Fast PCR by tandem primer embedded with ribonucleotide

Abstract

Described herein are methods and compositions for increasing amplification efficiency.

Description

[0001] Attorney Docket No. : 6010-0034W001

[0002] FAST PCR BY TANDEM PRIMER EMBEDDED WITH RIBONUCLEOTIDE SPECIFICATION

[0003] CROSS-REFERENCE APPLICATION

[0004] This application claims benefit of U.S. Application No. 63 / 721,594 filed November 18, 2024, the entirety of which is incorporated by reference in its entirety.

[0005] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE A Sequence Listing is provided herewith as file, “60100034PV01_sequencelisting_l 1152024. xml” created on 11 / 15 / 2024 and having a size of 5 kilobytes. The contents of the text file are incorporated by reference herein in their entirety.

[0006] STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0007] Not applicable.

[0008] FIELD

[0009] The methods and compositions described herein relate generally to the area of nucleic acid amplification. In particular, described herein are methods and compositions for increasing amplification efficiency.

[0010] BACKGROUND

[0011] A wide variety of nucleic acid amplification methods are available, and many have been employed in the implementation of sensitive diagnostic assays based on nucleic acid detection. Polymerase chain reaction (PCR) remains the most widely used DNA amplification and quantitation method. However, PCR in general has several limitations. PCR amplification can only achieve less than two-fold increase of the amount of target sequence at each cycle. It is still relatively slow. In addition, the sensitivity of this method is typically limited, making it difficult to detect a target that may be present at only a few molecules in a single reaction.

[0012] This disclosure is directed to processes and systems which will lead to much shorter turnaround time for molecular diagnostic assays based on PCR. Reducing the turnaround time for Attorney Docket No. : 6010-0034W001

[0013] PCR-based molecular diagnostics enhances the speed, precision, and effectiveness of medical decision-making, benefiting patients, healthcare providers, and public health efforts.

[0014] PCR-based assays are often used to detect pathogens (e.g., viruses, bacteria) in infectious diseases. Faster test results allow clinicians to make timely and informed decisions regarding treatment, isolation, or the need for further diagnostics. In situations such as viral outbreaks (e.g., COVID-19), timely diagnosis can be critical to prevent the spread of infection and initiate early treatment, reducing mortality and morbidity.

[0015] Rapid PCR results help healthcare providers to quickly adjust therapeutic strategies, particularly in critical care settings. For example, in sepsis or meningitis cases, immediate identification of the causative pathogen allows for targeted antibiotic or antiviral treatment, improving patient outcomes and avoiding the unnecessary use of broad-spectrum antibiotics.

[0016] Shorter turnaround times reduce patient wait times for diagnostic results, improving hospital workflow and efficiency. This minimizes delays in care, optimizes bed management, and reduces the risk of hospital-acquired infections by quickly identifying and isolating contagious patients when necessary.

[0017] In scenarios involving communicable diseases, such as influenza, tuberculosis, or COVID-19, rapid PCR assays help in quickly identifying and isolating infected individuals. This is crucial for controlling outbreaks and preventing the spread of disease within the community, particularly in high-risk environments like hospitals and schools.

[0018] Fast test results help reduce the anxiety that patients experience when waiting for critical diagnoses, particularly in cases of life-threatening conditions like cancer (e g., when using PCR-based assays for detecting specific genetic mutations or viral oncogenes like HPV).

[0019] Shorter turnaround times can lead to overall cost savings for both healthcare systems and patients. By enabling quicker diagnostic results, hospitals can reduce unnecessary hospital stays, prevent complications, and reduce the use of broad-spectrum antibiotics, all of which contribute to better cost management.

[0020] For mass screening purposes (e.g., during pandemics or large-scale disease surveillance), faster PCR-based assays allow labs to process more samples in a given time frame, contributing to better tracking of disease spread and more efficient use of laboratory resources.

[0021] The systems and processes as disclosed herein have distinct advantages over previously existing methods. These advantages can be achieved using the compositions and methods as Attorney Docket No. : 6010-0034W001

[0022] disclosed herein by replacing, for example, one or more deoxynucleotides that are positioned approximately in the middle of a PCR primer with one or more ribonucleotides. Primers comprising ribonucleotide(s) are referred to herein as Ribobase Aided Primers (RAP) or Fast PCR Primers. These Fast PCR Primers would dramatically increase per cycle amplification efficiency when strand displacement polymerase and Ribonuclease H2 are provided in the PCR reaction. Potential advantages of the disclosed method include that there is no complicated PCR design requiring additional primers compared to, for example, exponential amplification described in W02016100388A1; and because the primer design does not deviate too much from current multiplex assay design, converting current assays are potentially much easier. The methods and compositions as disclosed herein are applicable to any diagnostic enabled through the targeted detection of nucleic acids by PCR and subsequent nucleic acid base sequence recognition technologies with associated bioinformatic data processing. The Fast PCR Primer design as disclosed herein can improve the Time to Result (TTR) which is the total time required from the start of the PCR process to obtain a final, interpretable result. Reducing TTR is crucial in diagnostic and research settings, as faster TTR allows for quicker decision-making and improved workflow efficiency.

[0023] All references cited herein are incorporated herein by reference in their entireties.

[0024] BRIEF SUMMARY

[0025] Various embodiments contemplated herein may include, but need not be limited to, one or more of the following: Embodiment 1. A method for amplifying a target nucleic acid in a sample, the method comprising: (a) adding the sample to a polymerase chain reaction (PCR) mixture comprising: (i) An oligonucleotide primer for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, wherein the oligonucleotide primer comprises a first lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a first leading primer sequence, wherein the first lagging primer sequence is complementary to a sequence in the target nucleic acid, wherein the target-specific cleavage domain comprising the at least one ribonucleotide is complementary to a sequence in the first template strand, wherein the first leading primer sequence is complimentary to a sequence in the target nucleic acid, further wherein the oligonucleotide primer lacks a terminal 3' cap, and Attorney Docket No. : 6010-0034W001

[0026] (ii) an amplification buffer comprising a DNA polymerase with strand-displacement activity and a mixture of nucleotides; and an endonuclease enzyme (such as RNAse H2) capable of cleaving the oligonucleotide primer at the target-specific cleavage domain when the at least one ribonucleotide is hybridized to the target nucleic acid; (b) hybridizing the oligonucleotide primer to the first template strand of the target nucleic acid if present in the sample to form a first doublestranded substrate; (c) cleaving the oligonucleotide primer hybridized to the first template strand with the endonuclease enzyme at a point within or adjacent to the target-specific cleavage domain, thereby generating a 3 ’-OH group in the lagging sequence; (d) extending the leading sequence hybridized to the first template strand with the polymerase to form a first copy of the target nucleic acid; and (e) extending the lagging sequence hybridized to the first template strand of the with the polymerase to form a second copy of the target nucleic acid. Embodiment 2. The method of embodiment 1, further comprising: (f) hybridizing a complementary oligonucleotide primer to the second template strand, wherein the complementary oligonucleotide primer comprises a second lagging primer sequence which is adjacent to, and 5' of, a target- specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a second leading primer sequence, wherein the target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the second template strand, further wherein the oligonucleotide primer lacks a terminal 3' cap, to form a second double-stranded substrate; (g) cleaving the complementary oligonucleotide primer hybridized to the target nucleic acid with the endonuclease RNase H2 enzyme at a point within or adjacent to the target-specific cleavage domain, thereby generating a 3 ’-OH group in the lagging sequence; (h) extending the complementary leading sequence with the polymerase to form a third copy of the target nucleic acid; and (i) extending the complementary lagging sequence with the polymerase to form a fourth copy of the target nucleic acid. Embodiment 3. The method of any one of embodiment 1 - 2 wherein the DNA polymerase with strand-displacement activity lacks 5' — >3' exonuclease activity. Embodiment 4. The method of any one of embodiments 1 -3, wherein the first and second lagging primer sequences independently comprise 10 to 30 nucleotides. Embodiment 5. The method any one of embodiments 1 - 4, wherein the first and second lagging primer sequences independently comprise 12 to 20 nucleotides. Embodiment 6. The method of any one of embodiments 1 - 5, wherein the first and second leading primer sequences independently comprise 10 to 30 nucleotides. Embodiment 7. The method of any one of embodiments 1 - 6, Attorney Docket No. : 6010-0034W001

[0027] wherein the first and second leading primer sequences independently comprise 12 to 20 nucleotides. Embodiment 8. The method of any one of embodiments 1 - 7, wherein the second template strand is complementary to the first template strand. Embodiment 9. The method of any one of embodiments 1 - 8, wherein the first and second lagging primer sequences independently comprise deoxyribonucleotides. Embodiment 10. The method of any one of embodiments 1 - 9, wherein the first and second lagging primer sequence independently comprise at least one nonnatural nucleotide base. Embodiment 11. The method of any one of embodiments 1 - 10, wherein the first and second lagging primer sequence first and second comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino-8-(l’-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof. Embodiment 12. The method of any one of embodiments 1 - 11, wherein the target-specific cleavage domain comprises at least one natural ribonucleotide base. Embodiment 13. The method of any one of embodiments 1 - 12, wherein the target-specific cleavage domain comprises a single ribonucleotide. Embodiment 14. The method of any one of embodiments 1 - 13, wherein the target-specific cleavage domain comprises a plurality of ribonucleotides. Embodiment 15. The method of any one of embodiments 1 - 14, wherein the target-specific cleavage domain comprises 1 - 10 ribonucleotides. Embodiment 16. The method of any one of embodiments 1 - 15, wherein the first and second leading primer sequences independently comprise deoxyribonucleotides. Embodiment 17. The method of any one of embodiments 1 - 16, wherein the first and second leading primer sequences independently comprise at least one non-natural nucleotide base. Embodiment 18. The method of any one of embodiments 1 - 17, wherein the first and second leading primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lEI)-pyridone, 2-amino-8-(l’-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof.

[0028] Embodiment 19. An oligonucleotide primer for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, Attorney Docket No. : 6010-0034W001

[0029] wherein the oligonucleotide primer comprises a first lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a first leading primer sequence, wherein the first lagging primer sequence is complementary to a sequence in the target nucleic acid, wherein the target-specific cleavage domain comprising the at least one ribonucleotide is complementary to a sequence in the first template strand, wherein the first leading primer sequence is complimentary to a sequence in the target nucleic acid, further wherein the oligonucleotide primer lacks a terminal 3' cap. Embodiment 20. A complementary oligonucleotide primer to the second template strand of embodiment 19, wherein the complementary oligonucleotide primer comprises a second lagging primer sequence which is adjacent to, and 5' of, a target- specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a second leading primer sequence, wherein the target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the second template strand, further wherein the oligonucleotide primer lacks a terminal 3' cap, to form a second double-stranded substrate. Embodiment 21. The oligonucleotide primer of any one of embodiments 19 - 20, wherein the first and second lagging primer sequences independently comprises 10 to 30 nucleotides. Embodiment 22. The oligonucleotide primer of any one of embodiments 19 - 21, wherein the first and second lagging primer sequences independently comprises 12 to 20 nucleotides. Embodiment 23. The oligonucleotide primer of any one of embodiments 19 - 22, wherein the first and second leading primer sequences independently comprise 10 to 30 nucleotides. Embodiment 24. The oligonucleotide primer of any one of embodiments 19 - 23, wherein the first and second leading primer sequences independently comprise 12 to 20 nucleotides. Embodiment 25. The oligonucleotide primer of any one of embodiments 19 - 24, wherein the second template strand is complementary to the first template strand. Embodiment 26. The oligonucleotide primer of any one of embodiments 19 - 25, wherein the first and second lagging primer sequences independently comprise deoxyribonucleotides. Embodiment 27. The oligonucleotide primer of any one of embodiments 19 - 26, wherein the first and second lagging primer sequences independently comprise at least one non-natural nucleotide base. Embodiment 28. The oligonucleotide primer of any one of embodiments 19 - 27, wherein the first and second lagging primer sequence independently comprise a non-natural nucleotide base wherein the nonnatural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, Attorney Docket No. : 6010-0034W001

