from an individual. Wherein the individual is a mammal, the sample may be any suitable biological material, for example blood, plasma, saliva, serum, sputum, urine, semen, cerebral spinal fluid, cells, a cellular extract, a tissue sample, a tissue biopsy, a stool sample, a swab from any body site, and/or one or more organs. Wherein the individual is a mammal, the sample is typically from blood, serum, urine, saliva, semen and/or organ(s). The precise biological sample that is taken from the individual may vary, but the sampling preferably is minimally invasive and is easily performed by conventional techniques.

Wherein the individual is a tick, the sample is typically homogenised tick(s).

The biological sample may be taken from the individual before, during, and/or after treatment for Nairovirus. In one embodiment, the sample is taken after treatment or therapy for a Nairovirus infection has been initiated.

When samples are taken from an individual, extraction of Nairovirus RNA may be performed prior to testing according to the present invention. Accordingly, the methods of the present invention may comprise a nucleic acid extraction step (typically an RNA extraction step). Any suitable technique for nucleic acid extraction (typically RNA extraction) may be used. Suitable techniques are known in the art, for example spin column and precipitation techniques. Equally, an automated robotic system for nucleic acid extraction (typically RNA extraction extraction) may be used.

In some embodiments, the sample is a crude sample. A "crude sample" has undergone minimal or no purification. A crude sample may for example, have undergone centrifugation. Wherein the sample is a blood sample, a crude sample may have been left for sufficient time for the blood to clot. Wherein the sample is serum, urine or homogenised tick(s) the crude sample may be in a lysis buffer. Typically, a crude sample is not an isolated nucleic acid preparation, such as would typically be prepared for use in a conventional RT-PCR assay. For example, a crude sample has typically not undergone acid phenol/chloroform extraction, glass filter, or oligo (dT) chromatography.

In some embodiments, the sample (e.g. crude sample) is not diluted prior to performing the method of the invention. In some embodiments, the sample has been diluted, preferably with water or with a lysis buffer. For example, a sample may be diluted 1 in 10, 1 in 100, 1 in 1000, 1 in 10⁴, 1 in 10⁵, 1 in 10⁶, 1 in 10⁷, or more.

Thus, in one embodiment, compositions of the invention comprise a sample obtained from an individual.

In some embodiments, the sample comprises RNase inhibitor. Suitable RNAse inhibitors are known in the art.

As described herein, the presence or absence of Nairovirus in the sample is detected at the nucleic acid level (either quantitatively and/or semi-quantitatively and/or qualitatively). Typically, the detection is semi-quantitative. Thus, the Nairovirus nucleic acid may be detected as DNA and/or RNA and may be detected using any appropriate technique.

Typically the determination of the presence and/or amount of Nairovirus nucleic acid is carried out by amplifying said Nairovirus nucleic acid, or a target region of said Nairovirus nucleic acid or a fragment of said nucleic acid or target region. Amplification may be carried out using methods and platforms known in the art, for example recombinase polymerase amplification (RPA), PCR (for example, with the use of "Fast DNA Polymerase" , Life Technologies), such as real-time or quantitative PCR (qPCR), block-based PCR, ligase chain reaction, glass capillaries, isothermal amplification methods including loop-mediated isothermal amplification, rolling circle amplification transcription mediated amplification, nucleic acid sequence-based amplification, signal mediated amplification of RNA technology, strand displacement amplification, isothermal multiple displacement amplification, helicase- dependent amplification, single primer isothermal amplification, and circular helicase- dependent amplification. If employed, amplification may be carried using any amplification platform. In some embodiments, PCR, preferably q-PCR is used. In a preferred embodiment, an isothermal amplification technique is used. In a particularly preferred embodiment, RPA is used.

Primers of the invention are typically employed to amplify approximately 100-400, for example 100-300, 100-200 or 140-210 base pair regions of the Nairovirus nucleic acid. In RPA, in the presence of a suitable recombinase, single-stranded DNA binding proteins, strand-displacing polymerase and DNA precursors (dATP, dCTP, dGTP and dTTP), forward and reverse primers are extended in a 5' to 3' direction, thereby initiating the synthesis of new nucleic acid strands that are complementary to the individual strands of the target nucleic acid. The primers thereby drive amplification of the Nairovirus target nucleic acid, thereby generating amplification products comprising said target nucleic acid sequence. The RPA technique is isothermal, meaning that a thermal cycler is not required, making the technique particularly suitable for use as a low-cost point-of-care (POC) test.

In PCR, in the presence of a suitable polymerase and DNA precursors (dATP, dCTP, dGTP and dTTP), forward and reverse primers are extended in a 5' to 3' direction, thereby initiating the synthesis of new nucleic acid strands that are complementary to the individual strands of the target nucleic acid. The primers thereby drive amplification of Nairovirus target nucleic acid, thereby generating amplification products comprising said target nucleic acid sequence.

Primers of the invention are extended from their 3' ends (i.e. in a 5'-to-'3') direction.

In a preferred embodiment, the presence and/or amount of the Nairovirus nucleic acid is determined using RPA. Nucleic acid is isolated from a sample and primers of the invention are used to amplify the Nairovirus nucleic acid (if present in the sample).

References herein to determining the presence and/or amount of Nairovirus nucleic acid in a sample apply equally to determining the presence and/or amount of a region of the nucleic acid, and/or the presence and/or amount one or more fragment of said nucleic acid or target region thereof.

For example, primer pairs as disclosed herein may be used to amplify Nairovirus nucleic acid. The amplification products of (RPA or PCR) reaction may then be visualised by any appropriate means. For example, the amplification products may be separated and visualised by agarose gel electrophoresis. As the molecular weight of the Nairovirus nucleic acid will be known, the presence of the Nairovirus nucleic acid may be readily determined by the band size of the amplification products as run on an agarose gel. This method has the advantage of requiring standard equipment that will be present in most laboratories. Primer pairs of the invention may be used with RPA or other techniques such as qPCR to determine the presence and/or amount of the Nairovirus nucleic acid. Non-specific fluorescent dyes may be used in RPA or qPCR according to the present invention. Standard RPA and qPCR methods using such non-specific fluorescent dyes are known in the art. Preferably, a DNA probe specific for the Nairovirus nucleic acid in the sample, said probes further comprising a reporter, may be used. Said reporter may be a fluorescent reporter. Examples of fluorescent reporters (also referred to as fluorescent tags) are also described herein. Instruments enabling fast on-screen detection of genes by qPCR are commercially available (for example, Taqman®).

In some embodiments, DNA is extracted and/or isolated from a sample prior to analysis by a method of the present invention. Any appropriate method may be used to extract and/or isolate and/or purify the DNA. Standard techniques are known in the art and commercial kits are available.

