GB2596634A

GB2596634A - A SARS-CoV-2 molecular diagnostic test

Info

Publication number: GB2596634A
Application number: GB2106061.1A
Authority: GB
Inventors: Partington Matthew; Murton Heather
Original assignee: QuantumDx Group Ltd
Current assignee: QuantumDx Group Ltd
Priority date: 2020-04-28
Filing date: 2021-04-28
Publication date: 2022-01-05
Also published as: GB202006216D0; GB202106061D0

Abstract

A method of determining the presence of SARS-CoV-2 virus in a sample by reverse transcribing target RNA and amplifying the cDNA of three independent regions of the SARS-CoV-2 genome in a multiplex reaction. The three independent regions are the Orf1, the N and the S genes. The method can include the simultaneous amplification of a RNAseP control gene. Detection of the amplified products can be by probe hydrolysis, by hybridisation of a fluorescent tagged product and melt analysis, or by using probes that anneal to the amplicons. The method can comprise an RNA purification step. The method can have no RNA purification step. The patient samples can be nasopharyngeal wash or aspirates, nasopharyngeal swabs, oropharyngeal swabs, bronchoalveolar lavage, tracheal aspirate, pleural fluid tap, sputum, saliva or post-mortem tissue. Further aspects of the invention include kits and devices for performing the SARS-CoV-2 detection method. The device can be a cartridge or cassette. The device can be part of a point of care system with a means for visually imaging labelled amplicons.

Description

A SARS-CoV4 molecular diagnostic test The present invention relates to methods, kits and devices for assaying for a severe acute respiratory syndrome (SARS)-like coronavirus, designated SARS-CoV-2, in a patient. In particular, it relates to diagnostic tests that can be carried out rapidly and accurately and are appropriate for point-of-care testing with relatively low requirements for user technical knowledge.

A novel severe acute respiratory syndrome (SARS)-like coronavirus designated SARS-CoV-2 recently emerged as the causative agent of an infectious disease in humans, COVID-19 exhibiting rapid spread throughout the world.

The exact origin of SARS-00V-2 remains unclear; however, it is thought to have emerged from Wuhan province in China. It is currently hypothesised that the animal reservoir of the SARS-C0V-2 virus is bat due to molecular similarity with RaTG13 coronavirus that has been isolated from bats but, as yet, there is no confirmed link nor understanding about how the transition to humans may have occurred. There are also distinct similarities between SARS-00V-2 and SARS coronavirus (SARS-CoV), a virus identified in 2003. SARS-CoV is also thought to be an animal virus from an as-yet-uncertain animal reservoir, perhaps bats, that spread to other animals (civet cats) and first infected humans in the Guangdong province of southern China in 2002.

Coronaviruses are enveloped, non-segmented, positive-sense RNA viruses. Coronavirus virus particles contain four main structural proteins. These are the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins, all of which are encoded within the 3' end of the viral genome. In order to better understand and manage the spread of SARS-CoV-2 in humans it is essential that testing of individuals displaying symptoms occurs to allow monitoring and contact tracing. Molecular tests detecting viral RNA, indicating current viral infection, can being used to diagnose cases of COVID-19 and are an essential part of contact tracing and testing.

Primers for molecular tests for SARS-CoV-2 have been developed rapidly by the CDC in the US and WHO in Switzerland, however it appears that the majority of these are based around primers that were developed to test for the 2002 SAPS-Coy strain. As such, whilst they have been useful in the early stages of testing, they have limitations and are of limited suitability for multiplex assays. They also show iess specificity in distinguishing SAR-CoV-2 from other coronavirds strains.

It is understood that improved and 2 rternative methods, kits and devices for assaying SARSC0V-2 would be useful.

Throughout this document references to the SARS-CoV-2 viral genome refer to the reference sequences with NCBI references NC_045512, MN908947.3 and MT007544.1 According to the present invention there is provided a rapid method of determining the presence of SARS-00V-2 virus in a patent sample comprising; providing a sample of RNA from said patient sample; reverse transcribing target RNA in the sample in the presence of an appropriate reverse transcriptase to obtain cDNA; and amplifying substantially simultaneously at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, wherein the substantially simultaneous amplification occurs by annealing pairs of target oligonucleotide primers to the cDNA; simultaneously extending the annealed primers to synthesise an extension product, wherein each extension product after separation from the nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair of target oligonucleotide primers; and detecting the amplified products (amplicons), if present, to indicate the presence or absence of the target regions, wherein detection of said three independent, non-overlapping target regions is indicative of SARS-00V-2; characterised in that the pairs of target oligonucleotide primers are selected from the following list; Primer name Gene Sequence GC Tm Sequence ID covd-19 2431 Orf1 CGGCCGCTATAACAATACTAG 47.61904 59 SEQ ID 9 covd-19 2997 Orf1 GATGTTAGTTAGCCACTGCGA 47.61904 62 SEQ ID 10 covd-19 9517 Orf1 AGACGTTCTTGAGTGTAATGTGA 39.13043 61 SEQ ID 1 covd-19 9920 Orf1 AACCTAATACTCTAGATAATTCATTAGGT 27.58620 60 SEQ ID 2 covd-19 36227 S GCCAATAGGTATTAACATCACTAGG 40 61 SEQ ID 3 covd-19 36334 S CACATAATAAGCTGCAGCACCAG 47.82608 64 SEQ ID 4 covd-19 37795 5 AGGTGTTCTTACTGAGTCTAACAA 37.5 61 SEQ ID 11 covd-19 37907 S TCTCAAGTGTCTGTGGATCAC 47.61904 61 SEQ ID 12 covd-19 38692 S CAGACTAATTCTCCTCGGCG 55 61 SEQ ID 13 covd-19 38901 S TTCTGCACCAAGTGACATAGTGTAG 44 64 SEQ ID 14 covd-19 47059 N GCTTCTAAGAAGCCTCGGCA 55 64 SEQ ID 15 covd-19 47087 N CCTTGTCTGATTAGTTCCTGGT 45.45454 61 SEQ ID 16 covd-19 47295 N AACTGTGACTCTTCTTCCTGCT 45.45454 63 SEQ ID 5 covd-19 47392 N CTTGTGTGGTCTGCATGAGTT 47.61904 62 SEQ ID 6 with each pair of oligonucleotide primers comprising a forward primer and a reverse primer that are operable to bind genomic regions flanking a target region, and each pair selected such that the target regions that they flank do not overlap.

Tm = melting temperature.

Multiplexing allows for improved testing; however, such multiplexed reactions can be challenging due to non-specific binding between primers or primers interfering with each other. The primers identified allow a multiplexed reaction to be carried out. This in turn allows the assay to be carried out on a point of care (POC) system with the assay being carried out on a continuous flow device. The continuous flow device may be in the form of a cartridge or cassette.

The reverse transcription and amplification steps can be carried out together (e.g. sequentially, but in the same tube). The reverse transcription step utilises one of each of the primers in the selected primer pairs.

