Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes

Definitions

• the present disclosure relates to the field of biotechnology. More specifically, the disclosure relates to compositions, including kits and reagents, and methods for analysis of samples to detect viral pathogens, particularly influenza vims, coronavirus, and respiratory syncytial vims.
• Influenza is an acute respiratory' illness in humans caused by infection with the influenza (flu) vims, primarily types A and B. Influenza A viruses are further categorized into subtypes based on two major surface protein antigens, hemagglutinin (H), and neuraminidase (N). Influenza B viruses are not categorized into subtypes.
• the influenza vimses are RNA viruses in the family Orthomyxoviridae. Each of influenza types A and B (Flu A and Flu B, respectively) is a separate genus containing one species and many sub-species.
• Influenza epidemics occur yearly around the world. Although both flu types A and B circulate in the population, type A is usually dominant. These yearly epidemics are partly due to antigenic variation in the H and N surface proteins of the virus. Transmission of influenza is primarily via airborne droplet (coughing or sneezing). Symptoms arise on average 1 to 2 days post-exposure and include fever, chills, headache, malaise, cough, and coryza. Gastrointestinal symptoms such as nausea, vomiting, and diarrhea can occur, primarily in young children. Complications due to influenza include pneumonia, which can cause increased morbidity and mortality in pediatric, elderly, and immune-compromised populations.
• Coronaviruses are a family of RNA viruses that infect avians and mammals, including humans. Coronaviruses belong to the family Coronaviridae, which has four main sub-groupings, known as alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus.
• Human coronaviruses include alphacoronaviruses 229E and NL63 and betacoronaviruses OC43, HKU1, SARS-CoV and SARS-CoV-2 (the coronavirus that causes severe acute respiratory syndrome, or SARS), and MERS-CoV (the coronavirus that causes Middle East Respiratory Syndrome, or MERS).
• SARS-CoV-2 can cause severe lower respiratory tract infections (COVTD-19) and was declared a global emergency by the World Health Organization, (Huang et al., Lancet (2020) v395, issue 10223, p.497).
• Respiratory syncytial virus is the leading cause of lower respiratory tract infections in infants and children. Like influenza, RSV is an RNA virus. RSV is a member of the family Paramyxoviridae, in the genus Orthopneumo virus. There are 2 types of RSV, A and B, which are differentiated based on antigenic and surface protein variations. Most yearly epidemics contain a mix of RS V A and RSV B, but one subgroup can dominate during a season. RSV infection can cause severe respiratory illness among all ages but is more prevalent in pediatric, elderly, and immune-comprormsed populations. RSV can infect up to 80% of children less than 1 years of age.
• Bronchiolitis and pneumonia are the major clinical complications in infants and young children, resulting in an estimated 51,000-82,000 hospital admissions per year in the United States.
• RSV infection is also an important cause of severe respiratory disease and substantial number of deaths in the elderly, with an estimated annual cost of $150 million to $680 million for RSV pneumonia hospitalizations.
• compositions including kits and reagents, and methods for in vitro diagnostic analysis of influenza A virus (Flu A), influenza B virus (Flu B), SARS- CoV-2, respiratory syncytial virus type A (RSV A) or respiratory syncytial virus type B (RSV B) nucleic acids in a sample.
• influenza A virus influenza B virus
• SARS- CoV-2 respiratory syncytial virus type A
• RSV B respiratory syncytial virus type B
• nucleic acids in a sample.
• the in vitro diagnostic analysis utilizes polymerase chain reactions (PCR) or isothermal amplification reactions, though other in vitro assay methodologies are contemplated for use with the disclosed compositions.
• a particularly useful in vitro assay for use with the Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is a reverse transcription PCR (RT-PCR) assay, as these target nucleic acids are RNA viruses.
• RT-PCR reverse transcription PCR
• a particularly useful and convenient in vitro assay for use with the Flu A, Flu B, S ARS- CoV-2, RSV A or RSV B target nucleic acids is a real-time, RT-PCR assay.
• a particularly useful and convenient in vitro assay for use with the Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is a transcription-mediated amplification (TMA) reaction.
• TMA transcription-mediated amplification
• a particularly useful and convenient in vitro assay for use with the Flu A, Flu B, SARS-CoV-2, RSV A or RSV B target nucleic acids is a BiPhasic TMA reaction.
• the sample is a biological sample.
• the biological sample is a clinical sample
• the sample is a swab sample, for example, from nasopharyngeal (NP) swab specimens obtained from a patient.
• the compositions and methods can be used to aid in the differential diagnosis of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B infections. Negative results do not preclude such infection. Conversely, positive results do not rule-out bacterial infections or co-infections wi th other viruses. The use of additional laboratory testing and clinical presentation may also be considered to obtain the final diagnosis of respiratory viral infection.
• hybridization assay probes useful for detecting Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B target nucleic acid sequences.
• probe molecule species include a target hybridizing sequence that is substantially complementary to a probe target nucleic acid sequence in the viral genome, or an amplicon generated therefrom, being targeted for detection.
• the probe target nucleic acid sequence consists of about 17 to about 100 contiguous bases contained within targeted viral genome (or amplicon generated therefrom).
• the Flu A probe comprises a sequence that is preferably one of SEQ ID NOS:6 to 22.
• two probes, each separately selected from SEQ ID NOS: 6 to 22, are used in tandem to target two different regions of the Flu A genome or amplification products generated therefrom.
• the Flu B probe sequence is preferably one of SEQ ID NOS:30 to 57 and 59 to 66.
• the SARS-CoV-2 probe sequence is preferably one of SEQ ID NOs: 118, 128, 130, 133, 136, 139, 142, 145, 154, 193, 242, 246, 258, 261, and 265.
• two probes are used in tandem to target two different regions of the SARS-CoV-2 genome or amplification products generated therefrom [0015]
• the RSV A probe sequence is preferably one of SEQ ID NOS:71, 75 to 78, 80 to 87, 89 to 91, 97, and 98.
• nucleic acid molecules that are amplification primers for use in in vitro amplification of Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B target nucleic acid sequences.
• a related aspect of the disclosure relates to pairs of such primers that can be used to amplify desired amplicons that contain a target nucleic acid sequence.
• These primers include one or more of the following primers pairs: a first Flu A primer pair, a second Flu A primer pair that can be used to amplify a region of the Flu A target nucleic acid that is different from the region of the Flu A target nucleic acid that can be amplified using the first Flu A primer pair, a Flu B primer pair, a first SARS-CoV-2 primer pair, a second SARS-CoV-2 primer pair that can be used to amplify a region of the SARS-CoV-2 target nucleic acid that is from the region of the SARS-CoV-2 target nucleic acid that can be amplified using the first SARS-CoV- 2 primer pair, an RSV A primer pair, and an RSV B primer pair.
