Tandem isothermal amplification and polymerase chain reaction

Abstract

The present disclosure is directed to methods of amplifying one or more nucleic acid molecules. In particular, the methods of the present disclosure utilize a combination of (i) one or more isothermal amplification techniques, and (ii) polymerase chain reaction. In some embodiments, the methods of the present disclosure comprise performing an isothermal amplification for a predetermined amount of time at a predetermined temperature; and subsequently performing a predetermined number of PCR cycles.

Description

Attomey Docket No. P39522-WO-1TANDEM ISOTHERMAL AMPLIFICATION AND POLYMERASE CHAIN REACTIONCROSS REFERENCE TO RELATED PATENT APPLICATINOS

[0001] This patent application claims priority from U.S. Provisional Patent ApplicationNo. 63 / 730,666, filed December 11, 2024, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to methods of amplifying nucleic acid molecules, where the amplified nucleic acid molecules may be used for diagnostic purposes.BACKGROUND OF THE DISCLOSURE

[0003] Molecular biology techniques rely on samples containing many identical nucleic acid molecules to increase the sensitivity of the assay or to prepare enough material for further processing. Among the various nucleic acid amplification techniques, polymerase chain reaction (PCR) is the most common because of its sensitivity and efficiency at amplifying short nucleic acid sequences. PCR amplifies a target region of a nucleic acid molecule using a set of two primers with a thermal cycler to heat and cool for the denaturation, annealing and extension steps. PCR requires sophisticated temperature cycling instruments to complete the amplification process; and has an associated high cost and long reaction time.

[0004] Isothermal amplification techniques do not have any temperature cycling requirements. While PCR requires the lowering and rising of two or three working temperatures to facilitate annealing and denaturation of DNA strands and primers, and to permit strand displacement, isothermal amplification methods proceed at a single working temperature, which removes the need for a thermocycler. Isothermal amplification techniques include Nucleic Acid Sequence Based Amplification (NASBA), Rolling Circle Amplification (RCA), Sequence Mediated Amplification of RNA Technology (SMART), Strand Displacement Amplification (SDA), Transcription-Mediated Amplification (TMA), Loop- mediated Isothermal Amplification (LAMP), the Multiple Cross Displacement Amplification (MCDA), and Recombinase polymerase amplification (RPA).

[0005] RPA is an isothermal method that amplifies a target sequence using a set of two primers at a constant temperature (e.g., at about 37°C). As compared with PCR, RPA requires simple instrumentation, is rapid and sensitive. In fact, it can detect as little as 1 copy of S. Epidermidis plasmid within a 10-minute reaction time, permitting a decrease in assay time-to-Atorney Docket No. P39522-WO-1 result and improving the limits of detection. RPA has been demonstrated to amplify nucleic acids from crude samples including whole blood with no pre-processing or minimal preprocessing. RPA is known to have a high tolerance to hemoglobin, a common PCR inhibitor. One limitation of RPA, however, is its non-specificity, which can lead to inaccurate results. In comparison, PCR has remarkably high specificity due to well-designed PCR primers, and the steps of heat denaturation and annealing, but can take several hours to complete.

[0006] There remains a need to amplify nucleic acid molecules in a sample quickly while maximizing sensitivity and specificity.BRIEF SUMMARY OF THE DISCLOSURE

[0007] In view of the foregoing, Applicant has developed a workflow for amplifying one or more nucleic acid molecules, where the workflow has high specificity (such as compared with RPA); and a comparatively reduced time-to-result (such as compared with PCR). In some embodiments, the time-to-result is about 100 minutes or less, such as 90 minutes or less, such as 80 minutes or less, such as 70 minutes or less, such as 60 minutes or less, such as 50 minutes or less, such as 40 minutes or less, such as 30 minutes or less, etc.

[0008] The present disclosure provides for methods of amplifying one or more nucleic acid molecules in a sample by performing an isothermal amplification (e.g., LAMP, TMA, RPA, RCA, etc.), followed by PCR. In some embodiments, the isothermal amplification is performed for a predetermined amount of time and at a predetermined temperature. In some embodiments, the isothermal amplification is performed for a duration of less than about 120 minutes, such as less than about 90 minutes, such as less than about 80 minutes, such as less than about 70 minutes, such as less than about 60 minutes, such as less than about 50 minutes, such as less than about 40 minutes, such as less than about 30 minutes, such as less than about 20 minutes, such as less than about 10 minutes, etc. Following the performance of the one or more isothermal amplification techniques, PCR is performed for a predetermined number of cycles, such as for 30 cycles or less, such as between about 10 and 30 cycles. In some embodiments, the methods of the present disclosure may be performed within a cartridge, such as a cartridge including the components needed to perform the isothermal amplification and the PCR. In some embodiments, the one or more amplified nucleic acid molecules may then be detected, such as with an electrochemical detection technique, a fluorescent detection technique, or a colorimetric detection technique. In some embodiments, the detection technique is multimodal, e.g., a combination of two or more of electrochemical detection, fluorescent detection, colorimetric detection, etc.Atorney Docket No. P39522-WO-1

[0009] By way of example, if RPA is utilized for isothermally amplifying the one or more nucleic acids in the sample, in some embodiments the RPA may be performed for a time period ranging from between about 5 minutes to about 20 minutes, such as time period ranging from between about 5 minutes to about 10 minutes. Subsequently, less than 30 cycles of PCR may be conducted.

[0010] A first aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules in a sample, the method comprising: (a) performing an isothermal amplification on the sample including the one or more nucleic acid molecules for a predetermined amount of time and at a predetermined temperature, wherein the isothermal amplification provides a sample including one or more isothermally amplified nucleic acid molecules; and (b) performing a polymerase chain reaction on the sample including the one or more isothermally amplified nucleic acid molecules to provide a sample including the one or more amplified nucleic acid molecules, wherein less than 30 cycles of the polymerase chain reaction are performed. In some embodiments, less than 25 PCR cycles are performed. In some embodiments, less than 20 PCR cycles are performed. In some embodiments, the recombinase amplification technique comprises contacting the sample with a DNA polymerase having strand displacement activity.

[0010] In some embodiments, the isothermal amplification comprises loop-mediated isothermal amplification. In some embodiments, the predetermined temperature is between about 60°C to about 65°C. In some embodiments, the predetermined time ranges from between about 20 minutes to about 60 minutes. In some embodiments, the predetermined time ranges from between about 20 minutes to about 40 minutes. In some embodiments, the predetermined time is about 30 minutes.

[0011] In some embodiments, the isothermal amplification comprises recombinase polymerase amplification. In some embodiments, the predetermined temperature is between about 37°C to about 42°C. In some embodiments, the predetermined temperature is about 40°C. In some embodiments, the predetermined time ranges from between about 5 minutes to about 10 minutes. In some embodiments, the predetermined time is about 7 minutes. In some embodiments, the recombinase polymerase amplification comprises contacting the sample including the one or more nucleic acid molecules with one of Bsu DNA polymerase or Sau DNA polymerase. In some embodiments, the method further comprises contacting the sample with a single stranded binding protein and a recombinase. In some embodiments, the single stranded binding protein is T4 gp32. In some embodiments, the recombinase is selected fromAtorney Docket No. P39522-WO-1Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX.

[0012] In some embodiments, the isothermal amplification comprises rolling circle amplification. In some embodiments, the predetermined temperature is between about 35°C to about 40°C. In some embodiments, the predetermined temperature is about 37°C. In some embodiments, the predetermined time ranges from between about 60 minutes to about 120 minutes. In some embodiments, the predetermined time is about 60 minutes to about 90 minutes.

[0013] In some embodiments, the isothermal amplification comprises transcription- mediated amplification. In some embodiments, the predetermined temperature is between about 35°C to about 45°C. In some embodiments, the predetermined temperature is about 42°C. In some embodiments, the predetermined time is less than about 60 minutes.

[0014] In some embodiments, the method further comprises detecting the one or more amplified nucleic acid molecules. In some embodiments, the method further comprises detecting the one or more amplified nucleic acid molecules in the sample. In some embodiments, the detection comprises one or more of an electrochemical detection technique, a fluorescent detection technique, or a colorimetric detection technique. In some embodiments, the detection is multimodal.

[0015] A second aspect of the present disclosure is method of amplifying a plurality of target nucleic acid molecules in a sample, wherein the sample comprises the plurality of target nucleic acid molecules, and a plurality of non-target nucleic acid molecules, the method comprising: (a) performing an isothermal amplification on the sample for a predetermined amount of time and at a predetermined temperature, wherein the isothermal amplification is performed using a plurality of different target specific primers for the isothermal amplification, and wherein the isothermal amplification provides a plurality of isothermally amplified target nucleic acid molecules; and (b) performing a polymerase chain reaction on a sample including the plurality of isothermally amplified target nucleic acid molecules to provide a sample including a plurality of amplified target nucleic acid molecules, wherein less than 30 cycles of the polymerase chain reaction are performed, and wherein the polymerase chain reaction is performed with a plurality of different target specific PCR primers. In some embodiments, the method further comprises electrochemically detecting the plurality target nucleic acid molecules. In some embodiments, the method further comprises detecting the plurality target nucleic acid molecules with a fluorescent detection technique. In some embodiments, the method further comprises detecting the plurality target nucleic acid molecules with aAtorney Docket No. P39522-WO-1 colorimetric detection technique. In some embodiments, the recombinase amplification technique comprises contacting the sample with a DNA polymerase having strand displacement activity.

[0016] In some embodiments, the sample is derived from blood. In some embodiments, the plurality of target nucleic acid molecules is derived from bacteria. In some embodiments, the plurality of target nucleic acid molecules is derived from viruses. In some embodiments, the plurality of target nucleic acid molecules is derived from fungi. In some embodiments, the plurality of target nucleic acid molecules is derived from a gram-positive microorganism. In some embodiments, the plurality of target nucleic acid molecules is derived from a gramnegative microorganism. In some embodiments, the sample is derived from a tumor. In some embodiments, the sample is derived from a patient diagnosed with cancer. In some embodiments, the sample is derived from a patient suspected of having cancer. In some embodiments, the sample is derived from a patient diagnosed with a neurodegenerative disease. In some embodiments, the sample is derived from a patient suspected of having a neurodegenerative disease.

