Ultrafast amplification of nucleic acids

Abstract

Described herein are methods of amplifying nucleic acids via ultrafast thermal convection processes along with cartridge-based methods directed to the same. The methods are useful for polymerase chain reactions for detection and diagnosis of infectious diseases.

Description

Docket No. 68552WO01 (2025-25631 -P-WO)ULTRAFAST AMPLIFICATION OF NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 742,090, filed on January 6, 2025 and U.S. Provisional Patent Application No.63,791 ,042 filed April 18, 2025, the contents of which are hereby all incorporated by reference in their entirety into this disclosure.INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains one or more sequence listings that have been submitted in a computer readable format and are hereby incorporated by reference in its entirety. The computer readable file, created on January 3, 2025, is named 68552US01_SequenceListing.xml and is 38 kilobytes in size.FIELD OF THE INVENTION

[0003] This disclosure relates to methods of amplifying nucleic acids via ultrafast thermal convection processes along with cartridge-based methods directed to the same.BACKGROUND

[0004] Nucleic acid amplification and detection technologies have developed rapidly in recent years as with the appreciation of their value for detection of various diseases. In particular, the use of polymerase chain reaction (PCR) has been critical in the implementation of sensitive diagnostic assays based on nucleic acid detection. PCR technologies now are recognized as an essential diagnostic tool.

[0005] PCR was initially described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188. These patents describe amplification and detection methods wherein primers are hybridized to the strands of a targeted nucleic acid (considered the templates) in the presence of a nucleotide polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates. Under specified conditions, the result is the formation of primer extension products as nucleotides are added along the templates from the 3’-end of the primers. These products are then denatured and used as templates for more of the same primers in another extension reaction. WhenDocket No. 68552WO01 (2025-25631 -P-WO)this cycle of denaturation, hybridization and primer extension is carried out a number of times (for example 20 to 30 cycles), the process exponentially increases the original amount of targeted nucleic acid so that it is readily detected. The process exponentially increases the number of amplicons from the first cycle, and the number of amplicons may be increased by a factor of 2 in each cycle. Optical detection systems often require 20 to 30 cycles of amplification to detect a target. Once the targeted nucleic acid has been sufficiently amplified (that is, many times more copies of the molecule have been made after about 20+ cycles), various detection procedures can be used to detect it. For example, insolubilized or detectably labeled probes and gel electrophoresis are common detection methods.

[0006] A wide variety of nucleic acid amplification methods are currently available. These methods incorporate a wide range of times and temperatures are generally described with the specific combination of time and temperature largely dependent upon the type of DNA polymerase used, the complexity of the mixture of nucleic acids including the targeted nucleic acid, the length and specificity of the primers, the length of the targeted nucleic acid, pH and several other reaction conditions and components. While the overall process has become much faster than when it was initially envisioned, there is a continuing need for faster and more efficient amplification processes that are capable of maintaining high amplification efficiency in order to reduce the time for obtaining an analytical result.SUMMARY

[0007] In a first aspect, the disclosure provides a method for amplifying a target nucleic acid in an amplification reaction, the method comprising: providing a solution in a reaction chamber, the reaction chamber defined by two opposing major walls and side walls connecting the major walls to each other, and the solution comprising the target nucleic acid, a thermostable polymerase, and a primer pair configured for amplification of the target nucleic acid; maintaining a first region of the reaction chamber at a first temperature capable of denaturing the target nucleic acid sequence into a single-stranded nucleic acid template; maintaining a second region of the reaction chamber at a second temperature capable of annealing the primer pair to the single-stranded nucleic acid template; inducing a thermal convection cycle in the solution between the first region and the second region of the reaction chamber; wherein an average cycle time for the thermal convection cycle is 15 seconds or less;Docket No. 68552WO01 (2025-25631 -P-WO)and holding the first region at the first temperature and the second region at the second temperature, for a time sufficient for amplification of the target nucleic acid. In some embodiments, the time sufficient for amplification of the target nucleic acid is defined as a time sufficient to produce equal / greater level of amplification compared to at least 20 amplification cycles using conventional quantitative PCR (qPCR or also referred to as real-time PCR) by thermal cycling.

[0008] In some embodiments of the first aspect, the first region and second region of the reaction chamber are each defined by one of the two opposing major walls, respectively. In some embodiments, the major walls of the reaction chamber have a ratio of height to length of from 1 :1 to 1 :1 .5. In some embodiments, the length of the major walls of the reaction chamber is from 1 .4 to 20 mm; and / or the height of the major walls of the reaction chamber is from 1 to 15 mm; and / or the average distance between the two opposing major walls of the reaction chamber is from 0.5 to 5 mm.

[0009] In some embodiments of the first aspect, the solution further comprises at least one probe capable of hybridizing with the target nucleic acid, wherein the probe has a concentration of 500 nM or greater and a Tm below the first temperature. In some embodiments, the method further comprises detecting the presence of an amplified nucleic acid using a probe that has hybridized with the target single-stranded nucleic acid template.

[0010] In some embodiments of the first aspect, the difference in temperature between the first temperature and the second temperature is equal to or greater than 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C or from 25-37°C, 25-35°C, 27-35°C, 28-34°C. In some embodiments, the first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C. In some embodiments, the second temperature is from 60°G to 70°C, from 64°C to 68°C, or from 65°C to 67°C. In still other embodiments, the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C. In some embodiments, the amplification reaction is carried out over an amplification reaction time and wherein the first temperature and the second temperature each vary less than 1°C over the amplification reaction time.

[0011] In some embodiments of the first aspect, each primer has a melt temperature, Tm, from 7-13°G or from 8-10°C below the first temperature. In some embodiments, each primer has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, each primer has an annealing temperature, Ta, from 0-10°G or from 0-Docket No. 68552WO01 (2025-25631 -P-WO)6°C above the second temperature. In some embodiments, each primer has an annealing temperature, Ta, from 60-80°C, 60-75°C, or 60-70°G. In some embodiments, the primer is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM. In some embodiments, the primer has a length equal to or less than 45 nucleotides. In some embodiments, the solution comprises at least four, six, eight or ten sets of primer pairs and the amplification reaction comprises detecting at least four, six, eight, or ten target nucleic acids. In some embodiments, each primer comprises at least one stabilizing base and wherein, in some embodiments, the at least one stabilizing base comprises a stabilizing base at the 2ndor 3rdposition from the 3’ end of the primer. In some embodiments, the at least one stabilizing bases is selected from the group consisting of: locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0012] In some embodiments of the first aspect, the optional probe has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, the optional probe is present at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM. In some embodiments, the primer and / or probe comprise a detectable moiety. In some embodiments, the detectable moiety comprises a fluorescent dye and a quencher molecule. In some embodiments, the optional probe is present and comprises one or more destabilizing bases, wherein the destabilizing base may decrease the melt temperature of the target nucleic acid. In some embodiments, the one or more modified bases which is a destabilizing base is selected from the group consisting of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, 0(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0013] In some embodiments of the first aspect, the target nucleic acid has a melt temperature from 80-90°C, 81 -89°C, 82-88°G, 83-87°C, 84-86°C, or less than or equal to 90°C, 89°C, 88°C, 87°C or 86°C. In some embodiments, the target nucleic acid hasDocket No. 68552WO01 (2025-25631 -P-WO)a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 65-87, 67-87, or 69-86 nucleotides. In some embodiments, the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .

[0014] In some embodiments of the first aspect, the solution further comprises the optional reverse transcriptase, and the reverse transcriptase and thermostable polymerase are each present at concentrations of 2 U / pL or greater, 4 U / pL or greater, 6 U / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume. In some embodiments, the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited up to 55°C.

[0015] In some embodiments of the first aspect, the target nucleic acid sequences come from a biological sample selected from one or more of nasopharyngeal swab, nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix. In some embodiments, the biological sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater. In some embodiments, the biological sample volume is from 50 to 10,000 pL or from 50 to 5,000 pL.

[0016] A second aspect comprises a method for amplifying a target nucleic acid in an amplification reaction, the method comprising: providing a solution in a reaction chamber, the reaction chamber defined by two opposing major walls and side walls connecting the major walls to each other, and the solution comprising the target nucleic acid, a thermostable polymerase at a concentration of at least 4 U / pL of reaction volume, and a primer pair configured for amplification of the target nucleic acid, each primer at a concentration of at least 500 nM; maintaining a first region of the reaction chamber at a first temperature capable of denaturing the target nucleic acid sequence into a single-stranded nucleic acid template; maintaining a second region of the reaction chamber at a second temperature capable of annealing the primer pair to the single-stranded nucleic acid template; inducing a thermal convection cycle in the solution between the first region and the second region of the reaction chamber, holding the first region at the first temperature and the second region at the second temperature, for a time sufficient for amplification of the target nucleic acid. In some embodiments, the time sufficient for amplification of the target nucleic acid is defined as a time sufficient to produce equal / greater level of amplification comparedDocket No. 68552WO01 (2025-25631 -P-WO)to at least 20 amplification cycles using conventional quantitative PCR (qPCR or also referred to as real-time PCR) by thermal cycling. In some embodiments, the average cycle time for the thermal convection cycle is 15 seconds or less.

[0017] In some embodiments of the second aspect, the major walls of the reaction chamber each have a ratio of height to length of from 1 :1 to 1 :1.5. In some embodiments, the length of the major walls of the reaction chamber is from 1 .4 to 20 mm; and / or the height of the major walls of the reaction chamber is from 1 to 15 mm; and / or the average distance between the two opposing major walls of the reaction chamber is from 0.5 to 5 mm.

[0018] In some embodiments of the second aspect, the solution further comprises at least one probe capable of hybridizing with the target nucleic acid, wherein the probe has a concentration of 500 nM or greater and a Tm below the first temperature. In some embodiments, the method further comprises detecting the presence of an amplified nucleic acid using a probe that has hybridized with the target single-stranded nucleic acid template.

[0019] In some embodiments of the second aspect, the difference in temperature between the first temperature and the second temperature is equal to or greater than 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C or from 25-37°C, 25-35°C, 27-35°C, 28-34°C. In some embodiments, the first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C. In some embodiments, the second temperature is from 60°C to 70°C, from 64°C to 68°C, or from 65°C to 67°C. In still other embodiments, the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C. In some embodiments, the amplification reaction is carried out over an amplification reaction time and wherein the first temperature and the second temperature each vary less than 1°C over the amplification reaction time. The amplification reaction time can be defined as a time sufficient to produce equal / greater level of amplification compared to at least 20 amplification cycles using conventional quantitative PCR (qPCR or also referred to as real-time PCR) by thermal cycling.

[0020] In some embodiments of the second aspect, each primer has a melt temperature, Tm, from 7-13°C or from 8-10°C below the first temperature. In some embodiments, each primer has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, each primer has an annealing temperature, Ta, from 0-10°C or from 0-6°C above the second temperature. In some embodiments, eachDocket No. 68552WO01 (2025-25631 -P-WO)primer has an annealing temperature, T a, from 60-80°C, 60-75°G, or 60-70°C. In some embodiments, the primer is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM. In some embodiments, the primer has a length equal to or less than 45 nucleotides. In some embodiments, the solution comprises at least four, six, eight or ten sets of primer pairs and the amplification reaction comprises detecting at least four, six, eight, or ten target nucleic acids. In some embodiments, each primer comprises at least one stabilizing base and wherein, in some embodiments, the at least one stabilizing base comprises a stabilizing base at the 2ndor 3rdposition from the 3’ end of the primer. In some embodiments, the at least one stabilizing bases is selected from the group consisting of: locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0021] In some embodiments of the second aspect, the optional probe has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, the optional probe is present at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM. In some embodiments, the primer and / or probe comprise a detectable moiety. In some embodiments, the detectable moiety comprises a fluorescent dye and a quencher molecule. In some embodiments, the optional probe is present and comprises one or more destabilizing bases, wherein the destabilizing base may decrease the melt temperature of the target nucleic acid. In some embodiments, the one or more modified bases which is a destabilizing base is selected from the group consisting of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, 0(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2’-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0022] In some embodiments of the second aspect, the target nucleic acid has a melt temperature from 80-90°C, 81 -89°C, 82-88°G, 83-87°C, 84-86°C, or less than or equal to 90°C, 89°C, 88°C, 87°C or 86°C. In some embodiments, the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or fromDocket No. 68552WO01 (2025-25631 -P-WO)65-87, 67-87, or 69-86 nucleotides. In some embodiments, the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .

[0023] In some embodiments of the second aspect, the solution further comprises the optional reverse transcriptase, and the reverse transcriptase and thermostable polymerase are each present at concentrations of 5 U / j L or greater, 6 U / |xL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume. In some embodiments, the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited up to 55°C.

[0024] In some embodiments of the second aspect, the target nucleic acid sequences come from a biological sample selected from one or more of nasopharyngeal swab, nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix. In some embodiments, the biological sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater.

[0025] A third aspect comprises a cartridge-based method for amplifying and detecting a target nucleic acid in a sample via an amplification reaction, the method comprising: placing the sample a sample chamber in a self-contained cartridge, the self-contained cartridge further comprising: a reaction chamber in fluid communication with the sample chamber, the reaction chamber defined by two opposing major walls and side walls connecting the major walls to each other, and a filter disposed in a fluidic path between the sample chamber and the reaction chamber, isolating the target nucleic acid by passing it through the filter; forming a solution comprising the isolated target nucleic acid, a thermostable polymerase, and a primer pair configured for amplification of the target nucleic acid; transferring the solution into the reaction chamber; maintaining a first region of the reaction chamber at a first temperature capable of denaturing the target nucleic acid sequence into a single-stranded nucleic acid template; maintaining a second region of the reaction chamber at a second temperature capable of annealing the primer pair to the single-stranded nucleic acid template; inducing a thermal convection cycle in the solution between the first region and the second region, wherein an average cycle time for the thermal convection cycle is 15 seconds or less; and detecting the presence of the amplicon within 15 minutes of placing the sample within the self-contained cartridge. In some embodiments, theDocket No. 68552WO01 (2025-25631 -P-WO)amplification time is 15 minutes or less, 12 minutes or less, 9 minutes or less, or 8 minutes or less. In some embodiments, the total time for detecting the presence of the amplicon (including sample prep, reverse transcription, and amplification) is within 15 minutes, or within 14 minutes, or within 13 minutes, or within 12 minutes, or within 11 minutes, or within 10 minutes, of placing the sample within the self-contained cartridge.

[0026] In some embodiments of the third aspect, the filter comprises an amine modification for isolating nucleic acid from the biological sample. In some embodiments, the self-contained cartridge further comprises a lysis chamber, wherein the lysis chamber contains one or more lysis reagents for releasing nucleic acid. In some embodiments, each reaction chamber is configured to detect a single amplification product. In some embodiments, each reaction chamber is configured to detect a plurality of amplification products. In some embodiments, the self-contained cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.