In one preferred version, the at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome amplified in the multiplexed polymerase chain reaction include a region in the N gene, a region in ORF1 and a region in the S gene.

Optionally the step of providing a sample of RNA from said patient sample may refer to the provision of a raw patient sample i.e. a patient sample which has not had RNA extraction carried out.

Optionally the method may include the step of extracting RNA from a patient sample prior to the step of providing a sample of RNA from said patient sample.

Preferably the pairs of target oligonucleotide primers are;

Primer Name Sequence Description Sequence ID

covd-19 9517 F AGACGTTCTTGAGTGTAATGTGA Orfl__Span20_FPrimer SEQ ID 1 covd-19 9920 R AACCTAATACTCTAGATAATTCATTAGGT Orf1_Span2O_RPrimer SEQ ID 2 covd-19 36227 F GCCAATAGGTATTAACATCACTAGG sGene_Span3_Fprimer SEQ ID 3 covd-19 36334 R CACATAATAAGCTGCAGCACCAG sGene_Span3_Rprimer SEQ ID 4 covd-19 47295 F AACTGTGACTCTTCTTCCTGCT nGene_Spard_Fprimer SEQ ID 5 covd-19 47392 R CTTGTGTGGTCTGCATGAGTT nGene_Spard_Rprimer SEQ ID 6 In the table above the F and R notations in the primer name indicate forward or reverse primers.

Sequence ID 1 and Sequence ID 2 flank a target region in ORF1 of the SARS-00V-2 viral genome.

Sequence ID 3 and Sequence ID 4 flank a target region in the S Gene of the SARS-00V-2 viral genome.

Sequence ID 5 and Sequence ID 6 flank a target region in the N Gene of the SARS-CoV-2 viral 5 genome.

Advantageously, the above combination of primer pairs (Sequence ID 1 to 6) has the unexpected effect of reliably detecting the presence of variant strains of the SARS-00V-2 virus.

Preferably, the method includes testing the sample for the presence of a control gene, in this case human ribonuclease P gene (RNAseP) -GenBank accession number NM 006 Advantageously this acts as a control for PCR inhibitors, and to monitor nucleic acid extraction efficiency if utilised.

Preferably, whilst substantially simultaneously amplifying said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, a portion of the control gene (RNAseP) is also amplified, if present by including a pair of control oligonucleotide primers.

Preferably the control oligonucleotide primers are selected from the group; Primer name Gene Sequence GC Tm Sequence ID R N As e P4_F RNAseP GTTGGTCACATGGTGGTAAC SO 60 SEQ ID 7 R N As e P6_R RNAseP TGAAACATCTACAACACAGACATT 33.33333333 60 SEQ ID 8 CDC RNAsePF RNAseP AGATTTGGACCTGCGAGCG 57.89473684 64 SEQ ID 17 CDC RNAsePR RNAseP GAGCGGCTGTCTCCACAAGT 60 66 SEQ ID 18 Preferably the control oligonucleotide primers are;

Primer Name Sequence Description Sequence ID

RNA5eP4 F GTTGGTCACATGGTGGTAAC RNA5eP_Span2_Fprimer SEQ ID 7 RNA5eP6_R TGAAACATCTACAACACAGACATT RNA5eP_Span2_Rprimer SEQ ID 8 In the table above the F and R notations in the primer name indicate forward or reverse primers.

Alternatively, the primers have at least 80% sequence homology to the above primers.

Alternatively, the primers have at least 90% sequence homology to the above primers.

Alternatively, the primers have at least 95% sequence homology to the above primers.

Alternatively, the primers have at least 99% sequence homology to the above primers.

Preferably the primers can bind under stringent hybridisation conditions.

Preferably the PCR reaction takes less than 10 minutes, more preferably less than 9 minutes, more preferably less than 8 minutes.

Preferably, detection of the amplified products is by probe hydrolysis.

This methodology is well known in the art. Typically, probe hydrolysis utilises probes which incorporate a (e.g. 5') fluorescent reporter dye and a (e.g. 3') nonfluorescent quencher (NFQ).

Preferably, the probes are double-quenched probes such as BHQnovaTM probes which include an internal "Nova" quencher.

Alternatively, detection of the amplified products is by hybridisation of a fluorescent or otherwise tagged product and/and melt analysis.

Most preferably, the detection of the amplified products (amplicons) uses target probes to anneal to said amplicons.

Most preferably, the detection of the amplified products (amplicons) uses target probes to anneal to target amplicons and control probes to anneal to control amplicons.

Preferably the target probes for detection of the amplified products are selected from the following; Probe name Gene Sequence GC Tm Sequence ID covd-19 2515 Orf1 CAGTATTCACTGAGACTCATTGATGCTATG 40 66 SEQ ID 22 covd-19 2879 Orf1 TGAACAACACCACCTGTAATGTAGGC 46.15384 67 SEQ ID 23 covd-19 9620 Orfl TTACAGAAGAGGTTGGCCACACAGA 48 68 SEQ ID 19 covd-19 9787 Orf1 AAGACTAGAATTGTCTACATAAGCAGCCAT 36.66666 66 SEQ ID 24 covd- S TGTCCAACCTGAAGAAGAATCACCAGG 50 67 SEQ ID 20 19 36276 covd- S CAGCATCAGTAGTGTCAGCAATGTCTC 48.14814 67 SEQ ID 25 19 37846 covd- S CAATGATGGATTGACTAGCTACACTACG 44.82758 67 SEQ ID 26 19_38749 covd- N 42.85714 68 SEQ ID 27 19 47068

AGCATACAATGTAACACAAGLI I I CGGC

covd- N CAACAATCCATGAGCAGTGCTGA 50 68 SEQ ID 21 19 47356 Preferably the target probes are; Probe name Gene Sequence GC Tm Sequence ID covd-19 9620 Orf1 TTACAGAAGAGGTTGGCCACACAGA 48 68 SEQ ID 19 covd-19 36276 S TGTCCAACCTGAAGAAGAATCACCAGG 50 67 SEQ ID 20 covd-19 47356 CAACAATCCATGAGCAGTGCTGA 50 68 SEQ ID 21 It was identified by the inventors that by selecting probes that will anneal close to the 5) or 3' ends of a target region this increases the likelihood that they will be incorporated during amplification as they will included into even relatively short read lengths. Use of the selected probes will therefore increase the efficiency and speed of the assay.

Preferably the control probes are selected from the following; Sequence Probe name Gene Sequence GC Tm

ID

RNA5eP5 RNAseP CTCTTGCCAGTGCTGATGTCTCATG 52 67 SEQ ID 45 CDC RNAsePP RNAseP TTCTGACCTGAAGGCTCTGCGCG 60.86957 71 SEQ ID 46 Preferably the control probe is; Probe name Gene Sequence GC Tm Sequence ID RNAseP5 RNAseP CTCTTGCCAGTGCTGATGTCTCATG 52 67 SEQ ID 45 Preferably the probes can bind under stringent hybridisation conditions.

Preferably, the method is for the detection of a diagnostic target sARs-cov-2 RNA from patient samples and no step includes RNA purification.

