Patent Application
20250092448
The methods and compositions described herein relate generally to the area of nucleic acid amplification and detection. In particular, described herein are methods and compositions for increasing amplification efficiency.
A wide variety of nucleic acid amplification methods are available, and many have been employed in the implementation of sensitive diagnostic assays based on nucleic acid detection.
Polymerase chain reaction (PCR) remains the most widely used DNA amplification and quantitation method. However, PCR in general has several limitations. PCR amplification can only achieve less than two-fold increase of the amount of target sequence at each cycle. It is still relatively slow, and the sensitivity of PCR is limited. Similarly, isothermal amplification methods rely on the extension of DNA primers. Once extended, the primer is not further used to initiate another round of extension.
Primers are widely used in the various methods for nucleic acid amplification. They are generally DNA oligonucleotide designed to hybridize to target nucleic acid and extended by a polymerase. Once a primer is extended, it is incorporated into the extended amplicon and no longer used for amplification. The single-use of the standard primers place a cap on the efficiency of PCR and limits the rate of nucleic acid amplification and detection.
PCR primers for use in PCR amplification are typically all deoxynucleotides because DNA polymerases used in PCR typically do not use ribonucleotide as template. Even some polymerases are potentially capable of copying ribonucleotides (e.g., reverse transcriptase activity), the commonly held belief that substitution of a ribonucleotide for a deoxynucleotide typically increases primer cost, reduces primer stability and PCR efficiency leads to the avoidance of ribonucleotides in primers.
Efforts in increasing nucleic acid amplification efficiency have been focused on improving primer designs, polymerase functions, reaction conditions. For example, U.S. Pat. No. 8,252,558 teaches a form of nested PCR. US Patent Publication 2019/0203266A1 discloses methods to increase PCR efficiency through primer design and use of strand displacement DNA polymerase.
Described herein are methods, compositions and systems based on the use of primers that contain at least one ribonucleotide. They are designed to initiate a chain extension based on a template more than once in the presence of enzymes such as RNase H2 and polymerases.
In an aspect of the present disclosure, a nucleic acid primer set is provided. The primer set includes a first primer having a 3′ end and a 5′ end for amplifying a target nucleic acid in a sample, wherein the target nucleic acid includes a first template strand. The first primer comprises a first segment of DNA oligonucleotide and a second segment of DNA oligonucleotide, the first segment being at 5′ side of the second segment, the first segment and the second segment being linked by at least one first ribonucleotide. At least a portion of the second segment is capable of specifically hybridizing to the first template strand to initiate a nucleotide polymerization to produce an extension product complementary to the first template strand.
In some embodiments, the at least one first ribonucleotide of the first primer includes two or more ribonucleotides connected consecutively. In some embodiments, the at least one first ribonucleotide of the first primer includes two or more ribonucleotides that are spaced apart. In some embodiments, the at least one first ribonucleotide of the first primer consists of a single ribonucleotide.
In some embodiments, both the first segment and the second segment of the first primer specifically hybridize to the first template strand at a given condition. In some embodiments, the first segment does not specifically hybridize to the first template strand while the second segment specifically hybridizes to the first template strand. In some embodiments, only a portion of the second segment (and not the whole second segment) specifically hybridizes to the first template strand.
In some embodiments, the target nucleic acid further includes a second template strand complementary to the first template strand, and the primer set further comprises a second primer capable of specifically hybridizing to the second template strand. In certain embodiments, the second primer has a 3′ end and a 5′ end and comprises a third segment of DNA oligonucleotide and a fourth segment of DNA oligonucleotide, the third segment being at 5′ side of the fourth segment, the third segment and the fourth segment being linked by at least one second ribonucleotide, at least the fourth segment is capable of specifically hybridizing to the second template strand.
In another aspect of the present disclosure, a method for amplifying a target nucleic acid in a sample is provided, wherein the target nucleic acid includes a first template strand. The method includes: (a) contacting the sample with a first primer having a 3′ end and a 5′ end and comprising a first oligonucleotide segment and a second oligonucleotide segment, the first oligonucleotide segment being at 5′ side of and linked with the second oligonucleotide segment by at least one first ribonucleotide, under conditions where at least a portion of the second segment anneals to the first template strand; (b) producing a first amplicon complementary to the first template strand by extending the first primer using the first template strand as the template; and (c) cleaving the first primer at the position immediately 5′ to the at least one first ribonucleotide. The cleaving can be performed by using RNase H2.
In some embodiments, the first segment anneals to the first template strand under the conditions, the method further comprising producing a second amplicon complementary to the first template strand by extending the first segment of the first primer using the first template strand as a template while replacing the first amplicon from the first template strand.