Nairovirus nucleic acid from a sample (either purified or unpurified) may be labelled via any method (typically amplification) and used to interrogate one or more probe immobilised on a surface. The probe may be any length as defined herein.

Primers and probes of the invention bind specifically to CCHFV nucleic acid. By "specific", it will be understood that the primers and probes bind to CCHFV nucleic acid, with no significant cross-reactivity to any other molecule, particularly any other nucleic acid. Cross- reactivity of a nucleic acid or probe of the invention may be considered significant if the nucleic acid or probe binds to the other molecule at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to a nucleic acid from a related virus, that is not CCHFV. Preferably, the primers or probes of the invention bind to the other nucleic acid at less than 20%, less than 15%, less than 10% or less than 5%, less than 2% or less than 1 % the strength that it binds to CCHFV nucleic acid.

Primers of the invention do not consist of the following nucleic acid sequences:

AGGTGGTTTGAAGAGTTCA (SEQ ID NO: 1 17)

ACAAAACTTTGTTGCCTCC (SEQ ID NO: 1 18)

ATAGGAGTTTGTGAAGGTGT (SEQ ID NO: 119)

CCGATGATGCACAGAAGG (SEQ ID NO: 120)

TGGGAACACTCTCGCAAAAGGAAAAAGGAAATGGACTTGTGG (SEQ ID NO: 121) TGTGTTTCAGATGGCCAGTGCCGAGCAGATGCGTAGATGGAG (SEQ ID NO: 122) ACAGCCAAGAGGTACCAAGA (SEQ ID NO: 123) GCAGCATCATCAGGGTTGG (SEQ ID NO: 124)

TGCTGGAAAGAATCGTCGGCAATCTGCTGAGCACCCCAAT (SEQ ID NO: 125)

ATCTACATGCACCCTGCCGTGTCCCAAAGCAGACTCCCAT (SEQ ID NO: 126)

TCTGCTGAGCACCCCAAT (SEQ ID NO: 127)

TGCTGGAAAGAATCGTCGGCAA (SEQ ID NO: 128)

TCCCAAAGCAGACTCCCAT (SEQ ID NO: 129)

ATCTACATGCACCCTGCCGTG (SEQ ID NO: 130)

TCATAAAGTTTCTTCCCCCACTTC (SEQ ID NO: 131)

CTTACAGCAGGCAGAATCAGTG (SEQ ID NO: 132)

TCTCAAAGAAACACGTGCC (SEQ ID NO: 133)

CCTTTTTGAACTCTTCAAACC (SEQ ID NO: 134)

G GTTTG A AG AGTTC A A AA AG G (SEQ ID NO: 135)

In one embodiment, the method of the invention is not LAMP.

As described herein, the present invention provides a method for screening for the presence of Nairovirus in a sample, comprising determining the presence and/or amount of Nairovirus nucleic acid in a sample.

As described herein, the present invention provides a method for diagnosing a Nairovirus infection in an individual, comprising determining the presence and/or amount of Nairovirus nucleic acid in a sample obtained from the individual.

The method may comprise determining the presence and/or amount of Nairovirus nucleic acid in a first sample taken from the individual at a single initial point in time and multiple time points thereafter to monitor the efficacy of any treatment and disease resolution, and comparing the presence and/or amount of said Nairovirus nucleic acid in said first sample to the presence and/or amount of said Nairovirus nucleic acid in a reference or control sample. Said comparison may determine the status of Nairovirus infection in the individual with an accuracy, sensitivity and/or specificity of at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 90%, at least about 80%, at least about 70% or at least about 60%. Typically the accuracy, sensitivity and/or specificity is at least about 80% or at least about 90%.

The method may comprise determining the presence and/or amount of Nairovirus nucleic acid in a first sample from the individual; and comparing the presence or amount of the Nairovirus nucleic acid in the individual's first sample to the presence and/or amount of the Nairovirus nucleic acid in a sample from a reference or control population, said comparison being capable of classifying the individual as belonging to or not belonging to the reference or control population, wherein the comparison determines the status of Nairovirus infection in the individual.

The method may further comprise determining the presence and/or amount of Nairovirus nucleic acid in a second sample taken from the individual; and comparing the presence and/or amount of the Nairovirus nucleic acid in the individual's second sample to the presence and/or amount of the Nairovirus nucleic acid in the control or reference sample, wherein the second comparison is capable of classifying the individual as belonging to or not belonging to the control or reference population, and wherein the second comparison determines the status of Nairovirus infection in the individual.

The methods of the invention may be repeated at least once, at least twice, at least three times, at least four times, at least five times, or more. The presence and/or amount of the Nairovirus nucleic acid can be determined in a separate sample taken from the individual each time the method is repeated.

The methods of the invention may be used to diagnose, detect and/or predict Nairovirus infection. The methods of the invention may be used to distinguish between a Nairovirus infection and the absence of such an infection. The methods of the invention may be used to identify an individual with a Nairovirus infection and/or an uninfected individual. The methods of the invention may also be used to determine the status of a Nairovirus infection. Determining the status of a Nairovirus infection in an individual may comprise determining the progression or resolution of a Nairovirus infection. Determining the status of a Nairovirus infection in an individual may comprise determining the presence of a Nairovirus infection in an individual.

A Nairovirus infection may be diagnosed or predicted prior to the onset of clinical symptoms, and/or as subsequent confirmation after the onset of clinical symptoms. As set out by the WHO, the length of the incubation period depends on the mode of acquisition of the virus. Following infection by a tick bite, the incubation period is usually one to three days, with a maximum of nine days. The incubation period following contact with infected blood or tissues is usually five to six days, with a documented maximum of 13 days. Onset of symptoms is sudden, with fever, myalgia, (muscle ache), dizziness, neck pain and stiffness, backache, headache, sore eyes and photophobia (sensitivity to light). There may be nausea, vomiting, diarrhoea, abdominal pain and sore throat early on, followed by sharp mood swings and confusion. After two to four days, the agitation may be replaced by sleepiness, depression and lassitude, and the abdominal pain may localize to the upper right quadrant, with detectable hepatomegaly (liver enlargement). Other clinical signs include tachycardia (fast heart rate), lymphadenopathy (enlarged lymph nodes), and a petechial rash (a rash caused by bleeding into the skin) on internal mucosal surfaces, such as in the mouth and throat, and on the skin. The petechiae may give way to larger rashes called ecchymoses, and other haemorrhagic phenomena. There is usually evidence of hepatitis, and severely ill patients may experience rapid kidney deterioration, sudden liver failure or pulmonary failure after the fifth day of illness. The mortality rate from CCHF is approximately 30%, with death occurring in the second week of illness. In patients who recover, improvement generally begins on the ninth or tenth day after the onset of illness.

Accordingly, the present invention allows for more effective therapeutic intervention and/or diagnosis in the pre-symptomatic stage of infection.

