EP4168570A1

EP4168570A1 - Method for detecting the presence of coronavirus nucleic acid

Info

Publication number: EP4168570A1
Application number: EP21731538.1A
Authority: EP
Inventors: Heinrich Jehle; Peter Winklehner
Original assignee: 3a Diagnostics GmbH
Current assignee: 3a Diagnostics GmbH
Priority date: 2020-06-17
Filing date: 2021-06-17
Publication date: 2023-04-26
Also published as: WO2021255151A1

Abstract

The present invention relates to methods and kits for detecting the presence of SARS-CoV-2 nucleic acid in a sample.

Description

Method for Detecting the Presence of Coronavirus Nucleic Acid

FIELD OF THE INVENTION

The present invention provides kits and methods for detecting the presence of coronavirus nucleic acid in a sample. In particular, the invention is useful in diagnosing infection with SARS- CoV-2.

BACKGROUND

Coronaviridae (CoVs) are a family of enveloped, positive-sense, single-stranded RNA viruses. The current classification recognizes at least 39 species in 27 subgenera, five genera and two subfamilies that belong to the family Coronaviridae.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China, as the likely cause of severe pneumonia cases. The virus has since spread worldwide, affecting more than 7 million people and causing more than 400,000 deaths by 10 June 2020. The disease was named COVID-19. Taxonomic classification placed the novel virus within the subgenus Sarbecovirus, genus Betacoronavirus, family Coronaviridae, order Nidovirales. The genome size of the SARS-CoV-2 varies from 29.8 kb to 29.9 kb. More than two-thirds of the genome comprises orflab encoding orflab polyproteins, while the remaining part comprises genes encoding structural proteins including surface (S), envelope (E), membrane (M), and nucleocapsid N proteins. Additionally, the SARS-CoV-2 contains 6 accessory proteins, encoded by ORF3a, ORF6, ORF7a, ORF7b, and ORF8 genes.

SARS-CoV was found in sputum at mean concentrations of 1.2-2.8x10⁶ copies per mL (Drosten et al. J Clin Microbiol 42, 2043-2047, doi:10.1128/jcm.42.5.2043-2047.2004 (2004). For SARS-CoV-2, pharyngeal virus shedding was found to be very high during the first week of symptoms, with a peak at 7.11x10⁸ RNA copies per throat swab on day 4 (Wolfel et al. Nature 581, 465-469 (2020). https://doi.org/10.1038/s41586-020-2196-x).

In acute respiratory infections, RT-PCR is routinely used to detect causative viruses from respiratory secretions. RT-PCR protocols have been established to reliably detect SARS-CoV- 2, and further discriminate SARS-CoV-2 from SARS-CoV (Corman et al. Euro Surveill. 2020;25(3):pii=2000045. https://doi.orq/10.2807/1560-7917.ES.2020.25.3.2000045). While PCR tests have a high sensitivity and specificity, they must typically be carried out in a laboratory, and it takes several hours until a result is available. For these reasons, PCR tests cannot be used in a point-of-care testing format.

Antibody tests for detecting immunoglobulins directed against surface proteins of SARS-CoV- 2 are on the market. Tests detecting IgG antibodies may provide information as to whether the tested individual has had an infection in the past, but does not allow diagnosis of acute infections.

There is a need for a rapid test for diagnosing acute infections with SARS-CoV-2. Preferably, the test should be simple to use and provide a test result within 15 minutes or less.

SUMMARY OF THE INVENTION

The inventors surprisingly found that non-amplified SARS-CoV-2 RNA can be detected in samples from infected individuals using a lateral flow test which employs one or more probes targeting at least two different SARS-CoV-2-specific target regions, the two target regions being distantly located in the viral genome. Without wishing to be bound by any theory, it is believed that the use of one or more probes targeting multiple SARS-CoV-2-specific target regions in the viral genome confers sufficient sensitivity to allow detection of acute infections, in particular beginning infections, without the need for nucleic acid amplification. The invention further allows distinguishing between infection with SARS-CoV-2 and infections with other coronaviruses.

The present invention therefore relates to the subject matter defined in the following items 1 to 47:

[1] A test kit for detecting the presence of SARS-CoV-2 nucleic acid in a sample, wherein the test kit comprises a solid support comprising a first detection zone having immobilized thereto (i) a capture probe comprising a first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, and (ii) a capture probe comprising a second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, said second region being different from said first region.

[2] The test kit of item 1, wherein said first region and said second region are separated by at least 1,000 nucleotides within the genomic RNA of SARS-CoV-2.

[3] The test kit of item 1, wherein said first region and said second region are separated by at least 2,000 nucleotides within the genomic RNA of SARS-CoV-2. [4] The test kit of item 1, wherein said first region and said second region are separated by at least 3,000 nucleotides within the genomic RNA of SARS-CoV-2.

[5] The test kit of item 1, wherein said first region and said second region are separated by at least 4,000 nucleotides within the genomic RNA of SARS-CoV-2.

[6] The test kit of item 1, wherein said first region and said second region are separated by at least 5,000 nucleotides within the genomic RNA of SARS-CoV-2.

[7] The test kit of any one of the preceding items, wherein the length of the capture probe(s) is from 25 nt to 1 ,000 nt, or from 30 nt to 800 nt, or from 40 nt to 600 nt, or from 50 nt to 500 nt, or from 60 nt to 300 nt, or from 70 nt to 250 nt, or from 75 nt to 200 nt, or from 80 nt to 150 nt, or from 85 nt to 125 nt, or from 90 nt to 110 nt.

[8] The test kit of any one of the preceding items, wherein the first region within the genomic RNA of SARS-CoV-2 is specific for SARS-CoV-2, and the second region within the genomic RNA of SARS-CoV-2 is specific for SARS-CoV-2.