[0030] isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(l’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino-8-(l’-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof. Embodiment 29. The oligonucleotide primer of any one of embodiments 19 - 28, wherein the target-specific cleavage domain comprises at least one natural ribonucleotide base. Embodiment 30. The oligonucleotide primer of any one of embodiments 19 - 29, wherein the target-specific cleavage domain comprises a single ribonucleotide. Embodiment 31. The oligonucleotide primer of any one of embodiments 19 - 30, wherein the target-specific cleavage domain comprises a plurality of ribonucleotides. Embodiment 32. The oligonucleotide primer of any one of embodiments 19 -31, wherein the target-specific cleavage domain comprises 1 - 10 ribonucleotides. Embodiment 33. The oligonucleotide primer of any one of embodiments 19 - 32, wherein the first and second leading primer sequences independently comprise deoxyribonucleotides. Embodiment 34. The oligonucleotide primer of any one of embodiments 19 - 33, wherein the first and second leading primer sequences independently comprise at least one non-natural nucleotide base. Embodiment 35. The oligonucleotide primer of any one of embodiments 19 - 34, wherein the first and second leading primer sequence independently comprise a non-natural nucleotide base wherein the nonnatural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino-8-(l’-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof. Embodiment 36. A combination of at least two oligonucleotides according to any one of embodiments 19 - 35, wherein one oligonucleotide is a forward primer and one oligonucleotide is a reverse primer for amplifying a target nucleic acid. Embodiment 37. A composition for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the composition comprising: a polymerase chain reaction (PCR) mixture comprising: i) a set of oligonucleotide primers comprising: a forward oligonucleotide primer comprising comprises a first lagging primer sequence which is adjacent to, and 5' of, a targetspecific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a first leading primer sequence, wherein the lagging primer sequence is complementary to a sequence in the target nucleic acid, wherein the target-specific cleavage Attorney Docket No. : 6010-0034W001

[0031] domain comprises the at least one ribonucleotide and is complementary to a sequence in the target nucleic acid, wherein the first leading primer sequence is complimentary to a sequence in the target nucleic acid, further wherein the oligonucleotide primer lacks a terminal 3' cap; and a reverse oligonucleotide primer comprising comprises a second lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a second leading primer sequence, wherein the second lagging primer sequence is complementary to a sequence in the target nucleic acid, wherein the target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the target nucleic acid, wherein the second leading primer sequence is complimentary to a sequence in the target nucleic acid, further wherein the oligonucleotide primer lacks a terminal 3' cap, ii) an amplification buffer comprising a DNA polymerase and a mixture of nucleotides; iii) an endonuclease RNaseH2 enzyme capable of cleaving the oligonucleotide primer at the target-specific cleavage domain when the at least one ribonucleotide is hybridized to the target nucleic acid; wherein the at least one ribonucleotide is capable of forming a target-specific cleavage domain when hybridized to the target nucleic acid. Embodiment 38. The composition of embodiment 37, wherein the first and second lagging primer sequences independently comprises 10 to 30 nucleotides. Embodiment 39. The composition of any one of embodiments 37 - 38, wherein the first and second lagging primer sequences independently comprises 12 to 20 nucleotides. Embodiment 40. The composition of any one of embodiments 37 - 39, wherein the first and second leading primer sequences independently comprise 10 to 30 nucleotides. Embodiment 41. The composition of any one of embodiments 37 - 40, wherein the first and second leading primer sequences independently comprise 12 to 20 nucleotides. Embodiment 42. The composition of any one of embodiments 37 - 40, wherein the second template strand is complementary to the first template strand. Embodiment 43. The composition of any one of embodiments 37 - 45, wherein the first and second lagging primer sequences independently comprise deoxyribonucleotides. Embodiment 44. The composition of any one of embodiments 37 - 43, wherein the first and second lagging primer sequences independently comprise at least one non-natural nucleotide base. Embodiment 45. The composition of any one of embodiments 37 - 44, wherein the first and second lagging primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, Attorney Docket No. : 6010-0034W001

[0032] isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(l’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino-8-(l’-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof. Embodiment 46. The composition of any one of embodiments 37 - 45, wherein the target-specific cleavage domain comprises at least one natural ribonucleotide base. Embodiment 47. The composition of any one of embodiments 37 - 46, wherein the target-specific cleavage domain comprises a single ribonucleotide. Embodiment 48. The composition of any one of embodiments 37 - 47, wherein the target-specific cleavage domain comprises a plurality of ribonucleotides. Embodiment 49. The composition of any one of embodiments 37 - 48, wherein the target-specific cleavage domain comprises 1 - 10 ribonucleotides. Embodiment 50. The composition of any one of embodiments 37 - 49, wherein the target-specific cleavage domain is located near the 5’ end of the leading primer. Embodiment 51. The composition of any one of embodiments 37 - 50, wherein the targetspecific cleavage domain is located near the 3’ end of the lagging primer. Embodiment 52. The composition of any one of embodiments 37 - 51, wherein the first and second leading primer sequences independently comprise deoxyribonucleotides. Embodiment 53. The composition of any one of embodiments 37 - 52, wherein the first and second leading primer sequences independently comprise at least one non-natural nucleotide base. Embodiment 54. The composition of any one of embodiments 37 - 53, wherein the first and second leading primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4-ethylcytosine, 6-amino-5-nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino-8-(l’-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof. Embodiment 55. The oligonucleotide of any one of embodiments 19 - 36, or the composition of any one of embodiments 37 - 54, wherein the oligonucleotide, or composition is contained within a cartridge for detecting one or more target nucleic acids in a sample, the cartridge comprising: a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes: a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality; an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same; a reagent chamber comprising one or more of the oligonucleotide(s) and / or one or more of the combination(s); and Attorney Docket No. : 6010-0034W001

[0033] a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; and a filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel. Embodiment 56. The method of any one of embodiments 1 - 18, oligonucleotide of any one of embodiments 19 - 36, the composition of any one of embodiments 37 - 54, further comprising a probe. Embodiment 57. The method, oligonucleotide, or composition of embodiment 56, wherein the probe comprises a linear probe. Embodiment 58. The method, oligonucleotide, or composition of embodiment 56 or 57, wherein the probe comprises a fluorescent dye and a quencher molecule. Embodiment 59. The method, oligonucleotide, or composition of any one of embodiments 56 - 58, wherein the probe comprises a cycling probe. Embodiment 60. The method, oligonucleotide, or composition of any one of embodiments 56 - 59, wherein amplifying the target nucleic acid comprises a multiplex amplification reaction.

[0034] In certain embodiments as disclosed herein, the oligonucleotide primer further comprises a spacer adjacent to, and 3' of the first and / or second lagging primer sequence, which is adjacent to and 5' of the target-specific cleavage domain. In certain embodiments as disclosed herein, the oligonucleotide primer further comprises a spacer adjacent to, and 5' of the first and / or second lagging primer sequence, which is adjacent to and 5' of the target-specific cleavage domain. In certain embodiments as disclosed herein, the oligonucleotide primer further comprises a spacer sequence comprising restriction enzyme recognition sites, a GC-rich region, degenerate primer sequences, mismatched primer sequences, end stabilizing sequences, adapter sequences for high throughput sequence, or molecular bar code sequences. In certain embodiments as disclosed herein, the oligonucleotide primer further comprises a spacer adjacent to, and 5' of the first and / or second leading primer sequence, which is adjacent to and 3' of the target-specific cleavage domain. In certain embodiments as disclosed herein, the oligonucleotide primer further comprises restriction enzyme recognition sites, A GC-rich region, degenerate primer sequences, mismatched primer sequences, end stabilizing sequences, adapter sequences for high throughput sequence, or molecular bar code sequences.

[0035] BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

[0036] The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein: Attorney Docket No. : 6010-0034W001

[0037] Figure 1 is a schematic depicting Tandem Primer Design.

[0038] Figure l is a schematic representation of Amplification Steps.

[0039] Figure 3 is a schematic depicting Potential Isothermal Amplification of the Displaced Strand within Denaturation Cycle which can sustain base “4” amplification going into next denaturation cycle.

[0040] Figure 4 is a schematic showing primers for single-plex amplification of the SARS-CoV-2 N2 Gene. Forward RAP Primer (SEQ ID NO: 1); Forward on market reference assay primer (SEQ ID NO: 2); Reverse RAP Primer (SEQ ID NO: 3); Reverse on market reference assay primer (SEQ ID NO: 4).

[0041] Figure 5 are charts showing Amplification of SARS-CoV-2 N2 without PEG8000; Fig.5A shows the Individual Value plot for the N2_Ct; Fig. 5B shows the Individual Value plot for the N2_Ct.

[0042] Figure 6 are charts showing Anneal / Extension Temperature “Optimization.” Fig. 6A shows the Individual Value plot for the N2_Ct; Fig. 6B shows the Individual Value plot for the N2_Ct. Figure 7 are charts showing the PEG Effect Fig. 7A shows the Individual Value plot for the N2_Ct; Fig. 7B shows the amplification curve that shows the increase in fluorescence signal over the course of the PCR cycles.

[0043] Figure 8 are charts showing PCR in the presence of Ing / pL human genomic DNA. Fig.8A shows the Individual Value plot for the N2_Ct; Fig.8B shows the Individual Value plot for the EPF Ct.

[0044] Figure 9 are charts showing Evaluation of Non-specific PCR amplification by Eva Green Dye in Target-free Reactions. Fig.9A shows the Individual Value plot for the N2_Ct; Fig. 9B shows the Individual Value plot for the EPF Ct.

[0045] Figure 10 is a chart showing Performance at Higher Temperature and in Different amount of PEG8000.

[0046] Figure 11 is a chart showing that Additional Amount of Polymerase is not the Solution to Inhibition by Human gDNA.

[0047] Figure 12 are charts showing Evaluation of the Impact of Primer and MgCE Concentration on RAP Performance. Fig. 12A shows the Individual Value plot for N2_Ct; Fig. 12B shows the Individual Value plot for N2_Ct.

[0048] Figure 13 is a chart showing Additional RNase HII had Adverse Effect on Fast PCR. Attorney Docket No. : 6010-0034W001

[0049] Figure 14 is a schematic showing Potential Isothermal Amplification of the Displaced Strand within Denaturation Cycle, which can sustain base “4” amplification going into next denaturation cycle.

[0050] Figure 15 is a chart showing Addition of Limiting amount of Non-Ribobase Primer did not Release human gDNA Inhibition.

[0051] Figure 16 is a chart showing the Evaluation of the Lagging and Leading Primer Performance at Different Temperatures.

[0052] Figure 17 is a chart showing a Performance Comparison between on market PCR assay vs Ribobase-aided PCR .

[0053] DETAILED DESCRIPTION

[0054] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0055] The term “nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides. The term “nucleic acid” encompasses multi-stranded, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid templates described herein may be any size depending on the sample (from small cell-free DNA fragments to entire genomes), including but not limited to 50-300 bases, 100-2000 bases, 100-750 bases, 170-500 bases, 100-5000 bases, 50-10,000 bases, or 50-2000 bases in length. In some instances, templates are at least 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000 50,000, 100,000, 200,000, 500,000, 1,000,000 or more than 1,000,000 bases in length. Methods described herein provide for the amplification of nucleic acids, such as nucleic acid templates. Methods described herein additionally provide for the generation of isolated and at least partially purified nucleic acids and libraries of nucleic acids. Nucleic acids include but are not limited to those comprising DNA, RNA, circular RNA, cfDNA (cell free DNA), cfRNA (cell free RNA), siRNA (small interfering RNA), cffDNA (cell free fetal DNA), mRNA, tRNA, rRNA, miRNA (microRNA), synthetic polynucleotides, polynucleotide analogues, any other nucleic acid consistent with the specification, or any combinations thereof. The length of polynucleotides, when provided, are described as the number of bases and abbreviated, such as nt (nucleotides), bp (bases), kb (kilobases), or Gb (gigabases). The term Attorney Docket No. : 6010-0034W001

[0056] nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA. The term nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or tri pie- stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e, a double-stranded nucleic acid need not be double- stranded along the entire length of both strands).