[0017] In some embodiments, the isothermal amplification comprises recombinase polymerase amplification. In some embodiments, the predetermined temperature is between about 37°C to about 42°C. In some embodiments, the predetermined temperature is about 40°C. In some embodiments, the predetermined time ranges from between about 5 minutes to about 10 minutes. In some embodiments, the predetermined time is about 7 minutes. In some embodiments, the recombinase polymerase amplification comprises contacting the sample including the one or more nucleic acid molecules with one of Bsu DNA polymerase or Sau DNA polymerase. In some embodiments, the method further comprises contacting the sample with a single stranded binding protein and a recombinase. In some embodiments, the single stranded binding protein is T4 gp32. In some embodiments, the recombinase is selected from Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX.

[0018] A third aspect of the present disclosure is a cartridge comprising one or more reaction wells in communication with (a) a first chamber including one or more isothermal amplification reagents; and (b) a second chamber including one or more PCR reagents. In some embodiments, the one or more isothermal amplification reagents comprise a polymerase, a recombinase, and a single stranded binding protein. In some embodiments, the polymerase is Sau DNA polymerase or Bsu DNA polymerase. In some embodiments, the recombinase is selected from Cre recombinase, Hin recombinase, Tre recombinase. In some embodiments,Atorney Docket No. P39522-WO-1 the single stranded binding protein is T4 gp32. In some embodiments, the PCR reagents include a DNA polymerase and dNTPs. In some embodiments, the cartridge further comprises one or more additional chambers including one or more signal probes and one or more capture probes.

[0019] A fourth aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules in a sample, the method comprising: (a) performing an isothermal amplification on the sample including the one or more nucleic acid molecules for a predetermined amount of time and at a predetermined temperature, wherein the isothermal amplification provides a sample including one or more isothermally amplified nucleic acid molecules; and (b) performing a polymerase chain reaction on the sample including the one or more isothermally amplified nucleic acid molecules to provide a sample including the one or more amplified nucleic acid molecules, wherein less than 30 cycles of the polymerase chain reaction are performed. In some embodiments, the method further comprises detecting the one or more amplified nucleic acid molecules. In some embodiments, the detection comprises one or more of an electrochemical detection technique, a fluorescent detection technique, and / or a colorimetric detection technique. In some embodiments, the detection is multimodal. In some embodiments, the isothermal amplification comprises one of nucleic acid sequence-based amplification, "a simple method for amplifying RNA targets," loop-mediated isothermal amplification, strand displacement amplification, transcription mediated amplification, rolling circle amplification, multiple displacement amplification, or recombinase polymerase amplification.

[0020] In some embodiments, the sample is a bodily fluid or tissue-derived sample. In some embodiments, wherein the sample is derived from a tumor. In some embodiments, wherein the sample is derived from a subject diagnosed with cancer. In some embodiments, wherein the sample is derived from a subject suspected of having cancer. In some embodiments, wherein the sample is derived from a subject diagnosed with a neurodegenerative disease. In some embodiments, wherein the sample is derived from a subject suspected of having a neurodegenerative disease.

[0021] In some embodiments, the isothermal amplification comprises contacting the sample with a DNA polymerase having strand displacement activity. In some embodiments, the isothermal amplification is performed at a temperature ranging from between about 35 °C to about 75°C. In some embodiments, the isothermal amplification is performed at a temperature ranging from between about 35 °C to about 65 °C. In some embodiments, the isothermal amplification is performed at a temperature ranging from between about 40°C toAtorney Docket No. P39522-WO-1 about 60°C. In some embodiments, the isothermal amplification is performed at a temperature ranging from between about 37°C to about 60°C. In some embodiments, the isothermal amplification is performed for a time period ranging from between about 5 minutes to about 60 minutes. In some embodiments, the isothermal amplification is performed for a time period ranging from between about 5 minutes to about 55 minutes. In some embodiments, the isothermal amplification is performed for a time period ranging from between about 5 minutes to about 50 minutes. In some embodiments, the isothermal amplification is performed for a time period ranging from between about 5 minutes to about 45 minutes. In some embodiments, the isothermal amplification is performed for a time period ranging from between about 5 minutes to about 40 minutes.BRIEF DESCRIPTION OF THE FIGURES

[0022] For a general understanding of the features of the disclosure, reference is made to the drawings. In the drawings, like reference numerals have been used throughout to identify identical elements.

[0023] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office, upon request and payment of the necessary fee.

[0024] FIG. 1 depicts a workflow a method of amplifying one or more target nucleic acid molecules in a sample in accordance with one embodiment of the present disclosure.

[0025] FIG. 2 illustrates the PCR amplification of a S. epidermidis isothermally amplified diluted sample. In particular, FIG. 2 depicts that amplification is achieved using all of the RPA diluted samples.

[0026] FIG. 3 illustrates the PCR amplification of S. epidermidis template. In particular, FIG. 3 illustrates a standard curve of S. epidermidis template from about IxlO7cp / pL to about IxlO4cp / pL.

[0027] FIG. 4 depicts a about 1: 1000 Dilution RPA product versus about IxlO6cp / pL PCR reaction. The figure illustrates that about 1 : 1000 RPA pre-amplified product produced a melt curve temperature equivalent to the PCR sample.

[0028] FIG. 5 illustrates comparable amplification curves for each dilution of heated and unheated RPA sample, indicating that heating the crude RPA sample for about 10 minutes at about 65 °C does not impact PCR amplification performance.

[0029] FIGS. 6 A and 6B demonstrate a tandem amplification workflow in accordance with the present disclosure. FIG. 6A provides qPCR amplification curves of diluted RPAAtorney Docket No. P39522-WO-1 reaction products (1: 10 and 1: 100 dilutions of 7.5 min RPA reactions that contained 100 copies of B. subtilis plasmid) produce fluorescence at lower cycles than the direct PCR control where 1E2 copies of B. subtilis plasmid was added to PCR reactions. FIG. 6B illustrates that tandem amplification in accordance with the methods of the present disclosure reduced Ct values by about 20 cycles compared to PCR alone with 100 copies of starting template, indicating that RPA pre-amplification is compatible with subsequent PCR amplification. qPCR reactions were run in triplicate with FAM-labeled TaqMan™ probes providing the fluorescence readout. Mean Ct values with standard deviation error bars are presented for Ct data.

[0030] FIGS. 7A and 7B demonstrate that a tandem amplification workflow in accordance with the present disclosure exhibits high sensitivity with robust amplicon generation in less time than PCR alone and higher specificity than RPA alone. FIG. 7A illustrates that robust amplicon generation in 12 - 30% shorter total amplification time is observed with 7.5 min RPA + 15 cycle and 20 cycle PCR, when compared to standard 40 cycle direct PCR. FIG. 7B illustrates that NTCs from 10 min RPA + 15 cycle PCR exhibit fewer non-specific bands compared to 10 min and 20 min RPA NTCs, demonstrating higher specificity of tandem amplification than RPA alone. Input template was 1E2 copies of B. subtilis plasmid for each direct PCR reaction and each RPA reaction (1 : 100 RPA dilutions for tandem). All PCR reactions were run in triplicate from the same RPA dilution. The images displayed above represent simulated gel images obtained by running the PCR samples on an Agilent Bioanalyzer with a DNA 1000 kit.

[0031] FIG. 8 illustrates that a tandem amplification workflow in accordance with the methods disclosed herein maintains high sensitivity in the presence of common nucleic acid amplification inhibitors that result in complete inhibition of PCR. No direct PCR conditions amplified in the presence of inhibitors, while all tandem amplification conditions amplified while maintaining Ct values lower than direct PCR without inhibitors. Input template was 100 copies of B. subtilis plasmid for each direct PCR reaction and each RPA reaction (1: 100 RPA dilutions for tandem). Inhibitor concentrations referenced were either input into the RPA reaction in tandem conditions or directly into the PCR reactions. The blood culture matrix condition consisted of human blood incubated with BD BACTEC™ culture media and spiked into reactions at 5% by volume. qPCR reactions were run in triplicate with FAM-labeled TaqMan™ probes providing the fluorescence readout. Mean Ct values with standard deviation error bars are presented for Ct data.

[0032] FIG. 9 illustrates that a tandem amplification workflow in accordance with the methods disclosed herein results in about a 20 - 25 cycle reduction in mean Ct values comparedAttorney Docket No. P39522-WO-1 to PCR alone for two targets in a three-plex system (B. subtilis, S. epidermidis, and an internal control that was not read out). The input template was either 1000 copies of B. subtilis plasmid or S. epidermidis plasmid for each direct PCR reaction and each RPA reaction (1: 100 RPA dilutions for tandem). All RPA reactions contained 1000 copies of the internal control ssDNA sequence. Each PCR condition was run in triplicate with primer pairs that correspond to all three targets yet only one target in each reaction was read out via FAM-labeled TaqMan™ probe. Mean Ct values with standard deviation error bars are presented for Ct data.DETAILED DESCRIPTION

[0033] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0034] As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "includes" is defined inclusively, such that "includes A or B" means including A, B, or A and B.

[0035] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0036] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of "comprising" and is therefore interpreted to be an open term meaning "at least the following," and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, "aAtorney Docket No. P39522-WO-1 device having components a, b, and c" means that the device includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

[0037] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0038] As used herein, the term "amplicon" refers to the product of a polynucleotide amplification reaction; that is, a clonal population of polynucleotides, which may be single stranded or double stranded, which are replicated from one or more starting sequences. The one or more starting sequences may be one or more copies of the same sequence, or they may be a mixture of different sequences. In some embodiments, amplicons are formed by the amplification of a single starting sequence. In some embodiments, amplicons may be produced by a variety of amplification reactions whose products comprise replicates of the one or more starting, or target, nucleic acids. In some embodiments, amplification reactions producing amplicons are "template-driven" in that base pairing of reactants, either nucleotides or oligonucleotides, have complements in a template polynucleotide that are required for the creation of reaction products.

[0039] As used herein "amplification" refers to a process in which a copy number increases. Amplification may be a process in which replication occurs repeatedly over time toAttorney Docket No. P39522-WO-1 form multiple copies of a template. Amplification can produce an exponential or linear increase in the number of copies as amplification proceeds. Exemplary amplification strategies include polymerase chain reaction and any isothermal amplification technique (e.g., loop- mediated isothermal amplification, rolling circle replication, cascade-RCA, nucleic acid-based amplification, and the like). Also, amplification can utilize a linear or circular template. Amplification can be performed under any suitable temperature conditions, such as with thermal cycling or isothermally. Furthermore, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if any, in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be performed by thermal cycling between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing / extension temperature, or among three or more temperature setpoints, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, among others. PCR can be performed with a thermostable polymerase, such as Taq DNA polymerase. PCR produces an exponential increase in the amount of a product amplicon over successive cycles. PCR is described, for example, in U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,000,159; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,176,995), the disclosures of each are hereby incorporated by reference herein in their entirety.