The invention also provides the use of one or more primer and/or probe as defined herein in the manufacture of a diagnostic for a Nairovirus infection.

The invention also provides kits and devices that are useful in determining the presence and/or amount of Nairovirus in a sample.

In some embodiments (e.g. when employing TwistAmp Basic, TwistAmp Basic-RT, TwistAmp exo, TwistAmp exo-RT, TwistAmp fpg, and/or TwistAmp nfo), methods of invention can involve the use addition of magnesium acetate to initiate the amplification reaction. Thus, in one embodiment, kits and devices of the invention comprise magnesium acetate. Magnesium acetate is typically provided at a concentration of 200-360 rtiM, preferably 250-210 mM, most preferably 270-290 mM. In one embodiment, magnesium acetate is provided at 280mM.

The kits and devices of the present invention comprise at least one primer and/or probe of the invention. The primers and/or probes of the kit or device can be used to determine the presence and/or amount of Nairovirus nucleic acid in a sample, according to the present invention.

The primers and/or probes of the invention may be part of an array. The primers and/or probes of the invention may be packaged separately and/or individually. The primers and/or probes of the invention may be immobilised on an inert support. The kit or device may also comprise at least one internal standard to be used in generating profiles of the one or more Nairovirus nucleic acid according to the present invention. Likewise, the internal standards can be any of the classes of compounds described above.

The kits and devices of the present invention may also contain reagents that can be used to detectably label the Nairovirus nucleic acid contained in the samples from which the profiles of the Nairovirus nucleic acid are generated. For this purpose, the kit or device may comprise antibodies which bind to the probes and/or primers of the invention. The antibodies themselves may be detectably labelled. The kit or device also may comprise a specific binding component, such as an aptamer.

In a preferred embodiment, a kit or device of the invention comprises forward and reverse primers of the invention. In a preferred embodiment, a kit or device of the invention comprises a probe of the invention. Most preferably, a kit or device of the invention comprises forward and reverse primers of the invention and a probe of the invention.

The kits and devices of the present invention may also include other classes of compounds including, but not limited to, proteins (including antibodies), and fragments thereof, peptides, polypeptides, proteoglycans, glycoproteins, lipoproteins, carbohydrates, lipids, additional nucleic acids, organic and inorganic chemicals, and natural and synthetic polymers. The kits and devices of the present invention may also include pharmaceutical excipients, diluents and/or adjuvants. Examples of pharmaceutical adjuvants include, but are not limited to, preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic.

EXAMPLES

Design of primers and probes

Due to the urgent and unmet need for a rapid, simple and fieldable technology that would enable reliable identification of CCHFV, the inventors sought to identify primers and probes based on currently circulating CCHFV strains. To assist in designing primers and probes for the specific detection of CCHFV, the inventors gathered sequence information on a selection of CCHFV strains representing all 7 clades of CCHFV (Africa 1 , Africa 2, Africa 3, Asia 1 , Asia 2, Europe 1 and Europe 2). Having identified suitable CCHFV strains, the inventors attempted to manually identify genomic regions that are stable across the 7 clades, but they were hampered by significant variability between the CCHFV clade sequences. The inventors identified the CCHFV S-segment as their preferred target site (as summarised in Figure 1). These sites were then assessed for their potential suitability to bind a (cleavable) Exo probe, and then assessed to determine whether they would yield undesirable secondary structures. The bespoke set of primers and probes are detailed in Table 1 (see "CCHF RPA Fw", "CCHF RPA Rev", and "CCHF RPA Probe 1").

The primers and probes of the invention were specifically designed to be compatible with isothermal methods, particularly the RPA assay.

Table 1 :

Improved time to detection

Existing RT-PCR based methods for the detection of CCHFV suffer from slow detection times. The inventors sought to provide a method that is faster, and therefore better suited to high-throughput screening and field-use.

The primers and probes of the invention, employed in an RPA assay, outperformed in-house PCR experiments, with a shorter TTP (time to positive) for all samples tested. This desirable improvement in TTP is due in part to the lack of thermal cycling required by the RPA assay, which removes the need for temperature ramping and avoids the limit on the reaction kinetics of doubling only once every cycle. Also, the primers and probes of the invention allow the RPA assay to be performed at a temperature compatible with the reverse transcriptase enzyme, and so a separate reverse transcription step is not required.

Assay sensitivity

High sensitivity is extremely desirable for the detection of CCHFV. As shown in Table 2, the new rapid RPA assay, employing primers and probes of the invention, is (less than) 100 copies/μΙ, and a low (sub-threshold) detection is observed at 10 copies/μΙ (raw output data provided in Figure 2). The detection sensitivity of the tested primers and probes is highly desirable.

Table 2:

Moreover, the inventors believe that the signal strength of the assay may be further improved by further optimising the assay conditions. Specifically, the inventors believe that sensitivity may be further improved by slightly modifying the tested primer and probe, by "frame-shifting" the primers and/or probe by up to 6bp in the 5' or 3' direction or alter the size by +/- 15bp, preferably +/- 8bp (relative to the AY277672 DNA sequence shown in Figure 1 and SEQ ID NO: 11 1). Such primers and probes fall within the scope of the present invention.

Detection of CCHFV from different clades

The primers and probes of the invention were designed based on DNA sequences from 7 different clades of CCHFV. The ability to detect CCHFV from multiple clades would be highly desirable, as it would reduce the risk of false negatives.

The inventors therefore tested whether the primers and probe of the invention are capable of detecting CCHFV from all 7 clades of CCHFV, despite the above-discussed inter-clade sequence variability.

As shown in Table 3, the RPA assay employing primers and probes of the invention, detected representative strains of CCHFV from all 7 clades, at all dilutions tested. These data demonstrate that the invention provides a "PAN-CCHF" assay that is able to detect a divergence of CCHFV sequences. This is particularly surprising in view of the significant sequence variation between the 7 clades of CCHFV. Table 3. Cross-clade detection of CCHF

Table 3 : Table showing detection of CCHF viral extracts and synthetic CCHF RNA templates by CCHF RPA assay, in the form of time to positive (TTP; minutes) and number of replicates detected. The synthetic RNA templates were used at 5X10⁵ copies/reaction and viral extracts were confirmed positive by CCHF RT-PCR. The RT-PCR Ct values (cycle number) are included in the table. Note the RPA values shown are the mean of 3 independent experiments, each of which were performed with three replicates. The threshold was set at delta Rn 50,000. * indicates that the strain could not be detected by RT-PCR.

Performance using basic RT-RPA kit

The inventors tested whether the primers and probe of the invention are compatible with a basic kit (assay performed using a synthetic RNA fragments from a selection of CCHF strains, including AY277672, DQ21 1638, NC005302 and DQ211643). As shown in Figure 3, a single, strong dominant band is apparent around the expected size of 150bp, suggesting only the expected region is amplified. This indicates that the primers are highly specific for the target region.