Prefera.bly the patient samples are nasopharyngeal wash/aspirates, nasopharyngeal swabs, oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirate, pleural fluid tap, sputum, saliva and/or post-mortem tissue.

Preferably, said at least three independent, non-overlapping, target regions of the SARS-00V- 2 viral genome in a multiplexed polymerase chain reaction, are regions selected from the following target regions; Gene Sequence Sequence ID Orfl Spanl AAATTCCATAATCAAG ACTATTCAACCAAGGGTTGAAAAGAAAA AGCTTG ATGGCTTTATG GGTAGAATTCGATCTGTCTATCCAGTTG CGTCACCAAATG AATGCAACCAAATGTGCCTTTCAACTCTCATGA AGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTT GT SEQ ID 28 0 rf1_S p a n2 AAAGTCG GAG CATGTCACAATTCA GAAGTAGGACCTGAGCATAG TCTTGCCGAATACCATAATG AATCTGGCTTGAAAACCATTCTTCG TAAG G GTG G TCG CA CTATTG CCTTTG G AG G CTGTGTGTTCTCTTA TGTTGGTTG CCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAG CGCTAACA SEQ ID 29 0 rf1_S p a n4 TCCCGCGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTC ACTGAGACTCATTGATGCTATG ATGTTCACATCTGATTTGGCTAC TAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCA GTTGACTTCGCAGTGGCTAACTAACATCTTTG GCACTGTTTATGA AAAACTCAAACCCGT SEQ ID 30 Orf1_Spa n8 AAGAGTTTGAGCCATCAACTCAATATGAGTATGGTACTG AAGAT GATTACCAAGGTAAACCTTTG GAATTTGGTGCCACTTCTGCTGCT CTTCAACCTGAAGAAGAGCAAGAAG AAGATTGGTTAGATGATGA TAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATC AGA SEQ ID 31 0 rf1_S pa n15 TGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCT GAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAA GATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAG AATTT CTTAAG AG AG GTG ATAAAAGTGTATATTACACTA SEQ ID 32 Orf1_S pa n20 AAGACGTTCTTGAGTGTAATGTG AAAACTACCGAAGTTGTAG GA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAG AGTATTA G GT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 0 rf1a b_S p a n 0 GGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGC CTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTG ACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAAT ATG AC SEQ ID 34 0 rf1a b_S p a n AATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTC ACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATAT CCTGCTAATTCCATTGTTTGTAGATTTGACACTAGAGTGCTATCTA ACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTAT SEQ ID 35 0 rf1a b_S p a n 5 ATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAAT TAGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTC ATAGTCAGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAAC GTTTTAAGGAATCACCTITTGAATTAGAAG SEQ ID 36 Orf3a_Spa n1 AAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCA GCTGTACTCAACTCAATTGAGTACAGACACTGGTGTTGAACATGT TACCTTCTTCATCTACAATAAAATTGTTGATGAGCCTGAAGAACA TGTCCAAATTCACACAATCGACGGTTCATCCGGAGTTGTTAATCC AGTA SEQ ID 37 Orf8_Spa nO SEQ ID 38

TTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATGT AGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGAC CCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGG A GCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGC TGGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATAC AGTTTCCTGTTTACCTTTTACAATTAATTGCCAGGAACCTAAATTG GGTAGT

S_ge n e_S pa n 0 CTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAA CCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGG TGTTTATTACCCTGACAAACEITTCAGATCCTCAGTTTTACATTCA ACTCAG G ACTTG TTCTTACCTTTCTTTTC CAATG TTACTTG G TT CCA TGCTATACATGTCTCTGGGACCAATGGTACTAAGA SEQ ID 39 S_gene_Span GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA SEQ ID 40

GGTTGGACAGCTGEIGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGA

S_ge n e_S pa n GGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTT SEQ ID 41

CTGCC I I I CCAACAATTTGGCAGAGACATTGCTGACACTACTGAT GCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCA TGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACT TCTAA

S_ge n e_S pa n TGGTGCAGGTATATG CGCTAGTTATCAGACTCAGACTAATTCTCC TCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACAC TATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTC SEQ ID 42 N_ge n e_S p a nO CTGCTGAGGCTTCTAAGAAG CCTCG GCAAAAACGTACTGCCACT AAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGA ACAAACCCAAGGAAATTTTG GGGACCAGGAACTAATCAGACAAG GAACTGATTACAAACATTGGCCGCAA SEQ ID 43 N_ge n e_S p a n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTG GATGA TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG SEQ ID 44 Preferably, the at least three independent, non-overlapping, target regions of the SARS-CoV2 viral genome comprise the following target regions; Gene Sequence Sequence ID Orf1_Spa n20 AAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 S_gene_Span 3 GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA SEQ ID 40

GGTTGGACAGCTGEIGCTGCAGCTTATTATGTGGGTTATCTTCAA

CCTAGGACTTTTCTATTAAAATATAATGA GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC N_gene_Spa TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA n1 TAAACG SEQ ID 44 According to the present invention there is provided a rapid method of determining the presence of SARS-00V-2 virus in a patient sample comprising; providing a sample of RNA from said patient sample; reverse transcribing any RNA in the sample in the presence of an appropriate reverse transcriptase to obtain cDNA and substantially simultaneously amplifying said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, said regions selected from the following target regions; Gene Sequence Sequence ID Orfl_Spanl AAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAA AGCTTGATGGCTTTATGGGTAGAATTCGATCTGTCTATCCAGTTG CGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGA AGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTT GT SEQ ID 28 Orfl_Span2 AAAGTCGGAGCATGTCACAATTCAGAAGTAGGACCTGAGCATAG TCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCG TAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTA TGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAG CGCTAACA SEQ ID 29 Orfl_Span4 TCCCGCGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTC ACTGAGACTCATTGATGCTATGATGTTCACATCTGATTTGGCTAC TAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCA SEQ ID 30

GTTGACTTCGCAGTGGCTAACTAACATCTTTGGCACTGTTTATGA AAAACTCAAACCCGT

Orf1_Span8 AAGAGTTTGAGCCATCAACTCAATATGAGTATGGTACTGAAGAT GATTACCAAGGTAAACCTTTGGAATTTGGTGCCACTTCTGCTGCT CTTCAACCTGAAGAAGAGCAAGAAGAAGATTGGTTAGATGATGA TAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATC AGA 5E0 ID 31 0 rf1_S pa n15 TGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCT GAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAA GATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTT CTTAAG AG AG GTG ATAAAAGTGTATATTACACTA SEQ ID 32 0 rf1_S pa n20 AAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 0 rf 1a b_S pa n 0 GGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGC CTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTG ACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAAT ATG AC SEQ ID 34 0 rf 1a b_S pa n 2 AATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTC ACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATAT CCTG CTAATTCCATTG TTTGTAGATTTG ACACTAG AGTG CTATCTA ACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTAT SEQ ID 35 0 rf 1a b_S pa n 5 ATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAAT TAGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTC ATAG TCAG TTAGG TG GTTTAC ATCTACTG ATTG G ACTAG CTAAAC GTTTTAAGGAATCACCTTTTGAATTAGAAG SEQ ID 36 0 rf3a_Spa n1 SEQ ID 37

AAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCA GCTGTACTCAACTCAATTGAGTACAGACACTGGTGTTGAACATGT TACCTTCTTCATCTACAATAAAATTGTTGATGAGCCTGAAGAACA

TGTCCAAATTCACACAATCGACGGTTCATCCGGAGTTGTTAATCC AGTA

Orf8_Span0 TTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATGT AGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGAC CCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGGA GCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGC TGGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATAC AGTTTCCTGTTTACCTTTTACAATTAATTGCCAGGAACCTAAATTG GGTAGT SEQ ID 38 S_ge n e_S p a n 0 CTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAA CCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGG TGTTTATTACCCTGACAAAGITTICAGATCCTCAGTTTTACATTCA ACTCAG G ACTTG TTCTTACCTTTCTTTTCCAATG TTACTTGG TT CCA TGCTATACATGTCTCTGGGACCAATGGTACTAAGA SEQ ID 39 S_gene_Span 3 GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGA SEQ ID 40 S_ge n e_S p a n GGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTT CTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGAT GCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCA TGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACT TCTAA SEQ ID 41 S_ge n e_S pa n TGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCC TCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACAC TATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTC SEQ ID 42 N_gene_Spa nO CTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACT AAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGA ACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAG GAACTGATTACAAACATTGGCCGCAA SEQ ID 43 N_gene_Spa n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG SEQ ID 44 wherein the substantially simultaneous amplification occurs by annealing pairs of target oligonucleotide primers to the cDNA; simultaneously extending the annealed primers to synthesise an extension product, wherein each extension product after separation from the nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair of target oligonucleotide primers; and detecting the amplified products, if present, to indicate the presence or absence of the target regions, wherein detection of said three independent, non-overlapping target regions is indicative of SARS-00V-2.

Preferably, the at least three independent, non-overlapping, target regions of the SARS-00V- 2 viral genome comprise the following target regions: Gene Sequence Sequence ID Orf1_Span20 AAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 S_gene_Span 3 GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGA SEQ ID 40 N_gene_Spa n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA SEQ ID 44

TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG

According to another aspect of the present invention there is provided a kit for the detection of a diagnostic target SARS"-CoV-2 RNA from patient samples, the kit comprising: instructions describing a method for the direct detection of a diagnostic target RNA from a patient saMpie cornprising the steps described in the methods of the previous aspect of the invention; and an RT-PCR composition, wherein the RT-PCR composition comprises at east three pairs of target oligonucleotide primers selected from the following list; Primer name Gene Sequence GC Tm Sequence ID covd-19 2431 Orf1 CGGCCGCTATAACAATACTAG 47.61904762 59 SEQ ID 9 covd-19 2997 Orf1 GATGTTAGTTAGCCACTGCGA 47.61904762 62 SEQ ID 10 covd-19 9517 Orf1 AGACGTTCTTGAGTGTAATGTGA 39.13043478 61 SEQ ID 1 covd-19 9920 Orf1 AACCTAATACTCTAGATAATTCATTAGGT 27.5862069 60 SEQ ID 2 covd- S GCCAATAGGTATTAACATCACTAGG 40 61 SEQ ID 3 19 36227 covd- S CACATAATAAGCTGCAGCACCAG 47.82608696 64 SEQ ID 4 19 36334 covd- S AGGTGTTCTTACTGAGTCTAACAA 37.5 61 SEQ ID 11 19 37795 covd- S TCTCAAGTGTCTGTGGATCAC 47.61904762 61 SEQ ID 12 19 37907 covd- S CAGACTAATTCTCCTCGGCG 55 61 SEQ ID 13 19 38692 covd- S TTCTGCACCAAGTGACATAGTGTAG 44 64 SEQ ID 14 19 38901 covd- N GCTTCTAAGAAGCCTCGGCA SS 64 SEQ ID 15 19 47059 covd- N CCTTGTCTGATTAGTTCCTGGT 45.45454545 61 SEQ ID 16 19_47087 covd- 19 47295 AACTGTGACTCTTCTTCCTGCT 45.45454545 63 SEQ ID 5 covd- 19_47392 CTTGTGTGGTCTGCATGAGTT 47.61904762 62 SEQ ID 6 Preferably the pairs of target oligonucleotide primers are;

Primer Name Sequence Description Sequence ID

covd-19 9517 F AGACGTTCTTGAGTGTAATGTGA Orft_Span2O_FPrimer SEQ ID 1 covd-19 9920 R AACCTAATACTCTAGATAATTCATTAGGT Orf1_Span2O_RPrimer SEQ ID 2 covd-19 36227 F GCCAATAGGTATTAACATCACTAGG sGene_Span3_Fprimer SEQ ID 3 covd-19 36334 R CACATAATAAGCTGCAGCACCAG sGene_Span3_Rprimer SEQ ID 4 covd-19 47295 F AACTGTGACTCTTCTTCCTGCT nGene_Span1_Fprimer SEQ ID 5 covd-19 47392 R CTTGTGTGGTCTGCATGAGTT nGene_Spard_Rprimer SEQ ID 6 In the table above the F and R notations in the primer name indicate forward or reverse primers.

Preferably the kit further comprises one or more of; 2 polymerafie, preferably a heat activated thermosta hie polymerase; a reverse transcriptase enzyme; dNTIDs; and/ or appropriate buffers.

Preferably, the kit is for the detection of a diagnostic target SARS-CoV-2 RNA from patient samples and the instructions indicate that no step includes RNA purification.

According to an aspect of the present invention there is provided a device for rapidly determining the presence of SARS-CoV-2 virus in a patent sample comprising; a sample reception port; an amplification region for amplifying at least three independent, non-overlapping, target regions of the SARS-CoV-2 viral genome, said region in use containing pairs of oligonucleotide primers, each pair comprising a forward primer and a reverse primer, said pairs of target oligonucleotide primers being selected from the following list; Primer name Gene Sequence GC Tm Sequence ID covd-19 2431 Orfl CGGCCGCTATAACAATACTAG 47.61904762 59 SEQ ID 9 covd-19 2997 Orf1 GATGTTAGTTAGCCACTGCGA 47.61904762 62 SEQ ID 10 covd-19 9517 Orf1 AGACGTTCTTGAGTGTAATGTGA 39.13043478 61 SEQ ID 1 covd-19 9920 Orf1 AACCTAATACTCTAGATAATTCATTAGGT 27.5862069 60 SEQ ID 2 covd-19 36227 S GCCAATAGGTATTAACATCACTAGG 40 61 SEQ ID 3 covd-19 36334 S CACATAATAAGCTGCAGCACCAG 47.82608696 64 SEQ ID 4 covd-19_37795 S AGGTGTTCTTACTGAGTCTAACAA 37.5 61 SEQ ID 11 covd-19 37907 S TCTCAAGTGTCTGTGGATCAC 47.61904762 61 SEQ ID 12 covd-19 38692 S CAGACTAATTCTCCTCGGCG 55 61 SEQ ID 13 covd-19_38901 S TTCTGCACCAAGTGACATAGTGTAG 44 64 SEQ ID 14 covd-19 47059 N GCTTCTAAGAAGCCTCGGCA SS 64 SEQ ID 15 covd-19 47087 N CCTTGTCTGATTAGTTCCTGGT 45.45454545 61 SEQ ID 16 covd-19 47295 N AACTGTGACTCTTCTTCCTGCT 45.45454545 63 SEQ ID 5 covd-19 47392 N CTTGTGTGGTCTGCATGAGTT 47.61904762 62 SEQ ID 6 with each pair of oligonucleotide primers comprising a forward primer and a reverse primer that are operable to bind genomic regions flanking a target region, and each pair selected such that the target regions that they flank do not overlap; and a detection region, said detection region being downstream of the amplification region, for detecting the a mplicons to indicate the presence or absence of said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome.