In some embodiments, wherein the first segment of the first primer does not anneal to the first template strand under the conditions, the method further comprising: prior to cleaving at (c): separating the first amplicon from the first template strand; using a second primer to produce a nucleic acid strand complementary to the first amplicon, the new nucleic acid strand including a segment complementary to the first segment of the first primer; and after cleaving at (c): producing a third amplicon complementary to the new nucleic acid strand by extending the first segment of the first primer using the new nucleic acid strand as a template, while replacing the first amplicon from the new nucleic acid strand. In some of these embodiments, wherein at least a portion to 3′ side of the at least one first ribonucleotide and distal to the 3′ end of the first primer does not specifically hybridize to the first template strand.
In some embodiments, the target nucleic acid further includes a second template strand complementary to the first template strand, the method further comprises: (d) contacting the sample with a second primer comprising a third segment of DNA oligonucleotides and a fourth segment of DNA oligonucleotides, the third segment being at the 5′ side of and linked with the fourth segment by at least one second ribonucleotide, where at least a portion of the fourth segment anneals to the second template strand under the conditions; (e) producing a second amplicon complementary to the second template strand by extending the second primer using the second template strand as the template; and (f) cleaving the second primer at the position immediately 5′ to the at least one second ribonucleotide.
In some embodiments, the first segment and second segment both anneal to the first template strand under the conditions, the target nucleic acid further includes a second template strand complementary to the first template strand, the method further comprises: (d) contacting the sample with a second primer comprising a third segment of DNA oligonucleotides and a fourth segment of DNA oligonucleotides, the third segment being at the 5′ side of and linked with the fourth segment by at least one second ribonucleotide, where both the third segment and fourth segment anneal to the second template strand under the conditions; (e) producing a second amplicon complementary to the second template strand by extending the second primer using the second template strand as the template; (f) cleaving the second primer at the position immediately 5′ to the at least one second ribonucleotide; and (g) producing a third amplicon complementary to the first template strand by extending the first segment of the first primer using the first template strand as a template while replacing the first amplicon from the first template strand, and producing a fourth amplicon complementary to the second template strand by extending the second primer using the second template strand as the template while replacing the third amplicon from the second template strand. In some of these embodiments, the method can further comprise: (h) producing a fifth amplicon complementary to the first amplicon using another second primer; and producing a sixth amplicon complementary to the second amplicon using another first primer. Thermocycling can be performed to further amplify the amplification products obtained in the process, e.g., increasing temperature to separate double-stranded amplicons produced; and decreasing temperature to anneal further first primers and second primers to the separated amplicons.
In some embodiments of the methods described herein, the first primer is anchored on a solid surface or in a matrix.
In some embodiments of the methods described herein, wherein the first template strand is an RNA.
In some embodiments, the first segment cleaved from the first primer is used as a primer to initiate nucleotide polymerization using a second target nucleic acid as a template.
In a further aspect, method of detecting a nucleotide variation in a target nucleic acid in a sample is provided. The sample contains a first double-stranded DNA and a second double stranded DNA, the second double stranded DNA differing from the first double stranded DNA at at least one variance position. The method comprises:
In a further aspect, a method for amplifying a target nucleic acid in a sample is provided, wherein the target nucleic acid includes a first template strand. The method comprises:
In a further aspect, a molecular probe for detecting amplification of a nucleic acid is provided. The probe comprises a quencher portion, a fluorophore portion, and a linker portion linking the quencher portion and the fluorophore portion, the linker portion comprising a plurality of deoxyribonucleotides and a ribonucleotide, wherein no or low fluorescence is given by the fluorophore when then linker is intact. Accordingly, a method of detecting an amplicon product in an amplification system is provided, the method includes allowing such a molecular probe to specifically hybridize to an amplicon product in an amplification system; cleaving the ribonucleotide in the molecular probe; and detecting fluorescence given off by the fluorophore portion of the molecular probe.
Various embodiments contemplated herein may include, but need not be limited to, one or more of the following.
Terms used in the claims and specification are defined as set forth below unless otherwise specified.
The term “nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides.
The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; mRNA; and non-coding RNA.
The term nucleic acid encompasses double- or triple-stranded nucleic acid complexes, as well as single-stranded molecules. In double- or triple-stranded nucleic acid complexes, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).
The term nucleic acid also encompasses any modifications thereof, such as by methylation and/or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5-bromo-uracil, sugar-phosphate backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.
The nucleic acid(s) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.
As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides; i.e., if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid to form a canonical base pair, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
“Specific hybridization” or “specifically hydridizing” refers to the binding of a nucleic acid to a target nucleotide sequence in the absence of substantial binding to other nucleotide sequences present in the hybridization mixture under defined stringency conditions. Those of skill in the art recognize that relaxing the stringency of the hybridization conditions allows sequence mismatches to be tolerated.