Negative panel testing

To test whether the primers and probes of the invention are capable of specifically detecting CCHFV, the inventors performed an RPA assay using template nucleic acid from a number of viruses that are related to CCHFV (Negative viral strain collection was obtained from the Rare and Imported Pathogens Laboratory (RIPL), Public Health England; consisting of positive control extracts used in routine diagnostic PCR assays).

As shown in Table 4, the negative panel testing demonstrates that the RPA assay, performed using primers and probe of the invention, is highly specific for CCHFV, with all of the virus extracts from the control panel failing to produce a signal. Thus, primers and probes of the invention do not show cross-reactivity with other related virus strains endemic to the affected regions, viruses that are closely related to CCHFV virus, or to viruses that are more divergent.

Table 4. Negative viral panel testing.

Table 4: Table showing the results of a CCHF RPA run with a negative viral panel, consisting of a selection of strains from the Alphavirus, Arenavirus, Filovirus, Flavivirus, Hantavirus, Henipavirus and Nairovirus genii. Results represent 3 separate experiments, each performed with 3 replicates.

Robust detection - effect of crude samples

Existing RT-PCR based methods require intensive sample processing work, and are typically highly sensitive to sample quality. This increases sample preparation time and complexity, and typically requires the involvement of skilled technicians. The inventors therefore sought to provide a method that is more robust, and less sensitive to sample quality.

a) Testing the inhibitory effect of using dilutions of crude samples on the RPA assay, performed using primers and probe of the invention

The inventors tested the performance of the RPA assay, carried out using primers and probe of the invention, in the presence of crude samples (serum, urine and tick homogenate). 5 X10⁶ copies/reaction synthetic RNA template (Europe I strain AY277672) was tested, in the presence of a dilution series of crude samples. As shown in Table 5, the RPA assay performed using primers and probe of the invention tolerates crude sample preparations very well.

Table 5. Effect of crude sample dilution on detection in the RPA assay.

Table 5: *(Mean value where positive). RPA performed with 5X10⁶ copies/ reaction of synthetic RNA template of Europe I strain AY277672 and a serial (1 in 10) dilution of crude preparations of human male serum, urine and tick pool homogenate. b) Effect of crude samples on assay sensitivity

The inventors also tested the sensitivity of the RPA assay, carried out using the primers and probe of the invention, in the presence of crude samples (serum, urine and tick homogenate). 1 in 10 diluted crude samples added to a dilution series of synthetic RNA template (Europe I strain AY277672).

As shown in Table 6 (raw output data provided in Figure 8), the inhibitory effect of crude samples on sensitivity of the assay is minimal.

Table 6: Effect of crude material on assay sensitivity - limit of detection

Table 6: *(Mean value where positive). RPA performed with a serial (1 in 10) dilution of synthetic RNA template of Europe I strain AY277672 and 1 in 10 diluted crude preparations of serum and urine, or 1 in 100 diluted crude preparations of tick pool homogenate. The RPA results are shown as time to positive (TTP; minutes) and represent the mean of 3 independent experiments, each of which were performed with three replicates. The threshold was set at delta Rn 50,000. RT-PCR data is also included for comparison, with the values shown representing the mean time to positive (TTP; mins) of 2-3 separate experiments, each of which were performed with 2 replicates.

The addition of 1 in 10 diluted human serum shows a small inhibitory effect. The 1 in 100 diluted tick preparation and urine have had little-to-no impact on the detection limit. These data demonstrate that the primers and probes of the invention are advantageously suitable for testing of crude patient and tick samples with minimal sample preparation, particularly when used in the RPA assay.

Materials and Methods

Primer and probe preparation

Primers were prepared by Integrated DNA Technologies (IDT) and an RPA EXO probe by ATD BIO; all as HPLC purified material. Primer and probe stocks were prepared at 100μΜ in a Tris-EDTA buffer and diluted to 10μΜ in molecular grade dH₂0. A primer mix was prepared to 5μΜ (both forward and reverse primers) and both primer mix and probe stocks were frozen at -20 in single use aliquots.

Crude sample preparation

Crude samples included human serum male AB (Sigma H422-20ML), female Ixodes ricinus ticks (Charles River) and Surine standard -ve control urine (Sigma S-020-50ML). Sample preparation included aliqoting of the human male serum and urine standard into single use aliquots and storage at -20°C and fridge temperature respectively. The tick samples were prepared as tick pools (10 ticks) and frozen at -20 °C. The ticks were prepared by adding 300μΙ molecular grade water, transferral to Precellys-R tubes and were homogenised using a Precellys tissue homogeniser (3X 20 seconds, with 30 second breaks). The homogenate was centrifuged for 5 minutes at 5900xg and the supernatant retained. A serial dilution of each of the neat samples was prepared by diluting in molecular grade water.

Synthetic CCHF S-segment RNA template preparation

Synthetic CCHF S-segment DNA fragments from a selection of Europe group I and II strains and an Africa III laboratory control strain were prepared by IDT (AY277672, position 1 -1673, DQ21 1638, position 1-1659, DQ21 1643, position 1-1671 and NC005302, position 1-1672) with the addition of T7 and SP6 promoters at the 5' and 3' end respectively (see SEQ ID NOs: 112-1 16)). RNA templates were prepared from the synthetic DNA using a T7 High Yield RNA synthesis kit (NEB). Approximately ^g of DNA was added per reaction to an 0.2ml PCR tube with 2μΙ each of 100mM ATP, GTP, UTP and CTP, 2μΙ RNA polymerase mix, 2μΙ 10X reaction buffer and sufficient molecular grade dH₂0 to make the reaction up to 20μΙ. The reaction was incubated at 37°C for two hours in a thermocycler. The RNA template was then DNAse-treated to remove the original template contamination; 70μΙ nuclease free dH₂0 was added per tube with 10μΙ 10X DNAse I buffer and 2μΙ RNAse-free DNAse I (NEB). The tubes were mixed and incubated for 15 mins at 37 degrees. The RNA was purified using a Qiagen RNeasy minikit and quantified using a Qubit broad range RNA kit (Thermo-Fisher Scientific). EXO RT-RPA method

The CCHFV RPA assay was performed in a 50μΙ volume using a TwistAmp Exo-RT kit (TwistDx Cambridge UK). A mastermix was prepared, composed of the following/reaction; 4.2μΙ of a 5μΜ primer mix (forward and reverse primer), 0.6μΙ of the 10μΜ Exo-probe, 29.5μΙ rehydration buffer and sufficient distilled water to make the reaction up to 50μΙ after addition of all assay components. Where crude samples were used, 20 units (0.5μΙ) of an RNAse inhibitor was also included (RNAseOUT 401Ι/μΙ Invitrogen). The mastermix was distributed into the wells of a 96-well PCR plate. 1-5μΙ of template (together with 5μΙ crude sample if used) was added and the reaction mixture combined with the lyophilised enzyme pellet, before returning to the plate. The supplied magnesium actetate was diluted to 140mM with molecular grade dH₂0 and 5μΙ was added last and the plate briefly centrifuged before running at 40°C for 40 minutes on an Applied Biosystems 7500 real-time PCR system, with fluorescence detection every 60 seconds in the FAM channel. The threshold was set at 50,000 delta Rn.