[0027] In some embodiments of the third aspect, the major walls of the reaction chamber have a ratio of height to length of from 1 :1 to 1 :1 .5. In some embodiments, the length of the major walls of the reaction chamber is from 1.4 to 20 mm. In some embodiments, the height of the major walls of the reaction chamber is from 1 to 15 mm.

[0028] In some embodiments of the third aspect, the method further comprises at least one probe capable of hybridizing with the target nucleic acid, wherein the probe has a concentration of 500 nM or greater and a Tm below the first temperature. In some embodiments, the method further comprises detecting the presence of an amplified nucleic acid using a probe that has hybridized with the target single-stranded nucleic acid template.

[0029] In some embodiments of the third aspect, the difference in temperature between the first temperature and the second temperature is equal to or greater than 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 73°C or from 25-73°C, 25-35°C, 27-35°C, 28-34°C. In some embodiments, the first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C. In some embodiments, the second temperature is from 60°C to 70°C, from 64°C to 68°C, or from 65°C to 67°C. In some embodiments, the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C. In some embodiments, the amplification reaction is carried out over an amplification reaction time and whereinDocket No. 68552WO01 (2025-25631 -P-WO)the first temperature and the second temperature each vary less than 1°C over the amplification reaction time.

[0030] In some embodiments of the first aspect, each primer has a melt temperature, Tm, from 7-13°C or from 8-10°C below the first temperature. In some embodiments, each primer has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, each primer has an annealing temperature, Ta, from 0-10°G or from 0-6°C above the second temperature. In some embodiments, each primer has an annealing temperature, Ta, from 60-80°C, 60-75°C, or 60-70°C. In some embodiments, the primer is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM. In some embodiments, the primer has a length equal to or less than 45 nucleotides. In some embodiments, the solution comprises at least four, six, eight or ten sets of primer pairs and the amplification reaction comprises detecting at least four, six, eight, or ten target nucleic acids. In some embodiments, each primer comprises at least one stabilizing base and wherein, in some embodiments, the at least one stabilizing base comprises a stabilizing base at the 2ndor 3rdposition from the 3’ end of the primer. In some embodiments, the at least one stabilizing bases is selected from the group consisting of: locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, 0(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2’-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0031] In some embodiments of the first aspect, the optional probe has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, the optional probe is present at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM. In some embodiments, the primer and / or probe comprise a detectable moiety. In some embodiments, the detectable moiety comprises a fluorescent dye and a quencher molecule. In some embodiments, the optional probe is present and comprises one or more destabilizing bases, wherein the destabilizing base may decrease the melt temperature of the target nucleic acid. In some embodiments, the one or more modified bases which is a destabilizing base is selected from the group consisting of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, 0(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-Docket No. 68552WO01 (2025-25631 -P-WO)deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2’-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0032] In some embodiments of the first aspect, the target nucleic acid has a melt temperature from 80-90°C, 81 -89°C, 82-88°C, 83-87°C, 84-86°C, or less than or equal to 90°C, 89°C, 88°C, 87°C or 86°C. In some embodiments, the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 65-87, 67-87, or 69-86 nucleotides. In some embodiments, the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .

[0033] In some embodiments of the first aspect, the solution further comprises the optional reverse transcriptase, and the reverse transcriptase and thermostable polymerase are each present at concentrations of 6 U / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume. In some embodiments, the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited up to 55°C.

[0034] In some embodiments of the first aspect, the target nucleic acid sequences come from a biological sample selected from one or more of nasopharyngeal swab, nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix. In some embodiments, the biological sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 provides an embodied reaction vessel configured for receiving and holding a solution comprising the target nucleic acid to be amplified.

[0036] FIG. 2 shows an example reaction chamber having x, y, and z dimensions defined as length (x), width (y), and height (z).

[0037] FIG. 3 is a diagram of an embodied method of the present disclosure as described in Example 1.

[0038] FIG. 4 is a diagram of an embodied method of the present disclosure as described in Example 2.Docket No. 68552WO01 (2025-25631 -P-WO)

[0039] FIG. 5A and FIG. 5B present graphs demonstrating performance of primers and probes having different melt temperatures (Tms).

[0040] FIGS. 6A-6D present graphs demonstrating the output of the thermal convection assay across a range of temperatures varied by changing the lower temperature value.

[0041] FIGS. 7A-7D present graphs demonstrating the output of the thermal convection assay across a range of temperatures varied by changing the upper temperature value.

[0042] FIG. 8A and FIG. 8B present graphs demonstrating the influence cycle temperature range has on assay signal output as a function of time. TTR (Time-To-Result) was greatly improved with a wider temperature range of thermal convection cycling.

[0043] FIG. 9 is a schematic of the cartridge configuration for the rapid qualitative, multiplex real-time PGR in vitro test using thermal convection PGR technology.

[0044] FIGS. 10A-10E show graphs demonstrating performance of the rapid qualitative, multiplex real-time PGR in vitro test to detect Influenza A (Flu A), Influenza B (Flu B), SARS-CoV-2 (US CoV-2), Respiratory Syncytial Virus A (RSV A) and Respiratory Syncytial Virus B (RSV B) on the GeneXpert® RCC (revised cartridge C (ROC).

[0045] FIGS. 11A-11E shows graphs demonstrating performance of the rapid qualitative, multiplex real-time PGR in vitro test to detect Flu A, Flu B, US CoV-2, RSV A and RSV B on a prototype GeneXpert® cartridge (amine modified glass fiber filter cartridge.

[0046] FIG. 12 shows the time-to-result (TTR) and endpoint probe fluorescence (EPF) for PGR tests of the SAR-GoV-2 N2 and E genes using embodied assays.

[0047] FIG. 13 shows the time-to-result (TTR) and endpoint probe fluorescence (EPF) for PGR tests of the SAR-CoV-2 E / RdRp genes using embodied assay methods and commercial multiplex test beads for SARS-CoV-2, Flu A, Flu B, RSV A and RSV B.

[0048] FIG. 14 is a graph of the temperature cycle over time for a thermal cycling event in the embodied reaction chambers. The average cycle time is 15 seconds or less with a temperature cycle range from 64°C to 98°C and excellent stability across greater than 20 cycles. The probe fluorescence shows that detectable signal was seen at approximately 25 cycles with a rapid rise in intensity.Docket No. 68552WO01 (2025-25631 -P-WO)DESCRIPTION

[0049] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.Terms and Definitions

[0050] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0051] The terms “polymerase chain reaction,” or “PCR,” refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art (see, e.g., McPherson et al. eds (1995) PCR: A Practical Approach, 2nd Ed., IRL Press, Oxford; and the like). For example, in a conventional PCR using Taq DNA polymerase, a double stranded target nucleic acid may be denatured at a temperature greater than about 90°C, primers annealed at a temperature in the range of about 50°C to about 75°C, and primers extended at a temperature in the range of about 72°C to about 78°C. The term “PCR” encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. In various embodiments PCR reaction volumes can range from a few hundred nanoliters, e.g. 200 nl_, to a few hundred pL, e.g. 200 pL.

[0052] The term “real-time PCR” refers to a PCR for which the amount of reaction product, i.e. amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product (see, e.g., Gelfand et al. U.S. Pat. No. 5,210,015 (“TAQMAN™”);Docket No. 68552WO01 (2025-25631 -P-WO)Wittwer et al. U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al. U.S. Pat. No. 5,925,517 (molecular beacons); and the like). Detection chemistries for real-time PCR are reviewed, inter alia in Mackay et al. (2002) Nucl. Acids Res. 30: 1292-1305.

[0053] The terms “quantitative PCR” or “qPCR” refer to a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. Quantitative PCR includes both absolute quantitation and relative quantitation of such target sequences. Typically, quantitative measurements are made using one or more reference sequences that may be assayed separately or together with a target sequence. The reference sequence can be endogenous or exogenous to a sample or specimen, and in the latter case, may comprise one or more competitor templates. Typical endogenous reference sequences include, but are not limited to segments of transcripts of the following genes: p-actin, GAPDH, p2-microglobulin, ribosomal RNA, and the like. Techniques for quantitative PCR are well-known to those of ordinary skill in the art (see, e.g., Freeman etal. (1999) Biotechniques, 26: 112-126; Becker-Andre et al. (1989) Nucl. Acids Res. 17: 9437-9447; Zimmerman etal. (1996) Biotechniques, 21 : 268-279; Diviacco et al. (1992) Gene, 122: 3013-3020; and the like).

[0054] The term “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 viral RNA or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA. Nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triple-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).

[0055] 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-Docket No. 68552WO01 (2025-25631 -P-WO)uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

[0056] 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. 7:1817-195; Okamoto et al. (2020) “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. Patent Nos. 6,794,499, 6,670,461 , 6,262,490, and 6,770,748.

[0057] 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 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.

[0058] 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.

[0059] As used herein, the term “gene” encompasses coding sequences, introns, and any associated control sequences that participate in the expression of the coding sequences.

[0060] The term “sequence identity,” in the context of two or more amino acid or nucleotide sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.Docket No. 68552WO01 (2025-25631 -P-WO)

[0061] For sequence comparison to determine percent nucleotide or amino acid sequence identity, typically one sequence acts as a “reference sequence,” to which a “test” sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted using BLAST set to default parameters.

[0062] As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0063] The term “analyte” refers to any moiety that is to be detected and / or quantified. Analytes include, but are not limited to particular biomolecules (proteins, antibodies, nucleic acids (e.g., DNA and / or RNA), carbohydrates, lectins, etc.), bacteria or components thereof, bacterial toxins or components thereof, viruses or components thereof (e.g., coat proteins), fungi or components thereof, fungal toxins or components thereof, protozoa or components thereof, protozoal toxins or components thereof, drugs, other toxins, food pathogens, and the like.

[0064] “Selective hybridization” or “selective annealing” refers to the binding of a nucleic acid to a target nucleic acid in the absence of substantial binding to other nucleic acids 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.

[0065] As used herein, the Tm is the temperature at which a population of doublestranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g.,Docket No. 68552WQ01 (2025-25631 -P-WO)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)). As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm =81 .5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCI (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). In some embodiments, an estimate of the Tm is derived from using the Santa-Lucia nearest-neighboring algorithm.

[0066] 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.

[0067] A primer is said to “anneal to” or “hybridize 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.

[0068] 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 byDocket No. 68552WO01 (2025-25631 -P-WO)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.

[0069] 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 moiety 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.

[0070] 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.

[0071] The term “target nucleic acid” refers to nucleic acids to be detected and is generally used herein to refer to a segment of nucleic acid that is defined by a primer pair and that gives rise to an amplicon produced in an amplification reaction. The target nucleic acid can include any nucleic acid from an organism including prokaryotic cells, eukaryotic cells, virus(es), viroids, bacteria, fungi, spores, or tumor cells. Examples of nucleic acid include a DNA molecule, an RNA molecule, an miRNA molecule, or an mRNA molecule. Specific examples of organisms that target nucleic acids may come from include:• Viruses including, for example, a virus causing respiratory disease: influenza A virus (Flu A), influenza B virus (Flu B), Influenza C, respiratory syncytial virus A (RSV A), respiratory syncytial virus B (RSV B), Covid-19 virus, parainfluenza virus 1 (PIV 1), parainfluenza virus 2 (PIV 2), parainfluenza virus 3 (PIV 3), parainfluenza virus 4 (PIV 4), metapneumovirus (MPV), human enterovirus (HEV), human bocavirus (HBoV), human rhinovirus (HRV), coronavirus, and adenovirus. Other viruses include, for example, a virus causing gastrointestinal disease: norovirus, rotavirus, adenovirus, astrovirus, and sapovirus. As another example, the virus may include human papillomavirus (HPV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), dengue virus, herpes simplex virus (HSV), human herpes virus (HHV), herpesvirus 6, herpesvirus 7,Docket No. 68552WO01 (2025-25631 -P-WO)Epstein-Barr virus (EMV), varicella zoster virus (VZV), cytomegalovirus (CMV), parvovirus B19, parechovirus, mumps virus, chikungunya virus, zika virus, West Nile virus, hepatitis virus, such as hepatitis B virus (HBV), hepatitis C virus (HCV), poliovirus, human immunodeficiency virus type 1 (HIV-1), human T-Cell lymphotrophic virus type 1 (HTLV-1), human polyomavirus 2 (JC virus), ebola virus, monkeypox virus, rubella virus, human enteroviruses, or hantavirus; • Bacterial pathogens such as methicillin resistant Staphylococcus aureus, C.difficile, group B strep., chlamydia, gonorrhea, Mycobacteria tuberculosis, Rickettsia rickettsii, Ehrlichia chaffeensis, Borrelia burgdorferi, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis, Chlamydia pneumoniae, Mycoplasma pneumoniae, Mycoplasma sp., Legionella pneumophila, Legionella dumoffii, Mycoplasma fermentans, Ehrlichia sp., Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus pneumonia, S. agalactiae, or Listeria monocytogenes-,• Fungi such as Candida auris, Cryptococcus neoformans, Pneumocystis carinii, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, and Trichophyton rubrum. In some embodiments, the protozoa comprises one or more of Trypanosoma cruzi, Leishmania sp., Plasmodium, Entamoeba histolytica, Babesia microti, Giardia lamblia, Cyclospora sp., and Eimeria sp.• Other organisms including, for example, organisms associated with vulvovaginal candidiasis (VVC) and trichomoniasis (TV);• Tumor cells from eukaryotic organisms, such as humans, including cells associated with bladder cancer, lung cancer, breast cancer, colon cancer, and leukemia;• Eukaryotic organisms with chromosomal alterations, such as humans, including gene duplication, gene deletions or gene translocations, cells expressing specific cell surface markers such as CD4+ cells, detection of gene mutation / alterations such as single nucleotide polymorphisms (SNPs), antimicrobial resistant genes, and methylation status of genes may also be detected• Prokaryotic and eukaryotics organisms that produce potential biothreat analytes, such as anthrax (Bacillus anthracis) ricin, or other viruses, bacteria, fungi, or toxic substances; orDocket No. 68552WO01 (2025-25631 -P-WO)• Prokaryotic and eukaryotics organisms that produce potential health- associated infections (MRSA, C. Difficile, Vancomycin-resistant enterococcus (VRE), Norovirus), critical infectious diseases (MTB / RIF, Flu, RSV, EV), sexual health (CT / NG, GBS), oncology (e.g., breast or bladder cancer) and genetics (FII / FV).