• These primer pairs include first and second primers that can be used generate corresponding amplicons for Flu A, Flu B,
• Preferred first primers for generating a Flu B amplicon have the priming nucleotide sequence of SEQ ID NO:29 or SEQ ID NO:67.
• Preferred second primers for generating a Flu B amplicon have the priming nucleotide sequence of SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.
• Preferred second primers for generating a SARS-CoV-2 amplicon have the priming nucleotide sequence of SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 138, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 170, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:247, SEQ ID NO:254, SEQ ID NO:257, or SEQ ID NO:264.
• a probe and/or a primer contains one or more methylated cytosine bases.
• compositions that contain such probes, primers, and primer pairs. Such compositions include dry or liquid compositions. Dried compositions include lyophilized reagents containing one or more of a primer and a probe.
• kits that include primers and/or probes. Such kits can also include salts, enzymes, dNTPs, rNTPs, other substrates, and/or instructions for use of such materials.
• the primers, probes, salts, enzymes, dNTPs, rNTPs, and/or other substrates of the kit may be in a dried form or in an aqueous form.
• Embodiment 10 The composition or kit of embodiment 1, 2, 3, or 9, wherein the primer of (a)(ii) is SEQ ID NO: 117.
• Embodiment 11 The composition or kit of embodiment 1, 2, 3, 9, or 10, wherein the primer of (a)(ii) contains at least one nucleotide analog that is a 5-Me-C
• Embodiment 27 The composition or kit of embodiment 17, wherein the composition or kit further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the Flu B primer pair.
• Embodiment 28 The composition or kit of embodiment 27, wherein the detection probe oligomer is selected from the group consisting of: SEQ ID NOs:30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 60, 61, 62, 63, 64, 65, 66, 282, and 303.
• Embodiment 31 The composition or kit of embodiment 19, wherein the composition or kit further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the RSV B primer pair.
• Embodiment 32 The composition or kit of embodiment 31, wherein the detection probe oligomer is selected from the group consisting of: SEQ ID NOs:102, 103, 107, 108, 109, 110, 111, 112, 113, and 114.
• Embodiment 33 The composition or kit of any one of embodiments 12 to 14 and 22 to 32, wherein each detection probe oligomer comprises a nucleotide sequence that is from 0% to 100% nucleotide analogs.
• Embodiment 38 The composition or kit of any of embodiments 1 to 37, wherein the composition or kit further comprises a target capture oligonucleotide.
• Embodiment 39 The composition or kit of embodiment 38, wherein the target capture oligonucleotide is selected from the group consisting of: SEQ ID NOs:228, 229, 299, 300, 310, and 311.
• Embodiment 40 The composition or kit of any one of embodiments 1 to 37, wherein the composition or kit further comprises two or more target capture oligonucleotides, wherein each of the target capture oligonucleotide is independently selected from the group consisting of: SEQ ID NOs:228, 229, 299, 300, 310, and 311.
• Embodiment 41 The composition of any one of embodiments 1 to 40, wherein the composition is an aqueous formulation.
• Embodiment 42 The composition of embodiment 41, wherein the aqueous formulation further comprises a reagent for an amplification and/or a detection and/or a target capture reaction.
• Embodiment 43 The composition of any one of embodiments 1 to 40, wherein the composition is a dried formulation.
• Embodiment 44 The composition of embodiment 43, wherein the dried formulation further comprises a reagent for an amplification and/or a detection and/or a target capture reaction.
• Embodiment 45 A kit containing the composition of any one of embodiments 41 to 44.
• Embodiment 46 A kit or composition for the detection of amplicon generated in a nucleic acid amplification reaction of a biological sample suspected of containing SARS-CoV- 2, Flu A, Flu B, RSV A, and/or RSV B, wherein the kit or composition comprises: (a) for an amplicon generated with a SARS-CoV-2 primer pair, the probe molecule species comprises a nucleic acid sequence that is substantially identical to a sequence selected form the group consisting of: SEQ ID NOs:118, 128, 130, 133, 136, 139, 142, 145, 154, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 242, 246, 258, 261, 265, 277, 278, 279, 284, 285, 286, 287, 288, 289, 290, 304, 159, 160, 161, 162, 163, 164, 165, 166, 167, and 168;
• Embodiment 58 A method of determining whether a biological sample contains SARS-CoV-2, Influenza A (Flu A), Influenza B (Flu B), Respiratory Syncytial Virus A (RSV A), and/or Respiratory Syncytial Virus B (RSV B), comprising: (a) contacting under stringent hybridization conditions target nucleic acid molecules from the biological sample with one or more of the detection probe oligonucleotides of embodiment 50 or 51; (b) detecting the presence or absence of a detectable label, the presence indicating that a probe:target nucleic acid duplex formed in step (a); (c) based on whether the presence of a detectable label was detected in step (b), determining that the biological sample contains SARS-CoV-2, Flu A, Flu B, RSV A, and/or RSV B.
• Embodiment 59 The method of embodiment 58, wherein the detection probe oligonucleotides are distinguishably labeled, allowing for the determination of the presence or absence of each of SARS-CoV-2, Flu A, Flu B, and RSV A and RSV B at step (c).
• Embodiment 60 The method of embodiment 58 or 59, wherein target nucleic acid forming the probe:target nucleic acid duplex at step (b) is an amplicon generated using a primer pair.
• Embodiment 61 A method of determining whether a biological sample contains SARS-CoV-2, Influenza A (Flu A), Influenza B (Flu B), Respiratory Syncytial Virus A (RSV A), and/or Respiratory Syncytial Virus B (RSV B), comprising: (a) contacting target nucleic acid molecules from the biological sample with one or more primer pairs of one of embodiments 1 to 11 to form an amplification reaction mixture; (b) performing a nucleic acid amplification reaction to generate an amplicon from any target nucleic acid molecule in the amplification reaction mixture of step (a); and (c) determining whether an amplicon was generated at step (b), thereby determining whether the biological sample contains SARS-CoV- 2, Flu A, Flu B, and RSV A and RSV B.
• Embodiment 62 The method of embodiment 61, wherein, before step (a), a target capture reaction is performed to extract the target nucleic acid molecules from the biological sample.
• Embodiment 63 The method of embodiment 61 or embodiment 62, wherein steps (b) and (c) are performed simultaneously.
• Embodiment 64 The method of embodiment 61, 62, or 63, wherein the nucleic acid amplification reaction is a PCR reaction.