RPA with an RT-RPA basic kit

The RT-RPA basic assay was performed in a 50μΙ volume using a TwistAmp Basic-RT kit (TwistDx Cambridge UK). A mastermix was prepared, composed of the following/reaction; 4.2μΙ of 5μΜ primer mix (forward and reverse primer), 29.5μΙ rehydration buffer and sufficient distilled water to make the reaction up to 50μΙ after addition of template. The mastermix was distributed into 0.2ml PCR tube strips. 5μΙ of template was added and the reaction mixture combined with the lyophilised enzyme pellet, before returning to the wells. The supplied magnesium actetate was diluted to 140mM with molecular grade dH20 and 5μΙ was added last. The tube strip was then briefly centrifuged before incubating for 40 °C for 40 minutes on a thermocycler. The products of the RPA were purified using a QIAgen QIAquick PCR purification kit, then run on an Invitrogen 1 % Agarose gel (E-Gel EX with Sybr Gold II) with an Invitrogen E gel 1 Kb plus ladder.

RT-PCR

This method was adapted from the paper by Atkinson et al. 2012 (Development of a Real- Time RT-PCR Assay for the Detection of Crimean-Congo Hemorrhagic Fever Virus. Atkinson B., Chamberlain J., Logue C.H., Cook N., Bruce C, Dowall, S.D., Hewson R. 9, Sep 2012, Vector Borne Zoonotic Dis., Vol. 12, pp. 786-93). Briefly The CCHF RT-PCR assay was performed in a 20μΙ volume using a Superscript III Platinum One-step quantitative RT-PCR kit (Invitrogen). For each reaction a 15μΙ mastermix was prepared containing 10μΙ of the supplied 2X reaction mix, Ο.δμΙ of superscript III RT/Taq enzyme mix and a final concentration of each primer of 1.2μΜ and probe of Ο.δμΜ. Where crude samples were used, 20 units (0.5μΙ) of an RNAse inhibitor was also included (RNAseOUT 401Ι/μΙ Invitrogen) and the mastermix made up to 15μΙ with molecular grade water. The mastermix was distributed into the wells of a 96-well PCR plate. 3-5μΙ of template (together with 5μΙ crude sample if used) was added and the plate briefly centrifuged before running on an Applied Biosystems 7500 real-time PCR system. The PCR run parameters included a 10 minute RT step at 50 °C, followed by a 2 minute denaturation step at 95°C, an amplification stage composed of 45 cycles of denaturation; 95°C for 10 seconds, and annealing/extension at 60°C for 40 seconds, followed by a final extension at 40 °C for 20 seconds. Fluorescence was detected in the FAM channel, once each cycle during the amplification stage. The threshold was set at 250,000 delta Rn.

CCHF positive viral panel

A collection of CCHFV strains representing each of the following S segment clades: Asia I, Asia II, Africa II, Africa III and Europe I were cultured and viral RNA extracted using a standard RNA extraction kit (QIAamp viral RNA kit). Synthetic whole S-segment viral RNA was used to represent Africa I and Europe II clades.

Negative viral panel

Viral RNA extracts covering Arenavirus, Ebolavirus, Marburgvirus, Henipavirus, and the Orthohantavirus genera were donated by the Rare and Imported Pathogens Laboratory, PHE Porton from a collection of diagnostic assay positive controls. They are described as positive, with a Ct of approximately 30 in their respective assay where a real-time assay is available, or as having a clear band in a block-based PCR. The Orthonairovirus samples Hazara and Issyk-kul were prepared in-house by the Virology and Pathogenesis group and have also be confirmed positive by a block-based PCR.

Sequences of synthetic CCHF S-segment DNA fragments with SP6 and T7 promoters Shown are the DNA fragments (5'-3' orientation) designed to be a template for in vitro transcription to create the synthetic RNA templates used to test the RPA (see SEQ ID NOs: 112-1 16). The fragments are composed of a section of the S-segment of CCHF (lower case), a T7 promoter (CAPS) and an SP6 promoter (CAPS, underlined), flanked by GC-rich tails (CAPS, Italicised). Table 7: Sequences

a. A Y277672 S-segment DNA fragment (SEQ ID NO: 112)

GCGCTAATACGACTCACTATAGGGtctcaaagaaacacgtgccgcttacgcccacagtgttctcttgagtgtctg caaaatggaaaacaagatcgaggtgaacagcaaagatgagatgaacaaatggtttgaggagtttaaaaagggaaatggact tatggacactttcacaaactcctactccttttgtgagaatgtaccaaatctggataagtttgtgttccagatggccagcgccactgatg atgcacagaaggactccatctatgcatcggctctggtggaagcaaccaagttctgtgcacccatatatgaatgtgcctgggtcag ctctaccggcattgtgaagaaggggcttgagtggttcgagaagaattcaggaaccatcaaatcctgggatgagaactatgctga gctgaaggttgatgttcccaaaatagaacaactcgccaattaccagcaggctgctctcaagtggaggaaggacataggtttccgt gtcaatgcaaacacggcagccttaagcaacaaggtccttgcagaatataaagtccctggcgaaattgtgatgtctgttaaagaa atgctgtcagacatgattagaaggaggaatctaattctcaacaggggcggtgatgaaaatccgcgcggcccagtgagccgtga acatgtggagtggtgcagggaatttgtcaaaggcaagtacatcatggccttcaatccaccttggggggacatcaacaaaccagg ccgttcggggatagcacttgttgcaacaggccttgccaagcttgcagagaccgaggggaaaggagtttttgacgaagccaaga agaccgtggaggctctcaatgggtatttggacaagcacagggacgaagttgacaaagcaagtgctgacagcatgatagcaaa cctcctgaagcacattgccaaagcacaagagctttataaaaattcatctgctcttcgtgcacaaggtgcacagattgacaccccttt cagctcgttttactggctttacaaggccggtgtgactccagagaccttcccaactatctcacagttccttttcgaactggggaagcaa ccaagggggaccaagaaaatgaaaaaggcactcctgagcactccaatgaagtgggggaagaaactttatgagctctttgctg atgactctttccagcagaacagaatctacatgcaccctgctgtgttaacagccggtagaatcagtgaaatgggtgtctgctttggaa caatccctgttgccaatcctgatgacgctgctcagggatctggacataccaagtccattctcaaccttcggacaagcacagagac caacaatccatgcgccaagacaattgtcaaattgtttgaaatccaaaaaacaggatttaacatacaggacatggacattgtagcc tctgagcacctgctgcaccaatcccttgttggcaagcagtctccattccaaaatgcctataacgtcaagggcaatgccaccagtgc caacatcatctaaaactcaaggtgtttcatattcagcttttctcctcctgcatcactacttacagctatgactattaaccacatttatctta attgcttatgtaatgttgttttgctaattttatcttgctatctttcatttcaaatacttaaagggctgtgcggcaacgatatctttgaga^'TTCT