[0072] The term “amplification target” is also used herein to refer to this type of target nucleic acid. Primers and probes are also said to “target” nucleic acid sequences, and so these sequences can also be understood as “target nucleic acids.” Additionally, primers and probes are said to “target” or “be specific for” genes. In this usage, the primers and probes can be used to detect the presence of a particular gene by specifically hybridizing to a portion of the gene that indicates its presence. The meaning of “target” and “target nucleic acids” will be clear to one of skill in the art from the context in which the term is employed.

[0073] In some embodiments, the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 60-90, 60-87, 60-86, 60-85, 60-83, 60-80, 65-90, 65-87, 65-86, 65-85, 65-83, 65-80, 70-90, 70-87, 70-86, 70-85, 70-83, 70-80, 75-90, 75-87, 75-86, 75-85, 75-83, 75-80, 65-87, 67-87, or 69-86 nucleotides. In some embodiments, the target nucleic acid comprises a ratio of AT to GO content of greater than 1 .

[0074] The term “polymerase” refers to an enzyme that catalyze the synthesis of DNA or RNA polymers via the successive addition of nucleotides to a growing nascent nucleic acid strand (primer) by using the complementary template strand. “Thermostable polymerase” refers to polymerases that originate from a thermophile and are therefore thermostable, allowing for amplification via thermocycling.

[0075] The term “reverse transcriptase” refers to an RNA-dependent DNA polymerase enzyme that converts an RNA genome into DNA by creating a complementary strand of DNA based on the RNA sequence. Reverse transcriptase allows RNA templates to be amplified in the same manner as DNA. In some embodiments, the reverse transcriptase is present at concentrations of 1 U / pL or greater, 2 U / pL or greater, 3 U / pL or greater, 4 U / pL or greater, 5 U / pL or greater, 6 U / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume.

[0076] The term “U / pL” refers to a measure of the enzyme's catalytic power per microliter of a solution, where 1 U (unit) is the amount of enzyme that catalyzes theDocket No. 68552WO01 (2025-25631 -P-WO)conversion of one micromole (1 pmol) of substrate per minute. In some embodiments, 1 U / pL as described herein is equivalent to 40 nM Taq concentration.

[0077] As used herein, the term “thermostable polymerase” or “thermophilic polymerase” refers to an enzyme that is relatively stable to heat when compared, for example, to nucleotide polymerases from E. coli, and which catalyzes the templatedependent polymerization of nucleoside triphosphates. A “thermostable polymerase,” will, e.g., retain enzymatic activity for polymerization and exonuclease activities when subjected to the repeated heating and cooling cycles used in PCR. Preferably, a “thermostable nucleic acid polymerase” has optimal activity at a temperature above 45°C, or at a temperature ranging from 40°G to 80°C and more preferably from 55°C to 75°C. A representative thermostable polymerase enzyme isolated from Thermus aquaticus (Taq) is described in U.S. Pat. No. 4,889,818 and a method for using it in conventional PCR is described in 239 Science 487 (1988). Other thermostable DNA polymerases include, but are not limited to, DNA polymerases from thermophilic Eubacteria or Archaebacteria, for example, T. thermophilus, T. bockianus, T. flavus, T rubber, Thermococcus litoralis, Pyroccocus furiousus, P. wosei, Pyrococcus spec. KGD, Thermatoga maritime, Thermoplasma acidophilus, and Sulfolobus spec. In some embodiments, the thermostable polymerase is present at concentrations of 2 U / pL or greater, 3 U / pL or greater, 4 U / pL or greater, 5 U / pL or greater, 6 U / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume. In some embodiments, the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited at temperatures 55°C and below.

[0078] 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 (HDA), and the like. Descriptions of such techniquesDocket No. 68552WO01 (2025-25631 -P-WO)can be found in, among other sources, U.S. Pat. Nos. 5,830,711 , 6,027,889, 5,686,243, 6,027,998, and 6,605,451 , PCT Publ. Nos. WO97 / 31256, WO01 / 92579; WO0056927A3, and WO9803673A1 ; Ausubel et al., PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); 34 J. Clin. Micro. 501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); 4 Curr. Opin. Biotechnol. 41-7 (1993), 29 Genomics 152-162 (1995); 252 Science 1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); 18 Nature Biotechnology 561 -64 (2000); and 28 Infection 97-102 (2000); Belgrader, Barany, and LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; 88 Proc. Natl. Acad. Sci. USA 188-93 (1991); 25 Nucl. Acids Res.2924-2951 (1997); 27 Nucl. Acid Res. e40i-viii (1999); 99 Proc. Natl Acad. Sci. USA 5261-66 (2002); 109 Gene 1-11 (1991); 20 Nucl. Acid Res. 1691-96 (1992); 2 BMC Inf. Dis. 18 (2002); 13 Genome Res. 294-307 (2003); 241 Science 1077-80 (1988); 2 Expert Rev Mol Diagn. 542-8 (2002); 53 J. Microbiol. Methods 165074 (2003); 12 Curr. Opin. Biotechnol. 21-7 (2001).

[0079] A “nucleic acid template” or “template,” as used herein is a single-stranded DNA nucleic acid that contains the target sequence for copying via amplification. A template for replication can be from any DNA source, such as genomic DNA (gDNA), complementary DNA (cDNA), or plasmid DNA.

[0080] In some embodiments, amplification comprises at least one cycle, an “amplification cycle,” comprising the thermocycling procedure of: 1) denaturing dualstrand DNA into single-stranded DNA at elevated temperature which melts the hydrogen bonds between complementary bases to separate the strands; 2) annealing at a lower temperature of at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; and 3) extending or synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase. The cycle may or may not be repeated. If repeated, the amplification cycle is described as “X amplification cycles,” wherein X is a whole number defining said number of cycles.

[0081] As used herein, “in solution” means not immobilized on a substrate of any kind, for example, a bead or a surface in a cassette, such as a chamber wall.

[0082] A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.Docket No. 68552WO01 (2025-25631 -P-WO)

[0083] 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.

[0084] 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.

[0085] The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation and produces a detectable signal (e.g., a fluorescent signal).

[0086] The term “quencher,” as used herein generally refers to any organic or inorganic molecule that reduces the level of a detectable signal.

[0087] As used herein, the term “detecting” refers to “determining the presence of’ an item. The terms “detect”, “detecting” or “detection” may describe either the general act of discovering or discerning or the specific observation of a detectably labeled composition. As used herein, the term “detectably different” or “spectrally distinguishable” refers to a set of labels (such as dyes / fluorophores) that can be detected and distinguished simultaneously.

[0088] As used herein, the terms “patient” and “subject” are typically used interchangeably to refer to a human. In some embodiments, the methods described herein may be used on samples from non-human animals, e.g., a non-human primate, canine, equine, feline, porcine, bovine, lagomorph, and the like. Additionally, the term patient may be used for non-human animals in the veterinary context.

[0089] As used herein, “Clinical Laboratory Improvement Amendments (C LI A)” refers to The Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations in effect as of the original filing date of the present application. The CLIA regulations include federal standards applicable to all U.S. facilities or sites that test human specimens for health assessment or to diagnose, prevent, or treat disease. A “CLIA-Docket No. 68552WO01 (2025-25631 -P-WO)compliant” test is one that complies with these regulations. “CLIA-waived” tests include tests that does not comply with all of these regulations. For example, CLIA-waived tests include test systems cleared by the U.S. Food and Drug Administration for home use and those tests approved for waiver under the CLIA criteria.NUCLEIC ACID AMPLIFICATION METHODS

[0090] 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. Nucleic acid amplification typically includes multiple cycles of the sequential procedures in order to generate enough of the sample to be detectable. Amplification can comprise thermocycling or can be performed isothermally. Isothermal amplification typically requires the use of a nucleic acid polymerase that has strand displacement activity and / or some other means to effect strand separation. Thermocycling is standardly carried out by subjecting a PCR reaction mixture to three temperatures per cycle in the following sequence: denaturation, usually at about 95°C; annealing, usually at about 5°C below the Tm of the primers; and extension (e.g., at about 72°C). Some methods simplify this temperature / time course to two temperatures per cycle. For example, U.S. Patent No. 9,428,781 describes “oscillating amplification” in which a two-temperature cycle includes an upper temperature and a lower temperature that differ by no more than 20°C.Embodied Methods for Amplifying a Target Nucleic Acid

[0091] Embodied molecular testing methods utilize thermal cycling to cause a chemical reaction through a rapid polymerase chain reaction (PCR) process for amplification of a target nucleic acid. In order to maximize the speed and efficiency of such methods, the system needs to be able to accurately raise and lower sample temperatures with precision and rapidity. The methods described incorporate specific thermal regions in unique reaction vessel designs in order to maximize efficiency and reaction speed through thermal cycling at specific, well-controlled temperatures. The resulting reactions are unexpectedly faster than previously seen and allow for quantitative detection of target nucleic acids in minutes.Reaction Vessel

[0092] The reaction vessel is a container configured for receiving and holding a solution comprising the target nucleic acid to be tested (FIG. 1). An example of such a reaction vessel is disclosed in Int’l Pat. AppL Publ. No. WO2022 / 155304. The reaction vessel 100 comprises a fluid inlet port 110 for sample and solutionDocket No. 68552WO01 (2025-25631 -P-WO)introduction (dotted arrow denotes path into and out of the device), an inlet passage 120, and a reaction chamber entrance 130 that leads to the reaction chamber, 140.After the reaction has occurred, the materials can be removed via the outlet passage 150 and then out of the fluid outlet port, 160. The reaction chamber can be heated via one or more external devices that are situation orthogonally to the major axis of the reaction vessel and apply heat to the large faces of the reaction chamber, 170 and 171, to create a thermal cycling amplification process.

[0093] The reaction chamber of the reaction vessel can be a walled structure having X, Y, and Z dimensions defined as length (X), width (Y), and height (Z) (FIG. 2). In some embodiments, as shown in FIG.2, the reaction chamber 200 comprises a walled structure comprising two opposing major walls, 210 and 220, along with two side walls, 230 and 240. The example in FIG. 2 shows the opposing major walls 210 and 220 of the same length, but in some embodiments, they may be of different lengths. The example in FIG. 2 shows the side walls 230 and 240 of the same length, but in some embodiments, they may be of different lengths and further, may comprise multiple wall elements such that the overall structure has more than four sides. For example, the reaction chamber when viewed from the Z-axis can form a hexagon wherein the side walls 230 and 240 each comprise two side wall elements of the hexagon. In some embodiments the reaction chamber, when viewed from the Z-axis comprises a regular or irregular polygon of 4-, 5-, 6- , 7- , 8-, 9, or 10-sides. In some embodiments, the 3D shape of the analysis region of the reaction chamber is cylindrical, cube, spherical, rectangular, pyramidal, conical, or diamond. The angle between an opposing major wall and a side wall can be from 30° to 150°, 45° to 135°, 60° to 20°, 75° to 105°, or 90°±20°.

[0094] In some embodiments, the two opposing major walls 210 and 220 are directly or indirectly heated. The heating of major walls 210 and 220 are independent and can be done via known methods, such as via electricity (induction, resistance), acoustic, fluid (liquid, air), or light (infrared). The heating can be conductive, radiative, or convective or a combination thereof. The heating of the major walls 210 and 220 defines a first region at a first temperature and a second region at a second temperature, respectively, in the reaction chamber. The creation of a first region at a first temperature and a second region at a second temperature in the reaction chamber 200 induces a heat differential in the sample in the reaction chamber 200, which leads to a thermal amplification process.Docket No. 68552WO01 (2025-25631 -P-WO)

[0095] In addition to the thermal processes for amplification disclosed herein, it has unexpectedly been found that the dimensions of the reaction chamber - alone or in combination with other aspects of the disclosure - can have an impact on the speed and efficiency of the reactions disclosed herein. In some embodiments, the length of the two opposing major walls is from 1 to 25 mm, 1 .4 to 25 mm, 1 .6 to 25 mm, 1 .8 to 25 mm, 2 to 25 mm, 2.5 to 25 mm, 3 to 25 mm, 4 to 25 mm, 5 to 25 mm, 6 to 25 mm, 8 to 25 mm, 10 to 25 mm, 12 to 25 mm, 15 to 25 mm, 20 to 25 mm, 1 to 20 mm, 1 .4 to 20 mm, 1 .6 to 20 mm, 1.8 to 20 mm, 2 to 20 mm, 2.5 to 20 mm, 3 to 20 mm, 4 to 20 mm, 5 to 20 mm, 6 to 20 mm, 8 to 20 mm, 10 to 20 mm, 12 to 20 mm, 15 to 20 mm, 1 to 15 mm, 1.4 to 15 mm, 1 .6 to 15 mm, 1 .8 to 15 mm, 2 to 15 mm, 2.5 to 15 mm, 3 to 15 mm, 4 to 15 mm, 5 to 15 mm, 6 to 15 mm, 8 to 15 mm, 10 to 15 mm, 12 to 15 mm, 1 to 12 mm, 1.4 to 12 mm, 1.6 to 12 mm, 1.8 to 12 mm, 2 to 12 mm, 2.5 to 12 mm, 3 to 12 mm, 4 to 12 mm, 5 to 12 mm, 6 to 12 mm, 8 to 12 mm, 10 to 12 mm, 1 to 10 mm, 1.4 to 10 mm, 1.6 to 10 mm, 1.8 to 10 mm, 2 to 10 mm, 2.5 to 10 mm, 3 to 10 mm, 4 to 10 mm, 5 to 10 mm, 6 to 10 mm, 8 to 10 mm, 1 to 8 mm, 1 .4 to 8 mm, 1 .6 to 8 mm, 1 .8 to 8 mm, 2 to 8 mm, 2.5 to 8 mm, 3 to 8 mm, 4 to 8 mm, 5 to 8 mm, 6 to 8 mm, 1 to 6 mm, 1 .4 to 6 mm, 1 .6 to 6 mm, 1 .8 to 6 mm, 2 to 6 mm, 2.5 to 6 mm, 3 to 6 mm, 4 to 6 mm, 5 to 6 mm, 1 to 5 mm, 1 .4 to 5 mm, 1.6 to 5 mm, 1 .8 to 5 mm, 2 to 5 mm, 2.5 to 5 mm, 3 to 5 mm, 4 to mm, 1 to 4 mm, 1 .4 to 4 mm, 1 .6 to 4 mm, 1.8 to 4 mm, 2 to 4 mm, 2.5 to 4 mm, 3 to 4 mm, 1 to 3 mm, 1 .4 to 3 mm, 1 .6 to 3 mm, 1 .8 to 3 mm, 2 to 3 mm, 2.5 to 3 mm, 1 to 2.5 mm, 1 .4 to 2.5 mm, 1 .6 to 2.5 mm, 1 .8 to 2.5 mm, 2 to 2.5 mm, 1 to 2 mm, 1 .4 to 2 mm, 1.6 to 2 mm, 1.8 to 2 mm, 1 to 1.8 mm, 1 .4 to 1 .8 mm, or 1.6 to 1 .8 mm.