[0040] As used herein, the term "biological sample," "tissue sample," "specimen" or the like refers to any sample including a biomolecule (such as a protein, a peptide, a nucleic acid, a lipid, a carbohydrate, or a combination thereof) that is obtained from any organism including viruses. Other examples of organisms include mammals (such as humans; veterinary animals like cats, dogs, horses, cattle, and swine; and laboratory animals like mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies of tissue), cell samples (such as cytological smears such as Pap smears or blood smears or samples of cells obtained by microdissection), or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating their components by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (for example, obtained by a surgical biopsy or a needle biopsy), nipple aspirates, cerumen, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In certain embodiments, the term "biological sample" as used herein refersAttorney Docket No. P39522-WO-1 to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from a subject.

[0041] As used herein, the term "isothermal amplification reaction" means that the temperature does not significantly change during the reaction. In some embodiments, the temperature of the isothermal amplification reaction does not deviate by more than 10° C., preferably by not more than about 5°C, even more preferably not more than about 2°C.

[0042] As used herein, the terms "nucleic acid" or "nucleic acid molecule" as used herein, refer to a high-molecular-weight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The monomers from which nucleic acids are constructed are called nucleotides. Each nucleotide consists of three components: a nitrogenous heterocyclic base, either a purine or a pyrimidine (also known as a nucleobase); and a pentose sugar. Different nucleic acid types differ in the structure of the sugar in their nucleotides; DNA contains 2-deoxyribose while RNA contains ribose.

[0043] As used herein, the term "nucleotide" refers to a nucleoside-5 '-oligophosphate compound, or structural analog of a nucleoside-5 '-oligophosphate, which can act as a substrate or inhibitor of a nucleic acid polymerase. Exemplary nucleotides include, but are not limited to, nucleoside-5 '-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and dUTP); nucleosides (e.g., dA, dC, dG, dT, and dU) with 5 '-oligophosphate chains of 4 or more phosphates in length (e.g., 5'-tetraphosphosphate, 5'-pentaphosphosphate, 5'-hexaphosphosphate, 5'-heptaphosphosphate, 5'-octaphosphosphate); and structural analogs of nucleoside-5'-triphosphates that can have a modified base moiety (e.g., a substituted purine or pyrimidine base), a modified sugar moiety (e.g., an O-alkylated sugar), and / or a modified oligophosphate moiety (e.g., an oligophosphate comprising a thio-phosphate, a methylene, and / or other bridges between phosphates).

[0044] As used herein, the "polymerase" as used herein, refers to an enzyme that catalyzes the process of replication of nucleic acids. More specifically, DNA polymerase catalyzes the polymerization of deoxyribonucleotides alongside a DNA strand, which the DNA polymerase "reads" and uses as a template. The newly polymerized molecule is complementary to the template strand and identical to the template's partner strand.

[0045] As used herein, the term "primer" refers to an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence ofAttorney Docket No. P39522-WO-1 nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following reference that is incorporated by reference herein in its entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2ndEdition (Cold Spring Harbor Press, New York, 2003).

[0046] As used herein, the term "sample" refers to any sample that is suspected of containing a target agent to be detected. It is meant to include specimens or cultures (e.g., microbiological cultures), and biological and environmental specimens as well as non- biological specimens. Biological samples may comprise animal-derived materials, including fluid (e.g., blood, saliva, urine, lymph, etc.), solid (e.g., stool) or tissue (e.g., buccal, organspecific, skin, etc.), as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from, e.g., humans, any domestic or wild animals, plants, bacteria or other microorganisms, etc. Environmental samples can include environmental material such as surface matter, soil, water (e.g., contaminated water), air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items.

[0047] As used herein, the term "sequence," when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecules. In some cases, where different species of nucleotides are present in the nucleic acid molecule, the sequence includes an identification of the species of nucleotide (or base) at respective positions in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. The term can be used similarly to describe the order and positional identity of monomeric units in other polymers such as amino acid monomeric units of protein polymers.

[0048] As used herein, the term "sequencing" refers to the determination of the order and position of bases in a nucleic acid molecule. More particularly, the term "sequencing" refers to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNAAtorney Docket No. P39522-WO-1 sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modem automated DNA sequencing instruments.

[0049] OVERVIEW

[0050] The present disclosure provides for methods of amplifying one or more target nucleic acid molecules in a sample. In some embodiments, the amplifying of the one or more target nucleic acid molecules comprises performing a first amplification for a predetermined amount of time at a predetermined temperature to produce a sample including a first amplification product. In some embodiments, the first amplification comprises isothermal amplification. Subsequent to the first amplification, a second amplification is performed on the first amplification product, wherein the second amplification comprises a predetermined number of PCR cycles, e.g., less than about 50 PCR cycles, such as less than about 40 PCR cycles, such as less than about 30 PCR cycles. In some embodiments, the amplified one or more target nucleic acid molecules in the sample may be detected, such as using an electrochemical detection technique.

[0051] The present disclosure also provides for kits including (i) reagents for performing an isothermal amplification; and (ii) reagents for performing a predetermined number of PCR cycles. Additionally, the present disclosure also provides for a cartridge, such as a cartridge including (i) one or more compartments for performing isothermal amplification and PCR; (ii) reagents for performing an isothermal amplification; and (iii) reagents for performing a predetermined number of PCR cycles. In some embodiments, the kit further includes one or more probes to facilitate detection of the one or more target nucleic acid molecules following amplification. Any methods of detecting the one or more target nucleic acid molecules may be utilized but not limited to, electrochemical detection, fluorescence detection, luminescence detection, phosphorescence detection, colorimetric detection, radioactive detection, chemiluminescence detection, sample turbidity and surface plasmon waves (SPR), etc. In some embodiments, detection is multimodal, i.e., a combination of two or more different detection techniques.

[0052] In some embodiments, the method comprises one or more clean-up steps. In some embodiments, one or more clean-up steps may be performed after isothermal amplification and prior to PCR. In other embodiments, one or more clean-up steps may be performed after PCR. In yet other embodiments, one or more clean-up steps are performed both after isothermal amplification and after PCR.

[0053] These and other aspects of the disclosure are described further herein.Atorney Docket No. P39522-WO-1

[0054] METHODS OF AMPLIFYING ONE OR MORE NUCLEIC ACID MOLECULES IN A SAMPLE

[0055] One aspect of the present disclosure is directed to methods of amplifying one or more nucleic acid molecules. In particular, the methods of the present disclosure utilize a combination of (i) one or more isothermal amplification techniques, and (ii) polymerase chain reaction. It is believed that the combination of such amplification techniques permits high specificity and the ability to assay crude samples, while reducing the time-to-result as compared with using PCR alone. In some embodiments, the methods of the present disclosure comprise performing an isothermal amplification for a predetermined amount of time at a predetermined temperature; and subsequently performing a predetermined number of PCR cycles. In some embodiments, the isothermal amplification is LAMP. In other embodiments, the isothermal amplification is TMA. In yet other embodiments, the isothermal amplification is RCA. In yet other embodiments, the isothermal amplification is RPA. In some embodiments, the predetermined number of PCR cycles ranges from between about 5 cycles and about 50 cycles. In some embodiments, the predetermined number of PCR cycles ranges from between about 10 cycles and about 50 cycles. In some embodiments, the predetermined number of PCR cycles ranges from between about 10 cycles and about 45 cycles. In some embodiments, the predetermined number of PCR cycles ranges from between about 10 cycles and about 40 cycles. In some embodiments, the predetermined number of PCR cycles ranges from between about 10 cycles and about 35 cycles. In some embodiments, the predetermined number of PCR cycles ranges from between about 10 cycles and about 30 cycles.

[0056] It is believed that an isothermal amplification and PCR combination workflow (e.g., a tandem isothermal amplification - PCR workflow) permits high sensitivity and specificity while reducing the total time necessary to prepare an amplified sample to less than about 100 minutes, such as less than about 90 minutes, such as less than about 80 minutes, such as less than about 70 minutes, such as less than about 60 minutes, etc. It is also believed that the use of an isothermal amplification technique provides increased tolerance to inhibitors, such as certain concentrations of hemoglobin, heparin, mucin, urea, and / or other small molecule inhibitors. In addition, isothermal amplification techniques may be applied on crude samples, which may then be diluted prior to performing PCR.

[0057] Sample Preparation

[0058] In some embodiments, a sample comprising one or more nucleic acid molecules is obtained (step 101) and prepared for downstream amplification. In some embodiments, samples may be obtained from any source including a target nucleic acid molecule having oneAtorney Docket No. P39522-WO-1 or more modified nucleotides, e.g., tissue (including tumor tissue or formalin-fixed paraffin- embedded (FFPE) tissue), blood, skin, swab (e.g., buccal, vaginal), urine, saliva, etc. In some embodiments, the sample is a liquid biopsy sample.

[0059] In some embodiments, the sample is derived from a subject or a patient, such as a subject or a patient diagnosed with a disease or suspected of having a disease. In some embodiments, the sample is a blood sample. In some embodiments, samples include blood products, such as plasma, serum and the like.

[0060] In some embodiments, the sample may include a fragment of a solid tissue, or a tumor sample derived from the subject or the patient, e.g., by biopsy. As used herein, the term "tumor sample" encompasses samples prepared from a tumor or from a sample potentially including or suspected of comprising cancer cells, or to be tested for the potential presence of cancer cells, such as a lymph node. As used herein, the term "tumor" refers to a mass or a neoplasm, which itself is defined as an abnormal new growth of cells that usually grow more rapidly than normal cells and will continue to grow if not treated, sometimes resulting in damage to adjacent structures. Tumor sizes can vary widely. A tumor may be solid, or fluid filled. A tumor can refer to benign (not malignant, generally harmless), or malignant (capable of metastasis) growths. Some tumors can include neoplastic cells that are benign (such as carcinoma in situ) and, simultaneously, contain malignant cancer cells (such as adenocarcinoma). This should be understood to include neoplasms found in multiple locations throughout the body. Therefore, for purposes of the present disclosure, tumors include primary tumors, lymph nodes, lymphatic tissue, and metastatic tumors.