[0057] The term nucleic acid also encompasses any modifications thereof, such as by methylation and / or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5 -bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like. More particularly, in some embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino polymers (see, e g., Summerton and Weller (1997) “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Dev.

[0058] 7:1817-195; Okamoto et al. (20020) “Development of electrochemically gene-analyzing method using DNA-modified electrodes,” Nucleic Acids Res. Supplement No. 2:171-172), and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748, which are incorporated herein by reference in their entirety fortheir disclosure of LNAs.

[0059] The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation Attorney Docket No. : 6010-0034W001

[0060] from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

[0061] The term “complementary” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. Complementarity of nucleic acid strands means that the strands form a stabile duplex due to hydrogen bonding between their nucleobase groups. The complementary bases are in DNA, A with T and C with G, and, in RNA, C with G, and U with A. Nucleotides in respective strands are complementarity when they form one of these (Watson-Crick pairings) when the strands are maximally aligned. Nucleotides are mismatched when they do not form a complementarity pair when their respective strands are maximally aligned. Complementarity of strands can be perfect or substantial. Perfect complementarity between two strands means that the two strands can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. Substantial complementarity means most but not necessarily all bases in strands form Watson-Crick pairs to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0062] “Specific hybridization” refers to the binding of a nucleic acid to a target nucleotide sequence in the absence of substantial binding to other nucleotide sequences present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated. Attorney Docket No. : 6010-0034W001

[0063] In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase “stringent hybridization conditions” generally refers to a temperature in a range from about 5°C to about 20°C or 25°C below than the melting temperature (Tm) for a specific sequence at a defined ionic strength and pH. As used herein, the Tmis the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tmof nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by reference for their descriptions of stringent hybridization conditions). As indicated by standard references, a simple estimate of the Tmvalue may be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), as well as the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60° C. and a salt concentration of about 0.2 molar at pH7. Tmcalculation for oligonucleotide sequences based on nearest-neighbors thermodynamics can be carried out as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest -neighbor thermodynamics” lohn SantaLucia, Ir., PNAS Feb. 17, 1998 vol. 95 no. 4 1460-1465 (which is incorporated by reference herein for this description).

[0064] The term “oligonucleotide” is used to refer to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules. Attorney Docket No. : 6010-0034W001

[0065] The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from 10 to 30 nucleotides, or, in some embodiments, from 10 to 60 nucleotides, in length. In some embodiments, primers can be, e.g., 15 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.

[0066] A primer is said to anneal to another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence.

[0067] The term “primer pair” refers to a set of primers including a 5' “upstream primer” or “forward primer” that hybridizes with the complement of the 5' end of the DNA sequence to be amplified and a 3' “downstream primer” or “reverse primer” that hybridizes with the 3' end of the sequence to be amplified. As will be recognized by those of skill in the art, the terms “upstream” and “downstream” or “forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments.

[0068] A “probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable label to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size. Generally, probes are at least 7 to 15 Attorney Docket No. : 6010-0034W001

[0069] nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides long. Still other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides long. Yet other probes are longer still, and are at least 100, 150, 200 or more nucleotides long. Probes can also be of any length that is within any range bounded by any of the above values (e.g., 7-200 nucleotides in length).

[0070] The primer or probe can be perfectly complementary to the target nucleotide sequence or can be less than perfectly complementary. In some embodiments, the primer has at least 65% identity to the complement of the target nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a sequence in the range of 10-30 nucleotides, and, in some embodiments, over a sequence of at least 14-25 nucleotides, and, in some embodiments, has at least 75% identity, at least 85% identity, at least 90% identity, or at least 95%, 96%, 97%, 98%, or 99% identity. It will be understood that certain bases (e.g., the 3' base of a primer) are generally desirably perfectly complementary to corresponding bases of the target nucleotide sequence. Primer and probes typically anneal to the target sequence under stringent hybridization conditions.

[0071] As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions.

[0072] Amplification according to the present teachings encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction — CCR), helicase-dependent amplification (HD A), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 February; Attorney Docket No. : 6010-0034W001

[0073] 4(l):41-7, U.S. Pat. Nos. 6,027,998; 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 112579; Day et al., Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com / geneticidproc / ussymp6proc / blegrad.html-); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, NucL Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis.

[0074] 2:18-(2002); Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12(l):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

[0075] In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.

[0076] As used herein, the term “adjacent to” is used to refer to sequences that are in sufficiently close proximity for the methods to work. In some embodiments, sequences that are adjacent to one another are immediately adjacent, with no intervening nucleotides.

[0077] A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.

[0078] The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection. Attorney Docket No. : 6010-0034W001

[0079] A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.

[0080] The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates. As used herein, the term label refers to an agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, immunohistochemistry, immunofluorescence, microscopy, Northern analysis or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of labels include, but are not limited to, radioactive isotopes, nitroimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0081] As used herein, the term Marker or Label is an agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, immunohistochemistry, immunofluorescence, microscopy, Northern analysis or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby permitting detection of the nucleic acid molecule or protein. Examples of markers include, but are not limited to, radioactive isotopes, nitroimidazoles, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of markers appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Attorney Docket No. : 6010-0034W001

[0082] Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0083] The naturally occurring bases adenine, thymine, uracil, guanine, and cytosine, which make up DNA and RNA, are described herein as “unmodified bases” or “unmodified forms.” The term “modified base” is used herein to refer to a base that is not a canonical, naturally occurring base (e.g., adenine, cytosine, guanine, thymine, or uracil). Examples of modified bases are 2-thiothymine and 2-aminoadenine.

[0084] Nucleotides comprising modified bases are referred to herein as “modified nucleotides.” A DNA polymerase is said to be “stable” at a particular temperature if it provides a satisfactory extension rate in a nucleic acid amplification reaction.

[0085] Oligonucleotides including one or more "modified nucleotides" (e.g., pseudo-complementary nucleotides) are referred to herein as pseudo-complementary oligonucleotides (e.g., pseudo-complementary blocker oligonucleotide).

[0086] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, the term "patient" refers to an animal, preferably a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey and human), and most preferably a human. In some embodiments, the subject is a non-human animal such as a farm animal (e.g., a horse, pig, or cow) or a pet (e.g., a dog or cat). In a specific embodiment, the subject is an elderly human. In another embodiment, the subject is a human adult. In another embodiment, the subject is a human child. In yet another embodiment, the subject is a human infant. The patient or subject to be medicated according to the compositions and methods as disclosed herein may be any animal or human. In certain embodiments, animals may include vertebrates. The terms vertebrate or animals in this context is understood to comprise, for example fish, amphibians, reptiles, birds, and mammals including humans. One preferred group of vertebrates or animals according to the invention comprises warm-blooded animals including farm animals, such as cattle, horses, pigs, sheep and goats, poultry such as chickens, turkeys, guinea fowls and geese, fur-bearing animals such as mink, foxes, chinchillas, rabbits and the like, as well as companion animals such as ferrets, guinea pigs, rats, hamster, cats and dogs. A further group of preferred vertebrates or animals according to the disclosure comprises fish including salmonids, for example salmon, trout or whitefish. The subject is preferably mammalian. In some embodiments the subject is a human. In other embodiments the subject is an animal, more preferably a non-human mammal. The non- Attorney Docket No. : 6010-0034W001

[0087] human mammal may be a domestic pet, or animal kept for commercial purposes, e.g., a racehorse, or farming livestock or animals such as pigs, sheep or cattle. As such the disclosure may have veterinary applications. Non-human mammals include rabbits, guinea pigs, rats, mice or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, cattle (including cows or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primates. The subject may be male or female. The subject may be an adult or a child. The subject may be a patient.

[0088] As used herein, the term "about" when used in conjunction with a stated numerical value or range has the meaning reasonably ascribed to it by a person skilled in the art, i.e., denoting somewhat more or somewhat less than the stated value or range.

[0089] When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups and classes that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. As used herein, “and / or” means that one, all, or any combination of items in a list separated by “and / or” are included in the list; for example, “1, 2 and / or 3” is equivalent to “1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2, and 3”.

[0090] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition, in a description of a method, or in a description of elements of a device, is understood to encompass those compositions, methods, or devices consisting essentially of and consisting of the recited components or elements, optionally in addition to other components or elements. The disclosure as illustratively described herein suitably may be practiced in the absence of any element, elements, limitation, or limitations which is not specifically disclosed herein.

[0091] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a Attorney Docket No. : 6010-0034W001

[0092] method” includes a plurality of such methods and reference to “the nanoparticle” includes reference to one or more nanoparticles and equivalents thereof known to those skilled in the art, and so forth. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope as disclosed herein claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0093] All references throughout this application, for example patent documents, including issued or granted patents or equivalents and patent application publications, and non-patent literature documents or other source material are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference. None is admitted to being prior art. The term “template” and variations thereof refer to a nucleic acid sequence that is a target of nucleic acid sequencing reactions. A template sequence can comprise a naturally-occurring or synthetic nucleic acid sequence. A template sequence also can include a known or unknown nucleic acid sequence from a sample of interest. In various exemplary embodiments herein, a template sequence can be attached to a solid support, such as, for example, a bead, microparticle, flow cell, or any other surface or object.

[0094] As used herein, the term sample may be, for example, selected from tissue(s) samples, cells, biological fluid samples (e.g., blood, urine, saliva, lymphatic fluid, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbiota samples, viral preparations, soil samples, marine samples, freshwater samples, household or industrial samples, and combinations and isolates thereof. In one embodiment of the systems and methods as disclosed herein, the sample is a cell (e.g., an animal cell [e.g., a human cell], a plant cell, a fungal cell, a bacterial cell, and a protozoal cell). In one specific embodiment, the cell is lysed prior to the replication. In one specific embodiment, cell lysis is accompanied by proteolysis. In one specific embodiment, the cell is Attorney Docket No. : 6010-0034W001

[0095] selected from a cell from a preimplantation embryo, a stem cell, a fetal cell, a tumor cell, a suspected cancer cell, a cancer cell, a cell subjected to a gene editing procedure, a cell from a pathogenic organism, a cell obtained from a forensic sample, a cell obtained from an archeological sample, and a cell obtained from a paleontological sample. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a preimplantation embryo (e.g., a blastomere. In one specific embodiment, the method further comprises determining the presence of disease predisposing germline or somatic variants in the embryo cell. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a pathogenic organism (e.g., a bacterium, a fungus, a protozoan). In one specific embodiment, the pathogenic organism cell is obtained from fluid taken from a patient, microbiota sample (e.g., GI microbiota sample, vaginal microbiota sample, skin microbiota sample, etc.) or an indwelling medical device (e.g., an intravenous catheter, a urethral catheter, a cerebrospinal shunt, a prosthetic valve, an artificial joint, an endotracheal tube, etc.). In one specific embodiment, the method further comprises the step of determining the identity of the pathogenic organism. In one specific embodiment, the method further comprises determining the presence of genetic variants responsible for resistance of the pathogenic organism to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In one specific embodiment, the method further comprises determining the presence of one or more diagnostic or prognostic mutations. In one specific embodiment, the method further comprises determining the presence of germline or somatic variants responsible for resistance to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell subjected to a gene editing procedure. In one specific embodiment, the method further comprises determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the systems and methods as disclosed herein, the method further comprises determining the history of a cell lineage. In a related aspect, the invention provides a use of any of the systems and methods as disclosed herein for identifying low frequency sequence variants (e.g., variants which constitute >0.01% of the total sequences). The phrase “fragment library” refers to a collection of nucleic acid fragments, wherein one or more fragments are used as a sequencing template. A fragment library can be generated in numerous ways that are known in the art. As an example, a fragment library can be generated by cutting, shearing, restricting, or otherwise subdividing a larger nucleic acid into smaller Attorney Docket No. : 6010-0034W001

[0096] fragments. Fragment libraries can be generated from naturally occurring nucleic acids, such as, for example, from bacteria, cancer cells, normal cells, or solid tissue. Libraries comprising synthetic nucleic acid sequences can also be generated to create a synthetic fragment library.

[0097] The phrase “synthetic nucleic acid sequence” and variations thereof refers to a designed and synthesized sequence of nucleic acid. For example, a synthetic nucleic acid sequence can be designed to follow rules or guidelines.