In some embodiments, hybridizations are carried out under stringent hybridization conditions. The phrase “stringent hybridization conditions” generally refers to a temperature in a range from about 5° C. to about 20° C. or 25° C. below than the melting temperature (Tm) for a specific sequence at a defined ionic strength and pH. As used herein, the Tm is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands. Methods for calculating the Tm of nucleic acids are well known in the art (see, e.g., Berger and Kimmel (1987) METHODS IN ENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego: Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory), both incorporated herein by reference for their descriptions of stringent hybridization conditions). As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative Filter Hybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The melting temperature of a hybrid (and thus the conditions for stringent hybridization) is affected by various factors such as the length and nature (DNA, RNA, base composition) of the primer or probe and nature of the target nucleic acid (DNA, RNA, base composition, present in solution or immobilized, and the like), as well as the concentration of salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol). The effects of these factors are well known and are discussed in standard references in the art. Illustrative stringent conditions suitable for achieving specific hybridization of most sequences are: a temperature of at least about 60° C. and a salt concentration of about 0.2 molar at pH7. Tm calculation for oligonucleotide sequences based on nearest-neighbors thermodynamics can carried out as described in “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics” John Santa Lucia, Jr., PNAS Feb. 17, 1998 vol. 95 no. 4 1460-1465 (which is incorporated by reference herein for this description).
The term “oligonucleotide” refers to a nucleic acid that is relatively short, generally shorter than 200 nucleotides, more particularly, shorter than 100 nucleotides, most particularly, shorter than 50 nucleotides. Typically, oligonucleotides are single-stranded DNA molecules.
The term “DNA oligonucleotide” refers to an oligonucleotide whose components are all DNA or DNA analogues that are not RNA, for example, DNA derivatives including modified bases (such as methylated, hydroxymethylated, halo-substituted bases), LNA, etc.
The term “primer” refers to an oligonucleotide that is capable of hybridizing (also termed “annealing”) with a nucleic acid and serving as an initiation site for nucleotide (RNA or DNA) polymerization under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer or segments thereof depends on the intended use of the primer, but primers are typically at least 7 nucleotides long and, in some embodiments, range from 10 to 30 nucleotides, or, in some embodiments, from 10 to 60 nucleotides, in length. In some embodiments, primers can be, e.g., 15 to 50 nucleotides long. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.
A primer is said to anneal to another nucleic acid if the primer, or a portion thereof, hybridizes to a nucleotide sequence within the nucleic acid. The statement that a primer hybridizes to a particular nucleotide sequence is not intended to imply that the primer hybridizes either completely or exclusively to that nucleotide sequence. For example, in some embodiments, amplification primers used herein are said to “anneal to” or be “specific for” a nucleotide sequence.” This description encompasses primers that anneal wholly to the nucleotide sequence, as well as primers that anneal partially to the nucleotide sequence.
The term “ribonucleotide containing primer” (also referred to herein as “Ribo-Primer” or “R-Primer”) refers to a primer that includes one or more ribonucleotides. The “ribonucleotide containing primer” anneals with a nucleic acid and initiate nucleotide polymerization. The 3′ end of the primer can have 3′-hydroxyl group or be blocked but activatable. The ribonucleotide in the primer can be replicated by some polymerases, esp. reverse transcriptase or proof-reading DNA polymerases. When the primer is incorporated into a double strand DNA, the ribonucleotide can be recognized by a RNase H2 and cleaved at the 5′ side to form a 3′ hydroxyl upstream segment and a 5′ phosphate downstream segment. The ribonucleotide can be used to activate the primer when incorporated into a 3′-blocked primer or re-initiate the extension of the primer starting with the upstream segment.
The term “primer pair” refers to a set of primers including a 5′ “upstream primer” or “forward primer” that hybridizes with the complement of the 5′ end of the DNA sequence to be amplified and a 3′ “downstream primer” or “reverse primer” that hybridizes with the 3′ end of the sequence to be amplified. As will be recognized by those of skill in the art, the terms “upstream” and “downstream” or “forward” and “reverse” are not intended to be limiting, but rather provide illustrative orientations in some embodiments.
A “probe” is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, generally through complementary base pairing, usually through hydrogen bond formation, thus forming a duplex structure. The probe can be labeled with a detectable label to permit facile detection of the probe, particularly once the probe has hybridized to its complementary target. Alternatively, however, the probe may be unlabeled, but may be detectable by specific binding with a ligand that is labeled, either directly or indirectly. Probes can vary significantly in size. Generally, probes are at least 7 to 15 nucleotides in length. Other probes are at least 20, 30, or 40 nucleotides long. Still other probes are somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotides long. Yet other probes are longer still, and are at least 100, 150, 200 or more nucleotides long. Probes can also be of any length that is within any range bounded by any of the above values (e.g., 15-20 nucleotides in length).