[0061] Methods for isolating nucleic acid molecules from obtained samples and / or purifying the obtained samples are known (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001) and several kits are commercially available (e.g., High Pure RNA Isolation Kit, High Pure Viral Nucleic Acid Kit, and MagNA Pure LC Total Nucleic Acid Isolation Kit, DNA Isolation Kit for Cells and Tissues, DNA Isolation Kit for Mammalian Blood, High Pure FFPET DNA Isolation Kit, available from Roche). In the context of the presently disclosed methods, nucleic acid molecules, including genomic DNA, can be collected, purified, and / or isolated.

[0062] It will be appreciated that nucleic acid molecules may be isolated from obtained samples using any of a variety of procedures known in the art, for example, MagMAX™ DNA Multi-Sample Ultra Kit (Applied Biosystems, Thermo Fisher Scientific), the MagMAX™ Express-96 Magnetic Particle Processor and the KingFisher™ Flex Magnetic ParticleAttorney Docket No. P39522-WO-1Processor (Thermo Fisher Scientific), a RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE and PureLink™ FFPE RNA Isolation Kit (Ambion™, Thermo Fisher Scientific), the ABI Prism™ 6100 Nucleic Acid Prep Station and the ABI Prism™ 6700 Automated Nucleic Acid Workstation (Applied Biosystems, Thermo Fisher Scientific), and the like.

[0063] In some embodiments, the nucleic acid molecules within the obtained sample are selected from DNA molecules, genomic DNA molecules, cfDNA molecules, cDNA molecules, RNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof. In some embodiments, the plurality of nucleic acid molecules comprises single stranded polynucleotides. In some embodiments, the nucleic acid molecules are cfDNA molecules. In some embodiments, the nucleic acid molecules are cfDNA molecules; and the obtained sample us a liquid biopsy sample.

[0064] In some embodiments, the nucleic acid molecules within the obtained sample may be prepared for downstream processing (e.g., the ligation of one or more universal adapters) by fragmenting, cutting, or shearing the nucleic acids. In some embodiments, the fragmenting, cutting, and / or shearing may be accomplished using such procedures as mechanical force, sonication, restriction endonuclease cleavage, or any method known in the art. In other embodiments, no fragmentation is necessary (some genomic samples may already consist of appropriately sized fragments and will not require additional fragmentation).

[0065] In some embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 10 mer to about 1000 mer. In some embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 10 mer to about 550 mer. In other embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 500 mer. In yet other embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 450 mer. In further embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 400 mer. In even further embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 350 mer. In yet even further embodiments, following fragmentation the nucleic acid molecules within any obtained sample have a size ranging from between about 15 mer to about 300 mer. In some embodiments, the nucleic acid molecules within the sample are fragmented to a platform-specific size range.Attorney Docket No. P39522-WO-1

[0066] Isothermal Amplification

[0067] Following sample preparation (step 101), the one or more nucleic acid molecules in the sample are isothermally amplified to provide a sample including one or more isothermally amplified nucleic acid molecules (step 102). Any method of isothermal amplification (now known or later discovered or optimized) may be utilized to isothermally amplify the one or more nucleic acid molecules in the sample. Non-limiting examples of isothermal amplification techniques include, but are not limited to, Rolling Circle Amplification (RCA), Recombinase Polymerase Amplification (RPA), Strand Displacement Amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP), and Hybridization Chain Reaction (HCR). Other examples of isothermal amplification include nucleic acid sequence -based amplification (NASBA), transcription mediated amplification (TMA), and helicase dependent amplification (HD A). A further example of isothermal amplification includes multiple displacement amplification (MCDA) or a "simple method for amplifying RNA targets" (SMART). Yet another example of isothermal amplification is cross-priming amplification (CPA). CPA is described in more detail in Fang et al. (Cross-Priming Amplification for Rapid Detection of Mycobacterium tuberculosis in Sputum Specimens, Journal of Clinical Microbiology, March 2009, p. 845-847 Vol. 47, No. 3) and Xu et al. (Cross Priming Amplification: Mechanism and Optimization for Isothermal DNA Amplification, Scientific Reports, February 2012, Vol. 2 No. 246), the disclosures of which are hereby incorporated by reference in their entirety.

[0068] It is believed that isothermal amplification methods provide for the detection of a nucleic acid target sequence in a streamlined, exponential manner, and are not limited by the constraint of thermal cycling. Indeed, isothermal amplification conditions do not include any thermocycling steps in the amplification process. Instead, the reaction is kept at a substantially constant temperature, such as a temperature that does not fluctuate more than about 5°C, such as more than about 4° C, such as more than about 3°C, such as more than about 2°C, etc.

[0069] In some embodiments, isothermal amplification may be conducted atpredetermined temperature ranging from between about 35°C to about 75°C, such aspredetermined temperature ranging from between about 35°C to about 65°C, such as a predetermined temperature ranging from between about 37°C to about 65°C, such aspredetermined temperature ranging from between about 37°C to about 60°C, such as a predetermined temperature ranging from between about 40°C to about 65°C, such aspredetermined temperature ranging from between about 40°C to about 60°C, etc. In some embodiments, the predetermined temperature at which to conduct the isothermal amplificationAttorney Docket No. P39522-WO-1 depends on the type of isothermal amplification conducted (e.g., LAMP may be conducted at a temperature of about 60°C to 65°C; TMA may be conducted at a temperature of about 35°C to 45°C, such as at about 42°C; RPA may be conducted at a temperature of about 37°C to 42°C, such as at a temperature of about 40°C; SDA may be conducted at a temperature of about 37°C to 60°C; RCA may be conducted at a temperature of about 35°C to 40°C, such as at a temperature of about 37°C; and NASBA may be conducted at a temperature of about 41 °C).

[0070] In some embodiments, the isothermal amplification may be conducted for a predetermined length of time (e.g., 5 minutes to 60 minutes), wherein the predetermined length of time may depend on the particular isothermal amplification technique employed. For instance, RCA may be conducted for a greater time period as compared with LAMP, TMA, or RPA. In some embodiments, the isothermal amplification performed in accordance with presently disclosed workflow is performed for a duration which is shorter than that typically used for the type of isothermal amplification employed. For instance, if RPA is traditionally performed for a duration of about 10 to about 20 minutes, RPA may be performed for a shorter duration when combined with PCR (e.g., for about 5 to about 10 minutes). By way of another example, if LAMP is traditionally performed for a length of time of between about 30 to about 60 minutes, LAMP may be performed for a shorter duration when combined with PCR (e.g., for about 20 minutes to about 40 minutes, such as from about 15 minutes to about 30 minutes). By way of yet a further example, if RCA is traditionally performed for a length of time ranging from about 1 hour to about 2 hours, then RCA may be performed for a shorter duration when combined with PCR (e.g., for about 30 minutes to about 90 minutes).

[0071] In some embodiments, an isothermal amplification process is conducted over a length of time within about 120 minutes or less. In some embodiments, an isothermal amplification process is conducted over a length of time within about 60 minutes or less. In some embodiments, an isothermal amplification process is conducted over a length of time within about 40 minutes or less. In some embodiments, an isothermal amplification process is conducted over a length of time within about 30 minutes or less. In some embodiments, an isothermal amplification process is conducted over a length of time within about 20 minutes or less. In some embodiments, an isothermal amplification process is conducted over a length of time within about 10 minutes or less. For example, an isothermal amplification process may be conducted within about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20Atorney Docket No. P39522-WO-1 minutes. By way of example, LAMP may be conducted for between about 20 minutes and about 60 minutes; TMA may be conducted for about 60 minutes; RCA may be conducted for between about 60 minutes and about 120 minutes; HAD may be conducted for between about 30 minutes and about 120 minutes; SDA may be conducted for between about 60 minutes and about 120 minutes; and RPA may be conducted for between about 5 minutes and about 20 minutes).

[0072] In some embodiments, the isothermal amplification reactions of the present invention will be conducted such that the one or more nucleic acid molecules in the sample are amplified at least about 100-fold, such as at least about 200-fold, such as at least about 300- fold, such as at least about 400-fold, such as at least about 500-fold, such as at least about 1000- fold, such as at least about 2000-fold, such as at least about 5000-fold, such as at least about 10,000-fold, such as at least about 50,000-fold, such as at least about 100,000-fold, such as at least about 200,000-fold, such as at least about 300,000-fold, such as at least about 400,000- fold, such as at least about 500,000-fold, such as at least about 600,000-fold, such as at least about 700,000-fold, such as at least about 800,000-fold, such as at least about 900,000-fold, or so, up to and including at least about 1,000,000-fold, or such as at least about 2,000,000-fold, etc. In some embodiments, an input amount of one or more nucleic acid molecules may range from between about less than 1 copy to about greater than 1000 copies; and the output amount of one or more nucleic acid molecules following isothermal amplification may range from between about 10 ng to about 10,000 ng, such as about from 50 ng to about 2000 ng.

[0073] Instead of melting DNA strands apart at high temperatures, it is believed that isothermal amplification takes advantage of DNA polymerases with strand displacement activity, (e.g., Sau, Msu, Bst or phi29 DNA polymerases). As used herein, the term "strand displacement" refers to the ability of the enzyme to separate the DNA strands in a doublestranded DNA molecule during primer-initiated synthesis. In some embodiments, the enzyme can be a complete enzyme or a biologically active fragment thereof. In some embodiments, the enzyme can be isolated and purified or recombinant. In some embodiments, the enzyme is thermostable. It is believed that such an enzyme is stable at elevated temperatures (e.g., greater than 40°C) and heat resistant to the extent that it effectively polymerizes DNA at the temperature employed.

[0074] Non-limiting exemplary DNA polymerases having strand displacement activity include Bst DNA polymerase, Sau DNA polymerase, Bst 2.0 DNA polymerase, Bst 2.0 WarmStart™ DNA polymerase (New England Biolabs, Ipswich, Mass.), Phi29 DNA polymerase, Bsu DNA polymerase, OmniAmp™ DNA polymerase (Lucigen, Middleton,Atorney Docket No. P39522-WO-1Mich.), VentR® and Deep VentR® DNA polymerases (New England Biolabs), Klenow fragment of DNA polymerase I, DNA polymerase, phage M2 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, and Pol6. In some embodiments, the DNA polymerase is Bst 2.0 WarmStart™ DNA polymerase (New England Biolabs, Ipswich, Mass.). It is believed that San and Bsu operate at low temperatures (e.g., about 37°C to about 42°C) and are strand displacing. Bst polymerase is another common isothermal amplification polymerase, but it is believed that it operates at a higher temperature (e.g., about 65°C). Other suitable polymerases are described in United States Patent Nos. 8,921,072 and 5,602,011; and in United States Patent Publication No. 2008 / 0305142, the disclosures of which are hereby incorporated by reference herein in their entireties.