[0098] Every formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of materials are intended to be exemplary, as it is known that one of ordinary skill in the art can name the same material differently. It will be appreciated that methods, device elements, starting materials, and synthetic methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials, and synthetic methods are intended to be included in this invention. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0099] Primers

[0100] PCR Primers are designed using typical primer design parameters for the proposed applications to target DNA / RNA segments within, for example, a targeted organism (e.g., a specific bacteria, or virus), or neoplasia, that is specific to that organism within the reasonably expected organism that could be identified within that sample (meaning, if a PCR primer equally targets two organisms but one is only ever found in the arctic, it is specific to say a human saliva Attorney Docket No. : 6010-0034W001

[0101] sample) and are therefore diagnostic in nature. In addition, targeted segments which contain sequence variation within them add further value in sequence variation database development.

[0102] In one embodiment of any of the systems and methods as disclosed herein, amplification primers are, for example, between 4 and 70 nucleotides long. In one embodiment of any of the systems and methods as disclosed herein, the amplification products are between about 50 and about 2000 nucleotides in length. In one embodiment of any of the systems and methods as disclosed herein, the target nucleic acid is DNA (e.g., cDNA, or genomic DNA). In one embodiment of any of the systems and methods as disclosed herein, the target nucleic acid is RNA, such as messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), microRNA (miRNA), small nucleolar RNA (snoRNA), long non-coding RNA (IncRNA), or catalytic RNA (ribozymes). In one embodiment of any of the systems and methods as disclosed herein, the amplification primers are random primers. In one embodiment of any of the systems and methods as disclosed herein, the amplification primers comprise a barcode. In one specific embodiment, the barcode comprises a cell barcode. In one specific embodiment, the barcode comprises a sample barcode. In one embodiment of the systems and methods as disclosed herein, the amplification primers comprise a unique molecular identifier (UMI).

[0103] Target synthetics are designed by taking the targeted organismal PCR DNA / RNA sequence, breaking it into 4-mers and rearranging the 4-mers randomly to produce a new RNA / DNA segment that has the same base quality characteristics as the targeted organismal segment. These sequences are ordered from an oligonucleotide generating company as DNA / RNA synthetic oligos with the same primer sequences as the targeted PCR organismal PCR DNA / RNA sequence, that competes for amplification.

[0104] Primer Design

[0105] Primers suitable for nucleic acid amplification are sufficiently long to prime the synthesis of extension products in the presence of a suitable nucleic acid polymerase. The exact length and composition of the primer will depend on many factors, including, for example, temperature of the annealing reaction, source and composition of the primer, and where a probe is employed, proximity of the probe annealing site to the primer annealing site and ratio of primerprobe concentration. For example, depending on the complexity of the target nucleic acid sequence, an oligonucleotide primer typically contains in the range of about 10 to about 60 nucleotides, Attorney Docket No. : 6010-0034W001

[0106] although it may contain more or fewer nucleotides. The primers should be sufficiently complementary to selectively anneal to their respective strands and form stable duplexes.

[0107] In general, one skilled in the art knows how to design suitable primers capable of amplifying a target nucleic acid of interest. For example, PCR primers can be designed by using any commercially available software or open-source software, such as Primer3 (see, e.g., Rozen and Skaletsky (2000) Meth. Mol. Biol., 132: 365-386; www.broad.mit.edu / node / 1060, and the like) or by accessing the Roche UPL website. The amplicon sequences are input into the Primer3 program with the UPL probe sequences in brackets to ensure that the Primer3 program will design primers on either side of the bracketed probe sequence.

[0108] Primers may be prepared by any suitable method, including, for example, direct chemical synthesis by methods such as the phosphotri ester method of Narang et al. (1979) Meth. Enzymol.

[0109] 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; the solid support method of U.S. Pat. No. 4,458,066 and the like or can be provided from a commercial source. Primers may be purified by using a Sephadex column (Amersham Biosciences, Inc., Piscataway, N.J.) or other methods known to those skilled in the art. Primer purification may improve the sensitivity of the methods described herein.

[0110] Target Nucleic Acids

[0111] Any target nucleic acid that can be detected by nucleic acid amplification can be detected using the methods described herein. In typical embodiments, at least some nucleotide sequence information will be known for the target nucleic acids. For example, if the amplification reaction employed is PCR, sufficient sequence information is generally available for each end of a given target nucleic acid to permit design of suitable amplification primers.

[0112] The targets can include, for example, nucleic acids associated with pathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g., those for which over- or under-expression is indicative of disease, those that are expressed in a tissue- or developmental-specific manner; or those that are induced by particular stimuli; genomic DNA, which can be analyzed for specific polymorphisms (such as SNPs), alleles, or haplotypes, e.g., in genotyping. Of particular interest are genomic DNAs that are altered (e.g., amplified, deleted, and / or mutated) in genetic diseases or other pathologies; sequences that are associated with desirable or undesirable traits; and / or sequences that uniquely identify an individual (e g., in forensic or paternity determinations). Attorney Docket No. : 6010-0034W001

[0113] The disclosure provides systems and methods for use of Fast PCR to detect and / or quantify nucleic acid targets in a sample. In certain embodiments as disclosed herein, the nucleic acid targets may be, for example, a nucleic acid from a pathogenic infection selected from the group consisting of viral infection, bacterial infection, fungal infection, and / or parasitic infection. In certain embodiments as disclosed herein, the nucleic acid targets may be, for example, a nucleic acid from neoplasia.

[0114] In some embodiments, the target nucleic acid sample can be isolated from any source, or sample, such as solid tissue, tissue, cells, yeast, bacteria, or similar sources of nucleic acid samples. Methods for isolating nucleic acids from these sources are well known in the art. For example, the solid tissue or tissue can be weighed, cut, mashed, homogenized, and the nucleic acid can be isolated from the homogenized samples. In some embodiments, the target nucleic acid sample can be fragmented to prepare target nucleic acid fragments, using any procedure known in the art, including cleaving with and enzyme or chemical, or by shearing. Enzyme cleavage includes any type of restriction endonuclease, endonuclease, or transposase-mediated cleavage. In some embodiments, the biomolecules can be fragmented using well known methods, including enzymatic or chemical cleavage, or shearing forces.

[0115] Further provided herein are systems and methods wherein the sample, the amplification primers, the nucleic acid polymerase, and the mixture of nucleotides are contained in a microfluidic device. Further provided herein are systems and methods wherein the sample, the amplification primers, the nucleic acid polymerase, and the mixture of nucleotides are contained in a droplet.

[0116] Further provided herein are methods wherein the sample is selected from tissue(s) samples, cells, biological fluid samples, bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbiota samples, viral preparations, soil samples, marine samples, freshwater samples, household or industrial samples, and combinations and isolates thereof. Further provided herein are methods wherein the biological fluids are selected from blood, urine, saliva, lymphatic fluid, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, and aqueous humor. Attorney Docket No. : 6010-0034W001

[0117] Described herein are systems and methods of determining mutations in cells that are used for cellular therapy, such as but not limited to the transplantation of induced pluripotent stem cells, transplantation of hematopoietic or other cells that have not be manipulated, or transplantation of hematopoietic or other cells that have undergone genome edits. In a further embodiment, cells can be isolated from blastomeres that are created by in vitro fertilization. The cells can then undergo sequencing to determine the burden and combination of potentially disease predisposing genetic variants in each cell. The mutation profile of the cell can then be used to extrapolate the genetic predisposition of the blastomere to specific diseases prior to implantation.

[0118] In another embodiment, microbial cells (e.g., bacteria, fungi, protozoa) can be isolated from plants or animals (e.g., from microbiota samples [e.g., GI microbiota, skin microbiota, etc.] or from bodily fluids such as, e.g., blood, bone marrow, urine, saliva, cerebrospinal fluid, pleural fluid, pericardial fluid, ascites, or aqueous humor). In addition, microbial cells may be isolated from indwelling medical devices, such as but not limited to, intravenous catheters, urethral catheters, cerebrospinal shunts, prosthetic valves, artificial joints, or endotracheal tubes. The cells can then undergo sequencing to determine the identity of a specific microbe, as well as to detect the presence of microbial genetic variants that predict response (or resistance) to specific antimicrobial agents. These data can be used for the diagnosis of a specific infectious disease and / or as tools to predict treatment response.

[0119] Modified Bases

[0120] Modified bases useful in the primers and other oligonucleotides described herein include those wherein the modified base forms stable hydrogen-bonded base pairs with the natural complementary base but does not form stable hydrogen-bonded base pairs with its modified complementary base (e.g., pseudo-complementary bases). (For ease of discussion, complementary bases are also referred to herein as “partners.” Also, for ease of discussion, the following description relates to primers and primer pairs, but, as those of skill in the art, readily appreciate, this description also applies to the other oligonucleotides and oligonucleotide pairs described herein.) In some embodiments, this is accomplished when the modified base can form two or more hydrogen bonds with its natural partner, but only one or no hydrogen bonds with its modified partner. This allows the production of primer and other oligonucleotide pairs that do not form substantially stable hydrogen-bonded hybrids with one another, as manifested in a melting temperature (under physiological or substantially physiological conditions) of less than about 40° Attorney Docket No. : 6010-0034W001

[0121] C. The primers of the primer pair, however, form substantially stable hybrids with the complementary nucleotide sequence in a template strand (e.g., first template strand) of a single-or double-stranded target nucleic acid and with a strand complementary to the template strand (e.g., second template strand). In some embodiments, due to the increased (in some embodiments, double) number of hydrogen bonds in such hybrids, the hybrids formed with the primers of the present invention are more stable than hybrids that would be formed using primers with unmodified bases.

[0122] In accordance with well-established convention, the naturally occurring nucleotides of nucleic acids have the designation A, U, G and C, (RNA) and dA, dT, dG and dC (DNA). The following description applies to both ribonucleotides and deoxyribonucleotides, and therefore, unless the context otherwise requires, no distinction needs to be made in this description between A and dA, U and dT, etc.

[0123] Analogs of A that are modified in the base portion to form a stable hydrogen-bonded pair with T, (or U in the case of RNA) but not with a modified T are designated A*. Analogs of T that are modified in the base portion to form a stable hydrogen-bonded pair with A, but not with A* are designated T*. Analogs of G that are modified in the base portion to form a stable hydrogen-bonded pair with C, but not with a modified C are designated G*. Analogs of C that are modified in the base portion to form a stable hydrogen-bonded pair with G, but not with G* are designated C*, In some embodiments, the foregoing conditions are satisfied when each of the A*, T*, G*, and C* nucleotides (collectively, the modified nucleotides) form two or more hydrogen bonds with their natural partner, but only one or no hydrogen bonds with their modified partner. This is illustrated by Formulas la, lb, 2a, 2b, 3a, 3b, 4a and 4b below (and in FIG. 8A-8B), where the hydrogen bonding between natural A-T (or A-U in case of RNA) and G-C pairs, and hydrogen bonding between exemplary A*-T, T*-A, G*-C, C*-G, A*-T* and G*-C* pairs are illustrated. Attorney Docket No. : 6010-0034W001

[0124]

[0125] 4a 4b

[0126] In general, a sufficient number of modified nucleotides are incorporated into the primers described herein to preferentially increase the annealing of the primers to the template strands of a target nucleic acid, as compared to primer-to-primer annealing. It is not necessary to replace each natural nucleotide of the primer with a modified nucleotide in order to accomplish this. In some embodiments, the primers include, in addition to one or more modified nucleotides, one or more naturally occurring nucleotides and / or variants of naturally occurring nucleotides, provided that the variations do not interfere significantly with the complementary binding ability of the Attorney Docket No. : 6010-0034W001

[0127] primers, as discussed above. For example, primers including modified nucleotides can include pentofuranose moieties other than ribose or 2-deoxyribose, as well as derivatives of ribose and 2-deoxyribose, for example 3-amino-2-deoxyribose, 2-fluoro-2-deoxyribose, and 2 — O — Ci-6 alkyl or 2-O-allyl ribose, particularly 2-O-methyl ribose. The glycosidic linkage can be in the a or configuration. The phosphate backbone of the primer can, if desired, include phosphorothioate linkages.