[9] The test kit of any one of the preceding items, wherein the first detection zone has further immobilized thereto (iii) a capture probe comprising a third nucleic acid sequence capable of hybridizing to a third region within the genomic RNA of SARS-CoV-2, (iv) a capture probe comprising a fourth nucleic acid sequence capable of hybridizing to a fourth region within the genomic RNA of SARS-CoV-2, (v) a capture probe comprising a fifth nucleic acid sequence capable of hybridizing to a fifth region within the genomic RNA of SARS-CoV-2, and (vi) a capture probe comprising a sixth nucleic acid sequence capable of hybridizing to a sixth region within the genomic RNA of SARS-CoV-2, said first, second, third, fourth, fifth and sixth regions being different from each other.

[10] The test kit of item 9, wherein each of said first, second, third, fourth, fifth and sixth regions is specific for SARS-CoV-2.

[11] The test kit of any one of the preceding items, wherein the capture probes immobilized within the first detection zone do not hybridize to genomic RNA of SARS-CoV.

[12] The test kit of any one of the preceding items, wherein the capture probes immobilized within the first detection zone do not hybridize to genomic RNA of Coronaviridae other than SARS-CoV-2.

[13] The test kit of any one of the preceding items, wherein a first capture probe comprises the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, and a spacer linking said first nucleic acid sequence and said second nucleic acid sequence.

[14] The test kit of item 13, wherein the spacer consists of a nucleotide sequence having a length from 1 to 100 nt, preferably from 5 to 50 nt.

[15] The test kit of any one of items 1 to 12, wherein a first capture probe comprises the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS- CoV-2, and a second capture probe comprises the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2.

[16] The test kit of any one of the preceding items, wherein the first region and the second region within the genomic RNA of SARS-CoV-2, and optionally the third, fourth, fifth and sixth regions within the genomic RNA of SARS-CoV-2, are independently selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[17] The test kit of any one of the preceding items, wherein the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:1 ; and/or the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:2; and/or the third nucleic acid sequence capable of hybridizing to a third region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:3; and/or the fourth nucleic acid sequence capable of hybridizing to a fourth region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:4; and/or the fifth nucleic acid sequence capable of hybridizing to a fifth region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:5; and/or the sixth nucleic acid sequence capable of hybridizing to a sixth region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:6.

[18] The test kit of any one of the preceding items, wherein the length of the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV- 2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[19] The test kit of any one of the preceding items, wherein the length of the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS- CoV-2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[20] The test kit of any one of items 9-19, wherein the length of the third nucleic acid sequence capable of hybridizing to a third region within the genomic RNA of SARS-CoV-2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[21] The test kit of any one of items 9-20, wherein the length of the fourth nucleic acid sequence capable of hybridizing to a fourth region within the genomic RNA of SARS-CoV-2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[22] The test kit of any one of items 9-21 , wherein the length of the fifth nucleic acid sequence capable of hybridizing to a fifth region within the genomic RNA of SARS-CoV-2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[23] The test kit of any one of items 9-22, wherein the length of the sixth nucleic acid sequence capable of hybridizing to a sixth region within the genomic RNA of SARS-CoV-2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[24] The test kit of any one of the preceding items, wherein the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2 and the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, and optionally the third, fourth, fifth and sixth nucleic acid sequences capable of hybridizing to the respective regions within the genomic RNA of SARS-CoV-2, are each independently selected from the group consisting of SEQ ID NO:7-18. [25] The test kit of any one of items 9 to 23, wherein the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:7 or 13; the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:8 or 14; the third nucleic acid sequence capable of hybridizing to a third region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:9 or 15; the fourth nucleic acid sequence capable of hybridizing to a fourth region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:10 or 16; the fifth nucleic acid sequence capable of hybridizing to a fifth region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO: 11 or 17; and the sixth nucleic acid sequence capable of hybridizing to a sixth region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO: 12 or 18.

[26] The test kit of any one of the preceding items, wherein the solid support further comprises a second detection zone, having immobilized thereto at least one capture probe comprising a nucleic acid sequence capable of hybridizing to multiple genomes of different Coronaviridae.

[27] The test kit of item 26, wherein the nucleic acid sequence capable of hybridizing to multiple genomes of different Coronaviridae is selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.

[28] The test kit of item 26 or 27, wherein the at least one capture probe immobilized within the second detection area comprises at least two of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, optionally separated by a spacer.

[29] The test kit of item 28, wherein the at least one capture probe immobilized within the second detection area comprises SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, optionally separated by two spacers.

[30] The test kit of item 26, wherein the capture probe immobilized within the second detection area comprises the nucleotide sequence as shown in SEQ ID NO:22

[31] The test kit of any one of the preceding items, further comprising a labelled detection probe which comprises a nucleic acid sequence capable of hybridizing to a target region within the genomic RNA of SARS-CoV-2. [32] The test kit of item 31, wherein the target region within the genomic RNA of SARS- CoV-2 is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and/or wherein the nucleic acid sequence capable of hybridizing to a target region within the genomic RNA of SARS-CoV-2 is selected from the group consisting of SEQ ID NO:7-18.

[33] The test kit of item 31 or 32, wherein the length of the nucleic acid sequence capable of hybridizing to a target region within the genomic RNA of SARS-CoV-2 is from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

[34] The test kit of any one of items 31 to 33, wherein the nucleic acid sequence capable of hybridizing to a target region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:7-18.

[35] The test kit of any one of the preceding items, wherein the solid support is made of a porous material.

[36] The test kit of item 35, wherein the solid support is a porous membrane. [37] The test kit of any one of any one of items 1 to 34, wherein the solid support is made of a non-porous material.

[38] The test kit of item 37, wherein the solid support is part of microfluidic system.

[39] The test kit of any one of the preceding items, wherein the solid support is encased in a housing. [40] A method of detecting the presence of SARS-CoV-2 nucleic acid in a sample, comprising the following steps

(i) providing the sample and the test kit of any one of the preceding items;

(ii) contacting the sample with the labelled detection probe(s) so as to allow hybridisation of SARS-CoV-2 nucleic acid with the labelled detection probe(s), thereby obtaining a sample comprising the labelled detection probe(s);

(iii) contacting the sample comprising the labelled detection probe(s) with the first detection zone, so as to allow hybridisation of SARS-CoV-2 nucleic acid with the capture probe(s) immobilized in the first detection zone; (iv) contacting the sample comprising the labelled detection probe(s) with the second detection zone, so as to allow hybridisation of Coronavirus nucleic acid with the capture probe(s) immobilized in the second detection zone;

(v) detecting the label of the labelled detection probe(s) within the first detection zone; and (vi) detecting the label of the labelled detection probe(s) within the second detection zone.