Preferably the device is a continuous flow device. The device may be in the form of a cartridge or cassette.

Preferably the device is part of a point of care system. Preferably said point of care system is of an appropriate size to be portable. Ideally, it is of an appropriate size to be handheld.

Preferably the device comprises, or is associated with, a means for visually imaging bound amplicons. The means for visually imaging bound amplicons is a fluorescence reader.

In order to provide a better understanding of the present invention, embodiments will be descried by way of example only.

A reference to a patient encompasses individuals for whom a test is being carried out irrespective of whether they have symptoms of a disease.

Exemplary assay In one embodiment, a multiplexed PCR reaction can be performed using an RNA sample obtained from an individual.

RNA extraction may be carried out on a sample if required. This is carried out using standard extraction techniques well known in the art.

A polymerase chain reaction is then carried out. In this example the PCR reaction is carried out on a benchtop PCR machine, however it will be appreciated that the PCR could be carried out on a chip, cassette or similar which may be integrated with upstream and/or downstream processing in the same device.

PCR

The following benchtop PCR protocol is exemplary; In this embodiment a glycerol-free, lyophilised SARS-00V-2 assay V2 mastermix is used. The mastermix is formed from an initial mix provided with Taq polymerase, reaction buffer, dNTP, MgC12 and lyoexcipients and separate reverse transcriptase and dilution buffer. In order to produce room temperature lyophilized RT-qPCR reagents, the assay specific primers and probes (i.e. the target oligonucleotide primers, target probes, control oligonucleotide primers and control probe) are added to Lyo-Ready 1-Step RT-qPCR Mix and subsequently lyophilised.

In this embodiment, the mastermix volumes are; Reaction Mix, 2x -10 p.L 100x Lyo-compatible MMLV-RT* -0.2 jiL (preferably the concentration in the final reaction is 0.06 U/pL) Primer-Probe Mix, 20x -1 pL Water -x p.L Total volume -Up to 20 ML and the mix is freeze-dried. ;When running the assay the mix is rehydrated. ;An example for a 20u1 reaction (volumes can be adjusted if necessary, depending on reaction volume) PCR Reagent Volume 1X reaction (u1) Reconstituted SARS-CoV-2 assay v2 15 Dilution/positive control/TE 5 Total 20 The pairs of target o igonucleotide primers present in the assay mix are; ;Primer Name Sequence Description Sequence ID ;covd-19 9517 F AGACGTTCTTGAGTGTAATGTGA Orf1 Span20 FPrimer SEQ ID 1 covd-19 9920 R AACCTAATACTCTAGATAATTCATTAGGT Orf1_Span2O_RPrimer SEQ ID 2 covd-19 36227 F GCCAATAGGTATTAACATCACTAGG sGene_Span3_Fprimer SEQ ID 3 covd-19_36334_R CACATAATAAGCTGCAGCACCAG sGene_Span3_Rprimer SEQ ID 4 covd-19 47295 F AACTGTGACTCTTCTTCCTGCT nGene_Span1_Fprimer SEQ ID 5 covd-19 47392 R CTTGTGTGGTCTGCATGAGTT nGene_Spani_Rprimer SEQ ID 6 The control oligonucleotide primers present in the assay mix are; ;Primer Name Sequence Description Sequence ID ;RNAseP4 F GTTGGTCACATGGTGGTAAC RNAseP_Span2_Fprimer SEQ ID 7 RNAseP6_R TGAAACATCTACAACACAGACATT RNAseP_Span2_Rprimer SEQ ID 8 The pairs of target probes present in the assay mix are; Probe name Gene Sequence GC Tm Sequence ID covd-19 9620 Orf1 TTACAGAAGAGGTTGGCCACACAGA 48 68 SEQ ID 19 covd-19 36276 S TGTCCAACCTGAAGAAGAATCACCAGG 50 67 SEQ ID 20 covd-19 47356 N CAACAATCCATGAGCAGTGCTGA 50 68 SEQ ID 21 Each of the target probes is modified at the 5' end with a HEX fluorescent reporter, and at the 3' end with a Black Hole Quencher (BHQ) quencher. ;The control probe present in the mix is; Probe name Gene Sequence GC Tm Sequence ID RNAseP5 RNAseP CTCTTGCCAGTGCTGATGTCTCATG 52 67 SEQ ID 45 The control probe is modified at the 5' end with a FAM fluorescent reporter, and at the 3' end with a Black Hole Quencher (BHQ) quencher. ;All PCR constituents are mixed together, and amplification occurs on a bench-top real-time thermal cycler using the following protocol; Step Temperature Time (s) Cycles (°C) Reverse transcription 50 10 min 1 Initial denature 95 2 min 1 Denature 95 5 sec Anneal/Extension 63 20 sec When the target and control hydrolysis probes are intact, the fluorescence of the reporters are quenched due to its proximity to the quencher. The amplification reaction includes a combined annealing/extension step during which the probes hybridise to the targets if present, and the dsDNA-specific 5' 4 3' exonuclease activity of the enzyme (Taq or Tth) cleaves off the reporter. As a result, the reporter is separated from the quencher, resulting in a fluorescence signal that is proportional to the amount of amplified product (amplicon) in the sample. ;In this example, as all of the target probes are labelled with the same HEX fluorescent reporter the present of any target amplicon will give a positive result (with the present of all target amplicons giving a greater signal). As such a minimum fluorescent value can be set to give a threshold for a positive result. ;It will be understood that although HEX and FAM fluorescent labels are used here along with BHQ as the quencher, other appropriate labels and quenchers could be used. ;In certain embodiments, the probes may be of a double-quenched format, for example using the BHQnovaTM probe system. The BHQnovaTM probe system uses an internal "Nova2 quencher between bases 9 and 10 of the probe sequence, for example: Probe name Gene Sequence GC Tm Sequence ID Novacovd- Orli TTACAGAAG[Nova]AGGTTGGCCACACAGA 48 68 SEQ ID 19 19 9620 Novacovd- 19 36276 TGTCCAACC[Nova]MAAGAAGAATCACCAGG SO 67 SEQ ID 20 Novacovd- 19_47356 CAACAATCC[Nova]ATGAGCAGTGCTGA 50 68 SEQ ID 21 The inclusion of an internal Nova quencher improves the signal-to-noise ratio of longer probe sequences. ;As the probes in the assay anneal close to the 5' or 3' ends of a target region this increases the likelihood that they will be incorporated during amplification as they will be included into even relatively short read lengths. As such the cycle time and number can be reduced and the specificity of the assay will remain high. This is particularly useful for high throughput assays where multiple tests are to be carried out and also in a point of care setting where rapid sample to result times are important. ;It will be appreciated that features from one embodiment may be appropriately incorporated into another embodiment unless technically unfeasible to do so. ;With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. ;It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. ;However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a'' or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). ;It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims. *