[0096] In some embodiments, the height of the two opposing major walls is from 1 to 25 mm, 1 .4 to 25 mm, 1 .6 to 25 mm, 1 .8 to 25 mm, 2 to 25 mm, 2.5 to 25 mm, 3 to 25 mm, 4 to 25 mm, 5 to 25 mm, 6 to 25 mm, 8 to 25 mm, 10 to 25 mm, 12 to 25 mm, 15 to 25 mm, 20 to 25 mm, 1 to 20 mm, 1 .4 to 20 mm, 1 .6 to 20 mm, 1.8 to 20 mm, 2 to 20 mm, 2.5 to 20 mm, 3 to 20 mm, 4 to 20 mm, 5 to 20 mm, 6 to 20 mm, 8 to 20 mm, 10 to 20 mm, 12 to 20 mm, 15 to 20 mm, 1 to 15 mm, 1 .4 to 15 mm, 1 .6 to 15 mm, 1 .8 to 15 mm, 2 to 15 mm, 2.5 to 15 mm, 3 to 15 mm, 4 to 15 mm, 5 to 15 mm, 6 to 15 mm, 8 to 15 mm, 10 to 15 mm, 12 to 15 mm, 1 to 12 mm, 1 .4 to 12 mm, 1.6 to 12 mm, 1 .8 to 12 mm, 2 to 12 mm, 2.5 to 12 mm, 3 to 12 mm, 4 to 12 mm, 5 to 12 mm, 6 to 12 mm, 8 to 12 mm, 10 to 12 mm, 1 to 10 mm, 1 .4 to 10 mm, 1 .6 to 10 mm, 1 .8 to 10 mm, 2 to 10 mm, 2.5 to 10 mm, 3 to 10 mm, 4 to 10 mm, 5 to 10 mm, 6 to 10 mm, 8 to 10Docket No. 68552WO01 (2025-25631 -P-WO)mm, 1 to 8 mm, 1 .4 to 8 mm, 1.6 to 8 mm, 1 .8 to 8 mm, 2 to 8 mm, 2.5 to 8 mm, 3 to 8 mm, 4 to 8 mm, 5 to 8 mm, 6 to 8 mm, 1 to 6 mm, 1 .4 to 6 mm, 1 .6 to 6 mm, 1.8 to 6 mm, 2 to 6 mm, 2.5 to 6 mm, 3 to 6 mm, 4 to 6 mm, 5 to 6 mm, 1 to 5 mm, 1.4 to 5 mm, 1 .6 to 5 mm, 1 .8 to 5 mm, 2 to 5 mm, 2.5 to 5 mm, 3 to 5 mm, 4 to mm, 1 to 4 mm, 1 .4 to 4 mm, 1 .6 to 4 mm, 1.8 to 4 mm, 2 to 4 mm, 2.5 to 4 mm, 3 to 4 mm, 1 to 3 mm, 1.4 to 3 mm, 1 .6 to 3 mm, 1 .8 to 3 mm, 2 to 3 mm, 2.5 to 3 mm, 1 to 2.5 mm, 1.4 to 2.5 mm, 1 .6 to 2.5 mm, 1.8 to 2.5 mm, 2 to 2.5 mm, 1 to 2 mm, 1.4 to 2 mm, 1 .6 to 2 mm, 1 .8 to 2 mm, 1 to 1 .8 mm, 1 .4 to 1.8 mm, or 1 .6 to 1.8 mm.

[0097] In some embodiments, the average distance between the two opposing major walls is from0.25 to 10 mm, 0.5 to 10 mm, 1 to 10 mm, 2 to 10 mm, 3 to 10 mm, 5 to 10 mm, 7 to 10 mm, 0.25 to 7 mm, 0.5 to 7 mm, 1 to 7 mm, 2 to 7 mm, 3 to 7 mm, 5 to 7 mm, 0.25 to 5 mm, 0.5 to 5 mm, 1 to 5 mm, 2 to 5 mm, 3 to 5 mm, 0.25 to 3 mm, 0.5 to 3 mm, 1 to 3 mm, 2 to 3 mm, 0.25 to 2 mm, 0.5 to 2 mm, 1 to 2 mm, 0.25 to 1 mm, 0.5 to 1 mm, or 0.25 to 0.5. The two opposing major walls and the side walls form a volume within the reaction chamber. The volume of the reaction chamber can be from 0.5 pL to 6500 pL, 1 pL to 5000 pL, 5 pL to 5000 pL, 10 pL to 5000 pL, 25 pL to 5000 pL, 50 pL to 5000 pL, 75 pL to 5000 pL, 100 pL to 5000 pL, 250 pL to 5000 pL, 500 pL to 5000 pL, 1000 pL to 5000 pL, 1 pL to 1000 pL, 5 pL to 1000 pL, 10 pL to 1000 pL, 25 pL to 1000 pL, 50 pL to 1000 pL, 75 pL to 1000 pL, 100 pL to 1000 pL, 250 pL to 1000 pL, 500 pL to 1000 pL, 1 pL to 500 pL, 5 pL to 500 pL, 10 pL to 500 pL, 25 pL to 500 pL, 50 pL to 500 pL, 75 pL to 500 pL, 100 pL to 500 pL, 250 pL to 500 pL, 1 pL to 250 pL, 5 pL to 250 pL, 10 pL to 250 pL, 25 pL to 250 pL, 50 pL to 250 pL, 75 pL to 250 pL, 100 pL to 250 pL, 1 pL to 100 pL, 5 pL to 100 pL, 10 pL to 100 pL, 10 pL to 65 pL, 25 pL to 100 pL, 25 pL to 65 pL, 50 pL to 100 pL, 50 pL to 65 pL, 75 pL to 100 pL.

[0098] In some embodiments, the major walls of the reaction chamber have a ratio of height to length of from 0.5:1 to 1 :2, 0.75:1 to 1 :5, 1 :1 to 1 :1 .75, 1 :1 to 1 :1 .5 or 1 :1 to 1 :1.25. In some embodiments, the ratio of the length to height of the reaction chamber and / or the average distance between the two opposing major walls is such that an induced thermal convection cycle in a solution in the reaction chamber has an average cycle time of 15 seconds or less when the temperature difference between the two opposing major walls is from 25-37°C.

[0099] In some embodiments, the reaction chamber is part of a self-contained cartridge. The self-contained cartridge may be relatively small, such that it can beDocket No. 68552WO01 (2025-25631 -P-WO)easily hand-held, portable, and / or disposable. An example of such a cartridge is disclosed in U.S. Pat. No. 10,562,030. The sample cartridge can hold one or more reagents and / or chemicals that are used to process a sample, in order to ultimately detect some property of the sample. One example of such a process is PCR, which is used to amplify the presence of DNA. The sample cartridge can include a sample chamber, which is in fluid communication with the reaction chamber. Additionally, in some embodiments, a filter may be disposed in the fluidic path between the sample chamber and the reaction chamber. This filter can be used to isolate sample nucleic acids from the sample and may comprise an amine or other modification to maximize efficiency or effectiveness. The self-contained cartridge may further include additional chambers or fluidic connections to allow for chemical processes. In some embodiments, the self-contained cartridge comprises a lysis chamber, wherein the lysis chamber can comprise one or more lysis reagents for releasing nucleic acid. In some embodiments, the self-contained cartridge comprises additional chambers optionally in fluid communication with the sample chamber or the reaction chamber. For example, multiple reaction chambers may be in communication with a single sample chamber to allow for amplification of different target nucleic acids.

[0100] During the amplification process, there comprises two regions within the reaction chamber, a first and a second region. The first region is a sub-section of the volume of the reaction chamber wherein the solution is maintained at a first temperature, said first temperature in a range capable of denaturing a target nucleic acid sequence into a single-stranded nucleic acid template. In some embodiments, first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C. In some embodiments, the first temperature is maintained at a certain level with a variance of less than 3°, 2°, or 1 °C over the time of the amplification reaction.

[0101] The second region is a sub-section of the volume of the reaction chamber wherein the solution is maintained at a second temperature lower than the first temperature, said second temperature in a range capable of annealing a primer pair to a single-stranded nucleic acid template. In some embodiments, second temperature is from 60°C to 70°C, from 64°C to 68°C, or from 65°C to 67°C. In some embodiments, the second temperature is maintained at a certain level with a variance of less than 3°, 2°, or 1°C over the time of the amplification reaction. In some embodiments, the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C. In some embodiments, the difference in temperature between the firstDocket No. 68552WO01 (2025-25631 -P-WO)temperature and the second temperature is equal to or greater than 25°C, 26°G, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C or from 25-37°C, 25-35°C, 27-35°C, 28-34°C.

[0102] The temperature difference between the first and second regions creates a thermal convection cycle. The phrase “thermal convection cycle” or “thermal convection cycling” refers to the use of heat differentials to cyclically drive a reagent between different regions of the reaction chamber for spatially separate melting, annealing, and extending in a reaction chamber with constant heating temperatures at different locations. Convective PCR thermal cycling is implemented by inducing thermal convection inside the reaction chamber, which stratifies the reaction into spatially separate and stable melting, annealing, and extension zones created by the temperature gradient. Techniques for Convective PCR thermal cycling are described in Miao et al. (2020) 1108 Anal. Chim. Acta 177-197. In some embodiments, the thermal convection cycle forms an ordered or regular fluid flow pattern that is roughly cyclical. In some embodiments, the thermal convection cycle forms an irregular or turbulent flow pattern. In some embodiments, the thermal convection cycle is a type of Rayleigh-Bernard convection.

[0103] Methods of controlling the temperature of the reaction- chamber by a thermal control device are provided in, for example, U.S. Publ. AppL Nos. 2022 / 0253079 A1 and 2020 / 0116398 A1. As described above, the reaction- chamber has opposing major faces and an active thermal element adjacent to at least one major face of the reaction- chamber. In some embodiments, there comprises more than one active element, for example, two active thermal elements applied bilaterally on major faces on opposite sides of the reaction- chamber. One or more sensors may be incorporated and configured to measure the temperature of a portion of the active element(s). The one or more sensors can further include a second temperature sensor positioned and configured to measure the ambient temperature indicative of the thermal operating environment around the reaction- chamber.

[0104] In some embodiments, the thermal control device is configured as a removable module that can be coupled with a reaction-vessel extending from a sample analysis cartridge configured for detection of a nucleic acid target in a nucleic acid amplification test (NAAT), e.g., Polymerase Chain Reaction (PCR) assay. Preparation of a fluid sample in such a cartridge generally involves a series of processing steps, which can include chemical, electrical, mechanical, thermal, optical or acoustical processingDocket No. 68552WO01 (2025-25631 -P-WO)steps according to a specific protocol. Such steps can be used to perform various sample preparation functions, such as cell capture, cell lysis, purification, binding of analyte, and / or binding of unwanted material. Such a sample processing cartridge can include one or more chambers suited to perform the sample preparation steps. A sample cartridge suitable for use with the invention is shown and described in U.S. Pat. No. 6,374,684 and U.S. Pat. No. 8,048,386.

[0105] The number of amplification cycles in the reaction chamber can be fixed or vary depending on the target nucleic acid and detection methods. In some embodiments, the number of amplification cycles is from 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 20 to 50, 20 to 40, or 20 to 30. In some embodiments, the number of amplification cycles is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30. The average cycle time is the averaged time it takes for a target nucleic acid to go through the thermal amplification process steps of denaturation, annealing, and extension. In convective PCR, a ‘cycle’ exists only in an averaged sense, as reagents continuously follow different local trajectories. Measurement of the average cycle time can be done via standard methods. The average cycle time for the amplification methods described herein are, in seconds, from 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 40, 10 to 30, 10 to 20, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 20 to 50, 20 to 40, or 20 to 30 (seconds). In some embodiments, the average cycle time for the amplification methods described herein is, in seconds, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 (seconds).

[0106] In some embodiments, the amplification efficiency or time for the method to complete 20 amplification cycles is critical to performance. In some embodiments, the amplification efficiency is less than 20 minutes, less than 19 minutes, less than 18 minutes, less than 17 minutes, less than 16 minutes, less than 15 minutes, less than 14 minutes, less than 13 minutes, less than 12 minutes, less than 11 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, or less than 5 minutes. In some embodiments, the amplification efficiency is from 5 minutes to 20 minutes, 5 minutes to 18 minutes, 5 minutes to 15 minutes, 5 minutes to 12 minutes, 5 minutes to 10 minutes, 8 minutes to 20 minutes, 8 minutes to 18 minutes, 8 minutes to 15 minutes, 8 minutes to 12 minutes, 8 minutesDocket No. 68552WO01 (2025-25631 -P-WO)to 10 minutes, 10 minutes to 20 minutes, 10 minutes to 18 minutes, 10 minutes to 15 minutes, 10 minutes to 12 minutes, 12 minutes to 20 minutes, 12 minutes to 18 minutes, 12 minutes to 15 minutes, 15 minutes to 20 minutes, or 15 minutes to 18 minutes.

[0107] In some embodiments the reaction vessel and / or cartridge that comprises the reaction chamber is CLIA compliant. In some embodiments, the assays performed in the sample analyzers disclosed herein are performed in a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Accordingly, the module and cartridge may be Clinical Laboratory Improvement Amendments (CLIA)-compliant, operated in compliance with CLIA, operated by a CLIA-compliant laboratory, and / or operated in a CLIA-compliant location. The module and cartridge may be a Clinical Laboratory Improvement Amendments (CLIA)-certified device, operated by a CLIA-certified laboratory, operated in a CLIA-certified location, operated under the oversight of a CLIA-compliant laboratory, and / or operated under the oversight of a CLIA-certified laboratory. Advantageously, detection may be effected without transporting the sample from the site where the sample is collected (e.g., a POC diagnosis is preferably a hospital, an urgent care center, an emergency room, a physician's office, a health clinic, or a home).Starting Solution

[0108] The starting solution comprises an aqueous solution of compounds necessary to initiate an amplification reaction with a target nucleic acid. The starting solution may initially be separated into one or more dry components and an aqueous solution comprising one or more components, where the dry component can comprise materials that are sensitive to air, heat, light or have a limited lifetime in solution. The two components can be combined prior to entering the reaction chamber or one of the components may be initially present in the reaction chamber. In the case where the starting solution is a single aqueous solution, it can be present in the reaction chamber or added at some point before, after, or during the addition of the target nucleic acid.