[41] The method of item 40, wherein the SARS-CoV-2 nucleic acid is SARS-CoV-2 genomic RNA.

[42] The method of item 40 or 41, wherein the SARS-CoV-2 nucleic acid in the sample is non-amplified nucleic acid; and/or wherein the method does not comprise a nucleic acid amplification step.

[43] The use of a test kit according to any one of items 1 to 39 for detecting coronavirus nucleic acid.

[44] The use of a test kit according to any one of items 1 to 39 for detecting SARS-CoV-2 nucleic acid, preferably SARS-CoV-2 RNA. [45] The use of a test kit according to any one of items 1 to 39 for detecting or diagnosing an infection with a coronavirus.

[46] The use of a test kit according to any one of items 1 to 39 for detecting or diagnosing an infection with SARS-CoV-2.

[47] The use of a test kit according to any one of items 1 to 39 for differentiating infection with SARS-CoV-2 from infection with a different coronavirus.

DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the workflow of the method of the invention on a fluidic platform. Step 1 shows a swab with a sample in a tube with lysis solution added thereto. Step 2 shows the transfer of the lysed solution onto the application zone of the platform. Step 3 shows the application of enzyme solution. Step 4 shows a washing step to remove excess enzyme solution. Step 5 shows the application of dye solution followed by visual assessment of the result.

Figure 2 depicts the workflow of the method of the invention in a dip stick format. Step 1 shows a swab with a sample in a tube with lysis solution added thereto. Step 2 shows the transfer of the lysed solution onto the application zone of the dip stick. Step 3 shows the application of the detection solution followed by visual assessment of the result.

Figure 3 illustrates details of the embodiment depicted in Figure 1. Step 1 shows the hybridization of the labelling probe to the virus RNA. Step 2 shows the hybridization of the labelled virus RNA on the platform. Step 3 shows the binding of horseradish-peroxidase- streptavidin conjugate to the hybridized construct. Step 4 shows the generation of a visible precipitate by horseradish peroxidase after adding of TMB (tetramethylbenzidine). B=biotin; SA=streptavidin; HRP= horseradish peroxidase.

Figure 4 illustrates details of the embodiment depicted in Figure 2. Step 1 shows the hybridization of the labelling probe to the virus RNA. Step 2 shows the hybridization of the labelled virus RNA on the dip stick. Step 3 shows the binding of a streptavidin labelled Gold- nanoparticle to the hybridized construct generating a visible signal. B=biotin; SA=streptavidin; Au=gold nanoparticle.

Figure 5 schematically shows the six SARS-CoV-2-specific stretches and the non-specific conserved region in the SARS-CoV-2 genome.

Figure 6 shows the results obtained from an analysis according to Example 5. The lysed sample, which had ben lysed in accordance with Example 1, was applied to the application zone at the top. Three bands were observed. The band at the top represents a positive signal from the first detection zone, indicating the presence of SARS-CoV-2 in the sample. The band in the middle represents a positive signal from the second detection zone, indicating the presence of Coronaviridae in the sample. The band at the bottom represents a control that the assay is functional.

DETAILED DESCRIPTION

All terms used herein are intended to have the meanings commonly understood by the skilled person to which this invention pertains, unless otherwise defined. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

As used herein, the term “hybridizing” or “specifically hybridizing” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, 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, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Stringent hybridization conditions that can be used within the scope of the invention can include, e.g., hybridization in a buffer comprising 50% formamide, 5xSSC, and 1% SDS at 42° C, or hybridization in a buffer comprising 5xSSC and 1% SDS at 65° C, both with a wash of 0.2xSSC and 0.1% SDS at 65° C. Exemplary stringent hybridization conditions can also include a hybridization in a buffer of 40% formamide, 1 M NaCI, and 1% SDS at 37° C, and a wash in 1xSSC at 45° C. Yet additional stringent hybridization conditions include hybridization at 60° C or higher and 3xSSC (450 mM sodium chloride/45 mM sodium citrate) or incubation at 42° C in a solution containing 30% formamide, 1M NaCI, 0.5% sodium sarcosine, 50 mM MES, pH 6.5.

Another method to guarantee stringent hybridization conditions at room temperature is the use denaturating substances (e.g. guanidinium thiocyanate, guanidinium chloride, urea) in the hybridization buffer. For example, a buffer comprising 4 M guanidinium thiocyanate allows stringent room-temperature-hybridization (i.e. at 25° C) of nucleic acids with a melting point of 60 °C. Those of ordinary skill in the art will readily recognize that alternative but comparable hybridization and wash conditions can be utilized to provide conditions of similar stringency.

The term “SARS-CoV-2” refers to a coronavirus which is the causative agent of COVID-19. The genomic RNA sequence of a SARS-CoV-2 virus is shown in SEQ ID NO:23. This sequence is accessible under GenBank accession number NC_045512.2. The skilled person is aware that the genome of SARS-CoV-2 may undergo mutations.

The present invention relates to a test kit for detecting the presence of SARS-CoV-2 nucleic acid in a sample, wherein the test kit comprises a solid support comprising a first detection zone having immobilized thereto (i) a capture probe comprising a first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, and (ii) a capture probe comprising a second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, said second region being different from said first region.

In another aspect the present invention relates to a test kit for detecting the presence of SARS- CoV-2 nucleic acid in a sample, wherein the test kit comprises a solid support comprising a first detection zone, wherein a first capture probe is immobilized within the first detection zone, wherein said first capture probe comprises (i) a first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, and (ii) a second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS- CoV-2, said second region being different from said first region.