The primer or probe can be perfectly complementary to the target nucleotide sequence or can be less than perfectly complementary. In some embodiments, the primer has at least 65% identity to the complement of the target nucleotide sequence over a sequence of at least 7 nucleotides, more typically over a sequence in the range of 10-30 nucleotides, and, in some embodiments, over a sequence of at least 14-25 nucleotides, and, in some embodiments, has at least 75% identity, at least 85% identity, at least 90% identity, or at least 95%, 96%, 97%, 98%, or 99% identity. It will be understood that certain bases (e.g., the 3′ base of a primer) are generally desirably perfectly complementary to corresponding bases of the target nucleotide sequence. Primer and probes typically anneal to the target sequence under stringent hybridization conditions.
As used herein with reference to a portion of a primer or a nucleotide sequence within the primer, the term “specific for” a nucleic acid, refers to a primer or nucleotide sequence that can specifically anneal to the target nucleic acid under suitable annealing conditions.
Amplification according to the present teachings encompasses any means by which at least a part of at least one target nucleic acid is reproduced, typically in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Illustrative means for performing an amplifying step include PCR, nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), loop mediated amplification (LAMP), and the like, including multiplex versions and combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR), helicase-dependent amplification (HDA), and the like. Descriptions of such techniques can be found in, among other sources, Ausubel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al., Curr Opin Biotechnol. 1993 February; 4 (1): 41-7, U.S. Pat. Nos. 6,027,998; 6,605,451, Barany et al., PCT Publication No. WO 97/31256; Wenz et al., PCT Publication No. WO 01/112579; Day et al., Genomics, 29 (1): 152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available on the world wide web at: promega.com/geneticidproc/ussymp6proc/blegrad.html-); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27: e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18-(2002); Lage et al., Genome Res. 2003 February; 13 (2): 294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 November; 2 (6): 542-8., Cook et al., J Microbiol Methods. 2003 May; 53 (2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February; 12 (1): 21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.
In some embodiments, amplification comprises at least one cycle of the sequential procedures of: annealing at least one primer with complementary or substantially complementary sequences in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be repeated. Amplification can comprise thermocycling or can be performed isothermally.
A “multiplex amplification reaction” is one in which two or more nucleic acids distinguishable by sequence are amplified simultaneously.
The term “qPCR” is used herein to refer to quantitative real-time polymerase chain reaction (PCR), which is also known as “real-time PCR” or “kinetic polymerase chain reaction;” all terms refer to PCR with real-time signal detection.
A “reagent” refers broadly to any agent used in a reaction, other than the analyte (e.g., nucleic acid being analyzed). Illustrative reagents for a nucleic acid amplification reaction include, but are not limited to, buffer, metal ions, polymerase, reverse transcriptase, primers, template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, and the like. Reagents for enzyme reactions include, for example, substrates, cofactors, buffer, metal ions, inhibitors, and activators.
The term “label,” as used herein, refers to any atom or molecule that can be used to provide a detectable and/or quantifiable signal. In particular, the label can be attached, directly or indirectly, to a nucleic acid or protein. Suitable labels that can be attached to probes include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, electron dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.
The term “dye,” as used herein, generally refers to any organic or inorganic molecule that absorbs electromagnetic radiation.
A DNA polymerase is said to be “stable” at a particular temperature if it provides a satisfactory extension rate in a nucleic acid amplification reaction.
In one embodiment, the primer is designed to have two segments, the upstream segment at the 5′ end (Segment 1) and the downstream segment at the 3′ end (Segment 2), with a ribonucleotide joining them together (see
The disclosed methods make the use of a RNase H to cleave the ribonucleotide containing primer. When there is a single ribonucleotide surrounded by DNA sequences, RNase H2 is used due to its specificity for single ribonucleotide recognition and cleavage. A continuous stretch of ribonucleotides equal or more than two can also be used within the ribonucleotide containing primer, in which case RNase H is preferred to digest the RNA and make Segment 1 extendable.
Ribonuclease H is a family of non-sequence-specific endonuclease enzymes that catalyze the cleavage of RNA in an RNA/DNA substrate via a hydrolytic mechanism. RNase H2, aka. RNase H II, is a member of RNase H family. The enzyme binds to RNA-DNA duplexes and nicks 5′ to a ribonucleotide. It specifically cleaves at the ribonucleotide in an RNA-DNA hybrid. It does not hydrolyze the phosphodiester bonds when the sequence is single stranded or the ribonucleotide is mismatched. Upon cleavage, the enzyme leaves a 5′ phosphate end for the downstream segment and a 3′ hydroxyl end for the upstream segment. RNase H2 from different sources has different enzymatic activity. RNase H2 cloned from E. coli is available from New England Biolab (NEB) with optimal activity at 37° C. A thermostable RNase H2, cloned from Pyrococcus abyssi, is available from Integrated DNA Technologies (IDT). It has high activity at 70-95° C. but low activity below 50° C. In some embodiments, E. coli RNase H2 is included in the reaction. In some embodiments, RNase H2 active at low temperature is used in the reaction. If thermocycling or high temperature incubation is to be carried out (as in PCR and isothermal amplifications), the RNase H2 is preferably thermostable.