ATAGTGTCACCTAAATGCGC ^""""" ~ b. DQ211638 S-segment DNA fragment (SEQ ID NO: 113)

GCGCTAATACGACTCACTATAGGGtctcaaagaaacacgtgccgctcacgcccacagtgttatcttgagtgtaa gcaaaatggagaacaaaatcgaggtgaacagcaaagacgatctgaacaagtggtttgaggagttcaagaaaggaaatggg cttgtggacaccttcacaaactcatattctttttgtgaaaatgtgccgaacttggacagatttgtgtatcagatggccagtgcaaccga tgatgcacagaaggactctatatatgcatctgctcttgttgaagcaaccaagtactgtgcaccaatatatgagtgtgcctgggtcag ctctacaggcattgtaaagaaaggtttggagtggtttgaaaagaacacgagcactatcaaatcctgggacgagagctacactga gctgaaggttgacattcccaaaatagagcagctctctagttatcagcaggcagccctcaagtggagaaaggacataggtttccg catcaatgctaacacaacagcgctgagcaacaaagtgcttgcagagtacaaggttccaggagagatcttggtgcctgtcaaag agatgctgtcagacatgataaggagaaggaacattatccttaacagaggcagcgatgagaatccacgaggcccagtaagtca tgagcacattgagtggtgcagggagttcatcaagggaaagtacattatggccttcaatccaccttggggtgacatcaacaaatca ggtcgctccgggattgcacttgtcgcaaccggtcttgccaagctggcagagactgaaggtaaaggagtcttcgaagaagccaa aaagactgtggaagcactcaaggactaccttgacaaacacagagatgaagtcgacaagacaagtgctgacaacatggtgac aagccttttgaaacatattgccaaggctcaggaactctacaagaactcatctgcacttcgtgctcagggtgcacaaattgacactc ctttcagctcgttctactggctctacaaggcaggggtcactccagagacgttccccactgtttcccagtttctctttgagcttggaaagc agccaaggggcaccaagaaaatgaagaaagctctgctcagcactcccatgaagtggggaaagaagctctatgagctctttgc agatgactctttccagcagaacaggatctacatgcaccctgctgtgctgacagctggtaggatcagcgaaatgggtgtctgctttg ggacaattcctgtagccaacccagatgatgcagcccagggatctggacacaccaagtccattctaaatctaaggacaaacact gagtccaacaatccgtgtgccaggaccattgtcaagcttttcgaaattcagaagacgggctttgacataaaggacatggacatcg tggcctctgagcatctgctgcaccaatccctggttgggaagcagtcgcccttccagaatgcctacaatgtcaagggcaatgccac cagtgccaacatcatctagctgcccaggtgctctgcatcccacccacccaactccaagtcagtgctttcggctgcaactagtaatc atgcttgcttcaaatactgcttcactcaacttttattctttctt^ c. DQ211643 S-segment DNA fragment (SEQ ID NO: 114)

GCGCTAATACGACTCACTATAGGGtctcaaagaaacacgtgccgcttacgcccagtgttctcttgagtgtctgca aaatggaaaacaagatcgaggtgaacagcaaagatgagatgaataaatggtttgaggagtttaaaaaggaaaatggacttat ggacactttcacaaactcctactccttttgtgagaatgtaccaaatctggataagtttgtgttccagatggccagcgccactgatgat gctcagaaggactccatctatgcatcggctctggtggaagcaaccaagttctgtgcacccatatatgaatgtgcttgggtcagctct accggcattgtgaagaaggggcttgagtggttcgaaaagaattcaggaaccatcaaatcctgggatgagaactatactgagctg aaggttgatgttcccaaaatagagcaacttgccaattaccaacaggctgctctcaaatggaggaaggacataggtttccgtgtca atgcaaacacggcagccttaagcaacaaggtccttgcagaatataaagtccctggcgaaattgtgatgtctgttaaagaaatgct gtcagacatgattagaaggaggaatctaattctcaacaggggcggtgatgaaaatccgcgcggcccagtgagccgtgaacat gtggagtggtgcagggaatttgtcaaaggcaagtacatcatggccttcaatccaccttggggggacatcaacaaatcaggccgtt cgggaatagcacttgttgcaacaggccttgccaagcttgcagagaccgaggggaaaggagtttttgacgaagccaagaagac cgtggaggctctcaatgggtatttggacaagcacagagacgaagttgacaaagcaagtgctgacagcatgataacaaacctcc tgaagcacattgccaaagcacaagagctttataaaaattcatctgctcttcgtgcacaaggtgcacagattgacacccctttcagct cgttttactggctttacaaggccggtgtgactccagagaccttcccaactatctcacagttcctttttgaactggggaagcaaccaag ggggaccaagaaaatgaaaaaggcactcctgagcactccaatgaagtgggggaagaaactttatgagctctttgctgatgact ctttccagcagaacagaatctacatgcaccctgctgtgttgacagccggtagaatcagtgaaatgggtgtctgctttggaacaatc cctgttgccaatcctgatgacgctgctcagggatctggacataccaagtccattctcaaccttcggacaagcacagaaaccaaca atccttgcgccaagacaattgtcaaattgtttgaaatccaaaaaacaggatttaacatacaggacatggacattgtagcctctgag cacctgctgcaccaatcccttgttggcaagcagtctccattccaaaatgcctataacgtcaagggcaatgccaccagtgccaaca tcatctaaaactcaaggtgtttcatattcagcttttctcctcctgcatcactacttacagttataactattaatcacatttatcttaattgcttat gtaatgttgttttgctaattttatcttgctatcttttatttcaaatacttaaagg

d. NC005302 S-segment DNA fragment (SEQ ID NO: 115)