In yet another aspect the present invention relates to a test kit for detecting the presence of SARS-CoV-2 nucleic acid in a sample, wherein the test kit comprises a solid support comprising a first detection zone having immobilized thereto (i) a first capture probe comprising a first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, and (ii) a second capture probe comprising a second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, said second region being different from said first region.

Sample

The sample for testing in the present invention may be any sample expected to potentially include a coronavirus nucleic acid. The sample is typically obtained or derived from an individual suspected of being infected with a coronavirus, e.g. with SARS-CoV-2. The sample obtained from the individual includes, but is not limited to, saliva, nasopharyngeal secretions, sputum, mucus, stool, tissue, and body fluids. Preferably, the sample is obtainable using an oral swab, a nasal swab or a throat swab. Typically, the sample is processed before being applied to the test kit of the invention. The processing may include contacting the sample with a lysis buffer to break up or lyse the virus membrane and make the coronavirus RNA accessible. Suitable lysis buffers are known to the skilled person. Preferably, the lysis buffer includes a denaturing agent such as guanidinium thiocyanate or lauroylsarcosine. The sample is typically a liquid sample, preferably an aqueous sample, e.g. a solution or a dispersion. Swabs are typically removed prior to applying the sample to the solid support.

Solid support

The test kit may be a lateral flow device comprising a porous membrane. In this embodiment the liquid sample is applied to the sample application zone of the membrane, and the liquid sample is transported via capillary forces to the detection zone. The membrane may be any microporous membrane material which is lateral flow compatible, typically microporous cellulose or cellulose-derived materials such as nitrocellulose.

In another embodiment the test kit comprises or consists of a fluidic device, preferably a microfluidic device. In such a device the liquid sample is typically transported through microchannels. Suitable microfluidic device are described, e.g., in AT 505854 A1 ; Lui et al. , Sensors 2009, 9, 3713-3744 (doi:10.3390/s90503713); Choi et al. Microfluid Nanofluid (2011) 10:231-247; or in Jauset-Rubio, M. et al. Sci. Rep. 6, 37732 (doi: 10.1038/srep37732 (2016)). The solid support in accordance with this other embodiment is usually a non-porous material such as polycarbonate, polystyrene, polypropylene, poylstyrene-butadien copolymers, Poly(methyl methacrylate) (PMMA), methacryl-butadiene copolymers, or acrylonitrile butadiene styrene (ABS).

The test kit may be encased in a housing. Materials for use in the housing include, but are not limited to, transparent tape, plastic film, plastic, glass, metal and the like. Such housings preferably contain an opening or sample port for introducing sample, as well as at least one window permitting the visualization of the detection zone of the test kit.

Capture probe

The test kit comprises at least one capture probe immobilized within the first detection zone of the solid support. The at least one capture probe typically is an oligonucleotide, preferably a single-stranded DNA oligonucleotide. The oligonucleotide may comprise modified bases and/or modified internucleotide linkages and/or modified sugar moieties. In a specific embodiment the oligonucleotide does not comprise modifications but consists of DNA.

The length of the at least one capture probe is usually from 25 nt to 1 ,000 nt, or from 30 nt to 800 nt, or from 40 nt to 600 nt, or from 50 nt to 500 nt, or from 60 nt to 300 nt, or from 70 nt to 250 nt, or from 75 nt to 200 nt, or from 80 nt to 150 nt, or from 85 nt to 125 nt, or from 90 nt to 110 nt. Preferably, the length of the at least one capture probe is from 80 nt to 120 nt.

At least two different nucleotide sequences which are capable of hybridizing to different target regions within the genomic RNA of SARS-CoV-2, respectively, are immobilized to the first detection zone. The first nucleotide sequence is capable of hybridizing to a first target region, and the second nucleotide sequence is capable of hybridizing to second, different target region.

Preferably, at least three different nucleotide sequences, which are capable of hybridizing to at least three different target regions within the genomic RNA of SARS-CoV-2, respectively, are immobilized to the first detection zone. For example, three different nucleotide sequences, which are capable of hybridizing to three different target regions within the genomic RNA of SARS-CoV-2, respectively, may be immobilized to the first detection zone.

More preferably, at least four different nucleotide sequences, which are capable of hybridizing to at least four different target regions within the genomic RNA of SARS-CoV-2, respectively, are immobilized to the first detection zone. For example, four different nucleotide sequences, which are capable of hybridizing to four different target regions within the genomic RNA of SARS-CoV-2, respectively, may immobilized to the first detection zone.

More preferably, at least five different nucleotide sequences, which are capable of hybridizing to at least five different target regions within the genomic RNA of SARS-CoV-2, respectively, are immobilized to the first detection zone. For example, five different nucleotide sequences, which are capable of hybridizing to five different target regions within the genomic RNA of SARS-CoV-2, respectively, may be immobilized to the first detection zone.

Most preferably, at least six different nucleotide sequences, which are capable of hybridizing to at least six different target regions within the genomic RNA of SARS-CoV-2, respectively, are immobilized to the first detection zone. For example, six different nucleotide sequences, which are capable of hybridizing to six different target regions within the genomic RNA of SARS-CoV-2, respectively, may be immobilized to the first detection zone.

The different target regions within the genomic RNA of SARS-CoV-2 are regions within the SARS-CoV-2 genome that are separated from each other by at least 1,000 nucleotides (nt). Preferably the different target regions are separated by at least 2,000 nt, or by at least 3,000 nt, or by at least 4,000 nt, or by at least 5,000 nt. The number of nucleotides that are not part of a target region and are located between two given target regions in the genomic sequence of SARS-CoV-2 is at least 1 ,000 nt, or at least 2,000 nt, or at least 3,000 nt, or at least 4,000 nt, or at least 5,000 nt.