In some embodiments, the polymerase has strand displacement activity (
When the polymerase lacks strand displacement activity, Segment 1 does not displace Segment 2 and its extension product. However, Segment 1 can be designed to be used as a primer by itself once cleaved by the RNase.
In some embodiments, the position of the ribonucleotide within the primer is adjusted to make Segment 1 and Segment 2 to have different Tm. In one further embodiment, Segment 2 can be made short to have a Tm that is too low to hybridize to the template strand by itself.
In a further embodiment, more than one ribonucleotide is included in the primer. For example, when two ribonucleotides are included in the primer, a third segment can be further included in the primer. After cleavage, one primer generates up to three amplicons (
In another embodiment, Segment 1 is partially complementary to the template. As an example, Segment 1 has a portion on its 3′ end that is complementary to the template. The other portion can be any sequences of choices.
In some embodiments, more than one ribonucleotide are included in a R-primer. In one embodiment, the ribonucleotides are placed inside the primer, separated by deoxynucleotides (
RNases have variable activities and stabilities at different temperature. For isothermal amplification at low temperature, an RNase H or RNase H2 with optimal activity and stability at the targeted temperature can be used. When the amplification is carried out at elevated temperature an RNase with high activity at the targeted temperatures is needed. For amplification through thermocycling, a thermostable RNase H or RNase H2 with high activity at the extension temperature is required.
In some embodiments, RNA template is used with ribonucleotide containing primers. In
Nucleic acid template used in the invention can DNA or RNA or chimeric DNA and RNA. Nucleic acid-containing samples can be obtained from biological sources and prepared using conventional methods known in the art. In particular, nucleic acid useful in the methods described herein can be obtained from any source, including unicellular organisms and higher organisms such as plants or non-human animals, e.g., canines, felines, equines, primates, and other non-human mammals, as well as humans. In some embodiments, samples may be obtained from an individual suspected of being, or known to be, infected with a pathogen, an individual suspected of having, or known to have, a disease, such as cancer, or a pregnant individual.
Nucleic acids can be obtained from cells, bodily fluids (e.g., blood, a blood fraction, urine, etc.), or tissue samples by any of a variety of standard techniques. In some embodiments, the method employs samples of plasma, serum, spinal fluid, lymph fluid, peritoneal fluid, pleural fluid, oral fluid, and external sections of the skin; samples from the respiratory, intestinal genital, or urinary tracts; samples of tears, saliva, blood cells, stem cells, or tumors. Samples can be obtained from live or dead organisms or from in vitro cultures. Illustrative samples can include single cells, paraffin-embedded tissue samples, and needle biopsies. In some embodiments, the nucleic acids analyzed are RNA (
Nucleic acids of interest can be isolated using methods well known in the art. The sample nucleic acids need not be in pure form, but are typically sufficiently pure to allow the steps of the methods described herein to be performed.
Any target nucleic acid that can detected by nucleic acid amplification can be detected using the methods described herein. In typical embodiments, at least some nucleotide sequence information will be known for the target nucleic acids. For example, if the amplification reaction employed is PCR, sufficient sequence information is generally available for each end of a given target nucleic acid to permit design of suitable amplification primers.
The targets can include, for example, nucleic acids associated with pathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g., those for which over- or under-expression is indicative of disease, those that are expressed in a tissue- or developmental-specific manner; or those that are induced by particular stimuli; genomic DNA, which can be analyzed for specific polymorphisms (such as SNPs), alleles, or haplotypes, e.g., in genotyping. Of particular interest are genomic DNAs that are altered (e.g., amplified, deleted, and/or mutated) in genetic diseases or other pathologies; sequences that are associated with desirable or undesirable traits; and/or sequences that uniquely identify an individual (e.g., in forensic or paternity determinations).
In some embodiments, the targets contain the total of DNA from an organism (
Primers suitable for nucleic acid amplification are sufficiently long to prime the synthesis of extension products in the presence of a suitable nucleic acid polymerase. The exact length and composition of the primer will depend on many factors, including, for example, temperature of the annealing reaction, source and composition of the primer, and where a probe is employed, proximity of the probe annealing site to the primer annealing site and ratio of primer:probe concentration. For example, depending on the complexity of the target nucleic acid sequence, an oligonucleotide primer typically contains in the range of about 10 to about 60 nucleotides, although it may contain more or fewer nucleotides. The primers should be sufficiently complementary to selectively anneal to their respective strands and form stable duplexes.
In general, one skilled in the art knows how to design suitable primers capable of amplifying a target nucleic acid of interest. For example, PCR primers can be designed by using any commercially available software or open source software, such as Primer3 (see, e.g., Rozen and Skaletsky (2000) Meth. Mol. Biol., 132:365-386; www.broad.mit.edu/node/1060, and the like) or by accessing the Roche UPL website. The amplicon sequences are input into the Primer3 program with the UPL probe sequences in brackets to ensure that the Primer3 program will design primers on either side of the bracketed probe sequence.