[0109] In embodiments where the starting solution comprises a dry component, the nature, composition, and method of producing dried components is well-known to those of ordinary skill in the art as evidenced by the following references: U.S. Pat. Nos. 5,098,893, 5,102,788, 5,556,771 , 5,763,157, 6,294,365, and 5,413,732, U.S. Pat. AppL Publ. Nos. 2006 / 0068398 and 2006 / 0068399; and Pharm. Dev. TechnoL, 10: 151-173 (2005) and Pharm. BiotechnoL, 14: 281-360 (2002). Dried componentsDocket No. 68552WO01 (2025-25631 -P-WO)include, but are not limited to, solid and / or semi-solid particulates, powders, tablets, crystals, capsules, beads, spheres and the like, that are manufactured in a variety of ways.

[0110] In some embodiments, the starting solution comprises a thermostable polymerase, a primer pair that is optionally labeled, and optionally, one or more of: a probe, a reverse transcriptase, and additional reagents. In some embodiments, the starting solution comprises a reaction mixture including a thermostable polymerase, a primer pair, and optionally, a probe, a reverse transcriptase, and additional reagents. A “reaction mixture” means a solution containing all the necessary reactants for performing a reaction, which may include, but not be limited to, buffering agents to maintain pH at a selected level during a reaction, salts, co-factors, scavengers, and the like.

[0111] In some embodiments, the solution comprises at least four, at least six, at least eight, or at least ten sets of primer pairs. In such embodiments, the amplification reaction may comprise detecting at least four, at least six, at least eight, or at least ten target nucleic acids, respectively. In embodiments with multiple primer pair, the target nucleic acids may come from the same or different samples.Primers

[0112] Primers useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, a target RNA (genomic or transcript), a cDNA reverse transcribed from the target RNA, and / or an amplicon that has been amplified from genomic DNA, a target RNA, or a cDNA (collectively referred to as “template”), and, in the presence of the template, a polymerase and suitable buffers and reagents, can be extended to form a primer extension product. Primers are generally of a sufficient length to ensure selective hybridization to their target nucleic acids. Generally, primers of at least 15 nucleotides in length hybridize specifically in most contexts, and this length can be reduced, e.g., by including of affinity-enhancing modifications, such as those discussed above. Primers can but need not be exactly complementary to their target nucleic acids. Primers can have any degree of complementarity described above for exemplary polynucleotides. In illustrative embodiments, primers can be 8 to 45 nucleotides in length and at least 90% complementary to their target nucleic acids: 8 to 45 nucleotides in length and at least 95% complementary to their target nucleic acids: 8 to 45 nucleotides in length and at least 99% complementary to their target nucleic acids: 8 to 30 nucleotides in lengthDocket No. 68552WO01 (2025-25631 -P-WO)and at least 90% complementary to their target nucleic acids: 8 to 30 nucleotides in length and at least 95% complementary to their target nucleic acids: 8 to 30 nucleotides in length and at least 99% complementary to their target nucleic acids. In embodiments wherein a primer is less than 100% complementary to it target nucleic acid, having the 3' nucleotide in the primer be complementary to its target nucleic acid facilitates the production of an extension product.

[0113] In some embodiments, the primer is present at a concentration of at least 500 nM, 600 nM, 700 nM, or 800 nM. In some embodiments, the primer is present at a concentration of from 500 nM to 1000 nM, 500 nM to 900 nM, 500 nM to 800 nM, 600 nM to 1000 nM, 600 nM to 900 nM, 600 nM to 800 nM, 700 nM to 1000 nM, 700 nM to 900 nM, or 700 nM to 800 nM.

[0114] In some embodiments, a primer that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 5-fold greater affinity than to nontarget nucleic acid under the same assay conditions. In some embodiments, a primer that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 10-fold greater affinity than to non-target nucleic acid under the same assay conditions.

[0115] In some embodiments, a primer pair is designed to produce an amplicon that is 50 to 1500 nucleotides long, 50 to 1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to 150 nucleotides long.

[0116] The primers have a melt temperature, Tm. In some embodiments, the primer melt temperature is 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, the primer melt temperature is 13°C or less, 12°C or less, 11 °C or less, 10°C or less, 9°C or less, 8°C or less, or from 7-13°C or from 8-10°C below the first temperature in the reaction chamber.

[0117] The primers have an annealing temperature, Ta. In some embodiments, the primer annealing temperature is 60-80°C, 60-75°C, or 60-70°C. In some embodiments, the primer annealing temperature is 0°C or greater, 1°C or greater, 2°C or greater, 3°C or greater, 4°C or greater, 5°C or greaterj6°C or greater, 7°C or greater, 8°C or greater, 9°C or greater, or 10°C or greater, or from 0-10°C or from 0-6°C greater than the second temperature in the reaction chamber.Docket No. 68552WO01 (2025-25631 -P-WO)

[0118] In some embodiments, the primer further comprises a stabilizing base. A stabilizing refers to a greater tendency for the modified base to pair an unmodified complementary base, as compared to the tendency of canonical bases to form base pairs (e.g., A-T and G-C). The stabilizing base can be present at any location on the primer. However, in some embodiments, it is advantageous for at least one stabilizing base to be at the 2ndor 3rdposition from the 3’ end of the primer. Stabilizing bases can exert their effects by differences in, e.g., hydrogen bonding in base pairing, in base stacking, and effects on the secondary structure of sequences in which they appear. Stabilizing bases are known; some are known to exist in nature (e.g., inosine), and some have been produced by synthetically modifying a canonical base. Example stabilizing bases include locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, 0(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2 -deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0119] In some embodiments, the primer is labeled with a detectable moiety. The detectable moiety may comprise a fluorescent dye and a quencher molecule. In some embodiments, the primer is not labeled.Probes

[0120] In some embodiments, the starting solution further comprises one or more probes. Probes useful in the methods described herein are generally capable of selectively hybridizing to: genomic DNA, a target RNA (genomic or transcript), a cDNA reverse transcribed from the target RNA, and / or an amplicon that has been amplified from genomic DNA, a target RNA, or a cDNA (collectively referred to as “template”). Generally, probes of at least 15 nucleotides in length hybridize specifically in most contexts, and this length can be reduced, e.g., by including of affinity-enhancing modifications, such as those discussed above. Probes can but need not be exactly complementary to their target nucleic acids. Probes can have any degree of complementarity described above for exemplary polynucleotides. In illustrative embodiments, probes can be 8 to 45 nucleotides in length and at least 90% complementary to their target nucleic acids: 8 to 45 nucleotides in length and at least 95% complementary to their target nucleic acids: 8 to 45 nucleotides in length and at least 99% complementary to their target nucleic acids: 8 to 30 nucleotides in lengthDocket No. 68552WO01 (2025-25631 -P-WO)and at least 90% complementary to their target nucleic acids: 8 to 30 nucleotides in length and at least 95% complementary to their target nucleic acids: 8 to 30 nucleotides in length and at least 99% complementary to their target nucleic acids. In embodiments wherein a primer is less than 100% complementary to a target nucleic acid, any points or regions of non-complementarity are typically located so as not to disrupt the ability of the probe to selectively hybridize to its target nucleic acid.

[0121] In some embodiments, the probe is present at a concentration of at least 500 nM, 600 nM, 700 nM, or 800 nM. In some embodiments, the primer is present at a concentration of from 500 nM to 1000 nM, 500 nM to 900 nM, 500 nM to 800 nM, 600 nM to 1000 nM, 600 nM to 900 nM, 600 nM to 800 nM, 700 nM to 1000 nM, 700 nM to 900 nM, or 700 nM to 800 nM.

[0122] In some embodiments, a probe that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 5-fold greater affinity than to nontarget nucleic acid under the same assay conditions. In some embodiments, a probe that selectively hybridizes to its target nucleic acid hybridizes to its target nucleic acid with at least 10-fold greater affinity than to non-target nucleic acid under the same assay conditions.

[0123] The probes have a melt temperature, Tm. In some embodiments, the probe melt temperature is 76-82°C, 77-81 °C, or 78-80°C. In some embodiments, the probe melt temperature is 13°C or less, 12°C or less, 11 °C or less, 10°C or less, 9°C or less, 8°C or less, or from 7-13°C or from 8-10°C below the first temperature in the reaction chamber.

[0124] The probes have an annealing temperature, Ta. In some embodiments, the probe annealing temperature is 60-80°C, 60-75°C, or 60-70°C. In some embodiments, the probe annealing temperature is 0°C or greater, 1 °C or greater, 2°C or greater, 3°C or greater, 4°C or greater, 5°C or greaterj6°C or greater, 7°C or greater, 8°C or greater, 9°C or greater, or 10°C or greater, or from 0-10°C or from 0-6°C greater than the second temperature in the reaction chamber.

[0125] In some embodiments, the probe further comprises a destabilizing base. A destabilizing base refers to a lesser tendency for the modified base to pair an unmodified complementary base, as compared to the tendency of canonical bases to form base pairs (e.g., A-T and G-C). The destabilizing base can be present at any location on the primer. In some embodiments, the destabilizing base decreases the melt temperature of the target nucleic acid. Destabilizing bases can exert their effectsDocket No. 68552WO01 (2025-25631 -P-WO)by differences in, e.g., hydrogen bonding in base pairing, in base stacking, and effects on the secondary structure of sequences in which they appear. Destabilizing bases are known to exist in nature and have been produced by synthetically. Example destabilizing bases include locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

[0126] In some embodiments, the probe is labeled with a detectable moiety or label. Detectable moieties include directly detectable moieties, such as fluorescent dyes, and indirectly detectable moieties, such as members of binding pairs. When the detectable moiety is a member of a binding pair, in some embodiments, the probe can be detectable by incubating the probe with a detectable label bound to the second member of the binding pair. In some embodiments, a primer or probe is not labeled, such as when a primer or probe is immobilized, e.g., on a microarray or bead. A labeled primer is extendable, e.g., by a polymerase. In some embodiments, a probe is extendable. In other embodiments, a probe is not extendable. The following discussion centers on probes, as these are more typically employed for detecting in the methods described here, but those of skill in the art appreciate that the polynucleotide labeling strategies described below apply equally to the labeling of primers.

[0127] In some embodiments, the probe is a Fluorescence Resonance Energy Transfer (FRET) probe that, in some embodiments, is labeled at the 5'-end with a fluorescent dye (donor) and at the 3'-end with a quencher (acceptor), a chemical group that absorbs (i.e., suppresses) fluorescence emission from the dye when the groups are in close proximity (e.g., attached to the same probe). Thus, in some embodiments, the emission spectrum of the dye should overlap considerably with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the ends of the FRET probe.

[0128] Illustrative FRET probes, which include, but are not limited to, a TaqMan® probe, a Molecular Beacon probe and a Scorpion probe. A TaqMan® probe is a linear probe that typically has a fluorescent dye covalently bound at one end of the DNA and a quencher molecule covalently bound elsewhere, such as at the other end of theDocket No. 68552WO01 (2025-25631 -P-WO)DNA. The FRET probe comprises a sequence that is complementary to a region of the cDNA or amplicon such that, when the FRET probe is hybridized to the cDNA or amplicon, the dye fluorescence is quenched, and when the probe is digested during amplification of the cDNA or amplicon, the dye is released from the probe and produces a fluorescence signal. In some embodiments, the amount of target nucleic in the sample is proportional to the amount of fluorescence measured during amplification.

[0129] Like TaqMan® probes, Molecular Beacons use FRET to detect a PCR product via a probe having a fluorescent dye and a quencher attached at the ends of the probe. Unlike TaqMan® probes, Molecular Beacons remain intact during the PCR cycles. Molecular Beacon probes form a stem-loop structure when free in solution, thereby allowing the dye and quencher to be in close enough proximity to cause fluorescence quenching. When the Molecular Beacon hybridizes to a target nucleic acid, the stemloop structure is abolished so that the dye and the quencher become separated in space and the dye fluoresces. Molecular Beacons are available, e.g., from Gene Link™ (see www.genelink.com / newsite / products / mbintro.asp).

[0130] In some embodiments, Scorpion probes can be used as sequence-specific primers and for PCR product detection. Like Molecular Beacons, Scorpion probes form a stem-loop structure when not hybridized to a target nucleic acid. However, unlike Molecular Beacons, a Scorpion probe achieves both sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5'-end of the Scorpion probe, and a quencher is attached elsewhere, such as to the 3'-end. The 3' portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is linked to the 5'-end of the probe by a non-amplifiable moiety. After the Scorpion primer is extended, the target-specific sequence of the probe binds to its complement within the extended amplicon, thus opening up the stem-loop structure and allowing the dye on the 5'-end to fluoresce and generate a signal. Scorpion probes are available from, e.g., Premier Biosoft International (see www.premierbiosoft.com / tech notes / Scorpion.html).

[0131] In some embodiments, labels that can be used on the FRET probes include colorimetric and fluorescent dyes, such as Alexa Fluor dyes: BODIPY dyes, such as BODIPY FL, Cascade Blue, and Cascade Yellow: coumarin and its derivatives, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin: cyanine dyes, such as Cy3 and Cy5: eosins and erythrosins: fluorescein and its derivatives, such asDocket No. 68552WO01 (2025-25631 -P-WO)fluorescein isothiocyanate: macrocyclic chelates of lanthanide ions, such as Quantum Dye™: Marina Blue: Oregon Green: rhodamine dyes, such as rhodamine red, tetramethylrhodamine and rhodamine 6G: Texas Red: fluorescent energy transfer dyes, such as thiazole orange-ethidium heterodimer; and TOTAB.

[0132] Specific examples of dyes include, but are not limited to, those identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750; aminereactive BODIPY dyes, such as BODIPY 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650, BODIPY 650 / 655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR: Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2', 4', 5 ',7'-Tetrabromosulfonefluorescein, and TET.