The first target region within the genomic RNA of SARS-CoV-2 is specific for SARS-CoV-2, and the second target region within the genomic RNA of SARS-CoV-2 is specific for SARS- CoV-2. Accordingly, the at least one capture probe does not hybridize with genomic RNA of Coronaviridae other than SARS-CoV-2, e.g. with RNA from SARS-CoV, HCoV-HKlM , HCoV- OC43, HCoV-NL63 and HCoV-229E. This allows specific detection of SARS-CoV-2 infections. Preferably, each target region is specific for SARS-CoV-2. In particular, the first, second, third, fourth, fifth and sixth target regions are specific for SARS-CoV-2. Preferably, none of the capture probes immobilized within the first detection zone is capable of hybridizing with genomic RNA of Coronaviridae other than SARS-CoV-2, e.g. with RNA from SARS-CoV, HC0V-HKUI, HCOV-OC43, HCoV-NL63 or HCoV-229E.

The first target region and the second target region within the genomic RNA of SARS-CoV-2 are preferably selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The first target region and the second target region within the genomic RNA of SARS-CoV-2 preferably consist of an RNA sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

More preferable, the first, second and third target regions within the genomic RNA of SARS- CoV-2 are selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The first, second and third target regions within the genomic RNA of SARS-CoV-2 preferably consist of an RNA sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

More preferable, the first, second, third and fourth target regions within the genomic RNA of SARS-CoV-2 are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The first, second, third and fourth target regions within the genomic RNA of SARS-CoV-2 preferably consist of an RNA sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

More preferable, the first, second, third, fourth and fifth target regions within the genomic RNA of SARS-CoV-2 are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The first, second, third, fourth and fifth target regions within the genomic RNA of SARS-CoV-2 preferably consist of an RNA sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

Most preferably, the first, second, third, fourth, fifth and sixth target regions within the genomic RNA of SARS-CoV-2 are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The first, second, third, fourth, fifth and sixth target regions within the genomic RNA of SARS-CoV-2 preferably consist of an RNA sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In a specific embodiment, the first target region consists of the RNA sequence as shown in SEQ ID NO:1 , the second target region consists of the RNA sequence as shown in SEQ ID NO:2, the third target region consists of the RNA sequence as shown in SEQ ID NO:3, the fourth target region consists of the RNA sequence as shown in SEQ ID NO:4, the fifth target region consists of the RNA sequence as shown in SEQ ID NO:5, and the sixth target region consists of the RNA sequence as shown in SEQ ID NO:6.

Based on the identified specific target regions, the skilled person is able to design and prepare suitable specific probes. A capture probe may comprise at least two different nucleic acids capable of hybridizing to different target regions within the genome of SARS-CoV-2. In one embodiment, a first capture probe comprises two different nucleic acids capable of hybridizing to two different target regions within the genome of SARS-CoV-2, respectively. In another embodiment, a capture probe may comprise three different nucleic acids capable of hybridizing to three different target regions within the genome of SARS-CoV-2, respectively. In yet another embodiment, a capture probe may comprise four different nucleic acids capable of hybridizing to four different target regions within the genome of SARS-CoV-2, respectively.

The capture probe preferably comprises a spacer linking different nucleic acid sequences capable of hybridizing to different target regions. Typically, the spacer consists of a nucleotide sequence having a length from 1 nt to 100 nt, preferably from 5 nt to 50 nt.

In one embodiment, three capture probes are immobilized to the first detection zone, and each capture probe comprises two different nucleic acids targeting different target sequences within the genome of SARS-CoV-2, respectively. That is, the first capture probe comprises a first nucleic acid capable of hybridizing to a first region within the genome of SARS-CoV-2, and a second nucleic acid capable of hybridizing to a second region within the genome of SARS- CoV-2; the second capture probe comprises a third nucleic acid capable of hybridizing to a third region within the genome of SARS-CoV-2, and a fourth nucleic acid capable of hybridizing to a fourth region within the genome of SARS-CoV-2; and the third capture probe comprises a fifth nucleic acid capable of hybridizing to a fifth region within the genome of SARS-CoV-2, and a sixth nucleic acid capable of hybridizing to a sixth region within the genome of SARS-CoV- 2.

In other embodiments at least one capture probe comprises only one single nucleic acid capable of hybridizing to a given target region within the genome of SARS-CoV-2. For example, a first capture probe comprising a first nucleic acid capable of hybridizing to a target region within the genome of SARS-CoV-2 as the sole nucleic acid capable of hybridizing to a target region within the genome of SARS-CoV-2 may be immobilized to the first detection zone.

According to another example, a first capture probe comprising a first nucleic acid capable of hybridizing to a target region within the genome of SARS-CoV-2 as the sole nucleic acid capable of hybridizing to a target region within the genome of SARS-CoV-2, and a second capture probe comprising a second nucleic acid capable of hybridizing to a target region within the genome of SARS-CoV-2 as the sole nucleic acid capable of hybridizing to a target region within the genome of SARS-CoV-2, may be immobilized to the first detection zone. In another embodiment six different capture probes are immobilized onto first detection zone of the solid support, and each of the capture probes comprises only one single nucleic acid capable of hybridizing to a given target region within the genome of SARS-CoV-2.

The length of the first nucleic acid sequence within the capture probe capable of specifically hybridizing to a first region within the genomic RNA of SARS-CoV-2 is preferably from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt. Similarly, the length of the second nucleic acid sequence within the capture probe capable of specifically hybridizing to a second region within the genomic RNA of SARS-CoV-2 is preferably from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt. The same preferences apply to the length of the third, fourth, fifth and/or sixth nucleic acid sequence capable of hybridizing to a given target region within the genome of SARS-CoV-2.

In one embodiment, the first nucleic acid sequence capable of specifically hybridizing to a first region within the genomic RNA of SARS-CoV-2 is a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:1. The second nucleic acid sequence capable of specifically hybridizing to a second region within the genomic RNA of SARS-CoV-2 may be a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:2. The third nucleic acid sequence capable of specifically hybridizing to a third region within the genomic RNA of SARS-CoV-2 may be a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:3. The fourth nucleic acid sequence capable of specifically hybridizing to a fourth region within the genomic RNA of SARS-CoV-2 may be a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:4. The fifth nucleic acid sequence capable of specifically hybridizing to a fifth region within the genomic RNA of SARS- CoV-2 may be a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:5. The sixth nucleic acid sequence capable of specifically hybridizing to a sixth region within the genomic RNA of SARS-CoV-2 may be a DNA sequence which is the reverse complement of at least 10, or at least 12, or at least 15, or at least 20, or at least 25 contiguous nucleotides within the nucleotide sequence as shown in SEQ ID NO:6.