• Embodiment 65 The method of any one of embodiments 61 to 64, wherein the nucleic acid amplification reaction is a real-time nucleic acid amplification reaction.
• Embodiment 66 The method of any one of embodiments 61 to 65, wherein the detecting step (c) is performed using one or more detection probe oligonucleotides.
• Embodiment 67 The method of embodiment 66, wherein the detection probe oligonucleotides are labeled with donor/acceptor label pairs.
• Embodiment 68 The method of embodiment 66 or embodiment 67, wherein the detection probe oligonucleotides are differentially labeled to allow for the determination of which of SARS-CoV-2, Flu A, Flu B, and RSV A and RSV B are contained in the sample.
• Embodiment 69 The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen.
• Embodiment 70 The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen that is a nasopharyngeal specimen.
• Embodiment 71 The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen that is a bronchoalveolar specimen.
• Embodiment 72 The method of any one of embodiments 61 to 68, wherein the biological sample comprises a clinical specimen that is a lower respiratory' tract specimen.
• Embodiment 73 The method of any one of embodiments 61 to 72, wherein the biological sample is collected into a sample transport medium.
• Embodiment 74 The method of embodiment 73, wherein the sample transport media contains one or more of a buffer, a chelator, an anionic detergent and a degrative enzyme.
• Embodiment 75 A system for performing one or more steps of the method of any one of embodiments 61 to 74.
• Embodiment 76 The system of embodiment 75, wherein the system is an automated system.
• Embodiment 77 A system for performing one or more steps of the method of any one of embodiments 58 to 60.
• Embodiment 78 The system of embodiment 77, wherein the system is an automated system.
• Embodiment 78 A composition or kit or method for analysis of one or more of a plurality of pathogen-associated target nucleic acid molecule species that may be present in a biological sample, comprising: (a) a first SARS-CoV-2 primer pair for generating a first SARS- CoV-2 amplicon from the biological sample, the primer pair comprising (i) a first SARS-CoV- 2 primer and (ii) a second SARS-CoV-2 primer, wherein the primer of l(a)(i) comprises a target hybridizing sequence consisting of a nucleotide sequence that is 18 to 35 contiguous nucleotides in length contained within SEQ ID NO: 123 and containing SEQ ID NO: 122, and wherein the primer of 1 (a)(i) and the primer of 1 (a)(ii) generate an amplicon that contains SEQ ID NO: 124 and has a length of 57 to 89 contiguous nucleotides; and optionally, (b)
• Embodiment 79 The composition or kit or method of embodiment 78, wherein the primer of (a)(ii) comprises a target-hybridizing sequence consisting of a nucleotide sequence that is 22 to 25 nucleotides in length.
• Embodiment 80 The composition or kit or method of embodiment 78 wherein the target-hybridizmg sequence of the primer of (a)(i) is SEQ ID NO: 116 or SEQ ID NO: 119 or SEQ ID NO: 121.
• Embodiment 81 The composition or kit or method of embodiment 78, 79 or 80, wherein: the primer of (a)(i) contains at least one nucleotide analog; wherein the primer of (a)(i) contains at least one nucleotide analog that is a 5-Me-C; wherein the primer of (a)(ii) is 24 nucleotides in length and contains at least one nucleotide analog; wherein the primer of (a)(ii) contains at least one nucleotide analog that is a 5-Me-C; or combinations thereof.
• Embodiment 82 The composition or kit or method of embodiment 78 to 81 , wherein the targethybridizing sequence of the primer of (a)(ii) is SEQ ID NO:117.
• Embodiment 83 The composition or kit or method of any of embodiments 78 to 82, wherein the composition or kit further comprises a detection probe oligomer.
• Embodiment 84 The composition or kit or method of embodiment 83, wherein the composition or kit further comprises a detection probe oligomer comprising a target-hybridizing sequence consisting of a nucleotide sequence that is 25 to 40 contiguous nucleotides in length and is contained within SEQ ID NO: 124.
• Embodiment 85 The composition or kit or method of embodiment 84, wherein the detection probe oligomer target-hybridizing sequence 25 to 27 contiguous nucleotides in length and is contained within SEQ ID NO: 125.
• Embodiment 86 The composition or kit or method of embodiment 83, 84, or 85, wherein the detection probe oligomer target-hybridizing sequence is SEQ ID NO: 118.
• Embodiment 87 The composition or kit or method of any of embodiments 78 to 86, further comprising one or more of the second SARS-CoV-2 primer pair, the first Flu A primer pair, the second Flu A primer pair, the Flu B primer pair, the RSV A primer pair, and the RSV B primer pair, wherein: (a) for the second SARS-CoV-2 primer pair of 1 (b), the targethybridizing sequence of l(b)(i) and the target-hybridizing sequence of l(b)(ii) are selected from the groups consisting of: SEQ ID NOs: 146 and 170; SEQ ID NOs: 126 and 127; SEQ ID NOs:146 and 127; SEQ ID NOs:263 and 264; SEQ ID NOs: 131 and 132; SEQ ID NOs: 181 and 129; SEQ ID NOs: 131 and 129; SEQ ID NOs:262 and 257; SEQ ID NOs: 140 and 141;
• Embodiment 88 The composition or kit or method of embodiment 84, wherein each primer individually contains from 0 to 20 nucleotide analogs.
• Embodiment 89 The composition or kit or method of embodiment 88, wherein at least one of the nucleotide analogs is a 5-Me-C analog.
• Embodiment 90 The composition or kit or method of embodiment 78, wherein each primer individually contains from 0 to 20 nucleotide analogs.
• Embodiment 91 The composition or kit or method of embodiment 90, wherein at least one of the nucleotide analogs is a 5-Me-C analog.
• Embodiment 94 The composition or kit or method of embodiment 87, 92, or 93, wherein the composition or kit or method further comprises a detection probe oligomer that hybridizes under nucleic acid amplification conditions to an amplicon generated by the second Flu A primer pair, wherein the targethybridizing sequence of 1(d)(1), the target-hybridizing sequence of l(d)(ii), and the detection probe oligomer target-hybridizing sequence are selected from the groups consisting of: SEQ ID NOs:23, 25, and 9; SEQ ID NOs:23, 25, and 14; SEQ ID NOs:23, 25, 9, and 14; SEQ ID NOs:23, 6, and 9; SEQ ID NOs:23, 6, and 14; SEQ ID NOs:23, 6, 9, and 14;; SEQ ID NOs:23, 28, and 9; SEQ ID NOs:23, 28, and 14; SEQ ID NOs:23, 28, 9, and 14; SEQ ID NOs:23, 25, and 8;
• Embodiment 128 A forward primer and probe combination, wherein (i) the forward primer comprises a target hybridizing sequence that is from 13 to 28 nucleotides in length, is contained within SEQ ID NO: 169, and contains SEQ ID NO: 153, and (ii) the detection probe comprises a target hybridizing sequence that is from 17 to 40 nucleotides in length, is contained within SEQ ID NO: 169 and contains SEQ ID NO: 157.