[0133] Examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, fluorescein / tetramethylrhodamine: lAEDANS / fluorescein: EDANS / dabcyl: fluorescein / fluorescein: BODIPY FL / BODIPY FL; and fluorescein / QSY 7 or QSY 9 dyes. When the donor and acceptor are the same, FRET may be detected, in some embodiments, by fluorescence depolarization. Certain specific examples of dye / quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, Alexa Fluor 350 / Alexa Fluor 488: Alexa Fluor 488 / Alexa Fluor 546: Alexa Fluor 488 / Alexa Fluor 555: Alexa Fluor 488 / Alexa Fluor 568: Alexa Fluor 488 / Alexa Fluor 594: Alexa Fluor 488 / Alexa Fluor 647: Alexa Fluor 546 / Alexa Fluor 568: Alexa Fluor 546 / Alexa Fluor 594: Alexa Fluor 546 / Alexa Fluor 647: Alexa Fluor 555 / Alexa Fluor 594: Alexa Fluor 555 / Alexa Fluor 647: Alexa Fluor 568 / Alexa Fluor 647: Alexa Fluor 594 / Alexa Fluor 647: Alexa Fluor 350 / QSY35: Alexa Fluor 350 / dabcyl: Alexa Fluor 488 / QSY 35: Alexa Fluor 488 / dabcyl: Alexa Fluor 488 / QSY 7 or QSY 9; Alexa Fluor 555 / QSY 7 or QSY9: Alexa Fluor 568 / QSY 7 or QSY 9: Alexa Fluor 568 / QSY 21 : Alexa Fluor 594 / QSY 21 ; and Alexa Fluor 647 / QSY 21 . In some instances, the same quencher may be used for multiple dyes, for example, a broad spectrum quencher, such as an Iowa Black® quencher (Integrated DNA Technologies, Coralville, IA) or a Black Hole Quencher™ (BHQ™: Sigma-Aldrich, St. Louis, MO).Docket No. 68552WO01 (2025-25631 -P-WO)

[0134] Specific examples of fluorescently labeled ribonucleotides useful in the preparation of probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include, Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

[0135] Specific examples of fluorescently labeled deoxyribonucleotides useful in the preparation of probes for use in the methods described herein include Dinitrophenyl (DNP)-1 '-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630 / 650-14-dUTP, BODIPY 650 / 665-14-dUTP: Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, and Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially available and can be purchased from, e.g., Invitrogen.

[0136] As noted above, exemplary detectable moieties also include members of binding pairs. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, etc.Sample / Target Nucleic Acid

[0137] The target nucleic acid is obtained from a biological sample. The biological sample may be from one or more of a nasopharyngeal swab, a nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix. Illustrative biological samples include skin samples, lesion swabs, vesicular lesion fluid samples, pustular lesion fluid samples, rectal samples, and samples of bodily fluids, such as nasal aspirates, nasal washes, nasal swabs, nasopharyngeal swabs, saliva, oropharyngeal swabs, throat swabs, bronchoalveolar lavage samples, bronchial aspirates, bronchial washes, endotracheal aspirates, endotracheal washes, tracheal aspirates, nasal secretion samples, mucus samples, sputum samples, plasma samples, whole blood samples, etc.Docket No. 68552WO01 (2025-25631 -P-WO)

[0138] The sample to be tested is, in some embodiments, fresh (i.e., never frozen). In other embodiments, the sample is a frozen specimen. In some embodiments, the sample is a tissue sample, such as a formalin-fixed paraffin embedded sample. In some embodiments, the sample is a liquid cytology sample.

[0139] In some embodiments, a sample to be tested is contacted with a buffer after collection. For example, in the case of skin sample, lesion swab, vesicular lesion fluid sample, pustular lesion fluid sample, or rectal samples, a buffer (including, e.g., a preservative) can be added to the sample. In embodiments where the sample is a swab sample, the swab can simply be placed in a buffer. In some embodiments, that sample is contacted with the buffer immediately: in the case of a swab, the swab is immediately placed in the buffer. In some embodiments, the sample (e.g., including the swab) is contacted with buffer within 5 minutes, within 10 minutes, within 30 minutes, within 1 hour, or within 2 hours of sample collection.

[0140] In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, or less than 0.75 ml of sample or buffered sample are used in the present methods. In some embodiments, the sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater. In some embodiments, 0.01 ml to 1 ml of sample or buffered sample is used in the present methods.

[0141] In some embodiments, one or more additional steps are done to separate, purify, and / or isolate the target nucleic acid from the sample. Methods for separation, purification, and / or isolation techniques useful for the present disclosure and include, but are not limited to: filtration, organic (e.g., phenol-chloroform method), inorganic (e.g., salting out and proteinase K treatment), adsorption (silica-gel membrane), spin column, and magnetic bead extraction. In some embodiments, the purification step is done within immediately before amplification or as part of a cartridge-based method.

[0142] The target nucleic acid, as noted above, refers to nucleic acids to be detected and is generally used herein to refer to a segment of nucleic acid that is defined by a primer pair and that gives rise to an amplicon produced in an amplification reaction. In some embodiments, an assay can employ multiple target nucleic acids for one or more organisms and single target nucleic acids for one or more different organisms. In some embodiments, the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 60-90, 60-87, 60-86, 60-85, 60-83, 60-80, 65-90, 65-87, 65-86, 65-85, 65-83, 65-80, 70-90, 70-87, 70-86, 70-85, 70-83, 70-80, 75-90, 75-87, 75-86, 75-85, 75-83, 75-80, 65-87, 67-87, or 69-86 nucleotides. In someDocket No. 68552WO01 (2025-25631 -P-WO)embodiments, the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .

[0143] In some embodiments, the method, system, or cartridge includes at least four sets of primer and probes; alternatively, at least eight sets of primer and probes. In another aspect, the primer and probes are selected from a group consisting of: at least one primer pair and probe for detecting the presence of at least one influenza A gene in the sample selected from an influenza A PA gene, an influenza A PB2 gene, and an influenza A MP gene; at least one primer pair and probe for detecting the presence of at least one influenza B gene in the sample selected from an influenza B NS gene, an influenza B MP gene; at least one primer pair and probe for detecting the presence of an avian influenza gene in the sample selected from an avian influenza MP gene and an avian influenza HA gene; at least one primer pair and probe for detecting the presence of at least one SARS-CoV-2 gene in the sample selected from a SARS-Cov-2-E gene, a SARS-Cov-2-RdRp gene, and a SARS-Cov-2-N2 gene; and at least one primer pair for and probe detecting the presence of at least one respiratory syncytial virus (RSV) A gene in the sample and a respiratory syncytial virus (RSV) B gene.

[0144] The primers and probes used to detect the presence of target nucleic acids may bind to any number of genes in the disease vector. In some embodiments, the gene sequence in the disease vector is one of the ones listed in Table 1.Table 1 : Exemplary Nucleic Acid Target Gene SequencesDocket No. 68552WQ01 (2025-25631 -P-WO)

[0145] In some embodiments, the primer and probe are selected from a group consisting of at least two primer pairs detecting the presence of influenza A PA gene, influenza A PB 2 gene, and influenza A MP gene; at least two primer pairs and probes for detecting the presence of influenza B NS gene and an influenza B MP gene; at least three primer pairs and probes for detecting SARS-Cov-2-E gene, a SARS-Cov-2-RdRp gene, and a SARS-Cov-2-N2 gene; and a primer pair and probe for detecting the presence of at least one respiratory syncytial virus (RSV) A gene in the sample and a respiratory syncytial virus (RSV) B gene. In another aspect of the disclosure, the primer pair and probe for detecting the presence of an influenza A PA gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 1 , a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 1 ; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 1 , the primer pair and probe for detecting the presence of an influenza A MP gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 2, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 2; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 2, the primer pair and probe for detecting the presence of an influenza A PB 2Docket No. 68552WQ01 (2025-25631 -P-WO)gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 3, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 3; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 3, the primer pair and probe for detecting the presence of an influenza B NS gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 4, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 4; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 4, the primer pair and probe for detecting the presence of an influenza B MP gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 5, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 5; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 5, the primer pair and probe for detecting the presence of an SARS-CoV-2 E gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 6, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 6; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 6, the primer pair and probe for detecting the presence of an SARS-CoV-2 N gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 7, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 7; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 7, the primer pair and probe for detecting the presence of a SARS-CoV-2 RdRp gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 8, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 8; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 8, the primer pair and probe for detecting the presence of an RSV A gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 9, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 9; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQDocket No. 68552WO01 (2025-25631 -P-WO)ID NO: 9, the primer pair and probe for detecting the presence of an RSV B gene comprise a first primer comprising a sequence that is identical to at least 18 contiguous nucleotides of SEQ ID NO: 10, a second primer comprising a sequence that is complementary to at least 18 contiguous nucleotides of SEQ ID NO: 10; and a probe sequence that is identical or complementary at least 18 contiguous nucleotides of SEQ ID NO: 1O.Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading can occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0146] In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed step of doing J and a claimed step of doing K can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0147] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Docket No. 68552WO01 (2025-25631 -P-WO)

[0148] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and can include modification thereto and permutations thereof. Each embodiment described herein is envisaged to be applicable in each combination with other embodiments described herein.EXAMPLES

[0149] The present disclosure can be better understood by reference to the following examples which are offered by way of illustration. All reagents, starting materials, and solvents used in the following examples were purchased from commercial suppliers (for example, Sigma Aldrich, St. Louis, MO) and were used without further purification unless otherwise indicated.

[0150] Example 1 - FIG. 3 is a diagram of an embodied method of the present disclosure. A biological Sample 300 containing a target nucleic acid is obtained from a patient and added to a Sample Cartridge 310. Sample Cartridge 310 contains a number of spatial regions that allow for chemical processes to be done on the Sample 300, including isolation and filtration. A Reaction Vessel 320 containing a thermostable polymerase, a primer pair configured for amplification of the target nucleic acid, a reverse transcriptase and a fluorescent probe are added to the Sample Cartridge 310.The combination of the Sample Cartridge 310 and the Reaction Vessel 320 is placed in a Fast PCR Instrument 330.

[0151] The Fast PCR Instrument 330 provides the electronic and mechanical control for the Reaction Vessel 320. The Fast PCR Instrument 330 controls the electronic heating of two heaters, Heater 1 340 and Heater 2350, adjacent the Reaction Vessel 320 on opposite faces. Heaters 340 and 350 may be part of Reaction Vessel 320, part of Sample Cartridge 310, or Fast PCR Instrument 330. Heaters 340 and 350 are maintained at a first and second temperature, respectively, to create two regions with unique temperatures. Heater 1 340 creates a First Heated Region, 341, in theDocket No. 68552WO01 (2025-25631 -P-WO)Reaction Vessel 320 where the temperature is chosen to be from 94-100°C with a variance of 1°C over the amplification process time. Heater 2350 creates a Second Heated Region, 351, in the Reaction Vessel 320 where the temperature is chosen to be from 64-68°C with a variance of 1°C over the amplification process time.

[0152] The Sample 300 is filtered in the Sample Cartridge 310 to isolate the target nucleic acid. The isolated target nucleic acid enters the Reaction Vessel 320 and the Amplification Reaction 360 is initiated by heating the Reaction Vessel 320. The temperature difference between the First Heated Region 341 and Second Heated Region 351, and, in some cases, the structure of the Reaction Vessel 320 are such that the amplification rate is comparable to a regular thermal cycle time of 15 seconds or less. Twenty amplification cycles are completed in less than 15 minutes and the resulting amplified target nucleic acid is Detected 370 by fluorescent probe.

[0153] Example 2 - FIG. 4 is a diagram of an embodied method of the present disclosure. A biological Sample 400 containing a target nucleic acid is obtained from a patient and added to a Sample Cartridge 410. Sample Cartridge 410 contains a number of spatial regions that allow for chemical processes to be done on the Sample 300, including isolation and filtration. A Reaction Vessel 420 containing at least 4 U / pL of a thermostable polymerase, a primer pair configured for amplification of the target nucleic acid wherein each primer is at a concentration of at least 500 nM, a reverse transcriptase and a fluorescent probe are added to the Sample Cartridge 410. The combination of the Sample Cartridge 410 and the Reaction Vessel 420 is placed in a Fast PCR Instrument 430.

[0154] The Fast PCR Instrument 430 provides the electronic and mechanical control for the Reaction Vessel 320. The Fast PCR Instrument 430 controls the electronic heating of two heaters, Heater 1 440 and Heater 2450, adjacent the Reaction Vessel 320 on opposite faces. Heaters 440 and 450 may be part of Reaction Vessel 420, part of Sample Cartridge 410, or Fast PCR Instrument 430. Heaters 440 and 450 are maintained at a first and second temperature, respectively, to create two regions with unique temperatures. Heater 1 440 creates a First Heated Region, 441, in the Reaction Vessel 320 where the temperature is chosen to be from 94-100°C with a variance of 1°C over the amplification process time. Heater 2450 creates a Second Heated Region, 451, in the Reaction Vessel 420 where the temperature is chosen to be from 64-68°C with a variance of 1°C over the amplification process time.Docket No. 68552WO01 (2025-25631 -P-WO)

[0155] The Sample 400 is filtered in the Sample Cartridge 410 to isolate the target nucleic acid. The isolated target nucleic acid enters the Reaction Vessel 420 and the Amplification Reaction 460 is initiated by heating the Reaction Vessel 420. The temperature difference between the First Heated Region 441 and Second Heated Region 451, and, in some cases, the structure of the Reaction Vessel 420 are such that the cycle time is 15 seconds or less. Twenty amplification cycles are completed in less than 15 minutes and the resulting amplified target nucleic acid is Detected 470 by fluorescent probe.

[0156] Example 3 - Oligomers for an influenza B (Flu B) target were screened for the optimal Tm. Table 2 below shows the Tm of oligos tested. Some oligos included base modifications for Tm adjustment. Primers and probes having Tms between 78-80°C worked best, as shown in FIG 5A. Oligos with Tms outside this narrow range showed reduced performance, as shown in FIG 5B.Table 2: Flu B Oligomer Tm Testing

[0157] Example 4 - The effect of amplicon length and amplicon Tm on time to result (TTM) and effective positive fluorescence (EPF) was tested. Stronger targets generally have either low amplicon Tm (84-86°C), low amplicon length (~80nt) or both, as shown in Table 3, below. Sequence factors, such as folding and non-specific interactions, also contribute to performance. Results show that there is a need to balance primers Tm stabilization while keeping a low amplicon Tm.Table 3Docket No. 68552WO01 (2025-25631 -P-WO)

[0158] Example 5 - FIGS.6A-6D shows the assay is robust across a range of cycle temperatures by comparing relative fluorescent units over time as the assay is thermally cycled. FIG.6A shows the RFU signal over time with the assay cycled across temperatures from 64°C to 98°C. FIG. 6B is cycled from 65°C to 98°C, FIG. 6C is cycled from 66°C to 98°C and FIG.6D is cycled from 68°C to 98°C. As shown in FIGS.7A-7D, tests done lowering the upper temperature also provide evidence of the robustness of the assay. FIG. 7A shows the RFU signal over time with the assay cycled across temperatures from 64°C to 98°G. FIG. 7B is cycled from 64°C to 97°C, FIG. 7C is cycled from 64°C to 95°C and FIG. 7D is cycled from 64°C to 94°C.

