Compositions for the Multiplexed Detection of Viruses

Abstract

This specification discloses compositions of matter and processes that allow the detection of RNA from coronaviruses and other RNA viruses, in particular, compositions and processes that have the capacity to detect in multiplexed form many RNA targets within individual viruses, targets from multiple viruses, and other RNA molecules that can be used as positive controls.

Claims

1. A composition of matter that comprises the DNA molecules whose sequences comprise, at their 3′-ends, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 5, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, and SEQ ID NO 51.

2. The composition of claim 1, wherein DNA molecules whose sequences comprise, at their 3′-ends SEQ ID NO 46, SEQ ID NO 5, SEQ ID NO 48, and SEQ ID NO 49 are replaced by DNA molecules whose sequences comprise, at their 3′-ends SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 52, and SEQ ID NO 53 to give a composition comprising SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 50 and SEQ ID NO 51.

3. The composition of claim 2, wherein DNA molecules whose sequences comprise, at their 3′-ends SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 71, and SEQ ID NO 72 are replaced by DNA molecules whose sequences comprise, at their 3′-ends SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 63, SEQ ID NO 64, to give a composition comprising SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 50, and SEQ ID NO 51.

4. The composition of claim 3, wherein DNA molecules whose sequences comprise, at their 3′-their 3′-ends, SEQ ID NO 48 and SEQ ID NO 49 are replaced by SEQ ID NO 43 and SEQ ID NO 44 to give a composition comprising SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 63, SEQ ID, NO 64, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 50 and SEQ ID NO 51.

5. The composition of claim 4, to which are added one or more pairs of DNA molecules whose sequences comprise, at their 3′-ends, SEQ ID NO 117 and SEQ ID NO 118 in a pair, SEQ ID NO 119 and SEQ ID NO 120 in a pair, SEQ ID NO 121 and SEQ ID NO 122 in a pair, SEQ ID NO 123 and SEQ ID NO 124 in a pair, SEQ ID NO 125 and SEQ ID NO 126 in a pair, SEQ ID NO 127 and SEQ ID NO 128 in a pair.

6. The compositions of claim 1, 2, 3, 4, or 5, to which primers therein are added tags comprising nucleotides independently selected from an artificially expanded genetic information system.

7. The compositions of claim 6, wherein said primers to which are added tags comprising nucleotides independently selected from an artificially expanded genetic information system, comprise one or more of SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96, SEQ ID NO 95, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, ID NO 121, SEQ ID NO 122, ID NO 123, SEQ ID NO 124, SEQ ID NO 125, ID NO 126, SEQ ID NO 127, ID NO 128.

8. The composition of claim 1, 2, 3, 4, or 6, to which are added one or more pairs of DNA molecules that prime on a preselected region of an RNA target that is not selected from nCoV-2019.

9. The compositions of claim 8, wherein said primers to which are added comprising nucleotides independently selected from an artificially expanded genetic information system comprise one or more of SEQ ID NO 129, SEQ ID NO 130, SEQ ID NO 131, SEQ ID NO 132, SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137, SEQ ID NO 138, SEQ ID NO 139.

10. The compositions of claim 8, wherein said primers to which are added comprising nucleotides independently selected from an artificially expanded genetic information system comprise one or more of SEQ ID NO 140, SEQ ID NO 141, SEQ ID NO 142, SEQ ID NO 143, SEQ ID NO 144, SEQ ID NO 145, SEQ ID NO 146, SEQ ID NO 147, SEQ ID NO 148, SEQ ID NO 149, SEQ ID NO 150.

11. A process for detecting an RNA target in a mixture, wherein said process comprises a polymerase chain reaction, wherein the primers in said polymerase chain reaction comprise any of compositions of claims 1-9.

12. The process of claim 11, wherein said mixture comprises a viral transport medium.

13. The process of claim 11, wherein a polymerase used in said polymerase chain reaction is known in the art as, or is found in a kit known as TaqPath™, UltraPlex 1-Step ToughMix™, GoTaq® Probe 1-Step RT-qPCR System, SuperScript™ III Platinum™, One Step PrimeScript™, Luna® Universal Probe One-Step RT-qPCR Kit, and Reliance One-Step Multiplex RT-qPCR Supermix™.

14. The process of claim 11, wherein said mixture comprises saliva swabs, environmental swabs, and/or raw nasal swabs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1. Chemical structures of the nucleotide analogs, components of a self-avoiding molecular recognition system (SAMRS) that are incorporated into primers, primers that are comprised within compositions of the instant invention. Pairs between standard nucleobases (left). Pairs between standard nucleobases and their SAMRS complements (g, c, a, and t, middle). Pairs between SAMRS nucleobases and their formal SAMRS complements (right); these do not contribute substantially to duplex stability. Su=sugar backbone.

[0020] FIG. 2. Components of an artificially expanded genetic information system (AEGIS) presently preferred for incorporation into external tags in the tagged PCR of the instant invention. Su=sugar backbone.

[0021] FIG. 3. Speculated origins of primer dimers. Without wishing to be bound by theory, possible explanations for the failure of conventional primers and probes to give easy multiplying at high sensitivity. The N1, N2, and N3 sequences are from the CDC (SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9); the E and RdRp_SARSr are from Charité (SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19). Without wishing to be bound by theory, SAMRS components (lower case g, a, and c) may disrupt the indicated interactions, and this might be the mechanism by which they eliminate primer-primer and primer-probe interactions.

[0022] FIG. 4. Melting curves of the single-plex PCR from standard primers (gray color, SEQ ID NO 13, SEQ ID NO 14) or SAMRS primers (black color, SEQ ID NO 52, SEQ ID NO 53) targeting on E gene (BEI RNA at 1000 and 100 copies per reaction).

[0023] FIG. 5. Amplification curves of CDC standard primers (black) and SAMRS primers (gray) in quadruplex PCR targeting on N1, N2, N3, and RNAse P genes. The N1, N2, N3, and RNAse P standard primer and probe sequences are from the CDC (SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12). The N1, N2, N3, and RNAse P SAMRS modified sequences are from the Firebird (SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 5, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51). Synthetic Twist RNA was served as target at 10000, 1000, 100, and 10 copies per reaction.

[0024] FIG. 6A. Linear regression of standard primers in quadruplex PCR targeting on N3, RdRp-Hel, E, and RNAse P genes (BEI RNA at 400, 200, 100, 40, 20, and 10 copies per reaction).

[0025] FIG. 6B. Linear regression of SAMRS modified primers in quadruplex PCR targeting on N3, RdRp-Hel, E, and RNAse P genes (BEI RNA at 400, 200, 100, 40, 20, and 10 copies per reaction).

DESCRIPTION OF THE INVENTION

[0026] (i)(1) Sequences Used in the Process of Discovery

[0027] To create this invention, a process of discovery began with the primers and probes that were reported in the assays collected by the WHO from various individual entities developing coronavirus kits (the US CDC, the China CDC, Institute Pasteur, Hong Kong University, Charité, collectively the “WHO primers”). These are collected in Table 1. In addition, two primers were designed by adding three and four nucleotides (respectively) to the 5′-ends of SEQ ID NO 38 and SEQ ID NO 39 to give SEQ ID NO 40 and SEQ ID NO 41 (Table 1).

[0028] This discovery process continued by replacing nucleotides A, T, G, and C within those Table 1 primers by components of a self avoiding molecular recognition system (SAMRS, FIG. 1) designated by (in bold) a, t, g, and c. Many of the SAMRS-containing primers are in Table 2.

[0029] In addition, the inventors examined the coronavirus sequences in the database, as well as the sequences of related coronaviruses, and designed their own primers based on the comparisons of these. These are collected in Table 3, when they are built from entirely natural nucleotides. The sequences with SAMRS components, as well as sequences with components of an artificially expanded genetic information system (AEGIS), are collected Table 4.