Claims

Claims 1. A rapid method of determining the presence of SARS-00V-2 virus in a patient sample comprising; providing a sample of RNA from said patient sample; reverse transcribing target RNA in the sample in the presence of an appropriate reverse transcriptase to obtain cDNA; and amplifying substantially simultaneously at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, wherein the substantially simultaneous amplification occurs by annealing pairs of target oligonucleotide primers to the cDNA; simultaneously extending the annealed primers to synthesise an extension product, wherein each extension product after separation from the nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair of target oligonucleotide primers; and detecting the amplified products (amplicons), if present, to indicate the presence or absence of the target regions, wherein detection of said three independent, non-overlapping target regions is indicative of SARS-00V-2; characterised in that the pairs of target oligonucleotide primers comprise the primers in the following list: Primer name Gene Sequence GC Tm Sequence ID covd-19 9517 Orf1 AGACGTTCTTGAGTGTAATGTG A 39.13043 61 SEQ ID 1 covd-19 9920 Orf1 AACCTAATACTCTAGATAATTCA TTAGGT 27.58620 60 SEQ ID 2 Covd-1936227 S GCCAATAGGTATTAACATCACT AGG 40 61 SEQ ID 3 covd-19 36334 5 CACATAATAAGCTGCAGCACCA G 47.82608 64 SEQ ID 4 covd-19 47295 N AACTGTGACTCTTCTTCCTGCT 45.45454 63 SEQ ID 5 covd-19 47392 N CTTGTGTGGTCTGCATGAGTT 47.61904 62 SEQ ID 6 wherein each pair of oligonucleotide primers comprises a forward primer and a reverse primer that are operable to bind genomic regions flanking a target region, and each pair selected such that the target regions that they flank do not overlap.
2. A rapid method as in Claim 1, wherein the step of providing a sample of RNA from said patient sample may refer to the provision of a raw patient sample i.e. a patient sample which has not had RNA extraction carried out.
3. A rapid method as in Claim 1, further including the step of extracting RNA from a patient sample prior to the step of providing a sample of RNA from said patient sample.
4. A rapid method as in any of the previous Claims, wherein whilst substantially simultaneously amplifying said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, a portion of the control gene (RNAseP) is also amplified, if present by including a pair of control oligonucleotide primers.
5. A rapid method as in Claim 4, wherein the control oligonucleotide primers are selected from the group; Primer name Gene Sequence GC Tm Sequence ID RNA5eP4_F RNAseP GTTGGTCACATGGTGGTAAC 50 60 SEQ ID 7 RNAse P6_R RNAseP TGAAACATCTACAACACAGACATT 33.3333 60 SEQ ID 8 CDC RNAsePF RNAseP AGATTTGGACCTGCGAGCG 57.8947 64 SEQ ID 17 CDC RNAsePR RNAseP GAGCGGCTGTCTCCACAAGT 60 66 SEQ ID 18 6. A rapid method as in Claim 5, wherein the control oligonucleotide primers are;Primer Name Sequence Description Sequence ID
RNA5eP4 F GTTGGTCACATGGTGGTAAC RNA5eP_Span2_Fprimer SEQ ID 7 RNAseP6_R TGAAACATCTACAACACAGACATT RNAseP_Span2_Rprimer SEQ ID 8 In the table above the F and R notations in the primer name indicate forward or reverse primers.
7. A rapid method as in Claim 6, wherein, the primers have at least 80% sequence homology to the above primers, or wherein the primers have at least 90% sequence homology to the above primers, or wherein the primers have at least 95% sequence homology to the above primers, or wherein the primers have at least 99% sequence homology to the above primers.
8. A rapid method as in any of the previous Claims, wherein detection of the amplified products is by probe hydrolysis.
9. A rapid method as in any of Claims 1 to 7, wherein detection of the amplified products is by hybridisation of a fluorescent or otherwise tagged product and/and melt analysis.
10. A rapid method as in any of the previous Claims, wherein the detection of the amplified products (amplicons) uses target probes to anneal to target amplicons and control probes to anneal to control amplicons.
11. A rapid method as in any of the previous Claims, wherein the target probes for detection of the amplified products are selected from the following; Probe Gene Sequence GC Tm Sequence ID name covd- 0111 CAGTATTCACTGAGACTCATTGATGCTATG 40 66 SEQ ID 22 19 2515 covd- Orli TGAACAACACCACCTGTAATGTAGGC 46.15384 67 SEQ ID 23 19 2879 covd- Orf1 TTACAGAAGAGGTTGGCCACACAGA 48 68 SEQ ID 19 19_9620 covd- Orf1 AAGACTAGAATTGTCTACATAAGCAGCCAT 36.66666 66 SEQ ID 24 19 9787 covd- S TGTCCAACCTGAAGAAGAATCACCAGG 50 67 SEQ ID 20 19 36276 covd- S CAGCATCAGTAGTGTCAGCAATGTCTC 48.14814 67 SEQ ID 25 19 37846 covd- S CAATGATGGATTGACTAGCTACACTACG 44.82758 67 SEQ ID 26 19 38749 covd- N 42.85714 68 SEQ ID 27 19 47068AGCATACAATGTAACACAAGLI I ICGGCcovd- N CAACAATCCATGAGCAGTGCTGA 50 68 SEQ ID 21 19 47356 12.
A rapid method as in Claim 11, wherein the target probes are; Probe name Gene Sequence GC Tm Sequence ID covd-19 9620 Orf1 TTACAGAAGAGGTTGGCCACACAGA 48 68 SEQ ID 19 covd-19 36276 S TGTCCAACCTGAAGAAGAATCACCAGG 50 67 SEQ ID 20 covd-19 47356 N CAACAATCCATGAGCAGTGCTGA 50 68 SEQ ID 21 13.
A rapid method as in any of the previous Claims, wherein the control probe is; Probe name Gene Sequence GC Tm Sequence ID RNAseP5 RNAseP CTCTTGCCAGTGCTGATGTCTCATG 52 67 SEQ ID 45 1.4.
A rapid method as in any of Claims 8 to 13, wherein at least one of the target probes and/or at least one of the control probes is a double-quenched probe.
15. A rapid method as in any of the previous Claims, wherein the method is for the detection of a diagnostic target SARS-CoV-2 RNA from patient samples and no step includes RNA purification.
16. A rapid method as in any of the previous Claims, wherein the patient samples are nasopharyngeal wash/aspirates, nasopharyngeal swabs, oropharyngeal swabs, broncheoalveolar lavage, tracheal aspirate, pleural fluid tap, sputum, saliva and/or post-mortem tissue.