• Embodiment 129 A forward and reverse primer set, wherein the forward primer comprises a target hybridizing sequence that is from 13 to 28 nucleotides in length, is contained within SEQ ID NO: 151, and contains SEQ ID NO: 153, and wherein the reverse primer comprises a target hybridizing sequence that is from 15 to 35 nucleotides in length, is contained withing SEQ ID NO: 171, and contains SEQ ID NO: 172.
• Embodiment 136 An amplicon that is from 115 to 137 nucleotides in length and comprises SEQ ID NO:253.
• Embodiment 137. The amplicon of embodiment 136, wherein the amplicon sequence comprises SEQ ID NO:249.
• Embodiment 240 A combination comprising a SARS- CoV-2 reverse primer and SARS-CoV-2 detection probe for amplifying and detecting an amplicon from any one for embodiments 136 to 138, wherein the reverse primer comprises a target hybridizing sequence that is from 15 to 21 nucleotides in length, contained within SEQ ID NO:250, is not contained within SEQ ID NO:252, and hybridizes to SEQ ID NO:249, and wherein the detection probe comprises a target hybridizing sequence that is from 15 to 40 nucleotides in length, contained within SEQ ID NO:249, is not contained in SEQ ID NO:251, and hybridizes to SEQ ID NO: 250.
• the reverse primer comprises a target hybridizing sequence that is from 15 to 21 nucleotides in length, contained within SEQ ID NO:250, is not contained within SEQ ID NO:252, and hybridizes to SEQ ID NO:249
• the detection probe comprises a target hybridizing sequence that is from 15 to 40 nucleotides in length
• complement refers to a nucleic acid molecule that comprises a contiguous nucleotide sequence that is complementary to a contiguous nucleic acid sequence of another nucleic acid molecule (for standard nucleotides A:T, A:U, C:G).
• Two nucleic acid sequences are “sufficiently complementary” when, their respective contiguous nucleic acid sequences are at least 70% complementary. See, e.g., Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2 nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
• nucleic acid molecule comprises a contiguous nucleic acid sequence that is at least 70% homologous to a contiguous nucleic acid sequence of another nucleic acid molecule.
• sample or “biological sample” is any tissue or polynucleotide-containing material obtained from a human, animal, or environmental sample and which may contain a target nucleic acid.
• Biological samples include peripheral blood, mucus, plasma, serum, saliva, cerebrospinal fluid, urine, bone marrow, or other body fluid, biopsy tissue, or other materials of biological origin, as well as solutions or compositions containing materials of biological origin, for example, a bronchial lavage fluid. Samples can be obtained from several sources, including a clinical source wherein the sample is collected in order to determine the presence or absence of a target nucleic acid in the sample and in turn provide a patient with a diagnosis.
• a sample may be chemically and/or mechanically treated to disrupt tissue or cell structure, thereby releasing intracellular components into a solution.
• a “nucleotide” includes both nucleotides and nucleosides, including deoxyribonucleotides (e.g., dATP, dCTP, dGTP, dTTP), ribonucleotides (e.g., rATP, rCTP, rGTP, rUTP), and analogs thereof.
• Nucleotides comprise a purine or pyrimidine base linked glycosidically to a ribose or a deoxyribose sugar and a phosphate group attached to the ribose or deoxyribose sugar.
• Nucleosides comprise a purine or pyrimidine base linked glycosidically to a ribose or a deoxyribose sugar but lack the phosphate residues that are present in a nucleotide.
• Nucleotides and nucleosides, as used herein, refer to a monomer of DNA or RNA, respectively. See e.g., Kornberg and Baker, DNA Replication, 2 nd Ed. (Freeman, San Francisco, 1992).
• an analog in reference to a chemical compound, refers to compound having a structure like that of another one, but differing from it in respect of one or more different atoms, functional groups, or substructures that are removed or replaced with one or more other atoms, functional groups, or substructures.
• an analog refers to a compound that, like the nucleotide/side of which it is an analog, can be incorporated into a nucleic acid molecule (e.g., a primer, a probe and/or an amplification product).
• Nucleotide/side analogs are commonly added to synthetic oligonucleotides (such as primers and probes) using phosphorami dite chemistry techniques and devices. Nucleotide/side analogs are commonly added to amplification products by including the analog in a reaction mixture wherein a suitable polymerase, for example, a DNA polymerase, will incorporate the analog into the amplification product. Nucleotide/side (hereinafter “nucleotide”) analogs include synthetic nucleotides having modified base moieties and/or modified sugar moieties and/or modified phosphate groups.
• RNA oligonucleotides can be single-stranded (ssRNA) or doublestranded (dsRNA), and can include both single and double-stranded (or “duplex”) regions. Single-stranded DNA (or regions thereof) and ssRNA can, if sufficiently complementary, hybridize to form double-stranded complexes (or regions).
• RNA equivalent By “RNA equivalent,” “DNA equivalent,” “RNA equivalent bases,” and “DNA equivalent bases” is meant RNA and DNA molecules having similar complementary base pair hybridization properties. RNA and DNA equivalents have different sugar moieties (ribose versus deoxyribose) and may differ, for example, by the presence of uracil in RNA and thymine in DNA. The differences between RNA and DNA equivalents do not contribute to differences in homology (or sequence identity) because the equivalents have the same degree of complementarity' to a particular sequence.
• oligonucleotide means a multimeric compound comprising two or more joined RNA nucleotides, DNA nucleotides, analogs of RNA nucleotides, analogs of DNA nucleotides, or combinations thereof. Oligonucleotides can include other molecules that may be present in a joined sequence of nucleotides and that do not prevent hybridization of the polynucleotide with a second molecule having a complementary sequence.
• oligonucleotide or other nucleic acid
• A denotes adenosine (dATP/rATP) or an analog thereof
• C denotes cytidine (dCTP/rCTP) or an analog thereof
• G denotes guanosine (dGTP/rGTP) or an analog thereof
• U denotes uracil (rUTP) or an analog thereof
• T denotes thymidine (dTTP) or an analog thereof, unless otherwise noted.
• Oligonucleotides of the disclosure comprise the four natural nucleotides and often comprise non-natural nucleotide analogs.