TABLE-US-00002 TABLE 2 Primers created by replacing nucleotides A, T, G, and C in primers in Table 1 with components of a self-avoiding molecular recognition system (SAMRS, FIG. 1). The replacements are designated by (in lower case bold) a, t, g, and c. from the WHO list with simple SAMRS substitutions. Name SEQ ID SEQUENCE (5′-3′) N1-Fp/US CDC SEQ ID NO 43 GAC CCC AAA ATC AGC GAa AT N1-Rp/US CDC SEQ ID NO 44 TCT GGT TAC TGC CAG TTG AAT cTG N2-Fp-aa/US CDC SEQ ID NO 45 TTA CAA ACA TTG GCC GCa aA N2-Fp-a/US CDC SEQ ID NO 46 TTA CAA ACA TTG GCC GCA aA N2-Rp/US CDC SEQ ID NO 47 GCG CGA CAT TCC GAA GaA N3-Fp/US CDC SEQ ID NO 48 GGG AGC CTT GAA TAC ACC Aaa A N3-Rp/US CDC SEQ ID NO 49 TGT AGC ACG ATT GCA GCa TTG RNAseP-Fp/US SEQ ID NO 50 AGA TTT GGA CCT GCG AGc G CDC RNAseP-Rp/US SEQ ID NO 51 GAG CGG CTG TCT CCA CAA gT CDC E_Sarbeco_Fp/ SEQ ID NO 52 ACA GGT ACG TTA ATA GTT AAT AGc gT Charité E_Sarbeco_Rp/ SEQ ID NO 53 ATA TTG CAG CAG TAC GCA CAc A Charité RdRp_SARSr-Fp/ SEQ ID NO 54 GTG ARA TGG TCA TGT GTG GCg G Charité RdRp_SARSr-Rp-S/ SEQ ID NO 55 CAR ATG TTA AAS ACA CTA TTA GCa TA Charité RdRp_SARSr-Rp-A/ SEQ ID NO 56 CAR ATG TTA AAA ACA CTA TTA GCa TA Charité N Sarbeco Fp/ SEQ ID NO 57 CAC ATT GGC ACC CGC AaT C Charité N Sarbeco Rp/ SEQ ID NO 58 GAG GAA CGA GAA GAG GcT TG Charité ORF1ab-Fp/ SEQ ID NO 59 CCC TGT GGG TTT TAC ACT TaA China CDC ORF1ab-Rp/ SEQ ID NO 60 ACG ATT GTG CAT CAG CTg A China CDC N-Fp/China CDC SEQ ID NO 61 GGG GAA CTT CTC CTG CTA gAA T N-Rp/China CDC SEQ ID NO 62 CAG ACA TTT TGC TCT CAA GcT G RdRp-Hel_Fp/ SEQ ID NO 63 CGC ATA CAG TCT TRC AGg CT Hong Kong Univ. RdRp-Hel_Rp/ SEQ ID NO 64 GTG TGA TGT TGA WAT GAC ATG gTC Hong Kong Univ. ORF1b-Fp/ SEQ ID NO 65 TGG GGY TTT ACR GGT AAc CT Hong Kong Univ. ORF1b-Rp/ SEQ ID NO 66 AAC RCG CTT AAC AAA GCA cTC Hong Kong Univ. RdRp_IP2-Fp/ SEQ ID NO 67 ATG AGC TTA GTC CTg TTG Pasteur RdRp_IP2-Rp/ SEQ ID NO 68 CTC CCT TTG TTG TGT TgT Pasteur RdRp_IP4-Fp/ SEQ ID NO 69 GG TAA CTG GTA TGA TTT cG Pasteur-original RdRp_IP4-Rp/ SEQ ID NO 70 CTG GTC AAG GTT AAT ATa GG Pasteur-original RdRp_IP4-Fp */ SEQ ID NO 71 CAAT GG TAA CTG GTA TGA TTT cG Pasteur-extended RdRp_IP4-Rp */ SEQ ID NO 72 GCC CTG GTC AAG GTT AAT ATa GG Pasteur-extendeds * indicate the 5′ of primer is extended with a few more bases (Italic).

TABLE-US-00003 TABLE 3 Oligonucleotide primers designed by the inventors by analysis of the CoV19 genome with standard nucleotides. Name SEQ ID SEQUENCE (5′-3′) Tagged N1-Fp SEQ ID NO 73 CTCGACCGCTA GAC CCC AAA ATC AGC GAA AT Tagged N1-Rp SEQ ID NO 74 CTCGACCGCTA TCT GGT TAC TGC CAG TTG AAT CTG Tagged N2-Fp SEQ ID NO 75 CTCGACCGCTA TTA CAA ACA TTG GCC GCA AA Tagged N2-Rp SEQ ID NO 76 CTCGACCGCTA GCG CGA CAT TCC GAA GAA N4-Fp/Firebird SEQ ID NO 77 CGCGATCAAAACAACGTC N4-Rp/Firebird SEQ ID NO 78 CATCTGGACTGCTATTGG Tagged N4-Fp/ SEQ ID NO 79 CTCGACCGCTA CGCGATCAAAACAACGTC Firebird Tagged N4-Rp/ SEQ ID NO 80 CTCGACCGCTA CATCTGGACTGCTATTGG Firebird N4-Probe/Firebird SEQ ID NO 81 ATACTGCGTCTTGGTTCACC MERS-1-Fp/ SEQ ID NO 82 GGGTGTACCTCTTAATGCC Firebird MERS-1-Rp/ SEQ ID NO 83 GTCCAGTTCCAGTGTAGTAG Firebird MERS-2-Fp/ SEQ ID NO 84 CACTGATGCTCCTTCAAC Firebird MERS-2-Rp/ SEQ ID NO 85 AGATGATTGACTATTGCCTCC Firebird MERS-3-Fp/ SEQ ID NO 86 CACTTCTCCAGGTCCATC Firebird MERS-3-Rp/ SEQ ID NO 87 CAGCAGCATCTTTCTTAGTG Firebird SARS-1-Fp/ SEQ ID NO 88 CCAGATGGTACTTCTATTAC Firebird SARS-1-Rp/ SEQ ID NO 89 TTGCAACCCATACGATGC Firebird SARS-2-Fp/ SEQ ID NO 90 CGTCTTGGTTCACAGCTC Firebird SARS-2-Rp/ SEQ ID NO 91 TCATCTGGACCACTATTG Firebird SARS-3-Fp/ SEQ ID NO 92 CAGTACAACGTCACTCAAGC Firebird SARS-3-Rp/ SEQ ID NO 93 CCAAAGAATGCAGAGGCAC Firebird