In a preferred embodiment, the first nucleic acid sequence within the capture probe capable of specifically hybridizing to a first region within the genomic RNA of SARS-CoV-2 is selected from the group consisting of SEQ ID NO:7-18. The first nucleic acid sequence within the capture probe capable of specifically hybridizing to a first region within the genomic RNA of SARS-CoV-2 may consist of a nucleotide sequence selected from the group consisting of SEQ ID NO:7-18.

In another preferred embodiment, the second nucleic acid sequence within the capture probe capable of specifically hybridizing to a second region within the genomic RNA of SARS-CoV- 2 is selected from the group consisting of SEQ ID NO:7-18. The second nucleic acid sequence within the capture probe capable of specifically hybridizing to a second region within the genomic RNA of SARS-CoV-2 may consist of nucleotide sequence selected from the group consisting of SEQ ID NO:7-18. In another embodiment, the first nucleic acid sequence capable of specifically hybridizing to a first region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:7 or 13; the second nucleic acid sequence capable of specifically hybridizing to a second region within the genomic RNA of SARS-CoV- 2 consists of a nucleotide sequence as shown in SEQ ID NO:8 or 14; the third nucleic acid sequence capable of specifically hybridizing to a third region within the genomic RNA of SARS- CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:9 or 15; the fourth nucleic acid sequence capable of specifically hybridizing to a fourth region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO: 10 or 16; the fifth nucleic acid sequence capable of specifically hybridizing to a fifth region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO: 11 or 17; and the sixth nucleic acid sequence capable of specifically hybridizing to a sixth region within the genomic RNA of SARS-CoV-2 consists of a nucleotide sequence as shown in SEQ ID NO:12 or 18.

In one embodiment, at least one capture probe immobilized in the first detection zone comprises or consists of a nucleotide sequence as shown in SEQ ID NO:32, 33 or 34. In another embodiment, at least three capture probes are immobilized in the first detection zone, wherein the first capture probe comprises or consists of a nucleotide sequence as shown in SEQ ID NO:32, the second capture probe comprises or consists of a nucleotide sequence as shown in SEQ ID NO:33, and the third capture probe comprises or consists of a nucleotide sequence as shown in SEQ ID NO:34.

In another preferred embodiment the test kit of the invention allows the differential diagnosis as to whether the individual is infected with a coronavirus other than SARS-CoV-2. In this embodiment the solid support of the test kit further comprises a second detection zone, wherein at least one capture probe is immobilized within the second detection zone, wherein said at least one capture probe immobilized within the second detection zone comprises a nucleic acid sequence capable of hybridizing to multiple genomes of different Coronaviridae. That is, the at least one capture probe immobilized within the second detection zone is a universal probe recognizing genomes from multiple Coronaviridae. Preferably, the at least one capture probe immobilized within the second detection zone is capable to hybridizing with genomic RNA from each of SARS-CoV-2, SARS-CoV, HCoV-HKlM , HCoV-OC43, HCoV- NL63 and HCoV-229E.

In one embodiment, only one capture probe is immobilized within the second detection zone. In another embodiment, two capture probes are immobilized within the second detection zone. In another embodiment, three capture probes are immobilized within the second detection zone. In yet another embodiment, at least one capture probe immobilized within the second detection zone comprises two different nucleic acid sequences that are capable of hybridizing to multiple genomes of different Coronaviridae, optionally separated by a spacer. In yet another embodiment, at least one capture probe immobilized within the second detection zone comprises three different nucleic acid sequences that are capable of hybridizing to multiple genomes of different Coronaviridae, optionally separated by spacers.

In a preferred embodiment the capture probe immobilized within the second detection zone comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:20 and SEQ ID NO:21. In yet another preferred embodiment the capture probe immobilized within the second detection zone comprises at least two nucleotide sequences selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21 , optionally separated by a spacer. In yet another preferred embodiment the capture probe immobilized within the second detection zone comprises the nucleotide sequences SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21 , optionally separated by spacers. The spacer(s) may have the properties defined above with respect to the capture probe immobilized within the first detection zone.

In a specific embodiment the capture probe immobilized within the second detection zone comprises the nucleotide sequence as shown in SEQ ID NO:22.

Detection probe

Typically, the test kit comprises a detection probe which is labelled with a detectable label. The term “label” as used herein refers to any atom, atoms, molecule, or molecules, such as a fluorescent tag, attached to or associated with a nucleic acid and used to provide a detectable signal. Preferred labels include, but are not limited to, biotin, fluorescent tags, gold nanoparticles, colloidal gold, dyed latex beads, and digoxygenin. Methods of preparing labelled oligonucleotides are known to the skilled person.

According to a first embodiment the detection probe is not immobilized to the solid support, but is a separate component of the test kit of the invention. The detection probe may be added to the sample after adding the lysis buffer to the sample, but prior to applying the sample to a sample application zone of the solid support. If SARS-CoV-2 RNA is present in the sample, the detection probe will hybridize with the RNA to form a complex comprising SARS-CoV-2 RNA and labelled detection probe.

According to this embodiment, the method described hereinbelow may comprise the steps of (i) adding a lysis buffer to the sample obtained from an individual; (ii) contacting the detection probe with the sample; and/or (iii) applying the sample to a sample application zone of the solid support.

According to an alternative embodiment the detection probe may be adhered or immobilized to the solid support. Preferably, the detection probe is reversibly adhered or immobilized to the solid support at a site different from the detection zone. Upon application of the sample to the application zone the liquid sample gets contact with the detection probe so as to allow hybridization and release from the solid support.