• Oligonucleotides disclosed herein include, but are not limited to, amplification oligonucleotides (e g., primers and promoter primers), detection probes (e.g., linear probes, TaqMan probes, AE-labeled probes, molecular torches, and molecular beacons), target capture oligomers, amplicons, amplification products, and in vitro transcripts.
• the “backbone” of an oligonucleotide may be made up of a variety' of linkages known in the art, including one or more sugar-phosphodiester linkages, peptide-nucleic acid bonds (PNAs), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof.
• Sugar moieties of the oligonucleotide may be either ribose or deoxyribose, or similar compounds having known substitutions, such as, for example, 2'-(9-methyl ribose and 2' halide substitutions (e.g., 2'-O-Me or 2'-F).
• the nucleotide analogs in an oligonucleotide sequence can include inosine or “I,” 5-Me-dC, isoguanine, other derivatives of purine or pyrimidine bases, or abasic residues (e.g., nucleoside residues). See e.g., The Biochemistry of the Nucleic Acids, pages 5-36, Adams, et al., ed., 11 th ed., 1992; and PCT pub. No. WO 93/13121.
• a “probe” is an oligonucleotide that hybridizes specifically to a target nucleic acid sequence in a nucleic acid, preferably in an amplified nucleic acid, under conditions that promote hybridization, to form a detectable hybrid.
• Probe oligonucleotides comprise one or more of a contiguous nucleotide sequence, a target hybridizing sequence, a non-target hybridizing sequence, detectable labels, linkers, and nucleotide analogs. Probes preferably have oligonucleotide lengths from about 10 contiguous nucleotides up to 100 contiguous nucleotides and comprise RNA, DNA, analogs, modified backbones, and combinations thereof.
• a probe may comprise target-specific sequences and optionally other sequences that are non-target hybridizing sequences (e.g., a sequence that does not hybridize the nucleic acid to be detected).
• non-target hybridizing sequences can include, for example, sequences that contnbute to three-dimensional conformation of the probe as are common with molecular torches and molecular beacons. See, e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, and 6,361,945.
• a probe may contain a detectable label that is either attached to the end of the probe or attached internally on the probe.
• Detectable label refers to one or more atoms that can be specifically detected to indicate the presence of a substance to which the one or more atoms is attached.
• Labels include dyes, particles, chromophores (e.g., an atom or molecule that imparts a detectable color), combinatorial fluorescence energy transfer labels, electrophores, redox active moieties (e.g., transition metals), enzymes, haptens, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent moieties), fluorophores, mass labels, and radiolabels. Labels and related detections methods are well known. See, e.g., Styer and Haugland, (1967), Proc.
• fluorophore means a fluorescent chemical compound that can re-emit light upon light excitation
• Fluorophores include, for example, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, CalFluor RedTM, CalFluor OrangeTM, stilbene, Quasar dyes (e.g., Quasar 570, Quasar 670, Quasar 705), Lucifer Yellow, Cascade BlueTM, Texas Red, Alexa dyes, phycoerythin, Bodipy, and others known in the art.
• fluorescent lanthanide complexes including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, ery
• quencher is used to refer to a molecule that absorbs light. Quenchers are commonly used in combination with a light emitting label such as a fluorophore to absorb emitted light when in close proximity to the fluorophore. Quenchers are well-known in the art and include, e.g., Black Hole QuencherTM (or BHQTM, BHQ-1TM, or BHQ-2TM), Blackberry Quencher, Dabcyl, QSY, and TamraTM compounds, to name a few.
• Black Hole QuencherTM or BHQTM, BHQ-1TM, or BHQ-2TM
• Blackberry Quencher Dabcyl, QSY, and TamraTM compounds
• Linear probes, molecular torches, and beacons are preferably labeled with an interactive pair of detectable labels.
• detectable labels that are preferred as members of an interactive pair of detectable labels interact with each other by FRET or non- FRET energy transfer mechanisms, often referred to as “donor/acceptor pairs” wherein the donor initially absorbs and then transfers the energy, and the acceptor is the moiety to which the energy is subsequently transferred.
• donor/acceptor pairs wherein the donor initially absorbs and then transfers the energy, and the acceptor is the moiety to which the energy is subsequently transferred.
• donor/acceptor pairs include pairing a fluorophore such as fluorescein, IAEDANS, EDANS, coumarin, BODIPY FL, BODIPY, lucifer yellow, eosin, erythrosine, tetramethylrhodamine, CalOrange, CalRed, Quasar, Texas Red, CY5, or CY3 with a quencher such as tetramethylrhodamine, fluororescein, DABCYL, BHQ-1, BHQ- 2, QSY7, or BBQ. Labels are available from LGC Biosearch Technologies (Petaluma, CA); Glen Research (Sterling, VA); Integrated DNA Technologies (Skokie, IL); Thermo Fisher (Waltham, MA); and others.
• a fluorophore such as fluorescein, IAEDANS, EDANS, coumarin, BODIPY FL, BODIPY, lucifer yellow, eosin, erythrosine
• An “amplification primer” or “primer” is an optionally modified oligonucleotide that hybridizes to a target nucleic acid sequence, or its complement, and can participate in a nucleic acid amplification reaction.
• Primer oligonucleotides comprise one or more of a contiguous nucleotide sequence, a target hybridizing sequence, a non-target hybridizing sequence, linkers, and nucleotide analogs.
• Primers preferably have oligonucleotide lengths from about 10 contiguous nucleotides up to 100 contiguous nucleotides.
• the target nucleic acid sequence of a primer generally refers to both a sequence (or its complement) contained within the genetic information of an organism to be detected and a sequence (or its complement) contained within an amplified nucleic acid molecule that hybridizes specifically to at least a portion of the primer oligonucleotide using standard hydrogen bonding.
• Primers hybridize to a target nucleic acid sequence and typically have a 3' end that can be extended by a DNA polymerase (e.g., promoter providers are a type of primer that do not have an extendable 3' end; see, e.g., U.S. Pat. No.
• capture oligonucleotide is meant at least one nucleic acid oligonucleotide that allows for joining of a target nucleic acid and an immobilized oligonucleotide due to base pair hybridization (preferably resulting in an immobilized probe: capture oligonucleotide:target nucleic acid complex).
• a capture oligonucleotide preferably includes two binding regions: a target nucleic acid-binding region and an immobilized probe-binding region, usually contiguous on the same oligonucleotide, although the capture oligonucleotide may include a target nucleic acid-binding region and an immobilized probe-binding region that are present on two different oligonucleotides joined together by one or more linkers.