[0128] A general structure for a suitable class of the modified A analog, A*, shown as a d'phosphate (or phosphorothioate) incorporated into a primer, is provided by Formulas 5, 6, and 7, below, wherein:

[0129] X is N or CH;

[0130] Y is O or S;

[0131] Z is OH or CH3;

[0132] R is H, F, or OR2, where R2 is Ci-galkyl or allyl, or H in case of RNA; and

[0133] Riis Ci-4alkyl, C1-4 alkoxy, alkylthio, F, or NHR3, where Rais H, or C1-4 alkyl. An illustrative embodiment of A* has 2,6-diaminopurine (2-aminoadenine) as the base, as shown in Formula lb. The latter nucleotide can be abbreviated as 2-amA or d2-amA, as applicable.

[0134]

[0135] Attorney Docket No. : 6010-0034W001

[0136] A general structure for a suitable class of the modified T analog, T*, shown as a 3'-phosphate (or phosphorothioate) incorporated into the primer, is provided by Formula 8, wherein:

[0137] Y, Z, and R are defined as above; and

[0138] R4is H, Ci-6 alkyl, Ci-6 alkenyl, or Ci-ealkynyl. An illustrative embodiment of T* has 2-thio-4-oxo-5-methylpyrimidine (2-thiothymine) as the base, as shown in Formula 2b. The latter nucleotide can be abbreviated as 2-sT or d2-sT, as applicable.

[0139]

[0140] Formula 8

[0141] A general structure for a suitable class of the modified G analog, G*, shown as a 3°-phosphate (or phosphorothioate) incorporated into the primer, is provided by Formulas 9, 10 and 11, wherein:

[0142] Riis H, Ci-4 alkyl, Ci-4 alkoxy, Ci-4 alkylthio, F, or NHR3, where Rais defined as above; and X, Y, Z, and R are defined as above. An illustrative embodiment of G* has 6-oxo-purine (hypoxanthine) as the base, as shown in Formula 3b. The latter nucleotide can be abbreviated as I or di, as applicable. Attorney Docket No. : 6010-0034W001

[0143]

[0144] Formula 9 Formula 10 Formula 11

[0145] A general structure for a suitable class of the modified C analog, C*, shown as a 3'-phosphate (or phosphorothioate) incorporated into the primer, is provided by Formulas 12 and 13, wherein:

[0146] Y, Z, R, and R4 are defined as above;

[0147] Zi is O or NH; and

[0148] Rsis H or C1-4 alkyl. An illustrative embodiment of C* has pyrrolo-[2,3-d]pyrimidine-2(3H)-one as the base, as shown in Formula 4b. The latter nucleotide can be abbreviated as P or dP, as applicable.

[0149] The above-described modified bases and nucleotides are also described in U.S. Pat. No.

[0150] 5,912,340 (issued Jun. 15, 1999 to Kutyavin et al.), which is hereby incorporated by reference for this description. The hybridization properties of d2-amA and d2-sT are described in Kutyavin, et al. (1996) Biochemistry 35:11170-76, which is also hereby incorporated by reference for this description. The synthesis and hybridization properties of d / I and dP are described in Woo et al. (1996) Nucleic Acids Research 25(13) :2470-75, which is also hereby incorporated by reference for this description. Attorney Docket No. : 6010-0034W001

[0151] Additional examples of G* and C* include 7-alkyl-7-deazaguanine and N4-alkylcytosine (where alkyl methyl or ethyl), respectively, which are described in Lahoud et al. (2008) Nucleic Acids Research 36(10):3409-19 (hereby incorporated by reference for this description). Analogs tested in this study are shown in Formula 12.

[0152]

[0153] X = CH(CH3)2, Y = NH2(iProG) Y = NH2(mcvC)

[0154] X = CH2CH2OH, Y = NH2(hEtcG) X = CF3, Y = NH2(tiinC)

[0155] Formula 12

[0156] Further examples of G* and C* include 7-nitro-7-deazahypoxanthine (NitrocH) and 2-thiocytosine (sC), respectively, which are described in Lahoud et al. (2008) Nucleic Acids Research 36(22):6999-7008 (hereby incorporated by reference for this description). Hoshinka et al. (2010) Angew Chem Int Ed Engl. 49(32):5554-5557 describes the use of such bases (“SelfAvoiding Molecular Recognition Systems”), including 2 '-hypoxanthine as G* (this reference is hereby incorporated by reference for this description; see especially, FIG. 1 ); see also Yang et al. (2015) Chembiochem. 16(9): 1365-1367 (this reference is hereby incorporated by reference for this description; see especially, Scheme 1). The analogs tested in this study are shown in Formula 13. Attorney Docket No. : 6010-0034W001

[0157]

[0158] Formula 13

[0159] General Approach for Increasing Amplification Efficiency

[0160] U.S. Pat. No. 8,252,558 and Harris et al., BioTechniques 54:93-97 (February 2013) teach a form of nested PCR, termed “Polymerase Chain Displacement Reaction” (PCDR) (both documents are incorporated by reference herein for this description). In PCDR, when extension occurs from an outer primer, it displaces the extension strand produced from an inner primer because the reaction employs a polymerase that has strand displacement activity. In theory, this allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. In practice, every amplicon Attorney Docket No. : 6010-0034W001

[0161] created from a nested primer no longer contains a primer annealing site for the outer primer. Accordingly, PCDR cannot sustain a greater than 2-fold increase of amplification product for each amplification cycle for very many cycles. For this reason, PCDR offers only modest reduction in the number of amplification cycles (e.g., from about 23 to about 20) needed to detect a target nucleic acid. By contrast, Table 1 below shows that a sustained quadrupling per cycle ^number of cycles)s Ould halve the number of cycles needed to have the same amplification as a doubling per cycle. A sustained 6-fold replication per cycle should achieve in 15 cycles what would take 40 normal PCR cycles.

[0162] TABLE 1

[0163] ""

[0164]

[0165] Attorney Docket No. : 6010-0034W001

[0166]

[0167] In certain embodiments as disclosed herein, sustaining a greater than 2-fold increase of amplification product for each amplification cycle is provided by replacing at least one deoxynucleotide that is positioned, for example, approximately in the middle of a PCR primer, with at least one ribonucleotide which dramatically increases per cycle amplification efficiency when strand displacement polymerase and Ribonuclease H2 are provided in the PCR reaction. There are at least three advantages: There is no requirement for instrument change; Two: There is no complicated PCR design is required for additional primers in contrast to the use of nested primers; Three: Because the primer design does not deviate too much from current multiplex assay design, converting current assays using primers comprising at least one ribobase which can be cleaved by an endonuclease enzyme such as RNAse H2 (i.e., Fast PCR Primers), thereby splitting the primer into two separate primers, each of which can serve as a primer for amplification of target sequences are potentially much easier. The primer design as disclosed herein can improve the Time to Result (TTR) which is the total time required from the start of the PCR process to obtain a final, interpretable result. Reducing TTR is crucial in diagnostic and research settings, as faster TTR allows for quicker decision-making and improved workflow efficiency.

[0168] Figure 1 shows a scheme in which, according to an exemplary embodiment as disclosed herein, a primer comprises at the 5’ end a lagging primer sequence, where 3 ’of the lagging sequence is the ribobase sequence and 3’ of the ribobase sequence is a leading primer sequence. This type of tandem primer embedded with at least one ribobase as a cleavage domain is referred to herein as a Fast PCR primer. In certain embodiments as disclosed herein, the cleavage domain Attorney Docket No. : 6010-0034W001

[0169] is located near the 5’ end of the leading primer. In certain embodiments as disclosed herein, the target-specific cleavage domain is located near the 3’ end of the lagging primer. Figure 2 provides a schematic representation of the amplification steps using the Fast PCR primer as disclosed herein. In this scheme using the Fast PCR primer, the tandem primer binds to the target strand, and there is leading primer extension after RNase H2 cleavage of the ribobase sequence, there is extension from the lagging primer. The displaced target leading strand can be bound by reverse tandem primer binding with an extension from the reverse leading primer upon REH 2 cleavage of the ribobase sequence in the reverse tandem primer, extension can proceed from the lagging primer accordingly, in this schematic a total of 4 new copies of the amplicon are made per cycle.

[0170] Referring to Figure 3, which is a schematic showing potential isothermal amplification of the displaced strand within a denaturation cycle may be critical to sustain pace for amplification going into the next denaturation cycle. Isothermal amplification requires two events to take place sequentially: i) Extension of the Template strand using a ribonucleotide embedded tandem primer Fast primer as disclosed herein as template; ii) Cleavage of the tandem primer “AFTER” template extension to stabilize the lagging primer binding. This could be achieved by utilizing intrinsic RT activity of Taq polymerase. However, the initial primer / template duplex is not a substrate for Taq polymerase. The binding of Taq polymerase may also block RNase H2 access.

[0171] Figure 14 is a schematic showing Potential Isothermal Amplification of the Displaced Strand within Denaturation Cycle. This is necessary to sustain base “4” amplification going into next denaturation cycle. Isothermal amplification requires two events to take place sequentially: Extension of Template strand using ribonucleotide embedded tandem primer as template. Cleavage of the tandem primer “AFTER” template extension to stabilize the lagging primer binding. This could be achieved by utilizing intrinsic RT activity of Taq polymerase. The initial primer / template duplex is not a substrate for Taq polymerase. The binding of Taq polymerase will also likely block RNase H2 access.

[0172] Amplification

[0173] For amplification in any of the methods described herein, primers and any other appropriate oligonucleotides are contacted with sample nucleic acids under conditions wherein the primers anneal to their template strands, if present. In some embodiments, the amplification step is performed using PCR. Illustrative PCR reaction mixtures generally contain an appropriate buffer, a source of magnesium ions (Mg2+) in the range of about 1 to about 10 mM, e.g., in the Attorney Docket No. : 6010-0034W001

[0174] range of about 2 to about 8 mM, nucleotides, and optionally, detergents, and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM preferred. In one embodiment, the TRIS buffer concentration is 20 mM in the reaction mix double-strength (2X) form. The reaction mix can have a pH range of from about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 as typical. Concentration of nucleotides can be in the range of about 25 mM to about 1000 mM, typically in the range of about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 may also be included in the reaction mixture. Stabilizing agents such as dithiothreitol (DTT, Cleland's reagent) or 2-mercaptoethanol may also be included. In addition, master mixes may optionally contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A master mix is commercially available from Applied Biosystems, Foster City, Calif, (TaqMan® Universal Master Mix, cat. nos. 4304437, 4318157, and 4326708).

[0175] RNase H

[0176] RNase Hl cleaves the RNA strand in RNA-DNA hybrids. It requires at least four ribonucleotides in a row to bind and cleave efficiently. RNase Hl is crucial for mitochondrial DNA replication. It helps in the removal of RNA primers that are required to initiate DNA synthesis in mitochondria. In the nucleus, RNase Hl helps resolve R-loops (three-stranded nucleic acid structures composed of a DNA-RNA hybrid and a displaced single-stranded DNA), which are formed during transcription. Accumulation of R-loops can lead to genome instability, so RNase Hl plays a role in preventing DNA damage. It assists in removing RNA primers during DNA replication in both mitochondria and the nucleus.