The labelled detection probe preferably comprises a nucleic acid sequence capable of specifically hybridizing to a region within the genomic RNA of SARS-CoV-2. The region within the genomic RNA of SARS-CoV-2 may consist of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

The length of the nucleic acid sequence capable of specifically hybridizing to a third region within the genomic RNA of SARS-CoV-2 typically ranges from 10 nt to 50 nt, or from 15 to 40 nt, or from 20 to 30 nt.

In connection with the labelled detection probe, the preferred embodiments of the nucleic acid sequence capable of specifically hybridizing to a region within the genomic RNA of SARS- CoV-2 correspond to the preferred embodiments described above in connection with the capture probe immobilized in the first detection zone.

The nucleic acid sequence capable of specifically hybridizing to a region within the genomic RNA of SARS-CoV-2 preferably consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:7-18. In one embodiment the detection probe comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO:24-29. In another embodiment, the test kit comprises six labelled detection probes having the nucleotide sequences as shown in SEQ ID NO:24-29, respectively.

If the kit comprises a capture probe immobilized within the second detection zone as defined above (e.g. a universal probe detecting multiple coronavirus nucleic acids), the kit usually further comprises at least one further labelled detection probe. The further labelled detection probes are not specific for SARS-CoV-2 RNA, but can hybridize to RNA from various Coronaviridae.

In one embodiment the further labelled detection probe comprises the nucleotide sequence as shown in SEQ ID NO:30. In another embodiment, the further labelled detection probe comprises or consists of the nucleotide sequence as shown in SEQ ID NO:31.

Method

Another aspect of the invention is a method for detecting the presence of SARS-CoV-2 nucleic acid in a sample, comprising the following steps

(i) providing the sample and the test kit described herein;

(ii) contacting the sample with the labelled detection probe so as to allow hybridisation of SARS-CoV-2 nucleic acid with the labelled detection probe, thereby obtaining a sample comprising the labelled detection probe;

(iii) contacting the sample comprising the labelled detection probe with the first detection zone, so as to allow hybridisation of SARS-CoV-2 nucleic acid with the immobilized first capture probe;

(iv) contacting the sample comprising the labelled detection probe with the second detection zone, so as to allow hybridisation of Coronavirus nucleic acid with the immobilized second capture probe;

(v) detecting the label of the labelled detection probe that is immobilized within the first detection zone; and

(vi) detecting the label of the labelled detection probe that is immobilized within the second detection zone.

The method, kit or assay described herein preferably detects SARS-CoV-2 RNA in a sample without utilizing the polymerase chain reaction or other amplification steps designed to enhance sensitivity for the target nucleic acid. The methods and kits described herein are able to detect the target nucleic acids without amplifying the target nucleic acids. However, if the test analyte is already amplified, the amplified sample can still be used as a sample in the embodiments described herein. In some embodiments, the detected nucleic acids are also quantified. Some examples of how the nucleic acids may be quantified include, but are not limited to, quantification through visual gradations of the line intensity or through the use of an electronic optical reader.

Table 1. Summary of the sequence shown in the sequence listing

EXAMPLES

Sample Lysis and Labelling of RNA

Example 1 The oral swab was transferred into a tube with 100 pi of lysis solution (4 M

Guanidiniumthiocyanate, 500 mM Tris (pH = 7,4), 5% Lauroylsarcosin and detection probes (0,1 nM each)) and lysed by agitation for 1 min. After that, the swab was squeezed out and removed from the tube.

Example 2 The nasal swab was transferred into a tube with 100 mI lysis solution (4 M

Guanidiniumthiocyanate, 500 mM Tris (pH = 7,4), 5% Lauroylsarcosin and detection probes (0,1 nM each)) Lysis was supported by sonication for 1 min. Then the swab was squeezed out and removed from the solution

Example 3 The swab with stool sample was transferred into a tube with 100 mI of lysis solution (4 M Guanidiniumthiocyanate, 300 mM Tris (pH = 7,4), 8% Lauroylsarcosin and detection probes (0,05 nM each)). After agitation for 1 min the swab was removed. 50 mg of glass beads (1 mm diameter) were added to the tube. After vortexing for 1 min the sample is centrifuged at 100g for 1 min. Example 4

The oral swab was transferred into a tube with 500 mI of lysis solution (PBS) and lysed by agitation for 30 s. After that, the swab was squeezed out and removed from the tube.

A mix of six SARS-CoV-2-specific labelled detection probes were used in examples 1-3. The six detection probes have the nucleotide sequences as shown in SEQ ID NO:24-29, respectively. Optionally, a Coronaviridae-spec\f\c labelled detection probe having the nucleotide sequence as shown in SEQ ID NO:31 may further be included. Platform

In the following examples a platform having a solid support of polystyrene were used. The platform comprises a cover part comprising an inlet, a channel for sample flow, a waste reservoir and a vent. Three SARS-CoV-2-specific capture probes having the nucleotide sequences as shown in SEQ ID NO:32-34, respectively, were immobilized in a first detection zone of the solid support. Optionally, a Coronaviridae-spec\f c capture probe having the nucleotide sequence as shown in SEQ ID NO:22 may be immobilized in a second detection area of the platform.

Analysis procedure

Example 5

One drop of the lysed sample was transferred to a platform. After incubation for 15 min 1 drop Enzyme-solution (1 pg/ml horseradish-peroxidase-streptavidin-conjugate in 1% casein solution) was transferred to the platform. Aber incubation for another minute, 1 drop of Washing solution (0,1 X SSC) was added to the platform. Finally, after 1 min of incubation 1 drop of Colour reaction solution (1% TMB in 100mM Tris) was added. After 4 min the result was assessed visually. Positive signals appeared as blue lines.

Example 6

One drop of the lysed sample was transferred to the platform and incubated for 1 min. Then 1 drop of Detection solution (Gold-streptavidin-conjugate (40nm) at a concentration of OD=1 in 1% casein solution in PBS) was added to the platform and incubated for 10 min. Positive signals appeared as red lines.

Example 7

One drop of the lysed sample was transferred to the platform and incubated for 1 min. Then 1 drop of Detection solution (Gold-SarsCoV-2-Probe-conjugate (40nm) at a concentration of OD=1, Gold-universal-Probe-conjugate (120nm) at a concentration of OD=1, 1% casein solution in PBS) was added to the platform and incubated for 10 min. Positive signals for Sars CoV-2 appeared as red lines and positive signals for Coronaviridae appeared as blue lines.