• the target hybndizing region of a capture probe can be specific for the target nucleic acid (e.g., sufficiently complementary to the target nucleic acid sequence) or non-specific for the target nucleic acid.
• Target capture systems that include a capture oligonucleotide are described in U.S. Pat. Nos. 6,110,678, WO 2021/097358, and 9,051,601.
• immobilized probe or “immobilized nucleic acid” is meant a nucleic acid that joins, directly or indirectly, a capture oligonucleotide to an immobilized support.
• An immobilized probe is an oligonucleotide joined to a solid support that facilitates separation of bound target nucleic acids from unbound material in a sample.
• solid support means any suitable medium present in the solid phase to which an immobilized probe or other agent can be covalently or non-covalently affixed or immobilized.
• separating or “purifying” or “isolating” is meant that one or more components of the biological sample are removed from one or more other components of the sample.
• Sample components include nucleic acids in a generally aqueous solution phase that can also include other materials, for example, proteins, carbohydrates, lipids, and labeled probes.
• the separating, isolating, or purifying step removes at least about 70%, more preferably at least about 90% and, even more preferably, at least about 95% of the other components present in the sample.
• Amplicon refers to a DNA and/or RNA polynucleotide that is the product of a nucleic acid amplification or replication process. It can be formed using various methods, including polymerase chain reaction (PCR) and transcription-associated amplification (e.g., TMA).
• PCR polymerase chain reaction
• TMA transcription-associated amplification
• the term “multiplex,” when used to describe a PCR or TMA amplification reaction, is characterized in that two or more different amplification products, or amplicons, are generated by means of using two or more pairs of amplification primers in the same amplification reaction.
• target nucleic acid sequence (also referred to as “target nucleotide sequence,” “target sequence,” “target region,” “target nucleic acid molecule”) is meant a specific deoxyribonucleotide or ribonucleotide molecule or nucleotide sequence comprising all or part of the nucleotide sequence of a single-stranded nucleic acid molecule, and the deoxyribonucleotide or ribonucleotide sequence complementary thereto.
• Amplification Reagent is a Tris-buffered solution (pH 7-8) that includes magnesium chloride, potassium chloride, four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), four ribonucleotide triphosphates (rNTPs: ATP, CTP, GTP, and UTP), and one or more primers.
• an AMP Reagent contains one or more primers configured to hybridize to each target nucleic acid to be amplified and/or detected.
• Promoter Reagent is a Tris buffered solution that includes magnesium chloride, potassium chloride, four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), four ribonucleotide triphosphates (rNTPs: ATP, CTP, GTP, and UTP), one or more promoter primers, and one or more probes.
• the promoter primer in the Promoter Reagent targets the same target nucleic acid as the promoter primer in the TCR.
• the promoter primer may have the same sequence as the promoter primer in the TCR or it may have a sequence that is different from the promoter primer in the TCR.
• RNA polymerase can be, but is not limited to, T7 RNA polymerase.
• Enzyme Reagents used in amplification or pre-amplification reaction mixtures are HEPES- buffered solutions (pH 6.5-8) that include MMLV reverse transcriptase, T7 RNA polymerase, salts, and cofactors.
• compositions including kits and reagents, and methods for selectively detecting nucleic acids of various viral pathogens, specifically, influenza A (Flu A), influenza B (Flu B), S ARS-CoV-2, respiratory syncytial virus A (RSV A), and respiratory syncytial virus B (RSV B), in a sample.
• influenza A influenza A
• influenza B influenza B
• S ARS-CoV-2 respiratory syncytial virus A
• RSV A respiratory syncytial virus A
• RSV B respiratory syncytial virus B
• any primer and probe sequences specific for Flu A, Flu B, SARS-CoV-2, RSV A, RSV B and/or other pathogenic viral target may be used as primers or probes in any suitable primer/probe-based in vitro nucleic acid amplification method adapted for amplification of an intended target nucleic acid.
• the amplification primers are useful as components of singleplex or multiplex amplification reactions wherein amplicon species can be produced from target-specific primers in the reaction mixture.
• a multiplex amplification reaction includes pnmer pairs for amplifying two or more of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B, or additionally includes primers for one or more of Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B and one or more additional targets (e.g., human metapneumovirus, rhinovirus, adenovirus, parainfluenza virus, non-SARS-CoV-2 coronavirus, and/or Bordetella).
• additional targets e.g., human metapneumovirus, rhinovirus, adenovirus, parainfluenza virus, non-SARS-CoV-2 coronavirus, and/or Bordetella.
• Amplification methods useful in connection with the present disclosure include: Polymerase Chain Reaction (PCR); Transcription-Mediated Amplification (TMA), Nucleic Acid Sequence-Based Amplification (NASBA); Strand Displacement Amplification (SDA); and amplification methods using self-replicating polynucleotide molecules and replication enzymes such as MDV-1 RNA and Q-beta enzyme. Methods for carrying out these various amplification techniques respectively can be found in U.S. Pat. Nos. 4,965,188, 5,399,491, 5,455,166, and 5,472,840; published European patent application EP 0 525 882; and Lizardi, et al., BioTechnology' 6: 1197 (1988).
• Flu A, Flu B, RSV A, and RSV B nucleic acid sequences are amplified using real-time RT PCR or BiPhasic TMA.
• degenerate bases and non-Watson Crick (NWC) base pairing can, in some preferred embodiments, be included in a primer or probe oligonucleotide.
• a NWC position in an oligonucleotide refers to a position where the oligonucleotide is configured to hybridize to at least one target nucleic acid sequence with a non-Watson Crick pairing, such as G-U, G-T, or G-A (either the G or the U/T/A can be the base in the oligonucleotide) or using a nucleotide analog, such as G-inosine or G-5-MethylC, or .
• the NWC position is configured to hybridize via a wobble (G-U or G-T) or purine-purine (G-A) pair.
• multiple primer species or probe species may be synthesized to include all base combinations.
• Primers useful for conducting amplification reactions can have different lengths to accommodate the presence of extraneous sequences that do not participate in target binding and that may not substantially affect amplification or detection procedures.
• promoter primers useful for performing amplification reactions in accordance with the disclosure have at least a minimal sequence that hybridizes to the desired target nucleic acid sequence and a promoter sequence positioned upstream of that minimal sequence.
• insertion of sequences between the target binding sequence and the promoter sequence could change the length of the primer without compromising its utility in the amplification reaction.
• the lengths of the amplification primers and detection probes are matters of choice as long as the sequences of these oligonucleotides conform to the minimal essential requirements for hybridizing with the desired complementary target sequence.