TABLE-US-00004 TABLE 4 Primers created by replacing nucleotides A, T, G, and C in primers in Table 3 with components of a self-avoiding molecular recognition system (SAMRS, FIG. 1). The replacements are designated by (in lower case bold) a, t, g, and c. Name SEQ ID SEQUENCE (5′-3′) Tagged N1-Fp SEQ ID NO 94 CTCPACCPCTA GAC CCC AAA ATC AGC GAa AT Tagged N1-Rp SEQ ID NO 95 CTCPACCPCTA TCT GGT TAC TGC CAG TTG AAT cTG Tagged N2-Fp SEQ ID NO 96 CTCPACCPCTA TTA CAA ACA TTG GCC GCa aA Tagged N2-Rp SEQ ID NO 97 CTCPACCPCTA GCG CGA CAT TCC GAA GaA N4-Fp/Firebird SEQ ID NO 98 CGCGATCAAAACAACgTC N4-Rp/Firebird SEQ ID NO 99 CATCTGGACTGCTATTgG Tagged N4-Fp/ SEQ ID NO 100 CTCPACCPCTA CGCGATCAAAACAACgTC Firebird Tagged N4-Rp/ SEQ ID NO 101 CTCPACCPCTA CATCTGGACTGCTATTgG Firebird Tagged N1-Fp-samrs SEQ ID NO 102 P GAC CCC AAA ATC AGC GAa AT Tagged N1-Rp-samrs SEQ ID NO 103 P TCT GGT TAC TGC CAG TTG AAT cTG Tagged N2-Fp- SEQ ID NO 104 P TTA CAA ACA TTG GCC GCA aA samrs-a Tagged N2-Fp- SEQ ID NO 105 P TTA CAA ACA TTG GCC GCa aA samrs-aa Tagged N2-Rp-samrs SEQ ID NO 106 P GCG CGA CAT TCC GAA GaA Tagged N3-Fp-samrs SEQ ID NO 107 P GGG AGC CTT GAA TAC ACC Aaa A Tagged N3-Rp-samrs SEQ ID NO 108 P TGT AGC ACG ATT GCA GCa TTG Tagged RNAseP- SEQ ID NO 109 P AGA TTT GGA CCT GCG AGc G Fp-samrs Tagged RNAseP- SEQ ID NO 110  P GAG CGG CTG TCT CCA CAA gT Rp-samrs Tagged SEQ ID NO 111  P ACA GGT ACG TTA ATA GTT AAT AGc gT E_Sarbeco_Fp- samrs Tagged SEQ ID NO 112  P ATA TTG CAG CAG TAC GCA CAc A E_Sarbeco_Rp- samrs Tagged RdRp-Hel_Fp-samrs SEQ ID NO 113  P CGC ATA CAG TCT TRC AGg CT Tagged RdRp-Hel_Rp-samrs SEQ ID NO 114  P GTG TGA TGT TGA WAT GAC ATG gTC Tagged RdRp_IP4- SEQ ID NO 115  P CAAT GG TAA CTG GTA TGA TTT cG Fp-samrs * Tagged RdRp_IP4- SEQ ID NO 116  P GCC CTG GTC AAG GTT AAT ATa GG Rp-samrs * MERS-1-Fp/ SEQ ID NO 117 GGGTGTACCTCTTAATGcC Firebird MERS-1-Rp/ SEQ ID NO 118 GTCCAGTTCCAGTGTAgTaG Firebird MERS-2-Fp/ SEQ ID NO 119 CACTGATGCTCCTTCAaC Firebird MERS-2-Rp/ SEQ ID NO 120 AGATGATTGACTATTGCcTcC Firebird MERS-3-Fp/ SEQ ID NO 121 CACTTCTCCAGGTCcaTC Firebird MERS-3-Rp/ SEQ ID NO 122 CAGCAGCATCTTTCTTAgTG Firebird SARS-1-Fp/ SEQ ID NO 123 CCAGATGGTACTTCTaTTAC Firebird SARS-1-Rp/ SEQ ID NO 124 TTGCAACCCATACGATgC Firebird SARS-2-Fp/ SEQ ID NO 125 CGTCTTGGTTCACAGcTC Firebird SARS-2-Rp/ SEQ ID NO 126 TCATCTGGACCACTaTTG Firebird SARS-3-Fp/ SEQ ID NO 127 CAGTACAACGTCACTCAaGC Firebird SARS-3-Rp/ SEQ ID NO 128 CCAAAGAATGCAGAGGcaC Firebird * indicates the 5′ of primer is extended with a few more bases (Italic).
(i)(2) Reduction to Practice. Synthetic Procedures Used in the Process of Discovery

[0030] Standard and SAMRS-containing oligonucleotides (primers) were synthesized by standard solid phase phosphoramidite synthesis. Standard phosphoramidites were dimethylformamidine-dG, Acetyl-dC, Benzoyl-dA, and unprotected dT. SAMRS phosphoramidites were unprotected g, c protected as an acetylated derivative, and a protected as a dimethylformamidine derivative. SAMRS-containing oligonucleotides were deprotected in aqueous ammonium hydroxide (28%-33% NH.sub.3 in water) at 55° C. overnight (10-12 hours). They were then purified by ion-exchange HPLC (Dionex DNAPac PA-100, 22×250 mm column), and desalted over SepPak cartridges. The purity of each oligonucleotide component of the compositions of the instant invention was analyzed by analytical ion-exchange HPLC. For compositions, SAMRS-containing oligonucleotides were purified by ion-exchange HPLC to meet a purity standard >90%.

(i)(3) Targets Used to Test Compositions of the Instant Invention

[0031] Various CoV19 materials, simulants, and human analog materials, were used as PCR targets:

[0032] 1. A plasmid from Integrated DNA Technologies (IDT, Cat #10006625) was used to simulate the viral nucleocapsid N-gene. This product contains the complete N gene.

[0033] 2. The Hs_RPP30 plasmid from Integrated DNA Technologies (IDT, Cat #10006626), contains a portion of the human RNAse P gene, was used to simulate RNAse P gene.

[0034] 3. Synthetic full-length coronavirus RNA simulant from Twist. RNA Control 1 (MT007544.1)—SKU: 102019 and (MN908947.3)—SKU: 102024. This is an RNA target covering the entire CoV19 genome

[0035] 4. Heat-inactivated whole coronavirus from BEI. This material was isolated from an oropharyngeal swab from a patient (USA-WA1/2020) and heated at 65° C. for 30 minutes. The complete genome of SARS-CoV-2 has been sequenced after the isolation (GenBank: MN985325).

[0036] 5. SARS-CoV-2 RT-qPCR extraction control (BEI Resource, NR-52350) was isolated from a patient (BEI Resource, NR-52286, USA-WA1/2020) and diluted into Homo sapiens lung carcinoma cells (A549; ATCC® CCL-185™), for use as an extraction control in qPCR assays. 6. Human RNA Control (Fisher #4307281, 50 ng/μL) serves as human RNA background and internal control of the RNAse P gene.

(i)(4) Presently Preferred Samples

[0037] Without limitation, standard human specimens may be used with the compositions of the current invention. These include samples obtained from individuals by swabbing the nose or mouth. The swab may then be placed in a tube that may be filled with liquid (media) that maintains the sample for transport to the lab. RNA may be recovered from any of commercial available purification kits in a final volume of 30-50 μL. A portion (5 μL or 10 μL) of this “purified” RNA sample is added to a 20 μL or 25 μL of PCR assay.

(i)(5) Processes Used in the Process of Discovery with Singleplexed PCR, and Presently Preferred in the Use of Compositions of the Instant Invention.

[0038] SAMRS is used in the art in standard PCR, where intercalation dye (e.g. EvaGreen), TaqMan probes, or gel electrophoresis are commonly used. To test the compositions of the instant invention, they were used in a TaqMan architecture. Table 5 lists the reagents that are presently preferred. The TaqPath™ 1-Step RT-qPCR Master Mix can be replaced by the 4× enzyme mixture of the Quantabio UltraPlex™ 1-Step ToughMix® (Quantabio, 95166-01K) or replaced by the One Step PrimeScript™ III RT-PCR Kit (Takara Bio, RR600B), which is our presently preferred enzyme system.

TABLE-US-00005 TABLE 5 RT-PCR Enzyme Master mix Options Vendor Enzyme Mastermix Catalog No. ThermoFisher TaqPath ™ 1-Step RT-qPCR A15299 Master Mix, CG (4x) Quantabio UltraPlex 1-Step ToughMix ™ (4X) 95166-01K Promega GoTag ® Probe 1- Step A6121 RT-qPCR System (2x) ThermoFisher SuperScript ™ III Platinum ™ 11732088 One-Step qRT-PCR Kit (2x) Takara Bio One Step PrimeScript ™ III RR600B RT-PCR Kit (2x) New England Luna ® Universal Probe One-Step E3006X Biolab RT-qPCR Kit (2x) Bio-Rad Reliance One-Step Multiplex 12010220 RT-qPCR Supermix ™

[0039] The ability of various primers to support PCR was measured by real time PCR, including PCR whose results were quantitated by dye intercalation (e.g. EvaGreen), and by TaqMan style assays. SAMRS is used in the art in standard PCR by both TaqMan and intercalation dye.

[0040] Metrics for PCR performance included Ct, the number of cycles of PCR required to cross a threshold. Ct indicates the efficiency of amplification, with lower Ct values corresponding to higher efficiency. The signal at the end of the amplification was also used as a metric; higher signals are preferred, as they indicate that less of the PCR resources were diverted to off-target products. Finally, sensitivity (or limit of detection, LOD) was metricked by determining levels of targets that gave acceptable Ct values. A Ct of 40 or more is considered to be a “failed” assay. A series of monoplexed RT-PCR TaqMan experiments were performed to metric the ability of various standard primers and probes to support PCR amplification. In general, a total assay volume (20 μL) contained 4× master reaction mixture (5 μL, TaqPath™ 1-Step RT-qPCR Master Mix, ThermoFisher, A15299), forward and reverse primers (1.0-0.1 μM, final concentration), probe (0.05-0.3 μM), and RNA sample (5 μL). RT-PCR experiments were conducted on a Roche LightCycler® (models 96 or 480) with reverse transcription initiated at 53° C. for 5-10 min. Then, reverse transcriptase was inactivated at 95° C. for 0.5-2 min, and 40-50 cycles of PCR amplification were performed with (denaturing at 95° C. for 2-10 seconds and annealing/extending at 56-60° C. for 20-40 seconds). A representative sample of individual assays are described in individual examples. Results are collected in Table 6.