[0177] RNase H2 can cleave single ribonucleotides embedded in DNA, making it essential for resolving ribonucleotide misincorporations. It also cleaves RNA-DNA hybrids, though its substrate preference is different from RNase Hl. RNase H2 is crucial for the removal of single ribonucleotides that are mistakenly incorporated into DNA during replication. These ribonucleotides can destabilize the DNA structure if not removed, leading to genome instability. By excising misincorporated ribonucleotides, RNase H2 plays a major role in maintaining genome stability and preventing mutations. RNase H2 helps remove ribonucleotides from DNA to prevent stalling of the replication fork, which could lead to replication stress and DNA damage. Like RNase Hl, RNase H2 also processes R-loops, although it is more involved in the removal Attorney Docket No. : 6010-0034W001

[0178] of ribonucleotides embedded in genomic DNA. RNase H2 (Ribonuclease H2) is an enzyme that plays a crucial role in maintaining genome stability by degrading RNA molecules that are hybridized to DNA. Specifically, it recognizes and cleaves the RNA strand of RNA-DNA hybrids, which can arise during various cellular processes such as DNA replication, repair, and transcription. RNase H2 specifically cleaves the RNA in RNA-DNA hybrid structures, which can form during replication, reverse transcription, or during other cellular activities. One of the primary roles of RNase H2 is in the ribonucleotide excision repair (RER) pathway, where it recognizes ribonucleotides that are mistakenly incorporated into the DNA during replication. These ribonucleotides are excised to prevent DNA damage and maintain the integrity of the genome. In addition, RNase H2 has a role in DNA Replication and Repair, by clearing ribonucleotides from DNA, RNase H2 helps prevent replication stress and DNA strand breaks that can be caused by the presence of ribonucleotides in the genome.

[0179] RNase H2 is a heterotrimeric enzyme, meaning it consists of three subunits: RNase H2A, RNase H2B, and RNase H2C. Each of these subunits contributes to the enzyme’s activity and stability. RNase H2A is the catalytic subunit that carries out the cleavage reaction; RNase H2B and RNase H2C are accessory subunits that help with substrate recognition and stabilization.

[0180] RNase Hl primarily targets long RNA-DNA hybrids, while RNase H2 can cleave single ribonucleotides embedded within DNA, making it more versatile in handling different types of RNA-DNA hybrid structures. RNase Hl is more critical in the mitochondria, whereas RNase H2 is more involved in maintaining nuclear genome integrity through the ribonucleotide excision repair pathway. In bacteria, RNase H can be found in both types, similar to eukaryotic Hl and H2. In some bacteria, the RNase H activity is divided between two genes (RNase Hl and RNase H2), but in others, a single enzyme might have both activities.

[0181] Sample

[0182] In one embodiment of the systems and methods as disclosed herein, the sample is selected from tissue(s) samples, cells, biological fluid samples (e.g., blood, urine, saliva, lymphatic fluid, cerebrospinal fluid (CSF), amniotic fluid, pleural fluid, pericardial fluid, ascites, aqueous humor), bone marrow samples, semen samples, biopsy samples, cancer samples, tumor samples, cell lysate samples, forensic samples, archaeological samples, paleontological samples, infection samples, production samples, whole plants, plant parts, microbiota samples, viral preparations, soil samples, marine samples, freshwater samples, household or industrial samples, and Attorney Docket No. : 6010-0034W001

[0183] combinations and isolates thereof. In one embodiment of the systems and methods as disclosed herein, the sample is a cell (e.g., an animal cell [e.g., a human cell], a plant cell, a fungal cell, a bacterial cell, and a protozoal cell). In one specific embodiment, the cell is lysed prior to the replication. In one specific embodiment, cell lysis is accompanied by proteolysis. In one specific embodiment, the cell is selected from a cell from a preimplantation embryo, a stem cell, a fetal cell, a tumor cell, a suspected cancer cell, a cancer cell, a cell subjected to a gene editing procedure, a cell from a pathogenic organism, a cell obtained from a forensic sample, a cell obtained from an archeological sample, and a cell obtained from a paleontological sample. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a preimplantation embryo (e.g., a blastomere). In one specific embodiment, the method further comprises determining the presence of disease predisposing germline or somatic variants in the embryo cell. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell from a pathogenic organism (e.g., a bacterium, a fungus, a protozoan). In one specific embodiment, the pathogenic organism cell is obtained from fluid taken from a patient, microbiota sample (e.g., GI microbiota sample, vaginal microbiota sample, skin microbiota sample, etc.) or an indwelling medical device (e.g., an intravenous catheter, a urethral catheter, a cerebrospinal shunt, a prosthetic valve, an artificial joint, an endotracheal tube, etc ). In one specific embodiment, the method further comprises the step of determining the identity of the pathogenic organism. In one specific embodiment, the method further comprises determining the presence of genetic variants responsible for resistance of the pathogenic organism to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a tumor cell, a suspected cancer cell, or a cancer cell. In one specific embodiment, the method further comprises determining the presence of one or more diagnostic or prognostic mutations. In one specific embodiment, the method further comprises determining the presence of germline or somatic variants responsible for resistance to a treatment. In one embodiment of any of the systems and methods as disclosed herein, the sample is a cell subjected to a gene editing procedure. In one specific embodiment, the method further comprises determining the presence of unplanned mutations caused by the gene editing process. In one embodiment of any of the systems and methods as disclosed herein, the method further comprises determining the history of a cell lineage. In a related aspect, the invention provides a use of any of the systems and Attorney Docket No. : 6010-0034W001

[0184] methods as disclosed herein for identifying low frequency sequence variants (e.g., variants which constitute >0.01% of the total sequences).

[0185] Polymerase

[0186] As described herein, the disclosed methods make the use of a polymerase for amplification. In some embodiments, the polymerase is a DNA polymerase that lacks a 5' to 3' exonuclease activity. The polymerase is used under conditions such that the strand extending from a first primer can be displaced by polymerization of the forming strand extending from a second primer that is “outer” with respect to the first primer. Conveniently, the polymerase is capable of displacing the strand complementary to the template strand, a property termed “strand displacement.” Strand displacement results in synthesis of multiple copies of the target sequence per template molecule. In some embodiments, the DNA polymerase for use in the disclosed methods is highly processive. Exemplary DNA polymerases include variants of Taq DNA polymerase that lack 5' to 3' exonuclease activity, e.g., the Stoffel fragment of Taq DNA polymerase (ABI), SD polymerase (Bioron), mutant Taq lacking 5' to 3' exonuclease activity described in U.S. Pat. No. 5,474,920, Bea polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene). If thermocycling is to be carried out (as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase. Table 2 below lists polymerases available from New England Biolabs that have no 5' to 3’ exonuclease activity, but that have strand displacement activity accompanied by thermal stability.

[0187] In some embodiments, it can be advantageous to use a blend of two or more polymerases. For example, an illustrative polymerase blend includes a polymerase that is particularly proficient at initiating extension from a partially double-stranded DNA primer and a polymerase that is particularly proficient at strand displacement synthesis, since combining these properties may provide a net advantage in some embodiments. Alternatively or in addition, where it is desirable to use a Taqman-style probe to carry our real-time PCR, a polymerase blend can include a polymerase that has 5' to 3' exonuclease activity, provided the primer structure is designed so that it is not susceptible to “flap” endonuclease activity; indeed, the structures described herein may be inherently less susceptible to this activity because of the double-stranded nature of the “flap.” Taq DNA polymerase can, for example, be employed in such polymerase blends because, although it is described as including a 5' to 3' exonuclease activity, Taq DNA polymerase operates more like a flap endonuclease. US11352622B2, incorporated herein by reference, provides Attorney Docket No. : 6010-0034W001

[0188] several examples thermostable Stand-Displacing Polymerases Lacking 5' to 3' Exonuclease Activity.

[0189] In some embodiments, the DNA polymerase comprises a fusion between Taq polymerase and a portion of a topoisomerase, e.g., TOPOTAQ™ (Fidelity Systems, Inc.). Illustrative polymerase concentrations range from about 20 to 200 units per reaction, e.g., for SD polymerase. In various embodiments, the polymerase concentration can be at least: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 or more units per reaction. In some embodiments, the polymerase concentration falls within a range bounded by any of these values, e.g., 10-200, 10-150, 10-100, 10-50, 20-150, 20-100, 20-50, 50-200, 50-150, 50-100, 100-200, 100-150, etc. units per reaction. When polymerase blends are used, the total, combined polymerase concentration can be any of these values or fall within any of these ranges. Strand displacement can also be facilitated through the use of a strand displacement factor, such as a helicase. Any DNA polymerase that can perform strand displacement in the presence of a strand displacement factor is suitable for use in the disclosed method, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors useful in the methods described herein include BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68(2): 1158-1164 (1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91(22): 10665-10669 (1994)), single-stranded DNA binding proteins (SSB; Rigler and Romano, I. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem.

[0190] 267:13629-13635 (1992)). Helicase and SSB are available in thermostable forms and therefore suitable for use in PCR.

[0191] Amplification

[0192] The primer sets described above are contacted with sample nucleic acids under conditions wherein the primers anneal to their template strands, if present. The desired nucleic acid amplification method is carried out using a DNA polymerase lacking 5 '-3' exonuclease activity that is capable of strand displacement under the reaction conditions employed. This amplification produces amplicons that include the sequences of all primers employed in the amplification reaction. Attorney Docket No. : 6010-0034W001

[0193] In some embodiments, the amplification step is performed using PCR. For running realtime PCR reactions, reaction mixtures generally contain an appropriate buffer, a source of magnesium ions (Mg2+) in the range of about 1 to about 10 mM, e.g., in the range of about 2 to about 8 mM, nucleotides, and optionally, detergents, and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 30 mM preferred. In one embodiment, the TRIS buffer concentration is 20 mM in the reaction mix double-strength (2x) form. The reaction mix can have a pH range of from about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 as typical. Concentration of nucleotides can be in the range of about 25 mM to about 1000 mM, typically in the range of about 100 mM to about 800 mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 mM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 may also be included in the reaction mixture. Stabilizing agents such as dithiothreitol (DTT, Cleland's reagent) or mercaptoethanol may also be included. In addition, master mixes may optionally contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A master mix is commercially available from Applied Biosystems, Foster City, Calif, (TaqMan® Universal Master Mix, cat. nos. 4304437, 4318157, and 4326708).

[0194] Labeling Strategies

[0195] Any suitable labeling strategy can be employed in the methods described herein. Where the reaction is analyzed for presence of a single amplification product, a universal detection probe can be employed in the amplification mixture. In particular embodiments, real-time PCR detection can be carried out using a universal qPCR probe. Suitable universal qPCR probes include double-stranded DNA-binding dyes, such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, Org.), Eva Green (Biotium), ethidium bromide, and the like (see Zhu et al., 1994, Anal. Chem. 66:1941-48).

[0196] In some embodiments, one or more target-specific qPCR probes (i.e., specific for a target nucleotide sequence to be detected) is employed in the amplification mixtures to detect amplification products. By judicious choice of labels, analyses can be conducted in which the different labels are excited and / or detected at different wavelengths in a single reaction (“multiplex detection”). See, e.g., Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Attorney Docket No. : 6010-0034W001

[0197] Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed ). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992); and Linck et al. (2017) “A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species,” PLOS One 12(5).

[0198] In some embodiments, it may be convenient to include labels on one or more of the primers employed in in amplification mixture.

[0199] Exemplary Automation and Systems

[0200] In some embodiments, a target nucleic acid is detected using an automated sample handling and / or analysis platform. In some embodiments, commercially available automated analysis platforms are utilized. For example, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale, Calif.) is utilized.

[0201] The methods described herein are illustrated for use with the GeneXpert system. Exemplary sample preparation and analysis methods are described below. However, the present invention is not limited to a particular detection method or analysis platform. One of skill in the art recognizes that any number of platforms and methods may be utilized.

[0202] The GeneXpert® utilizes a self-contained, single use cartridge. Sample extraction, amplification, and detection may all be carried out within this self-contained “laboratory in a cartridge” (available from Cepheid — see www.cepheid.com).

[0203] Components of the cartridge include, but are not limited to, processing chambers containing reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. A valve enables fluid transfer from chamber to chamber and contains nucleic acids lysis and filtration components. An optical window enables real-time optical detection. A reaction tube enables very rapid thermal cycling.

[0204] In some embodiments, the GeneXpert® system includes a plurality of modules for scalability. Each module includes a plurality of cartridges, along with sample handling and analysis components.

[0205] After the sample is added to the cartridge, the sample is contacted with lysis buffer and released nucleic acid is bound to a nucleic acid-binding substrate such as a silica or glass substrate. The sample supernatant is then removed and the nucleic acid eluted in an elution buffer such as a Tris / EDTA buffer. The eluate may then be processed in the cartridge to detect target genes as Attorney Docket No. : 6010-0034W001

[0206] described herein. In some embodiments, the eluate is used to reconstitute at least some of the reagents, which are present in the cartridge as lyophilized particles.