[0075] In some embodiments, the isothermal amplification is multiplexed (e.g., 3-plex, 6-plex). For instance, in some embodiments, multiple sets of different primers (which may be linear or circular) are introduced during isothermal amplification, where each set of primers (which again may be linear or circular) is specific to a different target nucleic acid in the sample. For instance, isothermal amplification may utilize 2 or more different sets of primers, such as 4 or more different sets of primers, such as 6 or more different sets of primers, such as 8 or more different sets of primers, such as 10 or more different sets of primers, such as 12 or more different sets of primers, such as 16 or more different sets of primers, such as 20 or more different sets of primers, such as 24 or more different sets of primers, etc.

[0076] In some embodiments, a clean-up step is performed after isothermal amplification and prior to polymerase chain reaction. For example, in some embodiments, the sample including the one or more isothermally amplified nucleic acid molecules is "cleaned up" by following the QIAquick PCR Purification Kit Protocol. In some embodiments, the clean-up step comprises one of more of heating, centrifugation, detergent treatment (SDS, EDTA), different DNA polymerase, and enzymatic digestion (proteinase K).

[0077] Loop-Mediated Isothermal Amplification

[0078] Loop-mediated isothermal amplification (LAMP) is a method for amplifying DNA. The method is a single-step amplification reaction utilizing a DNA polymerase with strand displacement activity (e.g., Notomi et al., Nucl. Acids. Res. 28:E63, 2000; Nagamine et al., Mol. Cell. Probes 16:223-229, 2002; Mori et al., Biochem. Biophys. Methods 59: 145- 157, 2004). At least four primers, which are specific for eight regions within a target nucleic acid sequence, are typically used for LAMP. In some embodiments, the primers include a forward outer primer (F3), a backward outer primer (B3), a forward inner primer (FIP), and a backward inner primer (BIP). A forward loop primer (Loop F), and / or a backward loop primerAtorney Docket No. P39522-WO-1(Loop B) can also be included in some embodiments. The amplification reaction produces a stem-loop DNA with inverted repeats of the target nucleic acid sequence. In some embodiments, a strand-displacing DNA polymerase (e.g., Bst) initiates synthesis and two specially designed primers form "loop" structures to facilitate subsequent rounds of amplification through extension on the loops and additional annealing of primers.

[0079] In some embodiments, the sample is contacted with a suitable DNA polymerase having strand displacement activity, LAMP primers, dNTPs, and / or a reaction buffer. In some embodiments, the LAMP reaction is incubated at a predetermined temperature, such as at about 60°C to about 65°C. In some embodiments, LAMP is performed at a temperature of about 65°C. In some embodiments, LAMP is performed for a predetermined time ranging from between about 20 minutes to about 60 minutes. In some embodiments, LAMP is performed for a predetermined time period ranging from between about 20 minutes to 60 minutes; and at a predetermined temperature ranging from about 60°C to about 65°C. In other embodiments, LAMP is performed for a predetermined time period ranging from between about 20 minutes to 60 minutes; and at a predetermined temperature of about 65°C.

[0080] Recombinase Polymerase Amplification

[0081] RPA is a nucleic acid isothermal amplification technology developed by TwistDx Inc. in 2006. In RPA, a complex formed by recombinase combined with primers identifies homologous sequences in a template, which triggers a strand exchange reaction, initiates DNA synthesis after localization, and exponentially amplifies the target region of the template.

[0082] It is believed that pRPA technology can perform nucleic acid amplification rapidly at a predetermined constant temperature of about 25 °C to about 45 °C, such as about 30°C to about 42°C, such as about 35 °C to about 42°C, etc. In some embodiments, the predetermined temperature is about 37°C. In some embodiments, the predetermined temperature is about 38°C. In some embodiments, the predetermined temperature is about 39°C. In some embodiments, the predetermined temperature is about 40°C. In some embodiments, the predetermined temperature is about 41 °C. In some embodiments, the predetermined temperature is about 42°C.

[0083] In some embodiments, RPA is conducted for a predetermined time period ranging from between about 5 minutes to about 20 minutes. In some embodiments RPA is conducted for about 5 minutes, for about 6 minutes, for about 7 minutes, for about 8 minutes, 9 minutes, for about 10 minutes, for about 11 minutes, for about 12 minutes, for about 13Attorney Docket No. P39522-WO-1 minutes, for about 14 minutes, for about 15 minutes, for about 6 minutes, for about 17 minutes, for about 18 minutes, for about 9 minutes, for about 20 minutes, etc.

[0084] In some embodiments, RPA is conducted for a predetermined time period ranging from between about 5 minutes to about 20 minutes at a temperature ranging from about 37°C to about 42°C. In other embodiments, RPA is conducted for about 5 minutes, for about 6 minutes, for about 7 minutes, for about 8 minutes, 9 minutes, for about 10 minutes, for about 11 minutes, for about 12 minutes, for about 13 minutes, for about 14 minutes, for about 15 minutes, for about 6 minutes, for about 17 minutes, for about 18 minutes, for about 9 minutes, for about 20 minutes, etc. at a temperature ranging from about 37°C to about 42°C. In yet other embodiments, RPA is conducted for about 5 minutes, for about 6 minutes, for about 7 minutes, for about 8 minutes, 9 minutes, for about 10 minutes at a temperature ranging from about 37°C to about 42°C. In further embodiments, RPA is conducted for about 5 minutes, for about 6 minutes, for about 7 minutes, for about 8 minutes, 9 minutes, for about 10 minutes at a temperature of about 40°C.

[0085] RPA technology relies on three enzymes, namely a recombinase that can bind single-stranded nucleic acid molecules (oligonucleotide primer), a single-stranded DNA binding protein (SSB), and a strand displacing DNA polymerase. It is believed that single stranded binding proteins generally function to prevent premature annealing, protect the singlestranded DNA from being digested by nucleases, and / or remove secondary structure from DNA (e.g., destabilize helical duplexes) to enable action by other enzymes. One suitable single stranded binding protein is T4 gp32. In some embodiments, nucleic acid targets may be amplified without exposure to a single -strand DNA-binding protein.

[0086] As noted above, RPA utilizes a recombinase. Recombinases are enzymes involved in genetic recombination and sometimes are involved in nucleic acid repair (e.g., recombinational DNA repair). In some embodiments, recombinases can initiate strand exchange. Recombinases include, for example, Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX. In some embodiments, nucleic acid targets may be amplified without exposure to a recombinase accessory protein, such as, for example, a recombinase loading factor (e.g., T4 uvsY).

[0087] In some embodiments, the polymerase is a Sau DNA polymerase or a Bsu DNA polymerase. Bsu DNA polymerase has 5' to 3' DNA polymerase activity with its 5' to 3' exonuclease domain removed.Attorney Docket No. P39522-WO-1

[0088] In the first step, a recombinase binds to a primer and forms a recombinaseprimer complex. The formed recombinase-primer complex can search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequence, a strand exchange reaction occurs to form and initiate DNA synthesis. In particular, once the homologous sequence is found, the primers insert into the template chain to form a D-ring structure and start the chain replacement reaction. To prevent the inserted primer from being expelled through branch migration, the replaced template chain is bound to the single-stranded binding protein to maintain the stability of the single chain. Finally, the recombinase is isolated from the complex. In the presence of dNTPs, DNA polymerase is bound to the 3 '-OH end of the primer for chain elongation to form a new complementary chain.

[0089] In some embodiments, the sample including the one or more nucleic acid molecules to be amplified is contacted with a suitable DNA polymerase (Sau or Bsu), target specific primers (e.g., target specific primers having a length ranging from between about 30 nucleotides to about 35 nucleotides), dNTPs, a recombinase (e.g., RecA or UvsX), and a single stranded binding protein (e.g., T4 gp32). In other embodiments, the sample including the one or more nucleic acid molecules to be amplified is contacted with T4 UvsX protein, T4 UvsY protein, T4 gp32, Sau or Bsu, dNTPs, and forward and reverse primers.

[0090] Rolling Circle Amplification

[0091] In some embodiments, the isothermal amplification comprises Rolling Circle Amplification (RCA). Rolling circle amplification (RCA) is an isothermal process for generating multiple copies of a sequence. In rolling circle DNA replication in vivo, a DNA polymerase extends a primer on a circular template (Kornberg, A. and Baker, T. A. DNA Replication, W. H. Freeman, New Y ork, 1991). The product consists of tandemly linked copies of the complementary sequence of the template. RCA is a method that has been adapted for use in vitro for DNA amplification (Fire, A. and Si-Qun Xu, Proc. Natl. Acad. Sci. USA, 1995, 92:46414645; Lui, D., et al., Am. Chem. Soc., 1996,118: 1587-1594; Lizardi, P. M., et al., Nature Genetics, 1998, 19:225-232; U.S. Pat. No.5, 714, 320 to Kool). RCA can also be used in a detection method using a probe called a "padlock probe" (W O Pat. Ap. Pub. 95 / 22623 to Landegren; Nilsson, M., et al. Nature Genetics, 1997, 16:252-255, and Nilsson, M., and Landegren, U., in Landegren, U., ed., Laboratory Protocols for Mutation Detection, Oxford University Press, Oxford, 1996, pp. 135-138).

[0092] RCA operates at a predetermined temperature, e.g., from between about 25°C to about 60°C, such as from about 30°C to about 50°C, such as about 35 °C to about 40°C, for a predetermined about of time, e.g., between about 1 hour to about 2 hours. In someAtorney Docket No. P39522-WO-1 embodiments, RCA is performed at a predetermined temperature of about 37°C, such as for between about 1 hour and about 2 hours. In some embodiments, RCA is performed for less than 2 hours, such as less than 90 minutes.

[0093] Transcription-Mediated Amplification

[0094] Transcription mediated amplification (TMA) is an isothermal amplification method using RNA as a template, two primers, reverse transcriptase (RT), and RNA polymerase. Though primarily used for RNA detection, DNA can also be used as a starting material. In some embodiments, TMA is performed for a predetermined time period, such as less than about 60 minutes, such as less than about 50 minutes, such as less than about 40 minutes, etc. In some embodiments, TMA is performed for at least a predetermined temperature, such as at about 42°C.