TABLE-US-00006 TABLE 6 Ct of standard primers using TaqMan PCR Ct of standard primers using TaqMan PCR RNA copies/assay 10000 1000 100 10 N1 US CDC 27.0 30.5 34.5 36.5 SEQ ID NO 1, SEQ ID NO 2 N2 US CDC 27.7 31.0 35.6  35.6* SEQ ID NO 4, SEQ ID NO 5 N3 US CDC 28.0 31.0 35.3  36.2* SEQ ID NO 7, SEQ ID NO 8 RNAse P 28.8 28.7 28.9 28.3 SEQ ID NO 10, SEQ ID NO 11 E WHO 27.1 31.4 34.5 37.0 SEQ ID NO 13, SEQ ID NO 14 RdRp WHO 32.1 36.7 NA NA SEQ ID NO 16, SEQ ID NO 17 N WHO 28.9 32.6 36.2 NA SEQ ID NO 20, SEQ ID NO 21 ORF1ab CCDC 26.6 30.0  34.4* NA SEQ ID NO 23, SEQ ID NO 24 N CCDC 27.6 31.1 36.2  36.4* SEQ ID NO 26, SEQ ID NO 27 RdRp/Hel HK 29.2 32.8 36.2  37.6* SEQ ID NO 29, SEQ ID NO 30 ORF1b HK 29.7 32.8  36.4*  37.3* SEQ ID NO 32, SEQ ID NO 33 RdRp IP4 France 28.1 30.8 35.0 NA SEQ ID NO 38, SEQ ID NO 39 Two repeats for each assay. *indicate 1/2 gave signal. NA = No Amplification.
(i)(5)(A) SAMRS-Containing Primers Often Performed Worse or Failed Entirely in PCR

[0041] Initial experiments with primers containing SAMRS generally did not yield improved results, and in many cases yielded worse results than the standard primers; occasionally replacing standard nucleotides by SAMRS nucleotides caused failures. These are exemplified by three examples that targeted the N2 gene using primers recommended by the CDC, targeted the N4 gene by primers designed by the inventors, and targeted the RdRp-IP4 gene using primers from Pasteur.

[0042] For example, standard A was replaced by SAMRS a at positions 18 and 19 of the forward primer for the N2 gene (SEQ ID NO 4, from the CDC set) to give SEQ ID NO 45, and at position 17 of the reverse primer for the N2 gene (SEQ ID NO 5, from the CDC set) to give SEQ ID NO 47. The changes caused the Ct to worsen from 28.0 to 35.6 with 10000 copies of target (Table 7), and caused Ct of the amplification at 1000 copies to fall from Ct=31.6 to 40.8. A Ct >40 is considered a failure for 1000 copies of target per PCR (Table 7).

TABLE-US-00007 TABLE 7 Ct of TaqMan PCR using standard or SAMRS primers show that SAMRS often damages the performance of PCR targeting on N gene. Ct values of SAMRS vs standard primers in Ct values of SAMRS vs standard primers in single-plex PCR single-plex PCR Primer without Primer with tag sequence tag sequence RNA Target copies/reaction RNA Target copies/reaction Primer Types 10000 1000 NTC Primer Types 10000 1000 NTC N2 standard 28.0 31.6 N2 standard primers 29.9 32.1 primers with tag SEQ ID SEQ ID NO 4, NO 75, SEQ ID NO SEQ ID NO 5 76 N2 SAMRS 35.6 40.8 N2 SAMRS primers 31.2 34.9 primers with tag SEQ ID NO 45, SEQ ID NO 96, SEQ ID NO 47 SEQ ID NO 97 N4 standard 29.1 32.6 N4 standard primers 30.8 33.8 primers with tag SEQ ID NO 77, SEQ ID NO 79, SEQ ID NO 78 SEQ ID NO 80 N4 SAMRS 35.9 40.9 N4 SAMRS primers 32.8 36.6 primers with tag SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 99 SEQ ID NO 101

[0043] As a second example, N4 primers targeting the N gene were designed by the inventors using available knowledge in the art. For example, PCR with the standard N4 primers (SEQ ID NO 77 and SEQ ID NO 78, Table 3) gave amplifications with Ct values of 29.1 and 32.6 for 10000 and 1000 copies of target per reaction, respectively (Table 7). When standard G at positions 16 and 17 of N4 primers were replaced by SAMRS g to give SAMRS modified primers (SEQ ID NO 98 and SEQ ID NO 99, Table 4), the Ct values worsened to 35.9 and 40.9, the second considered to be failed amplification (Table 7).

[0044] As a third example, the Institute Pasteur offered two different primer pairs that targeted the RNA-dependent RNA polymerase (RdRp) gene (SEQ ID NO 35 and SEQ ID NO 36, and SEQ ID NO 38 and SEQ ID NO 39, respectively, Table 1). Experiments discovered that the sensitivity from the second pair of standard primers was better than the sensitivity from the first, which was therefore set aside. Then, C at position 18 replaced in SEQ ID NO 38 was replaced by SAMRS c to give SEQ ID NO 69, and the A at position 18 in SEQ ID NO 39 was replaced by SAMRS a to give SEQ ID NO 70 (Table 2). In single-plex PCR, the SEQ ID 69 and SEQ ID NO 70 primers produced lower PCR efficiency by ˜5 cycles relative to the standard primers in TaqMan PCR (Table 8). The SAMRS-containing (SEQ ID NO 69 and SEQ ID NO 70) failed to give signals in the PCR with EvaGreen (Table 8).

TABLE-US-00008 TABLE 8 Ct of TaqMan PCR using standard or SAMRS primers show that SAMRS often damages the performance of PCR targeting on RdRp gene. Comparing the performance of SAMRS primers to Pasteur standard primers Detection Method TaqMan Probe (detected EvaGreen (detected with with Hex setting) FAM setting) BEI viral RNA copies/reaction 1000 100 NTC 1000 100 NTC RdRp-IP4_Std 32.6 36 NS 38.9 40.1 40.0 SEQ ID NO 38, (100% SEQ ID NO39 dimer) RdRp-IP4_Std * 32.6 35.8 NS 34.5 37.1 41.22 SEQ ID NO 40, (50% SEQ ID NO 41 dimer) RdRp-IP4_samrs 37.6 40.4 NS NS NS NS SEQ lD NO 69, SEQ ID NO70 RdRp-IP4_samrs * 31.4 35.1 NS 34.7 37.2 NS SEQ ID NO 71, SEQ ID NO72 * indicate the 5′ of primer is extended with a few more bases (Table 1 and Table 2). NTC = No Target Control. NS = No Signal.

[0045] In some cases, the impact of adding SAMRS worsened performance without delivering failures. For example, standard A's were replaced in the N2 forward tagged primer (SEQ ID NO 75, Table 3) at positions 29 and 30 by SAMRS a to give SEQ ID NO 96 (Table 4), and at position 28 in the N2 reverse tagged primer (SEQ ID NO 76) to give SEQ ID NO 97. With 10000 copies, Ct worsened from 29.9 to 31.2; with 1000 copies, Ct worsened from 32.1 to 34.9 (Table 7). Likewise, when standard G's at positions 27 and 28 in the N4 standard tagged primers (SEQ ID NO 79 and SEQ ID NO 80, Table 3) were replaced by SAMRS g to give SEQ ID NO 100 and 101 (Table 4), the Ct values worsened from 30.8 and 33.8 to 32.8 and 36.6 (Table 7).

(i)(5)(B) Experiments were Done to Invent the Instantly Claimed Compositions

[0046] These results prompted a series of experiments to find useful combinations of SAMRS nucleotides, standard nucleotides, their positions, and the lengths of primers that contain them. For example, in some cases, improvement was seen by simply leaving out a SAMRS component. For example, to obtain useful N2 primers, a single A was replaced in the N2 forward primer (SEQ ID NO 4, Table 1) at site 19 by a single SAMRS a to give (SEQ ID NO 46, Table 2). Then, amplification was tested with a reverse primer that lacked any SAMRS component (SEQ ID NO 5). This pair of primers was shown to give approximately the same level of performance as the standard primers in single-plex PCR (Table 9).