[0159] FIG. 8A and FIG.8B compare RFU to time as a function of two different cycle temperature ranges. FIG. 8A shows the RFU vs time for an assay cycled between 69°C to 94°C, while FIG. 8B provides a graph of RFU for an assay cycled between 64°C to 98°C. The TTR for a Flu A target (PA) improved by 100 seconds with the wider temperature window shown in FIG. 8B. Narrow temperature ranges are major contributor to strict oligo design requirements. The wider cycle ranges greatly improve the assay. The 64-98°C temperature range is also operational at high ambient temperatures. A second Flu A target (MP1 ) is enabled with 64-98°C window and gives improved coverage and LoD compared to the narrower temperature window (not shown).

[0160] Example 6 - A rapid, qualitative, multiplex real-time PGR in vitro test using thermal convection cycling PGR technology was developed for de Cart AP+ and does not require PEG. Additionally, fewer on-board reagents requires fewer fluidic steps,Docket No. 68552WO01 (2025-25631 -P-WO)hence, contributing to faster sample prep time by ~2 minutes testing Influenza A (Flu A), Influenza B (Flu B), SARS-CoV-2, Respiratory Syncytial Virus A (RSV A) and Respiratory Syncytial Virus B (RSV B) and a control in individuals of all ages with signs and symptoms of respiratory tract infection (RTI). The sample type and sample collection process is a nasopharyngeal swab (NPS) and / or anterior nasal swab (NS) collected in universal transport medium (UTM) or viral transport medium (VTM). The TTR for the test was < 15 min, no EAT due to multiplexing. The test was run on a GeneXpert® system including the current GeneXpert® cartridge configuration (see FIG. 9) with thermal convection capabilities.

[0161] Table 4 provides a summary of the rapid, qualitative, multiplex real-time PGR in vitro test using thermal convection cycling PGR technology on the revised cartridge C (RGC) cartridge, referenced in W02021263101 A1 and WO2015013676 A1.Table 4: Target Summary for Fast PCR

[0162] Table 5 provides a summary of embodied nucleic acid target gene sequences used in the test.Table 5: Nucleic Acid Target Gene SequencesDocket No. 68552WO01 (2025-25631 -P-WO)

[0163] FIGS. 10A-10E show performance of the rapid, qualitative, multiplex real-time PCR in v / tro test using thermal convection cycling PCR technology on the GeneXpert® RCC. FIG. 10A compares the EPF over time for Flu A, FIG. 10B for Flu B, FIG. 10C for SARS-CoV-2, FIG. 10D for RSV A and FIG. 10E for RSV B. The assay was performed at 1 x LoD (referencing Xpert Xpress CoV-2 / Flu / RSV plus) on a nasopharyngeal swab (NPS) matrix. The assay showed a 100% hit rate (8 / 8) with a TTR of ~16 min on the GeneXpert® RCC with 65 pL reaction tube (also referenced herein as reaction vessel).

[0164] Similarly, FIGS. 11A-11D shows performance of the rapid, qualitative, multiplex real-time PCR in vitro test using thermal convection cycling PCR technology on a prototype GeneXpert® universal cartridge with amine modified glass fiber filter (as referenced in US20240035016A1 ). FIG. 11A compares the EPF over time for Flu A, FIG. 11B for Flu B, FIG. 11C for SARS-CoV-2, FIG. 11 D for RSV A and FIG. 11 E for RSV B. The assay was performed at 3 x LoD (referencing Xpert Xpress CoV-2 / Flu / RSV plus) on a nasopharyngeal swab (NPS) matrix. The prototype GeneXpert® universal cartridge with amine modified glass fiber filter does not require PEG. Additionally, fewer on-board reagents facilitate fewer fluidic steps, hence, contributing to faster sample prep time by ~2 minutes. Overall, the prototype GeneXpert® universal cartridge with amine modified glass fiber filter detected all five targets with the significant advantage of a fast sample prep time.

[0165] Example 7 - Solutions were created having 0, 1250, 6250, 12500, or 25000 copies per mL of intact SARS-CoV-2 virus as a target nucleic acid. A nasal matrix was created utilizing a nasal swab in 3 mL of collection buffer (20 mM Tris, pH 9, 0.1% Tween-20, 882 U RNAse inhibitor (CRI)). Target nucleic acid samples and PCR reaction mixtures / reagents were pre-mixed and transferred into SmartCycler tubes (Cepheid) where the embodied fast thermal cycling processes described in Examples 1 and 2 were applied. The sample was heated to 55°C for 60 seconds and introduced into the reaction vessel where it was cycled through a thermal convection cycle havingDocket No. 68552WO01 (2025-25631 -P-WO)an average cycle time of 15 seconds. The upper cycle temperature was set to 98°C and the lower cycle temperature was set to 62°C. Samples were optically monitored. At least 20 amplification cycles were done for each concentration of target nucleic acid and four replicates of each test were run.

[0166] FIG. 12 shows the time-to-result (TTR) and endpoint probe fluorescence (EPF) for PGR tests of the N2 and E genes of SAR-GoV-2. Four samples at each virus concentration were tested. A Cepheid Internal Control (CIC) was also measured to verify the effectiveness of on board sample processing, integrity of extracted nucleic acids, favorable reaction conditions for PCR performance, and absence of excess PCR inhibitors. The CIC is an exogenous (non-sample, non-analyte) nucleic acid pre-loaded in the cartridge that coextracts and co-amplifies along with the sample nucleic acids.

[0167] As shown in FIG. 12, at 6,250 copies of virus per mL, both the SAR-CoV-2 N2 and E PCR tests had 100% detection rates with average EPFs of 264 and 470, respectively. At 12,500 copies of virus per mL, the N2 and E PCR tests again had 100% detection rates with average EPFs of 321 and 909, respectively. Relative to conventional rapid antigen tests, the 6,250 virus / mL detection limit represents a one to two order of magnitude improvement in the detection limit. Additionally, the TTRs for 6,500 virus per mL N2 and E PCR tests were only about two and a half minutes to three minutes (167 and 194 seconds, respectively). Including the 60 second RT phase at 55 °C and the 15 second cycle time, the total runtime to detection was still under 4.5 minutes. Additional data are shown below in Table 3.Table 3 - Direct Detection of SARS-CoV-2 Virus

[0168] Example 8 - Solutions were created having 0, 1250, 6250, or 12500 copies per mL of intact SARS-CoV-2 virus as a target nucleic acid. Commercial multiplex test beads for SARS-CoV-2, Flu A, Flu B, RSV A and RSV B were used in direct detectionDocket No. 68552WO01 (2025-25631 -P-WO)assays of the viral solutions. The SARS-Cov-2-E gene and the SARS-Cov-2-RdRp gene were targeted in single channel tests.

[0169] Nasal swabs of the samples were inserted in 3 mL of collection buffer (20 mM Tris, pH 9, 0.1% Tween-20, 882 U RNAse inhibitor (CRI)). Target nucleic acid samples and PCR reaction mixtures / reagents were pre-mixed and transferred into SmartCycler tubes (Cepheid) where the embodied fast thermal cycling processes described in Examples 1 and 2 were applied. The sample was heated to 55°C for 60 seconds and introduced into the reaction vessel where it was cycled through a thermal convection cycle having an average cycle time of 15 seconds. The Tm was set to 98°C and the Ta was set to 62°C and the samples were optically monitored. At least 20 amplification cycles were done for each concentration of target nucleic acid and four replicates of each test were run.

[0170] FIG. 13 shows the time-to-result (TTR) and endpoint probe fluorescence (EPF) for PCR tests of the E / RdRp genes of SAR-CoV-2. A Cepheid Internal Control (CIC) was also measured. At 6,250 copies of virus per mL, the E / RdRp test had a 100% detection rate with for the four sample replicates that were run and an average EPF of 618 and a TTR of just 180 seconds. Additional data are shown below in Table 4.Table 4. Direct Detection of SARS-CoV-2 Virus

[0171] Example 9 - FIG. 14 is a graph of the temperature cycle over time for a thermal cycling event in the embodied reaction vessels. The target in this case is a GFP gene with sequences of unmodified F / R PCR primers and an unmodified FAM TaqMan® probe. The average cycle time is 15 seconds or less with a temperature cycle from 64°C to 98°C and excellent stability across greater than 40 cycles. The probe fluorescence shows that detectable signal was seen at approximately 25 cycles with a rapid rise in intensity.

Claims

1. Docket No. 68552WO01 (2025-25631 -P-WO)CLAIMSWhat is claimed is:

1. A method for amplifying a target nucleic acid in an amplification reaction, the method comprising:a) providing a solution in a reaction chamber,I. the reaction chamber defined by two opposing major walls and side walls connecting the major walls to each other, and II. the solution comprising the target nucleic acid, a thermostable polymerase, a primer pair configured for amplification of the target nucleic acid, optionally, a reverse transcriptase and optionally, a probe;b) maintaining a first region of the reaction chamber at a first temperature capable of denaturing the target nucleic acid sequence into a singlestranded nucleic acid template;c) maintaining a second region of the reaction chamber at a second temperature capable of annealing the primer pair to the single-stranded nucleic acid template;d) inducing a thermal convection cycle in the solution between the first region and the second region of the reaction chamber;wherein an average cycle time for the thermal convection cycle is 15 seconds or less; ande) holding both the first region at the first temperature and the second region at the second temperature for a time sufficient for amplification of the target nucleic acid.

2. The method of claim 1 , wherein the first region and second region of the reaction chamber are each defined by one of the two opposing major walls, respectively.

3. The method of claim 1 , wherein each of the major walls of the reaction chamber each has a ratio of height to length of from 1 :1 to 1 :1 .5.

4. The method of any one of claims 1 -3, wherein:Docket No. 68552WO01 (2025-25631 -P-WO)a) the length of the major walls of the reaction chamber is from 1.4 to 20 mm; and / orb) the height of the major walls of the reaction chamber is from 1 to 15 mm; and / orc) the average distance between the two opposing major walls of the reaction chamber is from 0.5 to 5 mm.

5. The method of claim 1 , wherein the solution further comprises at least one probe capable of hybridizing with the target nucleic acid, wherein the probe has a concentration of 500 nM or greater and a Tm below the first temperature.

6. The method of claim 5, further comprising detecting the presence of an amplified nucleic acid using a probe that has hybridized with the target singlestranded nucleic acid template.

7. The method of any of claims 1 -6, wherein the difference in temperature between the first temperature and the second temperature is equal to or greater than 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C or from 25-37°C, 25-35°C, 27-35°C, 28-34°C.

8. The method of any of claims 1 -7, wherein the first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C.

9. The method of any of claims 1 -8, wherein the second temperature is from 60°C to 70°G, from 64°C to 68°C, or from 65°C to 67°C.

10. The method of any one of claims 1 -9, wherein the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C.11 . The method of any of claims 1 -10, wherein the amplification reaction is carried out over an amplification reaction time, defined as a time sufficient to produce equal / greater level of amplification compared to at least 20 amplification cycles using conventional quantitative PGR (qPGR) by thermal cycling, and wherein the firstDocket No. 68552WO01 (2025-25631 -P-WO)temperature and the second temperature each vary less than 1 °C over the amplification reaction time.

12. The method of any of claims 1-11 , wherein each primer has a melt temperature, Tm, from 7-13°C or from 8-10°C below the first temperature.

13. The method of any of claims 1 -12, wherein each primer has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C.

14. The method of any of claims 1 -13, wherein each primer has an annealing temperature, Ta, from 0-10°C or from 0-6°C above the second temperature.

15. The method of any of claims 1-14, wherein each primer has an annealing temperature, Ta, from 60-80°G, 60-75°C, or 60-70°C.

16. The method of any of claims 1 -15, wherein each primer comprises at least one stabilizing base.

17. The method of claim 16, wherein the at least one stabilizing base comprises a stabilizing base at the 2ndor 3rdposition from the 3’ end of the primer.

18. The method of any of claims 1 -17, wherein the at least one stabilizing bases is selected from the group consisting of: locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

19. The method of any of claims 1 -18, wherein the probe has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C.

20. The method of any of claims 1 -19, wherein the primer and / or probe comprise a detectable moiety.Docket No. 68552WO01 (2025-25631 -P-WO)21 . The method of claim 20, wherein the detectable moiety comprises a fluorescent dye and a quencher molecule.

22. The method of any one of claims 5-21 , wherein a probe is present and the probe comprises one or more destabilizing bases.

23. The method of claim 22, wherein the one or more destabilizing base decreases the melt temperature of the target nucleic acid in double-stranded form.

24. The method of claim 22 or 23, wherein the one or more modified bases which is a destabilizing base is selected from the group consisting of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

25. The method of any of claims 1 -24, wherein each primer is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM.

26. The method of any of claims 5-25, wherein a probe is present and the probe is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM.

27. The method of any of claims 1 -26, wherein the target nucleic acid has a melt temperature from 80-90°C, 81-89°C, 82-88°C, 83-87°C, 84-86°C, or less than or equal to 90°C, 89°C, 88°C, 87°C or 86°C.

28. The method of any of claims 1 -27, wherein the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 65-87, 67-87, or 69-86 nucleotides.

29. The method of any of claims 1 -28, wherein the primer has a length equal to or less than 45 nucleotides.Docket No. 68552WO01 (2025-25631 -P-WO)30. The method of any one of claims 1 -29, wherein the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .31 . The method of any of claims 1 -30, wherein the solution further comprises a reverse transcriptase, and the reverse transcriptase and thermostable polymerase are each present at concentrations of 2 U / pL or greater, 4 U / pL or greater, 6 U / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume.

32. The method of any one of claims 1 -31 , wherein the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited up to 55°C.

33. The method of any of claims 1 -32, wherein the target nucleic acid sequences come from a biological sample selected from one or more of nasopharyngeal swab, nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix.

34. The method of claim 33, wherein the biological sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater.

35. The method of any one of claims 1 -34, wherein the amplification reaction is a multiplex amplification reaction and the solution comprises at least four sets of primer pairs for detecting at least four target nucleic acids.

36. The method of claim 35, wherein the solution comprises at least eight sets of primer pairs and the multiplex amplification reaction comprises detecting at least eight target nucleic acids.

37. A method for amplifying a target nucleic acid in an amplification reaction, the method comprising:Docket No. 68552WO01 (2025-25631 -P-WO)a) providing a solution in a reaction chamber,I. the reaction chamber defined by two opposing major walls and side walls connecting the major walls to each other, and II. the solution comprising the target nucleic acid, a thermostable polymerase at a concentration of at least 4 U / |iL of reaction volume, and a primer pair configured for amplification of the target nucleic acid, each primer at a concentration of at least 500 nM, optionally, a reverse transcriptase and optionally, a probe;b) maintaining a first region of the reaction chamber at a first temperature capable of denaturing the target nucleic acid sequence into a singlestranded nucleic acid template;c) maintaining a second region of the reaction chamber at a second temperature capable of annealing the primer pair to the single-stranded nucleic acid template;d) inducing a thermal convection cycle in the solution between the first region and the second region of the reaction chamber,e) holding the first region at the first temperature and the second region at the second temperature, for a time sufficient for amplification of the target nucleic acid.