[0207] In some embodiments, PCR is used to amplify and detect the presence of one or more target nucleic acids. In some embodiments, the PCR uses Taq polymerase with hot start function, such as AptaTaq (Roche).

[0208] In some embodiments, an off-line centrifugation is used to improve assay results with samples with low cellular content. The sample, with or without the buffer added, is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of supernatant, buffer, or other liquid. The resuspended pellet is then added to a GeneXpert® cartridge as previously described.

[0209] Kits

[0210] Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or any other material useful in sample processing, washing, or conducting any other step of the assay.

[0211] Kits preferably include instructions for carrying out one or more of the screening methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user can be employed. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.

[0212] The invention will be illustrated in more detail with reference to the following Examples, but it should be understood that the present invention is not deemed to be limited thereto.

[0213] EXAMPLES

[0214] Example 1 Attorney Docket No. : 6010-0034W001

[0215] Figure 4 shows the sequences of the Fast PCR primers as tested in this Example. Referring to Figure 5A and Figure 5B, a single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2_plasmid DNA) as a template was performed to amplify the SARS-CoV-2 N2 gene using PCR with a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. For this experiment, an on-market reference assay for the detection of Covid 19 N2 region, which was used to determine the effect on TTR using the ribobase-aided fast PCR for the detection of Covid 19 N2 region. The ribobase-aided fast PCR reaction is set up according to Table 1.

[0216] Table 1: PCR reaction setup

[0217]

[0218] N2_plasmid DNA: Plasmid containing the SARS-CoV-2 N2 gene sequence, used as the DNA template. N2 Primers: Primers specific to the SARS-CoV-2 N2 region, optimized for single-plex amplification. PCR Master Mix Components: included dNTPs: Deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) for DNA synthesis. MgCE: Magnesium chloride for enzyme activity and primer-template binding; Taq DNA Polymerase: Thermostable enzyme for DNA amplification; RNase H2; and PCR Buffer: Buffer to maintain pH and ionic strength for optimal enzyme activity. Add N2 plasmid DNA template (10 copies / pL in a 65pL reaction). PCR Thermal Cycling Conditions: PCR Cycling: 96C Is, 62C 8s, 45 cycles. Individual value plots for the threshold cycle (Ct) are provided forN2_Ct and N2_EndPoint. Controls included a notemplate control (NTC) to ensure no contamination or non-specific amplification. Attorney Docket No. : 6010-0034W001

[0219] Example 2

[0220] Figure 4 shows the sequences of the Fast PCR primers as tested in this Example. Referring to Figure 6, a single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2_plasmid DNA) as a template was performed to optimize the annealing / extension temperature. The ribobaseaided fast PCR reaction is set up according to Table 2.

[0221] Table 2: PCR reaction setup

[0222]

[0223] Two separate experiments to cover the temperature range. When tested at lOOcopies / pL, the average Ct was 3 Ct earlier than that at 10 copy / pL at 72C (data not shown)

[0224] Example 3

[0225] Referring to Figure 7A and Figure 7B, a single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2_plasmid DNA) as a template was performed, to amplify the SARS-CoV-2 N2 gene using PCR as a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. For this experiment, an on market reference assay for the detection of Covid 19 N2 region, which was used to determine the effect on TTR using the ribobase-aided fast PCR for the detection of Covid 19 N2 region. The ribobase-aided fast PCR reaction is set up according to Table 3.

[0226] Table 3: PCR reaction setup Attorney Docket No. : 6010-0034W001

[0227]

[0228] PCR Thermal Cycling Conditions: 96C Is, 72C 8s. Included a no-template control (NTC) to ensure no contamination or non-specific amplification.

[0229] Example 4

[0230] Referring to Figure 8, this experiment shows amplification in the presence of Ing / pL human genomic DNA. Referring to Figures 8A and Figure 8B, a single-plex PCR amplification of Ing / pL human genomic DNA, targeting the N2 gene sequence as a template was performed to amplify the N2 gene using PCR with a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. Fig. 8A shows the Individual Value plot for the N2_Ct; Fig. 8B shows the Individual Value plot for the External Positive Fluorescence Standards (EPF Ct). The ribobase-aided fast PCR reaction is set up according to Table 4.

[0231] Table 4: PCR reaction setup

[0232]

[0233] Attorney Docket No. : 6010-0034W001

[0234] KC1 65 mM

[0235]

[0236] Temperature profile: 96C Is, 72C 8s. NTC remained negative.

[0237] Example 5

[0238] Referring to Figure 9, an experiment evaluating non-specific PCR amplification using EvaGreen dye in target-free reactions assessing background amplification or primer-dimer formation in the absence of a specific DNA target was performed. EvaGreen, a fluorescent dye that binds to double-stranded DNA, can reveal any unintended amplification products or primer artifacts through changes in fluorescence. This experiment evaluates whether non-specific amplification occurs in PCR reactions that lack a template (target-free) but include primers, EvaGreen dye, and standard PCR reagents. The goal is to identify any amplification or primerdimer artifacts by observing fluorescence over multiple cycles, which would suggest non-specific amplification. The ribobase-aided fast PCR reaction is set up according to Table 5.

[0239] Table 5: PCR reaction setup

[0240]

[0241] 10% PEG8000 is the dominant contributor to non-specific signal accumulation by promoting primer-primer interaction. In a control reaction, Human genomic DNA did contribute to non Attorney Docket No. : 6010-0034W001

[0242] specific product accumulation only in the absence of 10% PEG8000. Elevated anneal / extension temperature reduced non-specific product amplification as expected.

[0243] Example 6

[0244] Referring to Figure 10, an experiment was conducted to Performance at Higher Temperature and in Different amount of PEG8000. A single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2_plasmid DNA) as a template was performed, to amplify the SARS-CoV-2 N2 gene using PCR as a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. The ribobase-aided fast PCR reaction is set up according to Table 6.

[0245] Table 6: PCR reaction setup

[0246]

[0247] The reaction in lower PEG preferred lower anneal / extension whereas higher PEG preferred higher anneal / extension. This is consistent with the Eva Green experiment result that higher concentration of PEG promote primer-primer interaction and high anneal / extension temp is required to counter that effect.

[0248] Example 7 Attorney Docket No. : 6010-0034W001

[0249] Referring to Figure 11, an experiment was performed which demonstrated that Additional Amount of Polymerase is not the Solution to Inhibition by Human gDNA. A single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2 plasmid DNA) as a template was performed, to amplify the SARS-CoV-2 N2 gene using PCR as a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. The ribobase-aided fast PCR reaction is set up according to Table 7.

[0250] Table 7: PCR reaction setup

[0251]

[0252] Temperature profile: 96C Is, 72C 8s. Doubling the TG46E enzyme to 2000 units / reaction had adverse effect on Fast PCR. Higher anneal / extension at 78C did not provide additional benefit by reducing non-specific amplification.

[0253] Example 8

[0254] Referring to Figure 12, an experiment was conducted to evaluate the Impact of Primer and MgCh Concentration on Fast PCR Performance. In Fig. 12A, samples contained 10% PEG, 1 ng / pL of human gDNA and 1 cp / pL of N2 plasmid. In Fig. 12B samples contained 600 nM primers, 1 cp / pL N2 primers 1 ng / pL human gDNA and 10% PEG 8000. 600nM primer and 7mM of MgCh concentration provided the best performance Attorney Docket No. : 6010-0034W001

[0255] Example 9

[0256] Referring to Figure 13, and experiment was conducted to determine the effect of additional RNase H2 on Fast PCR. 4 replicates, 1 copy / pL, Ing / pL human gDNA; additional RNase HII had adverse effect on RAP PCR.

[0257] Example 10

[0258] Referring to Figure 15, an experiment was conducted to determine the effect of the addition of limiting amount of non-ribobase primer on the inhibition by human gDNA of Fast PCR. A single-plex PCR amplification of Ing / pL human genomic DNA, targeting the N2 gene sequence as a template was performed to amplify the N2 gene using PCR with a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. The ribobase-aided fast PCR reaction is set up according to Table 8.

[0259] Table 8: PCR reaction setup

[0260]

[0261] Temperature profile: 96C Is, 72C 8s. Addition of limiting amount of non-ribobase primer did not release human gDNA inhibition.

[0262] Example 11

[0263] Referring to Figure 16 an experiment was conducted to evaluate the short Lag and Lead Primer Performance at Different Temperatures, a single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2_plasmid DNA) as a template was performed to amplify the SARS-CoV-2 N2 gene Attorney Docket No. : 6010-0034W001

[0264] using PCR with a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. A regular PCR reaction is set up according to Table 9. Table 9: PCR reaction setup

[0265]

[0266] PCR Thermal Cycling Conditions: 96C Is; 62 C or 68C or 72C; 8s. No PEG 8000 Example 12

[0267] Referring to Figure 17, a comparison of Fast PCR to on-market PCR assay was performed, a single-plex PCR amplification of the SARS-CoV-2 N2 gene, targeting the nucleocapsid (N) region, using a plasmid containing the N2 gene sequence (N2_plasmid DNA) as a template was performed to amplify the SARS-CoV-2 N2 gene using PCR with a DNA template and to evaluate the efficiency and specificity of the N2 primer set under optimized PCR conditions. For this experiment, an on market reference assay for the detection of Covidl9 N2 region, which was used to determine the effect on TTR using the ribobase-aided fast PCR for the detection of Covid 19 N2 region. The ribobase-aided fast PCR reaction is set up according to Table 12.

[0268] Table 12: PCR reaction setup

[0269]

[0270] Attorney Docket No. : 6010-0034W001

[0271]

[0272] N2_plasmid DNA: Plasmid containing the SARS-CoV-2 N2 gene sequence, used as the DNA template. N2 Primers: Primers specific to the SARS-CoV-2 N2 region, optimized for single-plex amplification. PCR Master Mix Components: included dNTPs: Deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) for DNA synthesis. MgCE: Magnesium chloride for enzyme activity and primer-template binding; Taq DNA Polymerase: Thermostable enzyme for DNA amplification; RNase H2; and PCR Buffer: Buffer to maintain pH and ionic strength for optimal enzyme activity. Add N2_plasmid DNA template (10 copies / pL in a 65 pL reaction). PCR Thermal Cycling Conditions: PCR Cycling: 96C Is, 62C 8s, 45 cycles. Individual value plots for the threshold cycle (Ct) are provided for N2_Ct and N2_EndPoint.

[0273] While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

Attorney Docket No. : 6010-0034W001CLAIMS WHAT IS CLAIMED IS:

1. A method for amplifying a target nucleic acid in a sample, the method comprising:(a) adding the sample to a polymerase chain reaction (PCR) mixture comprising:(i) An oligonucleotide primer for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, wherein the oligonucleotide primer comprises a first lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a first leading primer sequence, wherein the first lagging primer sequence is complementary to a sequence in the target nucleic acid,wherein the target-specific cleavage domain comprising the at least one ribonucleotide is complementary to a sequence in the first template strand,wherein the first leading primer sequence is complimentary to a sequence in the target nucleic acid,further wherein the oligonucleotide primer lacks a terminal 3' cap, and(ii) an amplification buffer comprising a DNA polymerase with stranddisplacement activity and a mixture of nucleotides; and an RNAse H2 endonuclease enzyme capable of cleaving the oligonucleotide primer at the target-specific cleavage domain when the at least one ribonucleotide is hybridized to the target nucleic acid;(b) hybridizing the oligonucleotide primer to the first template strand of the target nucleic acid if present in the sample to form a first double-stranded substrate;(c) cleaving the oligonucleotide primer hybridized to the first template strand with the endonuclease enzyme at a point within or adjacent to the target-specific cleavage domain, thereby generating a 3 ’-OH group in the lagging sequence;Attorney Docket No. : 6010-0034W001(d) extending the leading sequence hybridized to the first template strand with the polymerase to form a first copy of the target nucleic acid; and(e) extending the lagging sequence hybridized to the first template strand of the with the polymerase to form a second copy of the target nucleic acid.