TABLE-US-00009 TABLE 9 Ct of TaqMan PCR using standard N2 primers or primers with few SAMRS modification in single-plex PCR. Ct values of SAMRS vs standard primers in single-plex PCR RNA target copies/reaction 10000 1000 NTC Standard N2 forward and 26.8 30.3 standard N2 reverse primers SEQ ID NO 4, SEQ ID NO 5 SAMRS N2 forward primer and 27.0 30.6 standard N2 reverse primer SEQ ID NO 46, SEQ ID NO 5

[0047] In some cases, useful primers were obtained by lengthening the primers. Without wishing to be bound by theory, the melting temperatures (T.sub.ms) of the standard RdRp-IP4 primers (SEQ ID NO 38 and SEQ ID NO 39, Table 1) are ˜2° C. lower than the optimal 60° C. for TaqMan PCR. Here, for the Institute Pasteur primers recommended to target the RdRp-gene, four nucleotides (CAAT) were added to extend the 5′-end of the forward primer (SEQ ID NO 38) and three nucleotides (GCC) were added to extend the 5′-end of the reverse primer of the reverse primer (SEQ ID NO 39) to give extended primers for the RdRp target (SEQ ID NO 40 and SEQ ID NO 41); the sequences of these extensions were chosen to allow the 4 and 3 added nucleotides to be Watson-Crick complementary to the CoV19 consensus sequences. Separately, the same extensions were added to the SAMRS-containing SEQ ID NO 69 and SEQ ID NO 70 to give SEQ ID NO 71 and SEQ ID NO 72 (Table 2).

[0048] The performance of standard SEQ ID NO 38 and SEQ ID NO 39 was then compared with the performance of extended SEQ ID NO 40 and SEQ ID NO 41; the performance of SAMRS-containing SEQ ID NO 69 and SEQ ID NO 70 was then compared with the performance of SAMRS-containing extended SEQ ID NO 71 and SEQ ID NO 72. In single-plex PCR, the extended standard primers (SEQ ID NO 40 and SEQ ID NO 41) were more efficient than original standard primers SEQ ID NO 38 and SEQ ID NO 39 in PCR using TaqMan probe (Table 8, left) and in PCR with EvaGreen (Table 8, right). However, in the absence of target, SEQ ID NO 38 and SEQ ID NO 39 generated primer dimer in all duplicate experiments, while the extended primers SEQ ID NO 40 and SEQ ID NO 41 produced dimer in half of the duplicate experiments (Table 8, right).

[0049] In contrast, the SAMRS modification with lengthening rescued both standard RdRp-IP4 primers (SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, and SEQ ID NO 41, with or without extended sequences). The extended SAMRS primers (SEQ ID NO 71 and SEQ ID NO 72, RdRp-IP4_samrs*) gave faster and cleaner PCR than the standard primers, and produced no primer dimer in NTC (Table 8). Here was a useful advantage of SAMRS, but only with extended primers.

[0050] In some cases, the negative impact of SAMRS components was found to be mitigated by adding an external tag to the primer. This extension is not complementary to the coronaviral genes, but rather serves as a place where external primers can bind after PCR is initiated to “carry” the PCR. Thus, they can be any sequences. They may even incorporate components of an artificially expanded genetic information system (AEGIS) (FIG. 2).

[0051] Artificially expanded genetic information systems (AEGIS) are analogs of DNA and RNA (collectively xNA) that contain additional nucleotide pairs that recognize each other by an extended set of Watson-Crick complementarity rules. In standard DNA, the A: T and G:C fall two rules of complementarity: (a) size complementarity, where big purines pair with small pyrimidines, and (b) hydrogen bond complementarity, where hydrogen bond donors pair with hydrogen bond acceptors (FIG. 2). The added AEGIS still follow these two rules of Watson-Crick complementarity, but with rearranged hydrogen bond donor/acceptor groups. In principle, 12 building blocks form 6 orthogonal pairs are possible in AEGIS. The added eight nucleotide analogues forming four orthogonal pairs can complement nothing in natural biology, and therefore are highly orthogonal tags for tagged PCR.

[0052] Experimentation discovered that adding standard or AEGIS-containing tags to the primers allowed SAMRS-containing compositions of the instant invention to still better (Table 7). For example, in the N2 standard primers with tags (SEQ ID NO 75 and SEQ ID NO 76, Table 7, right) slow the tagged PCR by ˜1.7 cycles than the N2 standard primers without tags (SEQ ID NO 4 and SEQ ID NO 5, Table 7, left). In contrast, the N2 SAMRS primers with tags (SEQ ID NO 96 and SEQ ID NO 97, Table 7, right) speeded PCR by ˜5.8 cycles than N2 SAMRS primers without tag (SEQ ID NO 45 and SEQ ID NO 47, Table 7, left). This discovery was further demonstrated by the N4 standard and SAMRS primers with or without tag. The N4 standard primers with tag (SEQ ID NO 79 and SEQ ID NO 80, Table 7, right) slow the PCR by ˜1.5 cycles than N4 standard primers without tag (SEQ ID NO 77 and SEQ ID NO 78, Table 7, left). In contrast, the N4 SAMRS primers with tags (SEQ ID NO 100 and SEQ ID NO 101, Table 7, right) speeded PCR by ˜3.7 cycles than N4 SAMRS primers without tag (SEQ ID NO 98 and SEQ ID NO 99, Table 7, left).

[0053] In some cases, useful primers were obtained by introducing SAMRS components into sites that were speculated to problematically self-associate. For example, while not wishing to be bound by theory, analysis of the sequences of the primer/probe set of E gene from Charité suggested that the Charité E gene forward primer (SEQ ID NO 13, Table 1) might be susceptible to the formation of a self-dimer, and that the Charitè reverse primer for the E gene (SEQ ID NO 14, Table 1) can form dimers with primers targeting other genes (FIG. 3). We therefore explored various combinations of SAMR and standard nucleotides. This discovered that adding SAMRS components at specific sites to the forward and reverse primers of E gene (SEQ ID NO 52 and SEQ ID NO 53, Table 2) increased their performance. In several cases, this did not damage their singleplexed performance. For example, in singleplexed PCR, Charité E gene primers with one or two SAMRS components gave singleplexed PCR with approximately the same Ct (±0.3 cycles) as standard primers (Table 10). However, SAMRS primers gave stronger fluorescence intensity of the amplification curves than standard primers (FIG. 4). Further, at 100 copies of target per reaction, one third of the Charité standard primers produced primer dimer (Tm at ˜74° C.) and artifacts (Tm at ˜83° C.) in addition to the desired amplicon (Tm at 80° C., FIG. 4). SAMRS primers generated more desired products with amplicon Tm at ˜79.6° C. (FIG. 4), as shown by EvaGreen dye fluorescence and melting curve analysis (FIG. 4).

TABLE-US-00010 TABLE 10 Modification of the Charité's standard primers with SAMRS components improve the performance of single-plex PCR targeting on E gene. Comparing the performance of SAMRS primers to Charité's standard primers on E gene TaqMan Probe EvaGreen (detected with (detected Texas Red with FAM Detection Method setting) setting) BEI viral RNA copies/reaction 1000 100 1000 100 standard forward and standard 30.3 33.7 29.4 33.2* reverse primers, SEQ ID NO 13, SEQ ID NO 14 SAMRS forward and reverse primers, 30.4 34.0 29.4 33.1  SEQ ID NO 52, SEQ ID NO 53 Each assay has 3 repeats. *Standard primers form primer dimer (1 out 3 repeats) at low RNA concentrations (100 copies/assay).

[0054] These and other experiments are detailed in the examples. In many cases, SAMRS-containing primers have the same levels of efficiency as (Ct within ±0.5 cycles) the corresponding “well designed” standard primers in single-plexed PCR. Specifically: for the N1, N3, and RNAseP genes, SAMRS primers with SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, and SEQ ID NO 51 (Table 2), have the same or slightly better sensitivity (10 copies/reaction, Table 11) than the standard primers with SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, and SEQ ID NO 11 (Table 1).