[0095] One primer includes a T7 promoter sequence for the subsequent binding of RNA polymerase. This promoter binds to the RNA target and is extended via the DNA polymerase activity of reverse transcriptase (RT). An RNA-cDNA hybrid is generated through this process. The RNA strand of the RNA-cDNA hybrid is then digested with RNase H activity of the RT, leaving the newly created cDNA strand un-base paired. The second primer, non-T7 primer (i.e., the primer without the T7 promoter), binds to its complementary sequence on the cDNA, uses cDNA as a template, and extends by the DNA polymerase activity of the RT to synthesize another new DNA strand. This dsDNA contains the bacteriophage T7 RNA polymerase promoter sequence, which the bacteriophage T7 RNA polymerase can recognize. As a result, multiple copies of single -stranded RNA amplicons are transcribed. The RNA amplicon strands are opposite in polarity from the original RNA target and contain a region complementary to the probe used for amplicon detection. These RNA products reenter the cycle, forming more RNA-cDNA hybrids and then dsDNA that serve as templates for more RNA synthesis.

[0096] Polymerase Chain Reaction

[0097] Following the performing of the isothermal amplification for a predetermined amount of time at a predetermined temperature (step 102), a predetermined number of PCR cycles are conducted on the one or more isothermally amplified nucleic acid molecules in the sample (step 103). In some embodiments, less than 50 PCR cycles are conducted. In some embodiments, less than 40 PCR cycles are conducted. In other embodiments, less than 30 PCR cycles are conducted. In other embodiments, less than 25 PCR cycles are conducted. In yet other embodiments, less than 20 PCR cycles are conducted. In yet other embodiments, less than 15 PCR cycles are conducted. For instance, in some embodiments, 10 PCR cycles areAtorney Docket No. P39522-WO-1 conducted, such as 11 PCR cycles, such as 12 PCR cycles, such as 13 PCR cycles, such as 14 PCR cycles, such as 15 PCR cycles, such as 16 PCR cycles, such as 17 PCR cycles, such as 18PCR cycles, such as 19 PCR cycles, such as 20 PCR cycles, such as 21 PCR cycles, such as 22PCR cycles, such as 23 PCR cycles, such as 24 PCR cycles, such as 25 PCR cycles, such as 26PCR cycles, such as 27 PCR cycles, such as 28 PCR cycles, such as 29 PCR cycles, such as 30PCR cycles, such as 31 PCR cycles, such as 32 PCR cycles, such as 33 PCR cycles, such as 34PCR cycles, such as 35 PCR cycles, such as 36 PCR cycles, such as 37 PCR cycles, such as 38PCR cycles, such as 39 PCR cycles, such as 40 PCR cycles, such as 41 PCR cycles, such as 42PCR cycles, such as 43 PCR cycles, such as 44 PCR cycles, such as 45 PCR cycles, etc. In some embodiments, the number of PCR cycles depends on the type of isothermal amplification method utilized and / or the quantity of the one or more isothermally amplified nucleic acid molecules present in a sample following isothermal amplification.

[0098] In some embodiments, a pool of primers is introduced to the sample including the one or more isothermally amplified nucleic acid molecules, where the primers bind to and direct primer extension from a priming site. In some embodiments, the primers are different from those utilized during isothermal amplification. In some embodiments, the primers have a length ranging from between about 18 nucleotides to about 30 nucleotides. In some embodiments, the primers are universal primers.

[0099] In some embodiments, the PCR reaction is multiplexed. For instance, in some embodiments, multiple sets of different primers are introduced during PCR, where each set of primers is specific to a different target nucleic acid in the sample. For instance, PCR may utilize 2 or more different sets of primers, such as 4 or more different sets of primers, such as 6 or more different sets of primers, such as 8 or more different sets of primers, such as 10 or more different sets of primers, such as 12 or more different sets of primers, such as 16 or more different sets of primers, such as 20 or more different sets of primers, such as 24 or more different sets of primers, etc.

[0100] In some embodiments, amplification of the one or more isothermally amplified nucleic acid molecules is based on template directed oligonucleotide primer extension using one or more polymerases. Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerase, archaeal DNA polymerase, etc. In some embodiments, suitable polymerases may be derived from: archaea (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D 12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent,Atorney Docket No. P39522-WO-1GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811), Aeropyrum pemix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank: 029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictium occultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus sp. GE8 (GenBank: CAC12850), Thermococcus sp. JDF-3 (GenBank: AX135456; WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC 12849), Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23 (GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847), Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii (GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus (GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1), Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii (GenBank: Q58295), Desulforococcus species TOK, Desulforococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025) and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)), thermophilic bacteria Thermus species (e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus, Thermotoga maritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7 polymerases.

[0101] In some embodiments, to effectuate amplification, the one or more isothermally amplified nucleic acid molecules are heat denatured. Melting temperatures for heat denaturation are dependent upon several variables including the GC content of the nucleic acid molecule and / or the size of the nucleic acid molecule, but in general may be about 95 °C or higher, such as for about 15 seconds to about 2 minutes. Following heat denaturation, the primers are annealed to the primer binding sites of the one or more RPA amplified nucleic acid molecules at a lower temperature, (typically between about 40°C and about 60°C, such as for about 30 to about 60 seconds). The annealing temperature, like the heat denaturationAttorney Docket No. P39522-WO-1 temperature, is dependent upon the GC content and / or length of the primers. Subsequently, a corresponding nucleic acid strand to the template is synthesized from the primer through use of the polymerase and deoxynucleotide triphosphates (dNTPs) (also referred to as "primer extension"). In some embodiments, the temperature is raised for the polymerase, which in the case of commonly used thermostable polymerases is about 74° C, primer extension then lasts approximately 1 to 2 minutes. Reactions take place in a PCR master mixture which includes the one or more isothermally amplified nucleic acid molecules, a polymerase, primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium and / or optional additives.

[0102] Exemplary Methods of Amplifying One or More Nucleic Acid Molecules in a Sample

[0103] Tables 1 and 2 provides exemplary isothermal amplification and PCR conditions in accordance with the present disclosure.Table 1Attorney Docket No. P39522-WO-1Table 2

[0104] DETECTION OF THE ONE OR MORE AMPLIFIED NUCLEIC ACID MOLECULES

[0105] Another aspect of the present disclosure is directed to the detection of one or more amplified target nucleic acid molecules in a sample, where such target nucleic acid molecules are amplified in accordance with the methods described herein. In some embodiments the detection of the one or more target nucleic acid molecules is effectuated using any of one or more detection technique known in the art including, but not limited to, electrochemical detection, fluorescence detection, luminescence detection, phosphorescence detection, colorimetric detection, radioactive detection, chemiluminescence detection, etc. In some embodiments, the detection is multimodal.

[0106] Suitable electrochemical detection techniques are described in U.S. Patent Publication No. 2019 / 0185915; and in U.S. Patent Nos. 10,001,476, 10,670,591, 11,156,605, 10,495,656, 11,952,618, 10,864,522, 9,598,722, and 7,820,391, the disclosures of which are hereby incorporated by reference herein in their entireties. For instance, the electrochemical detection technique may utilize a separate signal probe or label probe having an electron transfer moiety (ETM). In some embodiments, the ETM is a metallocene. In some embodiments, the metallocene is a ferrocene. In some embodiments, the ferrocene is a ferrocene derivative. In some embodiments, the electrochemical detection technique determines the presence and / or quantity of a redox analyte through measurements of an electrical signal in a solution between a working electrode and a counter electrode, such asAtorney Docket No. P39522-WO-1 induced by a redox reaction or electrical potential from the release or absorption of ions. In some embodiments, the redox reactions take place at the working electrode, and which, for chemical detection, is typically constructed from an inert material such as platinum or carbon. The potential of the working electrode is measured against a reference electrode, which is typically a stable, well-behaved electrochemical half-cell such as silver / silver chloride. The electrochemical system can be used to support many different techniques for determining the presence and concentration of the target biomolecules including, but not limited to, various types of voltammetry, amperometry, potentiometry, coulometry, conductometry, and conductimetry such as AC voltammetry, differential pulse voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, anodic stripping voltammetry, cyclic voltammetry, and fast scan cyclic voltammetry. The electrochemical detection technique is further described in U.S. Pat. Nos. 9,557,295, 8,501,921, 6,600,026, 6,740,518, the disclosures of which are herein incorporated by reference in their entirety.

[0107] Another aspect of the present disclosure is directed to methods for identifying one or more amplified target nucleic acids in a sample. Yet another aspect of the present disclosure is directed to a method for identifying which of a plurality of amplified target nucleic acids are present within a sample. In some embodiments, a method of identifying one or more amplified target nucleic acids in a sample comprises providing a sample to a reaction vessel (such as a cartridge), providing isothermal amplification reagents (including, but not limited to, a polymerase, dNTPs, primers, etc.), and providing PCR regents (including, but not limited to primers, dNTPs, DNA polymerase, exonucleases, etc.). In some embodiments, the providing of the isothermal amplification reagents and the PCR reagents permits the amplification of one or more target nucleic acid molecules in a sample. In some embodiments, the providing of the isothermal amplification reagents and the PCR reagents permits the amplification of a locus from a different one of a plurality of target nucleic acid sequences in the sample. In some embodiments, the identification method comprises subjecting the sample to the amplification workflow disclosed herein, namely first subjecting the sample to an isothermal amplification reaction at a predetermined temperature for a predetermined amount of time, followed by the performance of a predetermined number of PCR cycles. In some embodiments, the identification method further comprises determining if the one or more target nucleic acids are hybridized to certain signal and / or capture probes (see, e.g., U.S. Patent Publication No. 2019 / 0185915, the disclosure of which is hereby incorporated by reference herein in its entirety).

[0108] KITSAttorney Docket No. P39522-WO-1

[0109] In some embodiments, the present disclosure provides a kit including components for amplifying one or more target nucleic acid molecules in a sample. In some embodiments, the kits include a first vessel or container including one or more components for performing an isothermal amplification reaction (e.g., one or more isothermal amplification reagents); and one or more components for performing PCR (e.g., one or more PCR reagents).