2. The method of claim 1, further comprising:(f) hybridizing a complementary oligonucleotide primer to the second template strand, wherein the complementary oligonucleotide primer comprises a second lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a second leading primer sequence,wherein the target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the second template strand, further wherein the oligonucleotide primer lacks a terminal 3' cap, to form a second double-stranded substrate;(g) cleaving the complementary oligonucleotide primer hybridized to the target nucleic acid with the endonuclease RNase H2 enzyme at a point within or adjacent to the target-specific cleavage domain, thereby generating a 3 ’-OH group in the lagging sequence;(h) extending the complementary leading sequence with the polymerase to form a third copy of the target nucleic acid; and(i) extending the complementary lagging sequence with the polymerase to form a fourth copy of the target nucleic acid.

3. The method of any one of claim 1 - 2 wherein the DNA polymerase with stranddisplacement activity lacks 5' — >3' exonuclease activity.

4. The method of any one of claims 1 - 3, wherein the first and second lagging primer sequences independently comprise 10 to 30 nucleotides.

5. The method any one of claims 1 - 4, wherein the first and second lagging primer sequences independently comprise 12 to 20 nucleotides.Attorney Docket No. : 6010-0034W0016. The method of any one of claims 1 - 5, wherein the first and second leading primer sequences independently comprise 10 to 30 nucleotides.

7. The method of any one of claims 1 - 6, wherein the first and second leading primer sequences independently comprise 12 to 20 nucleotides.

8. The method of any one of claims 1 - 7, wherein the second template strand is complementary to the first template strand.

9. The method of any one of claims 1 - 8, wherein the first and second lagging primer sequences independently comprise deoxyribonucleotides.

10. The method of any one of claims 1 - 9, wherein the first and second lagging primer sequence independently comprise at least one non-natural nucleotide base.

11. The method of any one of claims 1 - 10, wherein the first and second lagging primer sequence first and second comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4- ethylcytosine, 6-amino-5-nitro-3-(l ’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino- 8-(r-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof.

12. The method of any one of claims 1 - 11, wherein the target-specific cleavage domain comprises at least one natural ribonucleotide base.

13. The method of any one of claims 1 - 12, wherein the target-specific cleavage domain comprises a single ribonucleotide.

14. The method of any one of claims 1 - 13, wherein the target-specific cleavage domain comprises a plurality of ribonucleotides.

15. The method of any one of claims 1 - 14, wherein the target-specific cleavage domain comprises 1 - 10 ribonucleotides.

16. The method of any one of claims 1 - 15, wherein the first and second leading primer sequences independently comprise deoxyribonucleotides.

17. The method of any one of claims 1 - 16, wherein the first and second leading primer sequences independently comprise at least one non-natural nucleotide base.Attorney Docket No. : 6010-0034W00118. The method of any one of claims 1 - 17, wherein the first and second leading primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4- ethylcytosine, 6-amino-5-nitro-3-(l ’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino- 8-(r-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof.

19. An oligonucleotide primer for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, wherein the oligonucleotide primer comprises a first lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a first leading primer sequence,wherein the first lagging primer sequence is complementary to a sequence in the target nucleic acid,wherein the target-specific cleavage domain comprising the at least one ribonucleotide is complementary to a sequence in the first template strand,wherein the first leading primer sequence is complimentary to a sequence in the target nucleic acid,further wherein the oligonucleotide primer lacks a terminal 3' cap.

20. A complementary oligonucleotide primer to the second template strand of claim 19, wherein the complementary oligonucleotide primer comprises a second lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a second leading primer sequence,wherein the target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the second template strand,further wherein the oligonucleotide primer lacks a terminal 3' cap, to form a second double-stranded substrate.

21. The oligonucleotide primer of any one of claims 19 - 20, wherein the first and second lagging primer sequences independently comprises 10 to 30 nucleotides.Attorney Docket No. : 6010-0034W00122. The oligonucleotide primer of any one of claims 19 - 21, wherein the first and second lagging primer sequences independently comprises 12 to 20 nucleotides.

23. The oligonucleotide primer of any one of claims 19 - 22, wherein the first and second leading primer sequences independently comprise 10 to 30 nucleotides.

24. The oligonucleotide primer of any one of claims 19 - 23, wherein the first and second leading primer sequences independently comprise 12 to 20 nucleotides.

25. The oligonucleotide primer of any one of claims 19 - 24, wherein the second template strand is complementary to the first template strand.

26. The oligonucleotide primer of any one of claims 19 - 25, wherein the first and second lagging primer sequences independently comprise deoxyribonucleotides.

27. The oligonucleotide primer of any one of claims 19 - 26, wherein the first and second lagging primer sequences independently comprise at least one non-natural nucleotide base.

28. The oligonucleotide primer of any one of claims 19 - 27, wherein the first and second lagging primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4- ethylcytosine, 6-amino-5-nitro-3-(r-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino- 8-( 1 ’ -beta-D-2’ -ribofuranosyl)-imidazo[ 1 ,2-a]- 1 ,3 , 5-triazin-4(8H)-one, and combinations thereof.

29. The oligonucleotide primer of any one of claims 19 - 28, wherein the target-specific cleavage domain comprises at least one natural ribonucleotide base.

30. The oligonucleotide primer of any one of claims 19 - 29, wherein the target-specific cleavage domain comprises a single ribonucleotide.

31. The oligonucleotide primer of any one of claims 19 - 30, wherein the target-specific cleavage domain comprises a plurality of ribonucleotides.

32. The oligonucleotide primer of any one of claims 19 - 31, wherein the target-specific cleavage domain comprises 1 - 10 ribonucleotides.

33. The oligonucleotide primer of any one of claims 19 - 32, wherein the first and second leading primer sequences independently comprise deoxyribonucleotides.Attorney Docket No. : 6010-0034W00134. The oligonucleotide primer of any one of claims 19 - 33, wherein the first and second leading primer sequences independently comprise at least one non-natural nucleotide base.

35. The oligonucleotide primer of any one of claims 19 - 34, wherein the first and second leading primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4- ethylcytosine, 6-amino-5-nitro-3-(l ’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino- 8-(r-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof.

36. A combination of at least two oligonucleotides according to any one of claims 19 - 35, wherein one oligonucleotide is a forward primer and one oligonucleotide is a reverse primer for amplifying a target nucleic acid.

37. A composition for amplifying a target nucleic acid in a sample, wherein the target nucleic acid comprises a first template strand and, optionally, a second template strand, wherein the second template strand is complementary to the first template strand, the composition comprising:a polymerase chain reaction (PCR) mixture comprising:i) a set of oligonucleotide primers comprising:a forward oligonucleotide primer comprising comprises a first lagging primer sequence which is adjacent to, and 5' of, a target-specific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a first leading primer sequence, wherein the lagging primer sequence is complementary to a sequence in the target nucleic acid, wherein the target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the target nucleic acid, wherein the first leading primer sequence is complimentary to a sequence in the target nucleic acid, further wherein the oligonucleotide primer lacks a terminal 3' cap;anda reverse oligonucleotide primer comprising comprises a second lagging primer sequence which is adjacent to, and 5' of, a targetspecific cleavage domain comprising at least one ribonucleotide, wherein the cleavage domain is adjacent to, and 5' of, a second leading primer sequence, wherein the second lagging primer sequence is complementary to a sequence in the target nucleic acid, wherein theAttorney Docket No. : 6010-0034W001target-specific cleavage domain comprises the at least one ribonucleotide and is complementary to a sequence in the target nucleic acid, wherein the second leading primer sequence is complimentary to a sequence in the target nucleic acid, further wherein the oligonucleotide primer lacks a terminal 3' cap,ii) an amplification buffer comprising a DNA polymerase and a mixture of nucleotides;iii) an endonuclease RNaseH2 enzyme capable of cleaving the oligonucleotide primer at the target-specific cleavage domain when the at least one ribonucleotide is hybridized to the target nucleic acid;wherein the at least one ribonucleotide is capable of forming a target-specific cleavage domain when hybridized to the target nucleic acid.

38. The composition of claim 37, wherein the first and second lagging primer sequences independently comprises 10 to 30 nucleotides.

39. The composition of any one of claims 37 - 38, wherein the first and second lagging primer sequences independently comprises 12 to 20 nucleotides.

40. The composition of any one of claims 37 - 39, wherein the first and second leading primer sequences independently comprise 10 to 30 nucleotides.

41. The composition of any one of claims 37 -40, wherein the first and second leading primer sequences independently comprise 12 to 20 nucleotides.

42. The composition of any one of claims 37 - 40, wherein the second template strand is complementary to the first template strand.

43. The composition of any one of claims 37 - 45, wherein the first and second lagging primer sequences independently comprise deoxyribonucleotides.

44. The composition of any one of claims 37 - 43, wherein the first and second lagging primer sequences independently comprise at least one non-natural nucleotide base.

45. The composition of any one of claims 37-44, wherein the first and second lagging primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4- ethylcytosine, 6-amino-5-nitro-3-(l ’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino- 8-(r-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof.Attorney Docket No. : 6010-0034W00146. The composition of any one of claims 37 -45, wherein the target-specific cleavage domain comprises at least one natural ribonucleotide base.

47. The composition of any one of claims 37 - 46, wherein the target-specific cleavage domain comprises a single ribonucleotide.

48. The composition of any one of claims 37 - 47, wherein the target-specific cleavage domain comprises a plurality of ribonucleotides.

49. The composition of any one of claims 37 - 48, wherein the target-specific cleavage domain comprises 1 - 10 ribonucleotides.

50. The composition of any one of claims 37 - 49, wherein the target-specific cleavage domain is located near the 5’ end of the leading primer.

51. The composition of any one of claims 37 - 50, wherein the target-specific cleavage domain is located near the 3’ end of the lagging primer.

52. The composition of any one of claims 37 - 51, wherein the first and second leading primer sequences independently comprise deoxyribonucleotides.

53. The composition of any one of claims 37 - 52, wherein the first and second leading primer sequences independently comprise at least one non-natural nucleotide base.

54. The composition of any one of claims 37 - 53, wherein the first and second leading primer sequence independently comprise a non-natural nucleotide base wherein the non-natural nucleotide base is selected from the group consisting of thymine, inosine, xanthosine, isoguanosine, isocytosine, 2-aminopurine, 2-thiothymine, hypoxanthine, N4- ethylcytosine, 6-amino-5-nitro-3-(l ’-beta-D-2’-ribofuranosyl)-2(lH)-pyridone, 2-amino- 8-(r-beta-D-2’-ribofuranosyl)-imidazo[l,2-a]-l,3,5-triazin-4(8H)-one, and combinations thereof.

55. The oligonucleotide of any one of claims 19 - 36, or the composition of any one of claims 37 - 54, wherein the oligonucleotide, or composition is contained within a cartridge for detecting one or more target nucleic acids in a sample, the cartridge comprising:a cartridge body comprising a plurality of chambers therein, wherein the plurality of chambers includes:a sample chamber having at least a fluid outlet in fluidic communication with another chamber of the plurality;an optional lysis chamber in fluidic communication with the sample chamber, optionally wherein the sample chamber and lysis chamber are the same;a reagent chamber comprising one or more of the oligonucleotide(s) and / or one or more of the combination(s); andAttorney Docket No. : 6010-0034W001a reaction vessel fluidically coupled to the plurality of chambers of the cartridge body and configured for: i) amplification of nucleic acid and, optionally, ii) detection and identification of one or a plurality of amplification products; anda filter disposed in a fluidic path between the lysis chamber, if present, or the sample chamber, and the reaction vessel.

56. The method of any one of claims 1 - 18, oligonucleotide of any one of claims 19 - 36, the composition of any one of claims 37 - 54, further comprising a probe.

57. The method, oligonucleotide, or composition of claim 56, wherein the probe comprises a linear probe.

58. The method, oligonucleotide, or composition of claim 56 or 57, wherein the probe comprises a fluorescent dye and a quencher molecule.

59. The method, oligonucleotide, or composition of any one of claims 56 - 58, wherein the probe comprises a cycling probe.

60. The method, oligonucleotide, or composition of any one of claims 56 - 59, wherein amplifying the target nucleic acid comprises a multiplex amplification reaction.

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