TABLE-US-00011 TABLE 11 Results of comparing the performance of SAMRS primers to CDC standard primers in single-plex PCR targeting on N1, N2, N3, and RNAse P genes. Standard Primers or SAMRS Primers Ct values of single-plex PCR RNA Target Copies / reaction 10000 1000 100 10 NTC N1 Std Primer 26.9 30.7 33.6 36.1* SEQ ID NO 1, SEQ ID NO 2 N2 Std Primer 27.7 31.2 34.6 NA SEQ ID NO 4, SEQ ID NO 5 N3 Std Primer 27.9 31.0 34.1 NA SEQ ID NO 7, SEQ ID NO 8 RNAseP Std Primer 24.5 24.6 24.6 24.9  SEQ ID NO 10, SEQ ID NO 11 N1 SAMRS Primer 27.2 30.7 34.4 36.5* SEQ ID NO 43, SEQ ID NO 44 N2 SAMRS Primer 27.8 31.6 35.2 NA SEQ ID NO 46, SEQ ID NO 5 N3 SAMRS Primer 27.5 30.6 33.6 36.9* SEQ ID NO 48, SEQ ID NO 49 RNAseP SAMRS 23.8 24.0 23.9 24.2  SEQ ID NO 50, SEQ ID NO 51 Std indicates standard. Two repeats for each assay. *indicate 1/2 give signals. NA = No Amplification. RNAse P target was 1000 copies per reaction for all assays.

[0055] In the Hong Kong primer and probe sets that target the RdRp/Hel gene, the SAMRS-containing primers (SEQ ID NO 63 and SEQ ID NO 64) and the standard primers (SEQ ID NO 29 and SEQ ID NO 30) separately performed equally well in PCR with both the TaqMan readout and the EvaGreen readout in single-plex PCR.

(i)(5)(C) the Presently Preferred Compositions for Singleplexed PCR

[0056] These experiments delivered the presently preferred primers containing SAMRS that modified primers presented in the WHO collection. In summary:

[0057] To target the N1, N3, and RNAseP genes in a singleplexed format, SAMRS-containing primers with SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, and SEQ ID NO 51 are presently preferred. They have the approximately same levels of sensitivity (10 copies/assay) as their standard analogs, which are SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, and SEQ ID NO 11, respectively.

[0058] To target the N2 gene in a singleplexed format, SAMRS primers with SEQ ID NO 46 and SEQ ID NO 5.

[0059] To target the E gene in a singleplexed format, the presently preferred SAMRS primers are SEQ ID NO 52 and SEQ ID NO 53.

[0060] To target the RdRP gene in a singleplexed format, the presently preferred SAMRS primers are SEQ ID NO 63 and SEQ ID NO 64, or SEQ ID NO 71 and SEQ ID NO 72.

(i)(6) Experiments were Done to Invent the Compositions for Multiplexed PCR

[0061] This and other work with singleplexed PCR with primers including SAMRS supported the next level of experimentation. Here, SAMRS-containing primers developed as inventive compositions that performed adequately (or better) in singleplexed PCR performed well in multiplexed PCR. This contrasted with the standard primers, which frequently failed to perform in a multiplexed assay, even if they successfully performed in a singleplexed assay.

[0062] For example, the ability of the SAMRS-containing primers combined to support quadruplex TaqMan PCR was compared to the corresponding standard primers. Both quadraplex PCR targeted on the N, E, RdRp, and RNAse P genes. The RT-PCR (20 μL) contained 5 μL of 4× master reaction mixture (TaqPath™ 1-Step RT-qPCR Master Mix), 0.5 μM of forward and reverse primers, 0.125 μM of probe, and 5 μL of RNA sample. RT-PCR reactions were conducted on a thermal cycler (Roche LightCycler® 96 or 480) with the following conditions: Reverse transcription at 53° C. for 10 min, inactivation of reverse transcriptase at 95° C. for 2 min, 45-50 cycles of PCR amplification (denaturing at 95° C. for 3 s; annealing/extending at 58° C. for 30 s). Fluorescence was detected in real time during each annealing-extension cycle. LightCycler 96 or 480 software was used to obtain a cycle threshold (Ct).

[0063] The first quadruplex PCR experiments shown that if the CDC primers (SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11) for all four targets (N1, N2, N3, and RNAse P genes) without SAMRS were combined together in one PCR (Table 12). At low concentrations of target (10 copies/reaction), the N1 and N3 genes could not be amplified at all (Table 12). Further, the N2 target dropped out in half of the assays. However, the analogous primers containing SAMRS (SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 5, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51) could reliably amplify for detection the N1 and N3 targets; only the N2 gene experienced occasional dropout at these low target concentrations (Table 12).

TABLE-US-00012 TABLE 12 Results of comparing the performance of SAMRS primers to CDC standard primers in quadruplex PCR targeting on N1, N2, N3, and RNAse P genes. Standard Primers or SAMRS Primers RNA Target copies/ Ct values of Quadruplex PCR reaction 10000 1000 100 10 NTC N1 Std Primer 28.1 31.2 34.8 NA SEQ ID NO 1, SEQ ID NO 2 N2 Std Primer 27.9 31.1 33.3 34.3* SEQ ID NO 4, SEQ ID NO 5 N3 Std Primer 28.3 31.4 34.4 NA SEQ ID NO 7, SEQ ID NO 8 RNAseP Std Primer 21.6 24.6 28.4 31.6  SEQ ID NO 10, SEQ ID NO 11 N1 SAMRS Primer 27.7 30.8 34.6 35.5  SEQ ID NO 43, SEQ ID NO 44 N2 SAMRS Primer 27.9 31.4 33.6 34.4  SEQ ID NO 46, SEQ ID NO 5 N3 SAMRS Primer 27.9 31.3 34.8 37.5* SEQ ID NO 48, SEQ ID NO 49 RNAseP SAMRS 21.2 24.0 28.1 31.8  SEQ ID NO 50, SEQ ID NO 51 NA = No Amplification. *indicates 1/2 assay give signal.

[0064] At higher target concentrations (100 copies/reaction and above), the CDC primers for all four targets without SAMRS could detect all of the targets without drop-out (Table 12). However, the SAMRS primers produced faster (˜0.3 cycles) amplification and higher amplification curves than the standard CDC primers in the multiplex (FIG. 5). While not wishing to be bound by theory, the improve performance may be due to SAMRS preventing the loss of amplification resources into unproductive modes.

[0065] The improvements created by SAMRS became increasingly manifest in another quadruplex PCR. For example, both standard primers (SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 10, SEQ ID NO 11) and SAMRS primers (SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 50, SEQ ID NO 51) of the N1, RdRp-IP4, E, and RNAseP show similar efficiency and sensitivity at higher target concentrations (over 100 copies/reaction, Table 13). However, at lower target concentrations (10 copies/reaction), all standard primers had over 60% dropouts, while SAMRS primers had only ˜25% of dropouts (Table 13).

TABLE-US-00013 TABLE 13 Results of comparing the performance of SAMRS primers to standard primers (Ni, RdRp-IP4, E, and RNAse P) in quadruplex RT-PCR. Quadruplex PCR targeting on N, RdRp, E, and RNAse P genes Primer Types Standard primers SAMRS primers BEI viral RNA copies/reaction 1000 100 10 NTC 1000 100 10 NTC N1 target 30.9 33.3 NA 31.4 35.1 36.3 RdRp-IP4 target 32.0 34.0 NA 32.6 35.1 36.0* E target 31.2 33.4 36.6* 32.1 35.8 36.8* RNAse P target 28.5 28.3 27.8 28.4 28.6 28.2 *indicates 1 out of 2 repeats gave signal. NTC = No Target Control. NA = No Amplification.

[0066] Another example of a 4-plex PCR using SAMRS primers (SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 50, SEQ ID NO 51) or standard primers (SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 10, SEQ ID NO 11) that targeted the N3, RdRp-Hel, E, and RNAse P genes (Table 14). The SAMRS primers reliably detected 40 copies of RNA target per PCR reaction (20 μl) without any dropouts. At 20 and 10 copies targets per PCR, SAMRS primers gave dropouts in 19% and 50% of the trials (3 out of 16 and 8 out of 16, respectively). In contrast, standard primers gave 19% of dropouts at 100 copies of target per PCR. At 40, 20, and 10 copies of target, the standard primers gave 31%, 44%, and 63% of dropouts in the trials, respectively (Table 14).