GCC3CTAATACG.ACTC.ACTATAGGGtctcaaagaaacacgtgccgcttacgcccacagtgttctcttgagtgttag cagaatggaaaacaagatcgaggtgaataacaaagatgagatgaacaggtggtttgaagagttcaaaaaaggaaatggact tgtggacaccttcacaaactcctattccttttgcgagagtgttcccaatttggacaggtttgtgtttcagatggccagtgccaccgatga tgcacagaaggactccatctacgcatctgctctggtggaggcaacaaagttttgtgcacctatatatgagtgcgcatgggttagctc cactggcattgtaaaaaagggacttgaatggttcgagaaaaatgcaggaaccattaagtcctgggatgaaagttatactgagct aaaggtcgacgtcccgaaaatagagcagcttaccggttaccaacaagctgccttgaagtggagaaaagacataggtttccgtgt caatgccaacacagcagctctgagcaacaaagtcctcgcagaatacaaagtccctggtgagattgtgatgtctgtcaaagagat gctgtcagacatgattaggagaaggaacctgattctaaacaggggtggtgatgagaacccacgtggcccagtgagccatgag catgtagactggtgcagggagtttgtcaaaggcaaatacatcatggccttcaacccaccatggggggacatcaacaagtcagg ccgttcaggaatagcacttgttgcaacaggccttgctaagcttgcagagactgaaggaaagggaatatttgatgaagccaaaaa gactgtggaggccctcaacgggtatctggacaagcataaggacgaagttgatagagcaagcgccgacagcatgataacaaa ccttcttaagcatattgccaaggcacaggagctctataaaaattcatctgcacttcgtgcacaaagcgcacagattgacactgcttt cagctcatactattggctttacaaggctggcgtgactcctgaaaccttcccgacggtgtcacagttcctctttgagctagggaaaca gccaagaggtaccaagaaaatgaagaaggctcttctgagcaccccaatgaagtgggggaagaagctttatgagctctttgccg atgattctttccagcagaacaggatttacatgcatcctgccgtgcttacagctggtagaatcagtgaaatgggagtctgctttgggac aatccctgtggccaatcctgatgatgctgcccaaggatctggacacactaagtctattctcaacctccgtaccaacactgagacca ataatccgtgtgccaaaaccatcgtcaagctatttgaagttcaaaaaacagggttcaacattcaggacatggacatagtggcctct gagcacttgctacaccaatcccttgttggcaagcaatccccattccagaacgcctacaacgtcaagggcaatgccaccagtgct aacatcatttaaaatacaaactgctctgtactcaacttccttccttctgaaccgccatccataattgcaatacttaatcatgcttttttactt gcttatgtaaccttattttattaacctttctctattttctcttgttt

i O C

e. U88411 S-segment DNA fragment (SEQ ID NO: 116)

GCGCTAATACGACTCACTATAGGGtctcaaagaaacacgtgccgcttacgcccacagtgttaccttgagtgtta gcaaaatggagaacaaaatcgaagtgaacaacaaagatgagctgaacaaatggtttgaggagttcaagaaaggaaacggg cttgtggacactttcacaaactcctattctttctgtgagaacatgccgaaccttgacaggtttgtattccagatggctggtgcaaccgat gatgcacagaaagattccatctatgcatctgccctggttgaggctaccaagtactgtgctcccatatatgaatgtgcctgggttagct ccacaggtatagtgaaaagaggccttgagtggtttgaaaaaaatacaggaaccattaagtcttgggatgagagctacactgagc tgaaagtggatgtgcccaaaattgaacagcttgccaactaccagcaggccgctctcaagtggaggaaggacataggcttccgc gttaatgcaaacacagcggccctaagcaacaaagtcctctctgagtacaaggttcctggtgagattgtgatgtctgtcaaagagat gctttcagacatgattagaaggaggaacctgatccttaacagaggaggtgatgagaacccaagaggcccagtaagcaagga gcacatagaatggtgcagggagtttgtcaagggcaaatatataatggccttcaatccaccctggggtgatgtcaacaagtccggc cgctcaggaatagcactagttgctacaggtcttgccaaacttgcagaaacagaaggaaagggagtttttgaggaggcaaagaa gacagtggaggccctcaaggagtaccttgacaaacacaaagatgaggtggacaaggctagtgctgacagcatggtaacaaa cctcctcaagcacatcactaaggcccaggaactctacaaaaactcatcagcactgagagcacagggtgcacagattgacacc cctttcagctccttctactggctctataaagcgggcgtgactccagaaaccttccccaccgtctctcaattcctctttgagctgggaaa gcagccaagaggcactaaaaaaatgaagaaggcacttctgagcaccccaatgaaatggggaaagaagctttatgagctcttt gctgatgactctttccagcaaaacaggatctacatgcaccctgctgtgctgacagctggcagaattagtgagatgggtgtttgctttg gaaccatccctgttgccgatccagacgatgcagcccagggctcaggtcacaccaaatcaattctgaacctccgaacaagtagtg aaactaacaatccttgtgctaaaacgattgtcaaactcttcgaagttcaaaaaacaggattcaacatacaggacatggatattgtt gcctctgagcacctgctgcaccagtctcttgttggcaagcaatccccgttccaaaatgcttacaacgtcaaaggcaatgccacca gtgccaacattatctaaatctccagagtttttttctatttgttccagtttgtgcttctgcttctgaccataaccattaatcgcatttgctttttaca gttaccaagacctattttattttgctttattttattttgcttcatactattatattcttcttttacatgttgaagggctgtgcggcaacgatatcttt gag ajj u s J ^l^l^i^^li^ i ^ . c-

Gen Bank Accession AY277672 (SEQ ID NO: 111 )

tctcaaagaaacacgtgccgcttacgcccacagtgttctcttgagtgtctgcaaaatggaaaacaagatcgaggtgaacagcaa agatgagatgaacaaatggtttgaggagtttaaaaagggaaatggacttatggacactttcacaaactcctactccttttgtgaga atgtaccaaatctggataagtttgtgttccagatggccagcgccactgatgatgcacagaaggactccatctatgcatcggctctg gtggaagcaaccaagttctgtgcacccatatatgaatgtgcctgggtcagctctaccggcattgtgaagaaggggcttgagtggtt cgagaagaattcaggaaccatcaaatcctgggatgagaactatgctgagctgaaggttgatgttcccaaaatagaacaactcg ccaattaccagcaggctgctctcaagtggaggaaggacataggtttccgtgtcaatgcaaacacggcagccttaagcaacaag gtccttgcagaatataaagtccctggcgaaattgtgatgtctgttaaagaaatgctgtcagacatgattagaaggaggaatctaatt ctcaacaggggcggtgatgaaaatccgcgcggcccagtgagccgtgaacatgtggagtggtgcagggaatttgtcaaaggca agtacatcatggccttcaatccaccttggggggacatcaacaaaccaggccgttcggggatagcacttgttgcaacaggccttgc caagcttgcagagaccgaggggaaaggagtttttgacgaagccaagaagaccgtggaggctctcaatgggtatttggacaag cacagggacgaagttgacaaagcaagtgctgacagcatgatagcaaacctcctgaagcacattgccaaagcacaagagcttt ataaaaattcatctgctcttcgtgcacaaggtgcacagattgacacccctttcagctcgttttactggctttacaaggccggtgtgact ccagagaccttcccaactatctcacagttccttttcgaactggggaagcaaccaagggggaccaagaaaatgaaaaaggcac tcctgagcactccaatgaagtgggggaagaaactttatgagctctttgctgatgactctttccagcagaacagaatctacatgcac cctgctgtgttaacagccggtagaatcagtgaaatgggtgtctgctttggaacaatccctgttgccaatcctgatgacgctgctcag ggatctggacataccaagtccattctcaaccttcggacaagcacagagaccaacaatccatgcgccaagacaattgtcaaattg tttgaaatccaaaaaacaggatttaacatacaggacatggacattgtagcctctgagcacctgctgcaccaatcccttgttggcaa gcagtctccattccaaaatgcctataacgtcaagggcaatgccaccagtgccaacatcatctaaaactcaaggtgtttcatattca gcttttctcctcctgcatcactacttacagctatgactattaaccacatttatcttaattgcttatgtaatgttgttttgctaattttatcttgctat ctttcatttcaaatacttaaagggctgtgcggcaacgatatctttgaga