Primers may be prepared by any suitable method, including, for example, direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22:1859-1862; the solid support method of U.S. Pat. No. 4,458,066 and the like, or can be provided from a commercial source. Primers may be purified by using a Sephadex column (Amersham Biosciences, Inc., Piscataway, N.J.) or other methods known to those skilled in the art. Primer purification may improve the sensitivity of the methods described herein.
Ribonucleotide containing primers (shown in
“Stabilizing bases” include, e.g., stretches of peptide nucleic acids (PNAs) that can be incorporated into DNA oligonucleotides to increase duplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids (UNAs) are analogues of RNA that can be easily incorporated into DNA oligonucleotides during solid-phase oligonucleotide synthesis, and respectively increase and decrease duplex stability. Suitable stabilizing bases also include modified DNA bases that increase the stability of base pairs (and therefore the duplex as a whole). These modified bases can be incorporated into oligonucleotides during solid-phase synthesis and offer a more predictable method of increasing DNA duplex stability. Examples include AP-dC (G-clamp) and 2-aminoadenine, as well as 5-methylcytosine and C(5)-propynylcytosine (replacing cytosine), and C(5)-propynyluracil (replacing thymine).
“Destabilizing bases” are those that destabilize double-stranded DNA by virtue of forming less stable base pairs than the typical A-T and/or G-C base pairs. Inosine (I) is a destabilizing base because it pairs with cytosine (C), but an I-C base pair is less stable than a G-C base pair. This lower stability results from the fact that inosine is a purine that can make only two hydrogen bonds, compared to the three hydrogen bonds of a G-C base pair. Other destabilizing bases are known to, or readily identified by, those of skill in the art. Ribonucleotide containing primer in a primer set
A ribonucleotide containing primer can be employed with a simple conventional reverse primer or another ribonucleotide containing primer in a reaction.
Primer with Tag Sequence
A primer may be added one or more tag sequences at the 5′-end (
When the tag sequence in
In some embodiments, both forward primer and reverse primer are ribonucleotide containing primers (
The disclosed methods make the use of a polymerase for amplification. In some embodiments, the polymerase is a DNA polymerase that lacks a 5′ to 3′ exonuclease activity but has “strand displacement” activity. The polymerase is used under conditions such that the strand extending from a first primer can be displaced by polymerization of a second primer provided or generated by RNase H2 cleavage.
Conveniently, the polymerase is capable of displacing the strand complementary to the template strand, a property termed “strand displacement.” Strand displacement results in synthesis of multiple copies of the target sequence per template molecule. Exemplary polymerases with strand displacement activity include M-MuLV reverse transcriptase, phi29 DNA polymerase, DEEP VENT (exo-) DNA polymerase (all available from NEB), SD polymerase (Bioron). If thermocycling is to be carried out (as in PCR), the polymerase is preferably a thermostable polymerase.
Strand displacement can also be facilitated through the use of a strand displacement factor, such as a helicase. Any DNA polymerase that can perform strand displacement in the presence of a strand displacement factor is suitable for use in the disclosed method, even if the DNA polymerase does not perform strand displacement in the absence of such a factor. Strand displacement factors useful in the methods described herein include BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67 (12): 7648-7653 (1993)), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68 (2): 1158-1164 (1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67 (2): 711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91 (22): 10665-10669 (1994)), single-stranded DNA binding proteins (SSB; Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymus helicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)). Helicase and SSB are available in thermostable forms and therefore suitable for use in PCR.
In some embodiments, it can be advantageous to use a blend of two or more polymerases. For example, an illustrative polymerase blend includes a polymerase that is particularly proficient at initiating extension from a partially double-stranded DNA primer and a polymerase that is particularly proficient at strand displacement synthesis, since combining these properties may provide a net advantage in some embodiments. In some embodiments, a reverse transcriptase is combined with a DNA polymerase. The DNA polymerase and the reverse transcriptase may have strand displacement activity.
In some embodiments, the DNA polymerase for use in the disclosed methods is highly processive. Exemplary DNA polymerases include variants of Taq DNA polymerase that lack 5′ to 3′ exonuclease activity, e.g., the Stoffel fragment of Taq DNA polymerase (ABI), SD polymerase (Bioron), mutant Taq lacking 5′ to 3′ exonuclease activity described in U.S. Pat. No. 5,474,920, Bca polymerase (Takara), Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene). If thermocycling is to be carried out (as in PCR), the DNA polymerase is preferably a thermostable DNA polymerase.