17. A rapid method as in any of the previous Claims, wherein said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, are regions selected from the following target regions; Gene Sequence Sequence ID Orf1_Span1 AAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAA AGCTTGATGGCTTTATGGGTAGAATTCGATCTGTCTATCCAGTTG CGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGA AGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTT GT SEQ ID 28 Orf1_Span2 AAAGTCGGAGCATGTCACAATTCAGAAGTAGGACCTGAGCATAG TCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCG TAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTA TGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAG CGCTAACA SEQ ID 29 Orf1_Span4 TCCCGCGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTC ACTGAGACTCATTGATGCTATGATGTTCACATCTGATTTGGCTAC TAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCA SEQ ID 30GTTGACTTCGCAGTGGCTAACTAACATCTTTGGCACTGTTTATGA AAAACTCAAACCCGTOrf1_Span8 AAGAGTTTGAGCCATCAACTCAATATGAGTATGGTACTGAAGAT SEQ ID 31GATTACCAAGGTAAACCTTTGGAATTTGGTGCCACTTCTGCTGCT CTTCAACCTGAAGAAGAGCAAGAAGAAGATTGGTTAGATGATGA TAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATC AGA0 rf1_Spa n15 TGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAAACACCT GAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAA GATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTT CTTAAGAGAGGTGATAAAAGTGTATATTACACTA SEQ ID 32 0 rf1_S pa n20 AAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 0 rf1a b_S pa n 0 GGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGC CTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTG ACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAAT ATG AC SEQ ID 34 0 rf1a b_S pa n 2 AATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTC ACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATAT CCTGCTAATTCCATTGTTTGTAGATTTGACACTAGAGTGCTATCTA ACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTAT SEQ ID 35 0 rf1a b_S pa n 5 ATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAAT TAG AAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTC ATAGTCAGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAAC GTTTTAAGGAATCACCTTTTGAATTAGAAG SEQ ID 36 0 rf3a_Spa n1 SEQ ID 37AAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCA GCTGTACTCAACTCAATTGAGTACAGACACTGGTGTTGAACATGT TACCTTCTTCATCTACAATAAAATTGTTGATGAGCCTGAAGAACATGTCCAAATTCACACAATCGACGGTTCAT CCGGAGTTGTTAATCC AGTAOrf8_Span0 TTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATGT AGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGAC CCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGGA GCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGC TGGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATAC AGTTTCCTGTTTACCTTTTACAATTAATTGCCAGGAACCTAAATTG GGTAGT SEQ ID 38 S_ge n e_S p a n 0 CTTGTTTTATTGCCACTAGTCTCTAGTCAGTGTGTTAATCTTACAA CCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGG TGTTTATTACCCTGACAAAGITTICAGATCCTCAGTTTTACATTCA ACTCAG G ACTTG TTCTTACCTTTCTTTTCCAATG TTACTTGG TT CCA TGCTATACATGTCTCTGGGACCAATGGTACTAAGA SEQ ID 39 S_gene_Span 3 GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGA SEQ ID 40 S_ge n e_S p a n GGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTT CTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGAT GCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCA TGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACT TCTAA SEQ ID 41 S_ge n e_S pa n TGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCC TCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACAC TATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTC SEQ ID 42 N_gene_Spa nO CTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACT AAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGA ACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAG GAACTGATTACAAACATTGGCCGCAA SEQ ID 43 N_gene_Spa n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG SEQ ID 44
18. A rapid method as Claim 17, wherein the at least three independent, non-overlapping, target regions of the SARS-CoV-2 viral genome comprise the following target regions; Gene Sequence Sequence ID Orf1 Span20 AAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 S_gene_Span 3 GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGA SEQ ID 40 N_gene_Spa n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA SEQ ID 44TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG
19. A rapid method of determining the presence of SARS-00V-2 virus in a patient sample comprising; providing a sample of RNA from said patient sample; reverse transcribing any RNA in the sample in the presence of an appropriate reverse transcriptase to obtain cDNA and substantially simultaneously amplifying said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome in a multiplexed polymerase chain reaction, said regions selected from the following target regions; Gene Sequence Sequence ID 0 rf 1_5 p a n1 AAATTCCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAA AGCTTGATGGCTTTATG GGTAGAATTCGATCTGTCTATCCAGTTG CGTCACCAAATGAATGCAACCAAATGTGCCTTTCAACTCTCATGA AGTGTGATCATTGTGGTGAAACTTCATGGCAGACGGGCGATTTT GT SEQ ID 28 0 rf 1_5 p a n2 AAAGTCG G AG CATG TCACAATTCA G AAG TAG G AC CTG AG CATAG TCTTGCCGAATACCATAATGAATCTGGCTTGAAAACCATTCTTCG TAAG G GTG G TCG CA CTATTG CCTTTG G AG G CTGTGTGTTCTCTTA TGTTGGTTG CCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAG CGCTAACA SEQ ID 29 0 rf1_S p a n4 TCCCGCGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTC ACTGAGACTCATTGATGCTATGATGTTCACATCTGATTTGGCTAC TAACAATCTAGTTGTAATGGCCTACATTACAGGTGGTGTTGTTCA GTTGACTTCGCAGTGGCTAACTAACATCTTTG GCACTGTTTATGA AAAACTCAAACCCGT S EQ ID 30 0 rf 1_5 p a n8 AAG AGTTTG AG CCATCAACTCAATATG AGTATG GTACTG AAGAT GATTACCAAGGTAAACCTTTG GAATTTGGTGCCACTTCTGCTGCT CTTCAACCTGAAGAAGAGCAAGAAGAAGATTGGTTAGATGATGA TAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAGGACAATC AGA SEQ ID 31 0 rf 1_5 pa n15 TGTTACAG CGTATAATG GTTAT CTTACTTCTTCTTCTAAAACAC CT GAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAA GATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTT CTTAAGAGAGGTGATAAAAGTGTATATTACACTA SEQ ID 32 0 rf 1_5 pa n20 AAGACGTTCTTGAGTGTAATGTG AAAACTACC G AAG TTG TAG GA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAG GTTG G CCACACAGATCTAATG G CTG CTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAG AGTATTAG GT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 Orfla b_Span 0 GGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGC CTATATTAACCTTGACCAGGGCTTTAACTGCAGAGTCACATGTTG ACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAAT ATG AC SEQ ID 34 Orf1a b_S pa n AATAGAAGAATTATTCTATTCTTATGCCACACATTCTGACAAATTC ACAGATGGTGTATGCCTATTTTGGAATTGCAATGTCGATAGATAT CCTGCTAATTCCATTGTTTGTAGATTTGACACTAGAGTGCTATCTA ACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTAT SEQ ID 35 Orf1a b_S pa n 5 ATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAAT TAGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTC ATAGTCAGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAAC GHTTAAGGAATCACCTITTGAATTAGAAG SEQ ID 36 Orf3a_Spa n1 AAGACTGTGTTGTATTACACAGTTACTTCACTTCAGACTATTACCA GCTGTACTCAACTCAATTGAGTACAGACACTGGTGTTGAACATGT TACCTTCTTCATCTACAATAAAATTGTTGATGAGCCTGAAGAACA TGTCCAAATTCACACAATCGACGGTTCATCCGGAGTTGTTAATCC AGTA SEQ ID 37 Orf8_Spa nO SEQ ID 38TTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAATGT AGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGAC CCGTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGG A GCTAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGC TGGTTCTAAATCACCCATTCAGTACATCGATATCGGTAATTATAC AGTTTCCTGTTTACCTTTTACAATTAATTGCCAGGAACCTAAATTG GGTAGTS_ge ne_S pa n 0 CTTG TTTTATTG CCACTAG TCTCTAGTCAG TGTGTTAATCTTACAA CCAGAACTCAATTACCCCCTGCATACACTAATTCTTTCACACGTGG TGTTTATTACCCTG ACAAAGITTICAGATCCTCAGTMACATTCA ACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGGTTCCA TGCTATACATGTCTCTGGGACCAATGGTACTAAGA SEQ ID 39 S_gene_Span GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA SEQ ID 40GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGAS_gene_Span GGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTT CTGCCTTTCCAACAATTTGGCAGAGACATTGCTGACACTACTGAT GCTGTCCGTGATCCACAGACACTTGAGATTCTTGACATTACACCA TGTTCTTTTGGTGGTGTCAGTGTTATAACACCAGGAACAAATACT TCTAA SEQ ID 41 S_gene_Span TGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATTCTCC TCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACAC TATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTC SEQ ID 42 N_gene_Spa nO CTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACT AAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGA ACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAG GAACTGATTACAAACATTGGCCGCAA SEQ ID 43 N_gene_Spa n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG SEQ ID 44 wherein the substantially simultaneous amplification occurs by annealing pairs of target oligonucleotide primers to the cDNA; simultaneously extending the annealed primers to synthesise an extension product, wherein each extension product after separation from the nucleic acid serves as a template for the synthesis of an extension product for the other primer of each pair of target oligonucleotide primers; and detecting the amplified products, if present, to indicate the presence or absence of the target regions, wherein detection of said three independent, non-overlapping target regions is indicative of SARS-CoV-2.
20. A rapid method according to Claim 19, wherein the at least three independent, non-overlapping, target regions of the SARS-CoV-2 viral genome comprise the following target regions: Gene Sequence Sequence ID Orfl Spa n20 AAGACGTTCTTGAGTGTAATGTGAAAACTACCGAAGTTGTAGGA GACATTATACTTAAACCAGCAAATAATAGTTTAAAAATTACAGAA GAGGTTGGCCACACAGATCTAATGGCTGCTTATGTAGACAATTCT AGTCTTACTATTAAGAAACCTAATGAATTATCTAGAGTATTAGGT TTGAAAACCCTTGCTACTCATGGTTT SEQ ID 33 S_gene_Span 3 GGTAGATTTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATTCTTCTTCA GGTTGGACAGCTGGTGCTGCAGCTTATTATGTGGGTTATCTTCAA CCTAGGACTTTTCTATTAAAATATAATGA SEQ ID 40 N_gene_Spa n1 GAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGA TTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAAC TCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATA TAAACG SEQ ID 44
21. A kit for the detection of a diagnostic target S S-CoV-2 RNA from patient sampes, the kit comprising: instructions describing a method for the direct detection of a diagnostic target RNA from a patient sample comprising the steps described in the methods of the previous aspect of the invention; and an RT-PCR composition, wherein the RT-PCR composition comprises at least three pairs of target oligonucleotide primers, wherein said at least three pairs of target oligonucleotide primers comprise the primers in the fo lowing list: Primer name Gene Sequence GC Tm Sequence ID covd- Orf1 AGACGTTCTTGAGTGTAATGTGA 39.13043478 61 SEQ ID 1 19_9517 covd- Orli AACCTAATACTCTAGATAATTCATTAGGT 27.5862069 60 SEQ ID 2 19_9920 covd- S GCCAATAGGTATTAACATCACTAGG 40 61 SEQ ID 3 19_36227 covd- S CACATAATAAGCTGCAGCACCAG 47.82608696 64 SEQ ID 4 19_36334 covd- N AACTGTGACTCTTCTTCCTGCT 45.45454545 63 SEQ ID 5 19_47295 covd- N CTTGTGTGGTCTGCATGAGTT 47.61904762 62 SEQ ID 6 19_47392
22. A kit as in Claim 21 which further comprises one or more of; a polyrnerase, preferably a heat activated thermostable polymerase; a reverse transcriptase enzyme; ciNiliPs; and/ or appropriate buffers.
23. A kit as in any of Claims 21 to 22 which is for the detection of a diagnostic target SARSCoy-2 RNA from patient samples and the instructions indicate that no step includes RNA p
24. A device for rapidly determining the presence of SARS-CoV-2 virus in a patent sample comprising; a sample reception port; an amplification region for amplifying at least three independent, non-overlapping, target regions of the SARS-CoV-2 viral genome, said region in use containing pairs of oligonucleotide primers, each pair comprising a forward primer and a reverse primer, wherein said pairs of target oligonucleotide primers comprise the primers in the following list: Primer name Gene Sequence GC Tm Sequence ID covd- Orfl AGACGTTCTTGAGTGTAATGTGA 39.13043478 61 SEQ ID 1 19_9517 covd- Orf1 AACCTAATACTCTAGATAATTCATTAGGT 27.5862069 60 SEQ ID 2 19_9920 covd- S GCCAATAGGTATTAACATCACTAGG 40 61 SEQ ID 3 19_36227 covd- S CACATAATAAGCTGCAGCACCAG 47.82608696 64 SEQ ID 4 19_36334 covd- N AACTGTGACTCTTCTTCCTGCT 45.45454545 63 SEQ ID 5 19 47295 covd- N CTTGTGTGGTCTGCATGAGTT 47.61904762 62 SEQ ID 6 19 47392 with each pair of olieonucleotide primers comorisine a forward primer and a reverse primer that are operable to bind genomic regions flanking a target region, and each pair selected such that the target regions that they flank do not overlap; and a detection region, said detection region being downstream of the amplification region, for detecting the a mplicons to indicate the presence or absence of said at least three independent, non-overlapping, target regions of the SARS-00V-2 viral genome.
25. A device as in Claim 24 which is a continuous flow device that may be in the form of a cartridge or cassette.
26. A device as in Claim 25 which is part of a point of care system which may comprise, or be associated with, a means for visually imaging labelled amplicons.