Claims

1. A composition comprising: a) a nucleic acid probe comprising:

(i) nucleic acid sequence exhibiting at least 85% identity to any one of SEQ ID NOs: 29-110; or

(ii) nucleic acid sequence comprising 15 or more consecutive nucleic acids of the nucleic acid sequence of SEQ ID NO: 29-110; or

(iii) nucleic acid sequence of any one of SEQ ID NOs: 29-110; wherein: group "3" = modification that functions to block polymerase extension;

group "5 dT-fluorophore;

group "6 an abasic nucleotide analog; and

group "7 dT-quencher group (suitable for group "5");

and/or b) a forward nucleic acid primer and a reverse nucleic acid primer; the forward nucleic acid primer comprising: ii) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of SEQ ID NO: 2; or ii) nucleic acid sequence CGTGCCGCTTACGCC (SEQ ID NO: 2); and the reverse nucleic acid primer comprising: i) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of SEQ ID NO: 16; or ii) nucleic acid sequence TGTGAAAGTGTCCAT (SEQ ID NO: 16).

2. The composition of claim 1 , wherein:

group "3" = selected from (i) C₃-spacer, (ii) a phosphate, (iii) a biotin-TEG, or (iv) an amine;

group "5" = selected from (i) dT-fluorescein, (ii) TAMRA and (iii) Cy5;

group "6" = D-spacer; and

group "7" = selected from (i) dT-BHQ1 and (ii) dT-BHQ2 3) The composition of claim 2, wherein:

group "3"= propanol;

group "5"= dT-fluorescein;

group "6"= D-spacer; and

group "7"= dT-BHQ1 .

4) The composition of any one of the preceding claims, wherein the forward nucleic acid primer comprises or consists of:

(i) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1-14;

(ii) nucleic acid sequence comprising 15 or more consecutive nucleic acids of the nucleic acid sequence of any one of SEQ ID NOs: 1-14; or

(iii) the nucleic acid sequence of any one of SEQ ID NOs: 1-14.

5) The composition of any one of the preceding claims, wherein the reverse nucleic acid primer comprises or consists of:

(i) nucleic acid sequence exhibiting at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 15-28; or

(ii) nucleic acid sequence comprising 15 or more consecutive nucleic acids of any one of SEQ ID NOs: 15-28; or

(iii) the nucleic acid sequence of any one of SEQ ID NOs: 15-28.

6) The composition of any one of the preceding claims, comprising:

(i) forward nucleic acid primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1 ;

(ii) reverse nucleic acid primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15; and

(iii) nucleic acid probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29.

7) The composition of any one of the preceding claims, further comprising primer and/or probe for the detection of CCHFV and one or more other pathogen(s), optionally one or more other medically and/or agriculturally important pathogen(s).

8) The composition of claim 7, wherein said one or more other pathogen(s) includes more other haemorrhagic fever viruses. 9) A kit comprising a composition according to any one of the preceding claims.

10) A device comprising a composition according to any one of the preceding claims.

1 1) The kit of claim 9 or the device of claim 10, comprising part or whole of a TwistAmp Basic kit, TwistAmp Basic-RT kit, TwistAmp exo kit, TwistAmp exo-RT kit, TwistAmp fpg kit, and/or TwistAmp nfo kit.

12) Use of a composition, kit or device of any one of the preceding claims in a method of detecting CCHFV in a sample.

13) Use of a composition, kit or device of any one of the preceding claims in a multiplex method of detecting CCHFV and one or more other pathogen(s) in a sample, optionally one or more other medically and/or agriculturally important pathogen(s).

14) The use of a composition, kit or device of any one of the preceding claims, wherein said one or more other pathogen(s) includes one or more other haemorrhagic fever viruses.

15) A method for detecting the presence of CCHFV in a sample or detecting the absence of said CCHFV in said sample, said method comprising:

A) Combining said sample with a composition according to any one of claims 1-8;

B) Allowing nucleic acid present in the sample to contact the primers and/or probes within the composition; and

C) Performing a nucleic acid amplification technique;

wherein amplification of nucleic acid in the sample confirms that nucleic acid from CCHFV is present within the sample, and wherein the absence of amplification of nucleic acid in the sample confirms that nucleic acid from CCHFV is absent from the sample.

16) The method of claim 15, further comprising detecting the presence of one or more other pathogen(s) in a sample or detecting the absence of said one or more other pathogen(s) in a sample, optionally wherein said one or more other pathogen(s) is a medically and/or agriculturally important pathogen(s).

17) The method of claim 16, wherein said one or more other pathogen(s) includes one or more other haemorrhagic fever viruses. 18) The method of any one of claims 15-17, wherein the nucleic acid amplification technique is an isothermal nucleic acid amplification technique.

19) The method of claim 18, wherein the isothermal nucleic acid amplification technique is Recombinase Polymerase Amplification.

20) The method of any one of claims 15 to 19, wherein the sample is from an individual, typically an animal.

21) The method of claim 20, wherein the animal is a mammal, typically a human.

22) The method of claim 21 , wherein the sample is selected from blood, plasma, saliva, serum, sputum, urine, cerebral spinal fluid, semen, cells, a cellular extract, a tissue sample, a tissue biopsy, a stool sample, a swab from any body site and/or one or more organs; typically blood, serum, urine, saliva and/or organ(s).

23) The method of claim 20, wherein the animal is a tick.

24) The method of claim 23, wherein the sample is homogenised tick(s).

25) The method of any one of claims 15-24, wherein the sample is a crude sample.

26) The method of any one of claims 15-25, for use in surveillance of CCHFV prevalence.

27) The method of any one of claims 15-25, for use in diagnosing CCHFV infection in an individual.

28) The method of claim 27 wherein, upon identification of CCHFV infection in the individual, said individual is provided with an appropriate treatment or therapy.