In a different aspect, where it is desirable to use a Taqman-style probe to carry out real-time PCR, a polymerase blend can include a polymerase that has 5′ to 3′ exonuclease activity, provided the primer structure is designed so that it is not susceptible to “flap” endonuclease activity. In some embodiments, the probe has one or more ribonucleotides in the sequence (see an example shown in
In some embodiments, the ribonucleotide containing primers are used with DNA polymerases that lack strand displacement activity (
In some embodiments, the forward primer and the reverse primer are designed to each have a tag (
In some embodiments, the ribonucleotide containing primer is used to differentiate or enrich a variation in the target nucleic acid (
In some embodiments, the ribonucleotide containing primer can be used to initiate cascade of reactions (see
Illustrative polymerase concentrations range from about 0.1 to 200 units per reaction. In various embodiments, the polymerase concentration can be at least: 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 or more units per reaction. In some embodiments, the polymerase concentration falls within a range bounded by any of these values, e.g., 0.1-1, 1-10, 10-200, 10-150, 10-100, 10-50, 20-150, 20-100, 20-50, 50-200, 50-150, 50-100, 100-200, 100-150, etc. units per reaction. When polymerase blends are used, the total, combined polymerase concentration can be any of these values or fall within any of these ranges.
The primer sets described above are contacted with sample nucleic acids under conditions wherein the primers anneal to their template strands, if present. The desired nucleic acid amplification method is carried out using a DNA polymerase lacking 5′-3′ exonuclease activity under the reaction conditions employed. In some embodiments, strand displacement polymerase is advantageous. In other embodiments, non-strand displacement polymerase is advantageous. Reaction mixtures with appropriate components and buffer conditions are provided to support polymerase and RNase H activities. The primer sets can be conveniently added to the amplification mixture in the form of separate oligonucleotides. For example, the two-primer set can consist of one or two ribonucleotide containing primers. The reaction may be carried out in solution or with one or two primers fixed on a matrix (
In some embodiment, the amplification step is performed isothermally. Isothermal amplification may be performed as strand-displacement amplification, NEAR, LAMP, RT LAMP, NABSA, RPA (recombinase polymerase amplification). RNase H2 and ribonucleotide containing primers can be used in place of all-deoxynucleotide primers in their specific buffers.
In some embodiments, the amplification step is performed using PCR. For running real-time PCR reactions, reaction mixtures generally contain an appropriate buffer, a source of magnesium ions (Mg2+) in the range of about 1 to about 10 mM, e.g., in the range of about 2 to about 8 mM, nucleotides, and optionally, detergents, and stabilizers. An example of one suitable buffer is TRIS buffer at a concentration of about 5 mM to about 85 mM, with a concentration of 10 mM to 50 mM preferred. In one embodiment, the TRIS buffer concentration is 80 mM in the reaction mix double-strength (2×) form. The reaction mix can have a pH range of from about 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 as typical. Concentration of nucleotides can be in the range of about 25 μM to about 1000 μM, typically in the range of about 100 μM to about 800 μM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600, 700, and 800 μM. Detergents such as Tween 20, Triton X 100, and Nonidet P40 may also be included in the reaction mixture. Stabilizing agents such as dithiothreitol (DTT, Cleland's reagent) or mercaptoethanol may also be included. In addition, master mixes may optionally contain dUTP as well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A master mix is commercially available from Applied Biosystems, South San Francisco, Calif., (TaqMan Universal Master Mix, cat. nos. 4304437, 4318157, and 4326708).
Any suitable labeling strategy can be employed in the methods described herein. Where the reaction is analyzed for presence of a single amplification product, a universal detection probe can be employed in the amplification mixture. In particular embodiments, real-time PCR detection can be carried out using a universal qPCR probe. Suitable universal qPCR probes include double-stranded DNA-binding dyes, such as SYBR Green, Pico Green (Molecular Probes, Inc., Eugene, Org.), Eva Green (Biotium), ethidium bromide, and the like (see Zhu et al., 1994, Anal. Chem. 66:1941-48). In some embodiments, colorimetric dyes may be used to detect amplification. pH changes may be used to indicate amplification. A pH indicator may be used to show color changes when amplification causes pH shift. A colorimetric dye that is sensitive to free Mg2+ concentration changes may so be used. In some embodiments, luminescence may be used to detect the amplification.
In some embodiments, one or more target-specific qPCR probes (i.e., specific for a target nucleotide sequence to be detected) is employed in the amplification mixtures to detect amplification products. By judicious choice of labels, analyses can be conducted in which the different labels are excited and/or detected at different wavelengths in a single reaction (“multiplex detection”). See, e.g., Fluorescence Spectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); White et al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, New York, (1970); Berlman, Handbook of Fluorescence Spectra of Aromatic Molecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colour and Constitution of Organic Molecules, Academic Press, New York, (1976); Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland, Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene (1992); and Linck et al. (2017) “A multiplex TaqMan qPCR assay for sensitive and rapid detection of phytoplasmas infecting Rubus species,” PLOS One 12 (5).
In some embodiments, it may be convenient to include labels on one or more of the probes employed in in amplification mixture.
In some embodiments, a target nucleic acid is detected using an automated sample handling and/or analysis platform. In some embodiments, commercially available automated analysis platforms are utilized. For example, in some embodiments, real-time qPCR systems are utilized. However, the present invention is not limited to a particular detection method or analysis platform. One of skill in the art recognizes that any number of platforms and methods may be utilized.