• Hybridization assay probes useful for detecting Flu A, Flu B, SARS-CoV-2, RSV A, and RSV B nucleic acid sequences include a sequence of bases substantially complementary to the selected target nucleic acid sequence in the Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B genome (or amplicon representing the corresponding region and its flanking or surrounding regions). Such probes may optionally have additional bases outside of the targeted nucleic acid region, which may or may not be complementary to Flu A, Flu B, SARS-CoV-2, RSV A, or RSV B nucleic acid.
• Preferred probes are sufficiently homologous to the target nucleic acid to hybridize under stringent hybridization conditions corresponding to a designed amplification and detection reaction. For example, in PCR the extension and detection reactions are carried out such that an oligonucleotide would hybridize to its target nucleic acid sequence at a reaction temperature of about 60°C.
• Probes in accordance with the disclosure have sequences complementary to, or corresponding to, a pre-selected target region of a particular viral target nucleic acid targeted by the probe.
• Preferred probes have a probe sequence, which includes the target-hybridizing sequence of bases together with any base sequences that are not complementary to the nucleic acid that is to be detected, in the length range of from 10-100 nucleotides.
• TMA transcription-mediated amplification
• a T7 (promoter) primer and TCO are hybridized to the target sequence during target capture, followed by removal of excess T7 primer during a wash step prior to a first amplification reaction.
• a TCO is hybridized to the target sequence during target capture. Excess TCO may also be removed during a wash step prior to a first amplification reaction.
• Plate Setup In some embodiments, four different plates are set up for use on two automated Kingfisher devices. The reactions can also be carried out in tubes or other containers.
• Plate 1 TCR plate
• Target Capture Reagent e.g., 100 pL
• the TCO and T7 primer hybridize to target nucleic acid (e g., 400 pL sample).
• the TCO:target nucleic acid:T7 primer (pre-amplification hybrid) are captured using magnetic beads (immobilized probe on a solid support) and a magnet.
• plate 1 is placed into a heat block and heated to 60-65°C (e.g., 62°C) for 20-30 minutes followed by incubation at lower temperatures (e.g., 23°C) for 20 minutes to2 hours.
• T7 primer may be absent from the TCR mixture.
• sample is combined with TCR containing TCO and, for BiPhasic amplification, T7 primer.
• the mixture of sample and TCR is then removed from the plate or tube and beads are washed twice with wash buffer.
• Plate 2 is a deep-well plate and holds 200-500 pL/well wash buffer.
• the wash buffer contains detergent and alcohol used to wash the captured pre-amplification hybrid.
• Captured hybrid is then moved to plate 3, which contains 200-500 pL/well wash buffer and is used to provide a second wash of the captured pre-amplification hybrid.
• Captured hybrid is moved to plate 4, which contains 50 pL/well AMP reagent. Plate 4 is processed for real-time isothermal amplification and detection.
• Second Phase Amplification 25 pL of Promoter Reagent containing T7 primer and probe oligonucleotides is added to the first amplification product, and the reaction is incubated at 42-43°C for 30-60 minutes to allow formation of a second amplification product. Amplification product is detected in real time by recording fluorescent signal from the probe at regular intervals.
• a single-stranded hybridization probe for a given target is labeled with a donor/ acceptor pair of detectable labels.
• the donor e g., a fluorophore moiety
• the acceptor e.g., a quencher moiety
• the hybridization probe binds to the target DNA and is degraded by the 5'-3' exonuclease activity of the Taq polymerase during the subsequent elongation phase.
• RNA molecules typically involve first converting RNA to DNA (e.g., a “complementary” DNA or “cDNA”) using a reverse polymerase activity.
• the composition or reaction mixture typically comprises at least 2, preferably 3-8 detection probes.
• each detection probe comprises a donor/acceptor label pair.
• the detection probes each comprise a distinguishable donor/acceptor label pair.
• such a composition or reaction mixture also comprises several reagents, including one or more of the following: buffers designed for PCR, dNTPs, a template dependent DNA polymerase (preferably a thermostable DNA polymerase), and a reverse transcriptase.
• the reaction is monitored to detect stable hybridization between one or more of the distinguishably labeled probe species present in the reaction and its corresponding target nucleic acid sequence (carried in an amplicon generated using the corresponding primer pair for the particular viral (or other) pathogen to be detected). Based on whether the donor moieties from each of the different donor/acceptor pairs are detected, it can then be determined if the biological sample contains Flu A, Flu B, SARS-CoV-2, RSV A, and/or RSV B and/or such other pathogens as are targeted in the particular assay.
• kits will comprise one or more of a probe, a primer, a capture oligonucleotide, internal control oligonucleotides, other ancillary oligonucleotides, a buffer, dNTPs, DNA polymerase, reverse transcriptase, and instructions for using components of the kit (or a link to a website providing such instructions).
• amplifications were performed using a Panther Fusion instrument (Hologic, Inc.). Viral isolates used as amplification targets or controls were diluted in suitable media, e.g., Specimen Transport Media (Hologic, Inc., Cat. No. PRD-04423 or PRD-04339); Micro Test M4 Media (Remel, Inc. Cat. No. R12500), Micro Test M5 Viral Transport Medium (Remel, Inc. Cat. No. R12515), Micro Test M6 Viral Transport Medium (Remel, Inc. Cat. No. R12530), Micro Test M4RT Viral Transport Medium (Remel, Inc. Cat. No.
• suitable media e.g., Specimen Transport Media (Hologic, Inc., Cat. No. PRD-04423 or PRD-04339); Micro Test M4 Media (Remel, Inc. Cat. No. R12500), Micro Test M5 Viral Transport Medium (Remel, Inc. Cat. No. R12515), Micro Test M6 Viral Transport Medium (Remel, Inc. Cat. No. R
• a representative RT-PCR assay based on TaqMan reagent chemistry to provide for the detection of one or more target nucleic acids e.g., Flu A, Flu B, SARS-CoV-2, RSV A, and/or RSV B
• target nucleic acids e.g., Flu A, Flu B, SARS-CoV-2, RSV A, and/or RSV B
• a typical process begins by collecting a sample from a subject, for example, a nasopharyngeal swab specimen. Unless the sample is to be immediately assayed, the sample is typically placed in sealable container along with an appropriate volume of sample medium. Preferably, a Universal Internal Control (UIC) is also then added to the sample to monitor for inhibitors that may be present in the sample.