TABLE-US-00014 TABLE 14 Results of comparing the performance of SAMRS primers to standard primers (N3, RdRp-Hel, E, and RNAse P) in quadruplex RT-PCR in Example 4. Quadruplex PCR targeting on N3, RdRp-Hel, E, and RNAse P genes Primer Types Standard primers SAMRS primers BEI viral RNA copies/reaction 400 200 100 40 20 10 400 200 100 40 20 10 N3 target 30 30.9 32.7 *** 33.9 ** 34.7 ** 34.3 * 29.7 30 31.4 33.8 33.6 34.6 ** RdRp-Hel target 34.3 34.7 35.5 *** 37.1 ** 37.3 * NA 34.3 35.1 36 37.5 38.4 ** NA E target 32.9 33.6 35.4 *** 36.7 *** 37.4 ** 37.0 * 32.9 33.8 34.6 36.6 36.5 *** 37.8 ** RNAse P target 27.5 28.5 29.3 31.2 32 33.4 26.1 28.2 29 30.3 31.4 32.4 *** indicate 3 out of 4 repeats give signals, ** indicate 2/4 give signals and * indicate 1/4 give signals. NA = No Amplification.

[0067] Further, the same SAMRS primers generated faster amplification (by ˜0.3 cycles) and higher amplification curves than standard primers in this quadruplex PCR, indicating less wastage of amplification resources. The sensitivities of these SAMRS primers over standard primers are more pronounced at lower target concentrations (less 200 copies/assay). Linear regression was performed to obtain an averaged slope of −3.32±0.29, with R.sup.2=0.97±0.02 for SAMRS primers, compared to a slope of −3.05±0.47 and R.sup.2=0.94±0.05 for standard primers (FIG. 6a and FIG. 6b). The PCR amplification using SAMRS primers is close to perfect doubling per PCR cycle, and the R.sup.2 is higher than the R.sup.2 of standard primers. These represent useful improvements across all of the panels.

[0068] This improvement was surprisingly robust even at 10-plex PCR. For example, an assay that detects CoV19 would have special utility if it were also able to detect other coronaviruses, such as the coronavirus that caused the Middle East respiratory syndrome (MERS) and the coronavirus that cause the 2003 outbreak of SARS. Accordingly, three sets of the MERS-specific primers (SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87 for MERS, Table 3) and three sets of the SARS-specific primers (SEQ ID NO 88, SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93 for SARS, Table 3) were added to the presently most preferred quadruplex PCR. When six sets of the standard primers were added, the multiplex PCR with standard primers collapsed (Table 15). However, when the SAMRS modified primers SAMRS (SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121, SEQ ID NO 122 for MERS, SEQ ID NO 123, SEQ ID NO 124, SEQ ID NO 125, SEQ ID NO 126, SEQ ID NO 127, SEQ ID NO 128 for SARS, Table 4) were added, the multiplex PCR with SAMRS primers succeeded (Table 15). At 10-plex multiplex RT-PCR, the presence of additional six pairs of standard primers killed the PCR to detect CoV19 when using the UltraPlex 1-Step ToughMix (Quanta Bio). 10×-PCR with standard primers failed to detect 2500 copies of RNA for all three targets (N1, RdRp-Hel, and E genes). In contrast, 10×-PCR with SAMRS primers can successfully detect 500 copies of RNA for all four targets. At lower target concentrations, 100 copies of RNA, 10×-PCR with SAMRS primers can still detect all target, although with some dropouts (Table 15).

[0069] When the UltraPlex 1-Step ToughMix (Quanta Bio) was replaced by the One Step PrimeScript™ III Enzyme Mix (Takara Bio), the advantage of SAMRS primers to empower a flexible multiplex PCR is further demonstrated.

TABLE-US-00015 TABLE 15 Results of comparison of the performance of SAMRS primers to standard primers in 10-plex RT-PCR that includes primer pairs selected independently that target MERS and SARS. Comparison of the performance of SAMRS primers to standard primers in 10-plex RT-PCR Enzyme type UltraPlex 1-Step ToughMix Primer Type 10-plex standard primers 10-plex SAMRS primers BEI viral RNA copies/reaction 2500 500 100 NTC 2500 500 100 NTC N1 target 28.8 28.9 29.1 29 32 33.5 34.6 RdRp-Hel target NA NA NA 30.9 31.7 31.8* E target NA NA NA 31.3 32.1 32.5* RNAseP target 27.2 28.3 NA 29.6 31.1 32.7 Each target concentration has 4 repeats. *indicate 1/4 gave signal. NA = No Amplification.
(i)(7) the Performance of these Compositions were Robust with Respect to Different Presentations of the CoV19 Target, Different Enzymes, and Different Processes

[0070] The coronavirus targets can be presented to an assay either as DNA, or as RNA, with the RNA being generated either synthetically or derived from a natural virus. The performance of the compositions of the instant invention was robust with respect to alternative choices of target presentation. The analytical sensitivity of SAMRS-containing primers in quadruplex TaqMan RT-PCR was robust when with BEI viral RNA was used as targets. Further, the performance worked with enzymes from different vendors (Table 5).

[0071] For example, the TaqPath™ 1-Step RT-qPCR Master Mix can be replaced by the 4× enzyme mixture of the Quantabio UltraPlex™ 1-Step ToughMix® (Quantabio, 95166-01K), SuperScript™ III Platinum™ One-Step qRT-PCR Kit (ThermoFisher, 11732088), One Step PrimeScript™ III RT-PCR Kit (Takara Bio, RR600B), Luna® Universal Probe One-Step RT-qPCR Kit (NEB, E3006X), and enzymes from other vendors (Table 5). Various of primer concentrations from 1.0 μM to 0.1 μM and probe concentrations from 0.05 μM to 0.3 μM were also evaluated. In General, SAMRS primers have higher sensitivity and produce higher amplification signals than the standard primers with various of enzymes from different vendors.

[0072] The presently preferred SAMRS-containing primers combined in a 4-plexed PCR targets the N1, E, RdRp-Hel Hong Kong, and RNAse P genes, and comprises SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 50, SEQ ID NO 51. Experimental data shown that UltraPlex 1-Step ToughMix (Quanta Bio) are presently preferred for their sensitivity and efficiency over TaqPath™ 1-Step RT-qPCR Master Mix (ThermoFisher). At 10 copies of BEI viral RNA per reaction, only 1 out of 12 assay has dropout for the UltaPlex ToughMix, while, 5 out of 12 assays have dropouts for the TaqPath master mix. Over all, the PCR efficiency of the UltaPlex ToughMix is ˜1 cycle faster than the TaqPath master mix.

[0073] As further experimental data shown in Table 16, One Step PrimeScript™ III Enzyme Mix (Takara Bio) offers higher sensitivity than the UltraPlex 1-Step ToughMix (Quanta Bio). At 10 copies of BEI viral RNA per reaction, all 12 assays (no dropout) successfully give signals without dropout for the PrimeScript™ III Enzyme Mix, while, 1 out of 12 assays have dropouts for the UltraPlex 1-Step ToughMix Enzyme Mix. However, UltraPlex 1-Step ToughMix has higher PCR efficiency (˜1.5 cycles faster, Table 16).

TABLE-US-00016 TABLE 16 Results of evaluating the performance of SAMRS modified quadruplex RT-qPCR (N1, RdRp-Hel, E, and RNAse P) using Enzymes from different vendors. SAMRS modified Quadruplex RT-qPCR using Enzyme Mix from different vendors Enzyme Types One Step PrimeScript Enzyme Mix UltraPlex ToughMix Enzyme Mix BEI viral RNA copies/reaction 10000 1000 100 10 NTC 10000 1000 100 10 NTC N1 target 28.5 31.1 34.4 38.5 27.1 30.3 33.5 37.1 RdRp-Hel 29.9 32.4 36.5 38.4 27.3 30.6 33.5 35.3 *** target E target 28.7 31.8 34.8 37.8 27.5 30.7 33.9 36.6 RNAse P 29.2 30.4 34.0 37.5 26.9 29.6 33.0 36.1 Four repeats for each assay. *** indicates 3/4 give signals.