38. The method of claim 37, wherein each of the major walls of the reaction chamber has a ratio of height to length of from 1 :1 to 1 :1 .5.

39. The method of claim 37 or 38, wherein the length of the major walls of the reaction chamber is from 1 .4 to 20 mm.

40. The method of any one of claims 37-37, wherein the height of the major walls of the reaction chamber is from 1 to 15 mm.41 . The method of claim 37, further comprising at least one probe capable of hybridizing with the target nucleic acid, wherein the probe has a concentration of 500 nM or greater and a Tm below the first temperature.Docket No. 68552WO01 (2025-25631 -P-WO)42. The method of claim 41 , further comprising detecting the presence of an amplified nucleic acid using a probe that has hybridized with the target singlestranded nucleic acid template.

43. The method of any of claims 37-42, wherein the difference in temperature between the first temperature and the second temperature is equal to or greater than 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C or from 25-37°C, 25-35°C, 27-35°C, 28-34°C.

44. The method of any of claims 37-43, wherein the first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C.

45. The method of any of claims 37-44, wherein the second temperature is from 60°C to 70°G, from 64°C to 68°C, or from 65°C to 67°C.

46. The method of any one of claims 37-45, wherein the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C.

47. The method of any of claims 37-46, wherein an average cycle time for the thermal convection cycle is 15 seconds or less.The method of any of claims 37-46, wherein the amplification reaction is carried out over an amplification reaction time and wherein the first temperature and the second temperature each vary less than 1°C over the amplification reaction time.

48. The method of any of claims 37-47, wherein each primer has a melt temperature, Tm, from 7-13°C or from 8-10°C below the first temperature.

49. The method of any of claims 37-48, wherein each primer has a melt temperature, Tm, from 76-82°C, 77-81°C, or 78-80°C.

50. The method of any of claims 37-49, wherein each primer has an annealing temperature, Ta, from 0-10°C or from 0-6°C above the second temperature.Docket No. 68552WO01 (2025-25631 -P-WO)51 . The method of any of claims 37-50, wherein each primer has an annealing temperature, Ta, from 60-80°C, 60-75°C, or 60-70°C.

52. The method of any of claims 37-51 , wherein each primer comprises at least one stabilizing base.

53. The method of claim 52, wherein the at least one stabilizing base comprises a stabilizing base at the 2ndor 3rdposition from the 3’ end of the primer.

54. The method of any of claims 37-53, wherein the at least one stabilizing bases is selected from the group consisting of: locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, 0(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

55. The method of any of claims 37-54, wherein the probe has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C.

56. The method of any of claims 37-55, wherein the primer and / or probe comprise a detectable label.

57. The method of claim 56, wherein the detectable label comprises a fluorescent dye and a quencher molecule.

58. The method of any one of claims 37-57, wherein a probe is present and the probe comprises one or more destabilizing bases.

59. The method of claim 58, wherein the one or more destabilizing base decreases the melt temperature of the target nucleic acid.

60. The method of claim 58 or 59, wherein the one or more modified bases which is a destabilizing base is selected from the group consisting of locked nucleicDocket No. 68552WO01 (2025-25631 -P-WO)acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.61 . The method of any of claims 37-60, wherein each primer is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM.

62. The method of any of claims 41 -61 , wherein a probe is present and the probe is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM.

63. The method of any of claims 37-62, wherein the target nucleic acid has a melt temperature from 80-90°C, 81-89°C, 82-88°C, 83-87°C, 84-86°G, or less than or equal to 90°C, 89°C, 88°C, 87°C or 86°C.

64. The method of any of claims 37-63, wherein the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 65-87, 67-87, or 69-86 nucleotides.

65. The method of any of claims 37-64, wherein the primer has a length equal to or less than 45 nucleotides.

66. The method of any one of claims 37-65, wherein the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .

67. The method of any of claims 37-66, wherein the solution further comprises a reverse transcriptase, and the reverse transcriptase and thermostable polymerase are each present at concentrations of 5 U / pL or greater, 6 U / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume.Docket No. 68552WO01 (2025-25631 -P-WO)68. The method of any one of claims 37-67, wherein the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited up to 55°C.

69. The method of any of claims 37-68, wherein the target nucleic acid sequences come from a biological sample selected from one or more of nasopharyngeal swab, nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix.

70. The method of claim 69, wherein the biological sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater.71 . The method of any one of claims 37-70, wherein the amplification reaction is a multiplex amplification reaction and the solution comprises at least four sets of primer pairs for detecting at least four target nucleic acids.

72. The method of claim 71 , wherein the solution comprises at least eight sets of primer pairs and the multiplex amplification reaction comprises detecting at least eight target nucleic acids.

73. A cartridge-based method for amplifying and detecting a target nucleic acid in a sample via an amplification reaction, the method comprising:a) placing the sample a sample chamber in a self-contained cartridge, the self- contained cartridge further comprising:a. a reaction chamber in fluid communication with the sample chamber, the reaction chamber defined by two opposing major walls and side walls connecting the major walls to each other, andb. a filter disposed in a fluidic path between the sample chamber and the reaction chamber,b) isolating the target nucleic acid by passing it through the filter;Docket No. 68552WO01 (2025-25631 -P-WO)c) forming a solution comprising the isolated target nucleic acid, a thermostable polymerase, a primer pair configured for amplification of the target nucleic acid, optionally, a reverse transcriptase and optionally, a probe;d) transferring the solution into the reaction chamber;e) maintaining a first region of the reaction chamber at a first temperature capable of denaturing the target nucleic acid sequence into a single-stranded nucleic acid template;f) maintaining a second region of the reaction chamber at a second temperature capable of annealing the primer pair to the single-stranded nucleic acid template;g) inducing a thermal convection cycle in the solution between the first region and the second region, wherein an average cycle time for the thermal convection cycle is 15 seconds or less; andh) detecting the presence of the amplicon within 15 minutes of placing the sample within the self-contained cartridge.

74. The cartridge-based method of claim 73, wherein the filter comprises an amine modification for isolating nucleic acid from the biological sample.

75. The cartridge-based method of claim 73 or claim 74, wherein the self-contained cartridge further comprises a lysis chamber, wherein the lysis chamber contains one or more lysis reagents for releasing nucleic acid.

76. The cartridge-based method of any one of claims 73-75, wherein each reaction chamber is configured to detect a single amplification product.

77. The cartridge-based method of any one of claims 73-76, wherein each reaction chamber is configured to detect a plurality of amplification products.

78. The cartridge-based method of any one of claims 73-77, wherein the self-contained cartridge is a Clinical Laboratory Improvement Amendments (CLIA)-compliant cartridge.Docket No. 68552WO01 (2025-25631 -P-WO)79. The cartridge-based method of any one of claims 73-78, wherein each of the major walls of the reaction chamber each has a ratio of height to length of from 1 :1 to 1 :1.5.

80. The cartridge-based method of any one of claims 73-79, wherein the length of the major walls of the reaction chamber is from 1 .4 to 20 mm.81 . The cartridge-based method of any one of claims 73-80, wherein the height of the major walls of the reaction chamber is from 1 to 15 mm.

82. The cartridge-based method of claim 73, further comprising at least one probe capable of hybridizing with the target nucleic acid, wherein the probe has a concentration of 500 nM or greater and a Tm below the first temperature.

83. The cartridge-based method of claim 82, further comprising detecting the presence of an amplified nucleic acid using a probe that has hybridized with the target single-stranded nucleic acid template.

84. The cartridge-based method of any of claims 73-83, wherein the difference in temperature between the first temperature and the second temperature is equal to or greater than 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C, 36°C, or 73°C or from 25-73°C, 25-35°C, 27-35°C, 28-34°C.

85. The cartridge-based method of any of claims 73-84, wherein the first temperature is from 90°C to 100°C, from 94°C to 100°C, or from 97°C to 99°C.

86. The cartridge-based method of any of claims 73-85, wherein the second temperature is from 60°C to 70°C, from 64°C to 68°C, or from 65°C to 67°C.

87. The cartridge-based method of any one of claims 73-86, wherein the first temperature is from 97°C to 99°C and the second temperature is from 65°C to 67°C.

88. The cartridge-based method of any of claims 73-87, wherein the amplification reaction is carried out over an amplification reaction time and wherein the firstDocket No. 68552WO01 (2025-25631 -P-WO)temperature and the second temperature each vary less than 1 °C over the amplification reaction time.

89. The cartridge-based method of any of claims 73-88, wherein each primer has a melt temperature, Tm, from 7-13°C or from 8-10°C below the first temperature.

90. The cartridge-based method of any of claims 73-89, wherein each primer has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C.91 . The cartridge-based method of any of claims 73-90, wherein each primer has an annealing temperature, Ta, from 0-10°C or from 0-6°C above the second temperature.

92. The cartridge-based method of any of claims 73-91 , wherein each primer has an annealing temperature, Ta, from 60-80°C, 60-75°C, or 60-70°C.

93. The cartridge-based method of any of claims 73-92, wherein each primer comprises at least one stabilizing base.

94. The cartridge-based method of claim 93, wherein the at least one stabilizing base comprises a stabilizing base at the 2ndor 3rdposition from the 3’ end of the primer.

95. The cartridge-based method of any of claims 73-94, wherein the at least one stabilizing bases is selected from the group consisting of: locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

96. The cartridge-based method of any of claims 73-95, wherein the probe has a melt temperature, Tm, from 76-82°C, 77-81 °C, or 78-80°C.Docket No. 68552WO01 (2025-25631 -P-WO)97. The cartridge-based method of any of claims 73-96, wherein the primer and / or probe comprise a detectable label.

98. The cartridge-based method of claim 97, wherein the detectable label comprises a fluorescent dye and a quencher molecule.

99. The cartridge-based method of any one of claims 82-98, wherein a probe is present and the probe comprises one or more destabilizing bases.

100. The cartridge-based method of claim 99, wherein the one or more destabilizing base decreases the melt temperature of the target nucleic acid.101 . The cartridge-based method of claim 98 or 99, wherein the one or more modified bases which is a destabilizing base is selected from the group consisting of locked nucleic acids (LNAs), unlocked nucleic acids (UNAs), AP-dC (G-clamp), 2-aminoadenine, 5-methylcytosine, C(5)-propynylcytosine, C(5)-propynyluracil, 2-aminoadenine, 2-thiothymine, deoxyinosine, 7-alkyl-7-deazaguanine, 2'-hypoxanthine, or 7-nitro-7-deazahypoxanthine, 3-(2'-deoxy-beta-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one, N4-alkylcytosine, 2-thiocytosine, and combinations thereof.

102. The cartridge-based method of any of claims 73-101 , wherein each primer is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM.

103. The cartridge-based method of any of claims 82-102, wherein a probe is present and the probe is provided at a concentration of at least 500 nM, 600 nM, 700 nM or 800 nM.

104. The cartridge-based method of any of claims 73-103, wherein the target nucleic acid has a melt temperature from 80-90°C, 81-89°C, 82-88°C, 83-87°C, 84-86°C, or less than or equal to 90°C, 89°C, 88°C, 87°C or 86°C.Docket No. 68552WO01 (2025-25631 -P-WO)105. The cartridge-based method of any of claims 73-104, wherein the target nucleic acid has a length equal to or less than 90, 88, 87, 86, 85, 83, 82, 81 , or 80 nucleotides, or from 65-87, 67-87, or 69-86 nucleotides.

106. The cartridge-based method of any of claims 73-105, wherein the primer has a length equal to or less than 45 nucleotides.

107. The cartridge-based method of any one of claims 73-106, wherein the target nucleic acid comprises a ratio of AT to GC content of greater than 1 .

108. The cartridge-based method of any of claims 73-107, wherein the solution further comprises a reverse transcriptase, and the reverse transcriptase and thermostable polymerase are each present at concentrations of 2 U / pL or greater, 4 U / pL or greater, 6 LI / pL or greater, 7 U / pL or greater, or 8 U / pL or greater, of reaction volume.

109. The cartridge-based method of any one of claims 73-108, wherein the thermostable polymerase is inactive during reverse transcription and / or the activity of the thermostable polymerase is inhibited up to 55°C.

110. The cartridge-based method of any of claims 73-109, wherein the target nucleic acid sequences come from a biological sample selected from one or more of nasopharyngeal swab, nasal swab, blood, plasma, serum, semen, spinal fluid, tissue biopsy, tear, urine, stool, saliva, smear preparation, bacterial culture, mammalian cell culture, viral culture, human cell, bacteria, extracellular fluid, or in vitro nucleic acid modification reaction mix.

111. The cartridge-based method of claim 110, wherein the biological sample volume is 50, 75, 100, 125, 150, 175, 200, 250, or 300 pL or greater.

112. The cartridge-based method of any one of claims 73-111 , wherein the amplification reaction is a multiplex amplification reaction and the solution comprises at least four sets of primer pairs for detecting at least four target nucleic acids.Docket No. 68552WO01 (2025-25631 -P-WO)113. The cartridge-based method of claim 112, wherein the solution comprises at least eight sets of primer pairs and the multiplex amplification reaction comprises detecting at least eight target nucleic acids.

114. The method of any one of the proceeding claims, wherein the primer and probe are selected from a group consisting of:a primer pair and probe for detecting the presence of at least one influenza A gene in the sample selected from an influenza A PA gene, an influenza A PB2 gene, and an influenza A MP gene;a primer pair and probe for detecting the presence of at least one influenza B gene in the sample selected from an influenza B NS gene, an influenza B MP gene; a primer pair and probe for detecting the presence of an avian influenza gene in the sample selected from an avian influenza MP gene and an avian influenza HA gene;a primer pair and probe for detecting the presence of at least one SARS-CoV-2 gene in the sample selected from a SARS-Cov-2-E gene, a SARS-Cov-2-RdRp gene, and a SARS-Cov-2-N2 gene; anda primer pair for and probe detecting the presence of at least one respiratory syncytial virus (RSV) A gene in the sample and a respiratory syncytial virus (RSV) B gene.

115. The method of any one of the proceeding claims, wherein the primer and probe are selected from a group consisting of:at least two primer pairs detecting the presence of influenza A PA gene and influenza A MP gene;at least two primer pairs and probes for detecting the presence of influenza B NS gene and an influenza B MP gene;at least three primer pairs and probes for detecting SARS-Cov-2-E gene, a SARS- Cov-2-RdRp gene, and a SARS-Cov-2-N2 gene; anda primer pair and probe for detecting the presence of at least one respiratory syncytial virus (RSV) A gene in the sample and a respiratory syncytial virus (RSV) B gene.

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