[0110] In some embodiments, the one or more isothermal amplification reagents include a polymerase such as, but not limited to, Sau, Bsu, Bst, a Taq DNA polymerase, a Klenow fragment of DNA polymerase I, PhiPRDl DNA polymerase, phage M2 DNA polymerase, T4 DNA polymerase, Pol6, and T5 DNA polymerase. In some embodiments, the one or more isothermal amplification reagents further includes a recombinase and / or a single stranded binding protein. In other embodiments, the one or more isothermal amplification reagents further include a helicase.[oni] In some embodiments, the one or more PCR reagents include a polymerase. In some embodiments, the polymerase is selected from those described herein. One example of a polymerase is a Taq or Taq-derived polymerase (e.g., KAPA 2G polymerase from KAPA BIOSYSTEMS). Another example polymerase is a B-family DNA polymerase (e.g., KAPA HIFI polymerase from KAPA BIOSYSTEMS).

[0112] In some embodiments, the PCR reagents include nucleotides. In some embodiments, the PCR reagents include deoxynucleoside triphosphates (dNTPs), such as all the four naturally occurring deoxynucleoside triphosphates (dNTPs). In some embodiments, the PCR reagents include deoxyribonucleoside triphosphate molecules, including all dATP, dCTP, dGTP, dTTP. In some embodiments, the PCR reagents also include compounds useful in aiding the activity of the nucleic acid polymerase. For example, in some embodiments, the PCR reagent includes a divalent cation, e.g., magnesium ions. In some embodiments, the magnesium ions are provided in the form of magnesium chloride, magnesium acetate, or magnesium sulfate. In some embodiments, the PCR reagents further include a buffer or buffer solution. In some embodiments, each of the PCR reagents are provided alone. In other embodiments, each of the PCR reagents are provided in admixture.

[0113] In some embodiments, the kit further includes a reaction cartridge. Suitable reaction cartridges are described in U.S. Patent No. 10,005,080, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0114] CARTRIDGES

[0115] The present disclosure is also directed to a cartridge including one or more wells and / or fluid channels. In some embodiments, the cartridge includes a first well for performingAtorney Docket No. P39522-WO-1 isothermal amplification; and a second well for performing one or more PCR cycles. In some embodiments, the cartridge may further include one or more inlet portions, one or more outlet ports, one or valves, one or more mixing mechanisms, and other components for receiving, transporting, intermingling, mixing, and performing other processes on a sample introduced into the one or more wells of the cartridge. In some embodiments, the cartridge may further include one or more chambers, each including one or more reagents, fluids, signal probes, and / or capture probes, and one or more detection zones (such as one or more detection zones which each include one or more arrays of detection electrodes). For instance, the one or more chambers may include one or more isothermal amplification reagents and / or one or more PCR reagents. In some embodiments, the one or more chambers are fluidically coupled to the one or more wells through the one or more fluid channels. Suitable cartridges are described in U.S. Patent No. 10,005,080, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0116] EXAMPLES

[0117] Example 1 : PCR Amplification with Diluted RPA Products

[0118] PCR amplification with diluted RPA products was tested to evaluate the compatibility between the two amplification techniques. An outer RPA reaction with an outer primer set was run using IxlO3cp / pL of a plasmid that contains a S. epidermidis insert. The following dilutions were then performed on the S. epidermidis outer RPA crude product: about 1: 10, about 1: 100, and about 1: 1000. All dilutions were prepared in a reconstitution solution (ultrapure water with 0.2% Tween-20 and 0.05% sodium azide) to mimic and resemble an on- cartridge dilution step. The diluted RPA product was then used as template for a 30-cycle qPCR reaction (about 30 cycles of about 95°C denaturation for about 4 seconds followed by about 60°C annealing / extension for 20 seconds). Control qPCR reactions consisted of a negative control that used nuclease free water as template, as well as a positive control that used about IxlO3copies (cp) / pL of the aforementioned S. epidermidis plasmid. After the amplification cycles were completed, a melt curve analysis was performed on the about 1 : 1000 diluted RPA product and about IxlO6cp / pL template conditions to assess if the PCR reaction produced the correct target amplicon. FIG. 2 illustrates that amplification is achieved using all of the RPA diluted samples. Early amplification occurred in the about 1: 10 RPA diluted sample. No contamination is present in the qPCR mastermix as indicated by no amplification in the NTCs. FIG. 3 illustrates a standard curve of S. epidermidis template from about IxlO7cp / pL to about IxlO4cp / pL. Dropouts begin to occur for template concentration of about IxlO4cp / pL. FIG. 4 illustrates that about 1: 1000 RPA pre-amplified product produces a melt curveAttorney Docket No. P39522-WO-1 temperature equivalent to the PCR sample. RPA-PCR coupled amplification is achieved with all the dilutions of the crude RPA sample that were tested. The melt curve analysis shows the PCR sample, and the RPA product have equivalent melting temperatures. The qPCR results show compatibility between RPA-PCR amplification system.

[0119] Example 2: Effect on PCR Amplification Performance of Heating and Diluting RPA ProductsIt is believed that heating of the crude RPA products prior to PCR amplification has the potential to make the DNA more accessible for PCR amplification. Therefore, a heating step prior to PCR amplification was evaluated by heating the S. epidermidis outer RPA crude sample (same as in section 0106) prior to dilution. The following two conditions were evaluated: heating (H) and no heating (NH). The heating and no heating conditions were diluted in the reconstitution solution at the following ratios: about 1: 10, about 1: 100, about 1 :500, about 1 : 1000, about 1 : 1500 and about 1 :2000. In the H condition, the RPA sample was heated at about 65°C for about 10 minutes. A standard about 30-cycle qPCR (same cycling parameters as noted above) was performed on the heated and unheated RPA samples. FIG. 5 illustrates comparable amplification curves for each dilution of heated and unheated RPA sample, indicating that heating the crude RPA sample for about 10 minutes at about 65°C does not impact PCR amplification performance.

[0120] Example 3

[0121] Tandem amplification with a low starting template concentration of IxlO2copies (cp) / rxn was evaluated to demonstrate proof-of-principle. An outer RPA reaction with an outer primer set containing IxlO2cp / rxn of a plasmid that contains a B. subtilis insert was performed for 7.5 min. The following dilutions were then performed on the B. subtilis outer RPA crude product: about 1: 10, and about 1: 100. All dilutions were prepared in TE buffer (Tris + EDTA). The diluted RPA-generated amplicons were then included as template in 40- cycle qPCR reactions (about 40 cycles of about 96°C denaturation for about 4 seconds followed by about 60°C annealing / extension for 30 seconds). Control qPCR reactions consisted of a negative control that contained TE buffer as template, as well as a direct PCR positive control that included about IxlO2cp / rxn of the aforementioned B. subtilis plasmid. qPCR reactions were run in triplicate with FAM-labeled TaqMan™ probes providing the fluorescence readout. FIGS. 6A and 6B illustrate that RPA products do not inhibit subsequent PCR reactions, demonstrating the feasibility of a tandem isothermal and PCR reaction, and demonstrating that tandem amplification is feasible irrespective of biological target.

[0122] Example 4Attorney Docket No. P39522-WO-1

[0123] To demonstrate robust amplicon generation and high specificity of tandem amplification, an endpoint readout of tandem amplification was compared to direct PCR amplification. RPA reactions containing IxlO2cp / rxn of the aforementioned B. subtilis plasmid were performed for 7.5 minutes. RPA reactions were then diluted by a factor of about 1 : 100 in TE buffer. The diluted RPA-generated amplicons were then included as template for 15 cycle and 20 cycle PCR reactions (cycles consisting of about 96°C denaturation for about 4 seconds followed by about 60°C annealing / extension for 30 seconds). Negative controls consisted of running RPA reactions with TE buffer as template (RPA NTCs) followed by a dilution of about 1: 100 in TE buffer and subsequent PCR amplification for 15 cycles. Direct PCR controls consisted of 40-cycle PCR reactions (same cycling protocol mentioned above) that included about IxlO2cp / rxn of the aforementioned B. subtilis plasmid. Specificity comparison consisted of comparing RPA NTCs that were run for 10 min to tandem amplification NTCs (10 min RPA + 15 cycle PCR). All PCR and RPA products were analyzed and quantified on an Agilent 2100 Bioanalyzer system with a DNA 1000 Kit. FIGS. 7A and 7B illustrate comparable amplicon generation of tandem amplification in about 3 to about 7- minute shorter total amplification time than a standard 40-cycle PCR, and higher specificity of tandem amplification compared to RPA alone.

[0124] Example 5

[0125] Tandem amplification in the presence of common PCR inhibitors was evaluated to demonstrate improved tolerance to inhibitors compared to PCR alone. RPA reactions containing IxlO2cp / rxn of the B. subtilis plasmid were performed for 10 minutes. RPA reactions were then diluted by a factor of about 1 : 100 in TE buffer. The diluted RPA-generated amplicons were included as template in 40-cycle qPCR reactions (about 40 cycles of about 96°C denaturation for about 4 seconds followed by about 60°C annealing / extension for 30 seconds). Inhibitors evaluated included heparin at a concentration of 10 units / mL and hemoglobin at a concentration of 2 mg / mL, and negative blood culture matrix (human blood incubated with BD BACTEC™ culture media) at a concentration of 5% of the final reaction volume. The inhibitors were included at the aforementioned concentrations in the RPA reactions and, for comparison, in direct qPCR reactions containing IxlO2cp / rxn of the aforementioned B. subtilis plasmid. Negative controls consisted of qPCR reactions that were run with diluted RPA NTCs (TE buffer added in place of template). These RPA NTCs were run in the presence of the aforementioned inhibitors at the aforementioned concentrations for 10 min. Positive controls consisted of running direct qPCR reactions with IxlO2cp / rxn of the aforementioned B. subtilis plasmid in the absence of any added inhibitors and qPCR reactionsAttorney Docket No. P39522-WO-1 with the products of RPA reactions that did not contain any added inhibitors. qPCR reactions were run in triplicate with FAM-labeled TaqMan™ probes providing the fluorescence readout. FIG. 8 illustrates that direct PCR is completely inhibited by the concentrations of added inhibitors, while minimal inhibition is observed in tandem amplification.