Example 8

2 pi of a sample lysed according to Example 4 was transferred into 18 mI mastermix containing a commercial buffer and enzyme mix for RT-PCR and primers to amplify a region of SEQ ID NO:24-29 and SEQ ID NO:32-34. After mixing, the target region was amplified by the program: 3min at 50 °C, 5 s at 94 °C, 30 cycles of (2 s at 94 °C and 2 s at 53 °C). The PCR-product was mixed with a hybridization solution (4 M Guanidiniumthiocyanate, 500 mM Tris (pH = 7.4), 5% Lauroylsarcosin and detection probes out of SEQ ID NO:24-29 (0.1 nM each)). After denaturation at 95 °C for 1 min, 20 pi were transferred to the platform. After incubation for 1 min 1 drop Enzyme-solution (1pg/ml horseradish-peroxidase-streptavidin-conjugate in 1% casein solution) was transferred to the platform. Aber incubation for another minute, 1 drop of Washing solution (0,1 X SSC) was added to the platform. Finally, after 1 min of incubation 1 drop of Colour reaction solution (1% TMB in 100mM Tris) was added. After 1 min the result was assessed visually. Positive signals appeared as blue lines.

Claims

1. A test kit for detecting the presence of SARS-CoV-2 nucleic acid in a sample, wherein the test kit comprises a solid support comprising a first detection zone having immobilized thereto (i) a capture probe comprising a first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, and (ii) a capture probe comprising a second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, said second region being different from said first region.

2. The test kit of claim 1, wherein said first region and said second region are separated by at least 1,000 nucleotides within the genomic RNA of SARS-CoV-2.

3. The test kit of claim 1 or 2, wherein the length of the capture probe(s) is from 40 nt to 150 nt.

4. The test kit of any one of the preceding claims, wherein the first region within the genomic RNA of SARS-CoV-2 is specific for SARS-CoV-2, and the second region within the genomic RNA of SARS-CoV-2 is specific for SARS-CoV-2.

5. The test kit of any one of the preceding claims, wherein the first detection zone has further immobilized thereto (iii) a capture probe comprising a third nucleic acid sequence capable of hybridizing to a third region within the genomic RNA of SARS-CoV-2, (iv) a capture probe comprising a fourth nucleic acid sequence capable of hybridizing to a fourth region within the genomic RNA of SARS-CoV-2, (v) a capture probe comprising a fifth nucleic acid sequence capable of hybridizing to a fifth region within the genomic RNA of SARS-CoV-2, and (vi) a capture probe comprising a sixth nucleic acid sequence capable of hybridizing to a sixth region within the genomic RNA of SARS-CoV-2, said first, second, third, fourth, fifth and sixth regions being different from each other.

6. The test kit of claim 5, wherein each of said first, second, third, fourth, fifth and sixth region is specific for SARS-CoV-2.

7. The test kit of any one of the preceding claims, wherein a first capture probe comprises the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, the second nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2, and a spacer linking said first nucleic acid sequence and the second nucleic acid sequence.

8. The test kit of any one of claims 1 to 6, wherein a first capture probe comprises the first nucleic acid sequence, and a second capture probe comprises the second nucleic acid sequence.

9. The test kit of any one of the preceding claims, wherein the solid support is a porous membrane.

10. The test kit of any one of the preceding claims, wherein the first region and the second region within the genomic RNA of SARS-CoV-2, and optionally the third, fourth, fifth and sixth region within the genomic RNA of SARS-CoV-2, are independently selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

11. The test kit of any one of the preceding claims, wherein the first nucleic acid sequence capable of hybridizing to a first region within the genomic RNA of SARS-CoV-2 and the second nucleic acid sequence capable of hybridizing to a second region within the genomic RNA of SARS-CoV-2, and optionally the third, fourth, fifth and sixth nucleic acid sequence capable of hybridizing to the respective region within the genomic RNA of SARS-CoV-2, are each independently selected from the group consisting of SEQ ID NO:7-18.

12. The test kit of any one of the preceding claims, wherein the solid support further comprises a second detection zone, having immobilized thereto a capture probe comprising a nucleic acid sequence capable of hybridizing to multiple genomes of different Coronaviridae viruses.

13. The test kit of claim 12, wherein the nucleic acid sequence capable of hybridizing to multiple genomes of different Coronaviridae viruses is selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.

14. The test kit of any one of the preceding claims, further comprising a labelled detection probe which comprises a nucleic acid sequence capable of hybridizing to a target region within the genomic RNA of SARS-CoV-2.

15. The test kit of claim 14, wherein the target region within the genomic RNA of SARS-CoV-2 is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; and/or wherein the nucleic acid sequence capable of hybridizing to a target region within the genomic RNA of SARS-CoV-2 is selected from the group consisting of SEQ ID NO:7-18.

16. A method of detecting the presence of SARS-CoV-2 nucleic acid in a sample, comprising the following steps

(i) providing the sample and the test kit of claim 14 or 15;

(iii) contacting the sample comprising the labelled detection probe with the first detection zone, so as to allow hybridisation of SARS-CoV-2 nucleic acid with the capture probe(s) immobilized in the first detection zone;

(iv) contacting the sample comprising the labelled detection probe with the second detection zone, so as to allow hybridisation of Coronavirus nucleic acid with the capture probe(s) immobilized in the second detection zone;

(v) detecting the label of the labelled detection probe within the first detection zone; and

(vi) detecting the label of the labelled detection probe within the second detection zone.

17. The method of claim 16, wherein the SARS-CoV-2 nucleic acid is SARS-CoV-2 genomic RNA.

18. The method of claim 16 or 17, wherein the SARS-CoV-2 nucleic acid in the sample is non- amplified nucleic acid.

19. The method of any one of claims 16 to 18, wherein the method does not comprise a nucleic acid amplification step.