• UICC Universal Internal Control
• Nucleic acids in the sample are isolated on the automated Panther Fusion system (Hologic, Inc.), or other system such as, for example, a KingFisher Flex Magnetic Particle Processor (ThermoFisher), a MagNA Pure LC System (Roche) and a MagNA Pure Total Nucleic Acid Isolation Kit (Roche; Cat. No. 03038505001), or aNuchSENS easy MAG System (bioMerieux) and Automated Magnetic Extraction Reagents (bioMerieux). Purified nucleic acids are then added to a PCR reaction mix along with a thermostable DNA polymerase and a reverse transcriptase.
• Hologic, Inc. or other system such as, for example, a KingFisher Flex Magnetic Particle Processor (ThermoFisher), a MagNA Pure LC System (Roche) and a MagNA Pure Total Nucleic Acid Isolation Kit (Roche; Cat. No. 03038505001), or aNuchSENS easy MAG System (bio
• the reaction mix contains oligonucleotide primer pairs and targetspecific oligonucleotide probes for each of each of the target nucleic acids to be assayed, as well as a buffer containing dNTPs (dATP, dCTP, dGTP, dTTP (or dUTP)), MgCh, stabilizers, and bovine serum albumin.
• dNTPs dATP, dCTP, dGTP, dTTP (or dUTP)
• MgCh stabilizers
• bovine serum albumin bovine serum albumin
• an RNase inhibitor e.g., RNase Inhibitor II
• control nucleic acids may also be included. Such controls may be, for example, non-infectious in vitro transcribed RNA of specific viral sequences and/or non-infectious plasmid DNA containing control sequences.
• Detection probe species are often dual-labeled with a distinguishable reporter dye and a quencher (donor/acceptor) which work in conjunction to provide real-time results.
• Reverse transcription of RNA into cDNA and subsequent amplification of DNA may be performed, for example, on a Panther Fusion automated instrument (Hologic, Inc.) or a Cepheid SmartCycler II instrument (Cepheid, Sunnyvale, CA).
• the probe species anneals specifically to the target sequence of the target nucleic acid molecule (e.g., a specific region of the Flu A genome), followed by primer extension and amplification.
• the TaqMan reagent chemistry utilizes the 5'-3' exonuclease activity of the Taq polymerase to cleave the probe, thus separating the reporter dye from its quencher.
• This example describes screening experiments testing primer and probe combinations for the real-time PCR amplification and detection of coronavirus. Reactions are generally prepared and performed as presented herein and as follows.
• NP nasopharyngeal
• lysis and target nucleic acid isolation reactions are generally described in U.S. Pat. Nos. 6,110,678, WO 2021/097358, and 9,051,601.
• Target nucleic acids isolated from each clinical specimen were then eluted from the capture reaction to provide individual eluates corresponding to each of the NP swab specimens.
• Nucleic acid amplification and detection reactions were performed as follows: 5 pL of eluate from each sample condition was added to a well of a multiwall plate. Also contained within each well was 20 pL of a rehydrated real-time PCR reaction mixture. Components of the real-time PCR reaction mixture are described above and comprised an experimental PPR mix (0.6 pM of each primer at 1 x concentration and 0.4 pM of each probe at 1 x concentration).
• Primers and probes in the PPR mix for this example were SEQ ID NOs:5, 9, 14, 20, 23, 25, 26, 27, 59, 67, 68, 75, 79, 92, 101, 102, 115, 117, 118, 119, 176, 193, 239, 272, 273, and 274.
• Probes for detecting Flu A amplification products were labeled with FAM, probes for detecting Flu B amplification products were labeled with Quasar 670, probes for detecting RSV A and RSV B amplification products were labeled with Cal Orange 560, probes for detecting SARS- CoV-2 were labeled with Cal Red 610, probes for detecting internal control nucleic acids were labeled with Quasar 705, and all probes contained a BHQ-1 or a BHQ-2 quencher (labels available from LGC Biosearch Technologies, Petaluma, CA).
• This PPR mix showed a sensitivity of 100% (22/22) and a positive predictive value (compared with commercial assay) of 100% (22/22), including with the samples diluted to about 3X LoD.
• Comparable Cts for the specimen tested neat using both the experimental system and the commercial system shows that detection of clinical titers of SARS-CoV-2 was not delayed by the presence of the Flu A, Flu B, RSV A, and RSV B primers and probes.
• Cts for the dilute specimen (3X LoD) were about 1 Ct earlier than estimated (calculation not shown), indicating that detection of dilute samples is not delayed in the multiplex system.
• NP swab specimens from Flu A, Flu B, RSV A, or RSV B positive individuals were analyzed using the above described PPR mix. These NP swab samples were also tested with the Panther Fusion Flu A/B/RSV assay (Cat. No. PRD-04328), for performance comparisons. Nucleic acid extraction, amplification, and detection reactions were set up and performed as is generally described above. Ct and RFU data was collected, and the data obtained with the PPR mix was compared to the data obtained with the commercial assay. Results are presented in Table 4, below.
• the purpose of this experiment was to test a multiplex PPR mix containing a modified Flu A primer and probe combination.
• the primer and probe mixture (PPR mix) was prepared to contain SEQ ID NOs:5, 9, 14, 23, 25, 67, 68, 75, 79, 92, 101, 102, 115, 117, 118, 119, 176, 193, 239, 266, 267, 269, 272, 273, and 274.
• the target nucleic acids were prepared from a stock panel to provide 1E4, 1E3, and 1E2 copies per reaction of each target. Each serial dilution was combined with 1E4 copies/mL of HeLa cells (each condition was run in triplicate). Nucleic acid extraction and real-time amplification and detection reactions were set up and performed on a Panther Fusion device, as is generally described above. Ct and RFU data were collected and analyzed.
• a multiplex reaction was prepared to test the performance of primers and probes for amplifying and detecting two regions of a SARS-CoV-2 target nucleic acid in the presence of primers and probes for detecting influenza target nucleic acids.
• the oligonucleotides used in this experiment were as follows: in the target capture reagent were SEQ ID NOs: 310.
• T7 promoter primers in the amplification reagent were SEQ ID NOs: 305, 306, 283, and 184 (non-T7 primers); and in the promoter reagent were SEQ ID NOs: 307, 308, 193, and 309 (T7 promoter primers) and SEQ ID NOs: 301 , 302, 303, 289, and 304 (torch oligomers).
• SEQ ID NOs: 291 and 292 were used as target nucleic acids (5 copies/mL, 10 copies/mL, 20 copies/mL, 30 copies/mL, and 100 copies/mL). Negative reactions were sample transport media alone. Each reaction condition was run in replicates of twenty (20).
• the limit of detection for the multiplex reaction is 13.9 copies/mL of SEQ ID NO: 291 and 17.2 copies/mL of SEQ ID NO: 292, as determined by Probit analysis with a 95% probability.

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