[0126] Example 6

[0127] Tandem amplification was evaluated in 3-plex to demonstrate that it can be used for multiplexing. Multiplex primer pools consisting of RPA outer primers targeting B. subtilis, S. epidermidis, and an internal control sequence were prepared and included in the RPA reactions along with the internal control template at a concentration of around IxlO3cp / rxn. The RPA reactions containing IxlO3cp / rxn of either the B. subtilis plasmid or the S. epidermidis plasmid were run for 10 minutes. RPA reactions were then diluted by a factor of about 1: 100 in TE buffer. The diluted RPA-generated amplicons were included as template in 40-cycle multiplex qPCR reactions (about 40 cycles of about 96°C denaturation for about 4 seconds followed by about 60°C annealing / extension for 30 seconds). The multiplex qPCR reactions included primers targeting B. subtilis, S. epidermidis, and the internal control sequence. Positive controls consisted of direct multiplex qPCR reactions containing IxlO3cp / rxn of either the B. subtilis plasmid or the S. epidermidis plasmid. Negative controls consisted of qPCR reactions that were run with diluted RPA NTCs (TE buffer added in place of template). These RPA NTCs, which contained all three of the aforementioned primer pairs and the internal control template, were run for 10 minutes. Multiplex qPCR conditions were run in triplicate with one FAM-labeled TaqMan™ probe sequence providing the fluorescence readout for one target per condition. FIG. 9 illustrates the feasibility of tandem amplification in multiplex.

[0128] Although the present disclosure has been described with reference to several illustrative embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More particularly, reasonable variations and modifications are possible in the component parts and / or arrangements of the subject combination arrangement within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the disclosure. In addition to variations and modifications in the component parts and / or arrangements, alternative uses will also be apparent to those skilled in the art.

Claims

Attorney Docket No. P39522-WO-1CLAIMS1. A method of amplifying one or more nucleic acid molecules in a sample, the method comprising: (a) performing an isothermal amplification on the sample including the one or more nucleic acid molecules for a predetermined amount of time and at a predetermined temperature, wherein the isothermal amplification provides a sample including one or more isothermally amplified nucleic acid molecules; and (b) performing a polymerase chain reaction on the sample including the one or more isothermally amplified nucleic acid molecules to provide a sample including the one or more amplified nucleic acid molecules, wherein less than 30 cycles of the polymerase chain reaction are performed.

2. The method of claim 1, wherein the isothermal amplification comprises loop-mediated isothermal amplification.

3. The method of claim 2, wherein the predetermined temperature is between about 60°C to about 65°C.

4. The method of any one of claims 2-3, wherein the predetermined time ranges from between about 20 minutes to about 60 minutes.

5. The method of claim 3, wherein the predetermined time ranges from between about 20 minutes to about 40 minutes.

6. The method of claim 1, wherein the isothermal amplification comprises recombinase polymerase amplification.

7. The method of claim 6, wherein the predetermined temperature is between about 37°C to about 42°C.

8. The method of claim 6, wherein the predetermined temperature is about 40°C.

9. The method of any one of claims 6-8, wherein the predetermined time ranges from between about 5 minutes to about 10 minutes.

10. The method of any one of claims 6-8, wherein the predetermined time is about 7 minutes.

11. The method of any one of claims 6-10, wherein the recombinase polymerase amplification comprises contacting the sample including the one or more nucleic acid molecules with a DNA polymerase having strand displacement activity.

12. The method of claim 11, further comprising contacting the sample with a single stranded binding protein and a recombinase.

13. The method of claim 12, wherein the single stranded binding protein is T4 gp32.Atorney Docket No. P39522-WO-114. The method of claim 12, wherein the recombinase is selected from the group consisting of Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX.

15. The method of claim 1, wherein the isothermal amplification comprises rolling circle amplification.

16. The method of claim 15, wherein the predetermined temperature is between about 35°C to about 40°C.

17. The method of claim 15, wherein the predetermined temperature is about 37°C.

18. The method of any one of claims 15-17, wherein the predetermined time ranges from between about 60 minutes to about 120 minutes.

19. The method of any one of claims 15-17, wherein the predetermined time is about 60 minutes to about 90 minutes.

20. The method of claim 1, wherein the isothermal amplification comprises transcription- mediated amplification.

21. The method of claim 20, wherein the predetermined temperature is between about 35 °C to about 45°C.

22. The method of claim 20, wherein the predetermined temperature is about 42°C.

23. The method of any one of claims 20-22, wherein the predetermined time is less than about 60 minutes.

24. The method of any one of claims 1-23, wherein less than 25 PCR cycles are performed.

25. The method of any one of the preceding claims, further comprising detecting the one or more amplified nucleic acid molecules.

26. The method of claim 25, wherein the detection comprises one or more of an electrochemical detection technique, a fluorescent detection technique, or a colorimetric detection technique.

27. The method of any one of the preceding claims, wherein the sample is a bodily fluid or tissue-derived sample.

28. A method of amplifying a plurality of target nucleic acid molecules in a sample, wherein the sample comprises the plurality of target nucleic acid molecules, and a plurality of non-target nucleic acid molecules, the method comprising: (a) performing an isothermal amplification on the sample for a predetermined amount of time and at a predetermined temperature, wherein the isothermal amplification is performed using a plurality of different target specific primers for the isothermal amplification, and wherein the isothermal amplification provides a plurality of isothermally amplified target nucleic acid molecules; andAttorney Docket No. P39522-WO-1(b) performing a polymerase chain reaction on a sample including the plurality of isothermally amplified target nucleic acid molecules to provide a sample including a plurality of amplified target nucleic acid molecules, wherein less than 30 cycles of the polymerase chain reaction are performed, and wherein the polymerase chain reaction is performed with a plurality of different target specific PCR primers.

29. The method of claim 28, wherein the sample is derived from a bodily fluid.

30. The method of any one of claims 28-29, wherein the plurality of target nucleic acid molecules is derived from bacteria.

31. The method of any one of claims 28-29, wherein the plurality of target nucleic acid molecules is derived from viruses.

32. The method of any one of claims 28-29, wherein the plurality of target nucleic acid molecules is derived from fungi.

33. The method of any one of claims 28-29, wherein the plurality of target nucleic acid molecules is derived from a gram-positive microorganism.

34. The method of any one of claims 28-29, wherein the plurality of target nucleic acid molecules is derived from a gram-negative microorganism.

35. The method of claim 28, wherein the sample is derived from a tumor.

36. The method of claim 28, wherein the sample is derived from a subject diagnosed with cancer.

37. The method of claim 28, wherein the sample is derived from a subject suspected of having cancer.

38. The method of claim 28, wherein the sample is derived from a subject diagnosed with a neurodegenerative disease.

39. The method of claim 28, wherein the sample is derived from a subject suspected of having a neurodegenerative disease.

40. The method of any one of claims 28-39, wherein the isothermal amplification comprises recombinase polymerase amplification.

41. The method of claim 40, wherein the predetermined temperature is between about 37°C to about 42°C.

42. The method of claim 40, wherein the predetermined temperature is about 40°C.

43. The method of any one of claims 40-41, wherein the predetermined time ranges from between about 5 minutes to about 10 minutes.

44. The method of any one of claims 40-43, wherein the predetermined time is about 7 minutes.Atorney Docket No. P39522-WO-145. The method of any one of claims 40-41, wherein the recombinase polymerase amplification comprises contacting the sample including the one or more nucleic acid molecules with a DNA polymerase having strand displacement activity.

46. The method of claim 45, further comprising contacting the sample with a single stranded binding protein and a recombinase.

47. The method of claim 46, wherein the single stranded binding protein is T4 gp32.

48. The method of claim 46, wherein the recombinase is selected from the group consisting of Cre recombinase, Hin recombinase, Tre recombinase, FLP recombinase, RecA, RAD51, RadA, and T4 uvsX.

49. The method of any one of claims 28-48, further comprising detecting the one or more amplified nucleic acid molecules with one or more of an electrochemical detection technique, a fluorescent detection technique, and / or a colorimetric detection technique.

50. A cartridge comprising one or more reaction wells in communication with (a) a first chamber including one or more isothermal amplification reagents; and (b) a second chamber including one or more PCR reagents.

51. The cartridge of claim 50, wherein the one or more isothermal amplification reagents comprise a polymerase, a recombinase, and a single stranded binding protein.

52. The cartridge of claim 51, wherein the polymerase is Sau DNA polymerase or Bsu DNA polymerase.

53. The cartridge of claim 51, wherein the recombinase is selected from the group consisting of Cre recombinase, Hin recombinase, Tre recombinase.

54. The cartridge of claim 51, wherein the single stranded binding protein is T4 gp32.

55. The cartridge of claim 50, wherein the PCR reagents include a DNA polymerase and dNTPs.

56. The cartridge of claim 50, further comprising one or more additional chambers including one or more signal probes and one or more capture probes.

57. A method of amplifying one or more nucleic acid molecules in a sample, the method comprising: (a) performing an isothermal amplification on the sample including the one or more nucleic acid molecules for a predetermined amount of time and at a predetermined temperature, wherein the isothermal amplification provides a sample including one or more isothermally amplified nucleic acid molecules; and (b) performing a polymerase chain reaction on the sample including the one or more isothermally amplified nucleic acid molecules to provide a sample including the one or more amplified nucleic acid molecules, wherein less than 30 cycles of the polymerase chain reaction are performed.Atorney Docket No. P39522-WO-158. The method of claim 57, further comprising detecting the one or more amplified nucleic acid molecules.

59. The method of claim 58, wherein the detection comprises one or more of an electrochemical detection technique, a fluorescent detection technique, or a colorimetric detection technique.

60. The method of claim 57, wherein the sample is a bodily fluid or tissue-derived sample.

61. The method of claim 57, wherein the sample is derived from a tumor.

62. The method of claim 57, wherein the sample is derived from a subject diagnosed with cancer.

63. The method of claim 57, wherein the sample is derived from a subject suspected of having cancer.

64. The method of claim 57, wherein the sample is derived from a subject diagnosed with a neurodegenerative disease.

65. The method of claim 57, wherein the sample is derived from a subject suspected of having a neurodegenerative disease.

66. The method of any one of claims 57-65, wherein the isothermal amplification comprises contacting the sample with a DNA polymerase having strand displacement activity.

67. The method of any one of claims 57-66, wherein the isothermal amplification is performed at a temperature ranging from between about 35°C to about 75°C.

68. The method of any one of claims 57-66, wherein the isothermal amplification is performed at a temperature ranging from between about 35 °C to about 65 °C.

69. The method of any one of claims 57-66, wherein the isothermal amplification is performed at a temperature ranging from between about 37°C to about 60°C.

70. The method of any one of claims 57-66, wherein the isothermal amplification is performed for a time period ranging from between about 5 minutes to about 60 minutes.

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