[0074] Over all, the One Step PrimeScript™ III Enzyme Mix (Takara Bio) is the presently preferred enzyme among all the enzymes tested. The analytical sensitive is below ˜10 copies of BEI viral RNA per reaction with One Step PrimeScript™ III Enzyme Mix.

(i)(8) the Performance of these Compositions were Robust with Respect to the CoV19 Target in Different Media.

[0075] As is shown in Table 17, the assay was also robust in detecting CoV19 targets when presented directly from viral transport media (VTM) without RNA extraction and purification. Here, BEI viral RNA samples were spiked into Corning™ Transport Medium (VTM, Fisher Scientific, MT25500CV), then, the BEI RNA with VTM was directly added into RT-PCR. The performances of the RT-PCR with SAMRS modified primers were compared to the standard primers.

TABLE-US-00017 TABLE 17 Results of evaluating the performance of Standard and SAMRS primers in quadruplex RT-qPCR using BEI viral RNA in VTM. Enzyme type Quantabio UltraPlex EnzymeMix Primer Type Standard primers SAMRS primers BEI viral RNA in VTM copies/reaction 320 80 20 NTC 320 80 20 NTC N1 target 31.4 31.3*** 32.2*** 33.3 34.4 35.6*** RdRp-Hel target NA NA NA 32.6 32.7** 33.9** E target NA NA NA 33.4 33.5** 35.4** RNAseP target NA NA NA 34.3 34.8*** NA Each target concentration has 4 repeats. **indicate 2/4 gave signal. ***indicate 3/4 gave signals. NA = No Amplification.

[0076] The presence of VTM inhibited RT-PCR when using the UltraPlex 1-Step ToughMix (Quanta Bio). PCR with standard primers failed to detect 320 copies of RNA with VTM for RdRp-Hel, E, and RNAseP targets. In contrast, PCR with SAMRS primers can successfully detect 320 copies of RNA with VTM for all four targets. At lower target concentrations, 80 and 20 copies of RNA with VTM per reaction, the dropout rates were 5 out of 16 assays (31%) and 9 out of 16 assays (56%), respectively (Table 17).

[0077] When the UltraPlex 1-Step ToughMix (Quanta Bio) was replaced by the One Step PrimeScript™ III Enzyme Mix (Takara Bio), the presence of VTM still inhibit the RT-PCR as the results shown in Table 17. However, the Takara PrimeScript™ III enzyme has higher tolerance of VTM than the Quanta Bio UltraPlex enzyme mix.

(i)(9) the Performance of these Compositions was Further Improved by Adding an AEGIS Tag to the 5′ of SAMRS Primers.

[0078] When add an AEGIS tag to the 5′ of the SAMRS primers. The performance of the AEGIS-SAMRS primers (SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 115, SEQ ID NO 116, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 109, SEQ ID NO 110, Table 4) were compared to SAMRS primers (SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 71, SEQ ID NO 72, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 50, SEQ ID NO 51, Table 2) in a quadruplex PCR targeting on N1, RdRp-IP4, E, and RNAse P genes.

[0079] For a quadruplex TaqMan RT-PCR (Table 18), the RT-PCR assay (20 μL) contained 5 μL of 4× master reaction mixture (TaqPath™ 1-Step RT-qPCR Master Mix), 0.5 μM of SAMRS modified forward and reverse primers, 0.125 μM of probe, and 5 μL of RNA sample. For the AEGIS-tagged SAMRS primers, dZTP (0.05 mM final) need to be included into the PCR. RT-PCR reactions were conducted on a thermal cycler (Roche LightCycler® 480) with the following conditions: Reverse transcription at 53° C. for 10 min, inactivation of reverse transcriptase at 95° C. for 2 min, 50 cycles of PCR amplification (Denaturing at 95° C. for 5 s; Annealing/Extending at 58° C. for 30 s). Fluorescence was detected in real time during each annealing-extension cycle. LightCycler 480 software was used to obtain a cycle threshold (Ct).

TABLE-US-00018 TABLE 18 Results of comparison of the performance of SAMRS primers to AEGIS-SAMRS primers in 4-plex RT-PCR. Ct values of SAMRS primers vs AEGIS-SAMRS primers in quadruplex PCR Primer Types SAMRS Primers SAMRS Primers with AEGIS tag RNA Target copies / reaction 400 100 40 10 400 100 40 10 N1 target 34.3 36.6 37.1** 37.6* 34.6 35.3 37.2** 37.9* RdRp-lP4 target 35.5 37.0 37.0* NA 36.0 36.6 37.4** 38.7* E target 35.0 37.9 NA NA 36.7 38.3 39.4** NA RNAseP target 29.2 31.3 32.5 34.5 29.8 31.7 32.9 34.7 Three repeats for each RNA concentration. *indicate one out of three give signal. **indicate two out of three give signal. NA indicate No Amplification.

[0080] As the results shown in Table 18, the assay was robust in tagged PCR carried by AEGIS-containing external primers. The performance of the AEGIS-SAMRS primers were compared to untagged SAMRS primers in a quadruplex PCR (Table 18). As shown in Table 18, the AEGIS tagged SAMRS primers (AEGIS-SAMRS primers) gave higher sensitivity than the SAMRS primers without AEGIS tag.

[0081] To expand the assay to influenza, RSV, or other RNA targets, which may be, without limitations, viral and non-viral targets and control targets, pairs of primers containing SAMRS may be added to any of the multiplexes described herein. The presently preferred primers for influenza A and influenza B are shown in Table 19.

TABLE-US-00019 TABLE 19 Presently preferred SAMRS-containing primers to be included in coronavirus-targeted multiplexes that target influenza and RSV, as pathogens that give symptoms that can be confused with coronavirus symptoms. Y = C + T, V = G + A + C Oligo Name SEQ ID SEQUENCE (5′-3′) 1-InfA-F1_a SEQ ID NO 129 CAA GAC CAA TCY TGT CAC CTC TGa C 2-InfA-F2_a SEQ ID NO 130 CAA GAC CAA TYC TGT CAC CTY TGa C 3-InfA-R1-V_a SEQ ID NO 131 GCA TTY TGG ACA AAV CGT CTa CG 4-InfA-R1-I_a SEQ ID NO 132 GCA TTY TGG ACA AAg CGT CTa CG 5-InfA-R2-G_a SEQ ID NO 133 GCA TTT TGG AYA AAG CGT CTa CG 6-InfB-F_c SEQ ID NO 134 TCC TCA AYT CAC TCT TCG AGc G 7-InfB-R_g SEQ ID NO 135 CGG TGC TCT TGA CCA AAT Tg G 8-RSV-A-F_c SEQ ID NO 136 CGT CTT AAT GTA GCA GAA TTc AC 9-RSV-A-R_a SEQ ID NO 137 ATC AAT CCC ATT CTA ACA AGa TC 10-RSV-B-F_a SEQ ID NO 138 GGA AAC ATA CGT GAA CAa GC 11-RSV-B-R_ga SEQ ID NO 139 GAT GAC TGG AAC ATA GgC aC 1-InfA-F1_a SEQ ID NO 140 P CAA GAC CAA TCY TGT CAC CTC TGa C 2-InfA-F2_a SEQ ID NO 141 P CAA GAC CAA TYC TGT CAC CTY TGa C 3-InfA-R1-V_a SEQ ID NO 142 P GCA TTY TGG ACA AAV CGT CTa CG 4-InfA-R1-I_a SEQ ID NO 143 P GCA TTY TGG ACA AAg CGT CTa CG 5-InfA-R2-G_a SEQ ID NO 144 P GCA TTT TGG AYA AAG CGT CTa CG 6-InfB-F_c SEQ ID NO 145 P TCC TCA AYT CAC TCT TCG AGc G 7-InfB-R_g SEQ ID NO 146 P CGG TGC TCT TGA CCA AAT Tg G 8-RSV-A-F_c SEQ ID NO 147 P CGT CTT AAT GTA GCA GAA TTc AC 9-RSV-A-R_a SEQ ID NO 148 P ATC AAT CCC ATT CTA ACA AGa TC 10-RSV-B-F_a SEQ ID NO 149 P GGA AAC ATA CGT GAA CAa GC 11-RSV-B-R_ga SEQ ID NO 150 P GAT GAC TGG AAC ATA GgC aC

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