In some embodiments, a target nucleic acid is detected using a lateral flow device. A sample contains a target nucleic acid is added to the cartridge, the sample is contacted with lysis buffer and released nucleic acid is shifted along the lateral flow cartridge. The nucleic acid may then contact with reaction mixes deposited in the cartridge to initiate amplification. The amplified products may be then detected by fluorescence, luminescence or color changes.
Components of the cartridge include, but are not limited to, spots or lines containing lysis reagents, filters, and capture technologies useful to extract, purify, and amplify target nucleic acids. An optical window enables detection.
Also contemplated is a kit for carrying out the methods described herein. Such kits include one or more reagents useful for practicing any of these methods. A kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as an admixture where the compatibility of the reagents will allow. The kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.
In some embodiments, a kit can include one or more ribo-primers as disclosed herein, a polymerase described herein, and a RNase as described herein.
Kits preferably include instructions for carrying out one or more of the amplification methods described herein. Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user can be employed. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.
Covid-19 N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Catalog #10006625) was amplified with primers that targeted the N gene sequence within the plasmid. Standard primers (Forward primer: ACT GAG GGA GCC TTG AAT ACA (SEQ ID NO:1); Reverse primer: TGC AGC ATT GTT AGC AGG AT (SEQ ID NO:2)) was compared to R-primer (same standard primer; R-primer: ACT GAG GGA GCrC TTG AAT A+CA, +: LNA (SEQ ID NO:3)) in the amplification of the target sequence. In each 10 ul reaction, 500 nM of forward primer and 500 nM of reverse primer were used to amplify 2000 copies of N gene plasmid in 1× Q5 Hot Start High-Fidelity Master Mix (NEB, Catalog #M0494) in the presence of 1×SYBR and 30 nM ROX. For reactions with RNase H2, 2.6 mU/ul of RNase H2 (IDT, catalog #Nov. 3, 2002-02) was added in the reaction. The reactions were thermos cycled on a StepOnePlus Real Time PCR System (Thermo Fisher Scientific, Catalog #4376600) and the amplification was monitored at SYBR fluorescence channel. The thermocycles were 95° C./30 sec, 40 cycles of 95° C./15 sec and 65° C./1 min. The amplification curves were plotted as SYBR fluorescence intensity vs cycle number in Multicomponent Plot view, see
As shown in
Melt curves of the amplification products with R-primer in Example 1 were collected on the StepOnePlus Real Time PCR System after the thermocycling steps. See
Human genomic DNA was amplified with a pair of primers, one of which was a F-primer. Primers were designed to amplify the sequences of human EGFR gene exon 21. The forward R-primer (CAA GAT CAC AGA TTT TGG GCrU GGC (SEQ ID NO:4)) was designed to have the ribonucleotide base located at the base that is mutated from wildtype T to cancerous G. The R-primer was used with reverse primer (TTT CTC TTC CGC ACC CAG (SEQ ID NO: 5)) in 1×Q5 Hot Start High Fidelity Master Mix to amplify EFGR exon 21 sequence. The reaction consisted of 500 nM each of the forward R-primer and reverse primer, 2 ng human genomic DNA in 1× master mix and 1×SYBR. For the reaction with RNase H2, 2.6 mU/ul of RNase H2 was added to each reaction. The reactions were thermo cycled at the conditions of 95° C./2 min, 40 cycles of 95° C./15 sec, 60° C./30 sec and 72° C./1 min on a StepOnePlus Real Time PCR System (Thermo Fisher Scientific). As shown in
LAMP assay was designed to amplify SARS-CoV-2 N gene. One ribonucleotide was incorporated into each of two of the primers, B3 and BIP (B3: TGC AGC ATT GrUT AGC AGG+AT (SEQ ID NO:6); BIP: AGA CGG CAT rCAT ATG GGT TGC ACG GGT GCC AAT GTG AT+C T (SEQ ID NO:7)), while the other four, F3, FIP, LF and LB, were standard primers (F3: TGG CTA CTA CCG AAG AGC T (SEQ ID NO:8); FIP: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO:9); LF: GGA CTG AGA TCT TTC ATT TTA CCG T (SEQ ID NO:10); LB: ACT GAG GGA GCC TTG AAT ACA (SEQ ID NO:1)). In the presence of RNase H2, the R-primer reduced the time of detection (
Primer concentrations in the final reaction were 200 nM for F3 and B3, 1.6 μM FIP and BIP, and 400 nM LF and LB. The primers were pre-annealed with 2000 copies of Covid-19 N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Catalog #10006625) with 0.5×SYBR in a volume of 8.31 ul by heating up to 90° C. and cooling to 20° C. with a StepOnePlus Real Timer PCR System. The primers and plasmid mixtures in each reaction well on a 96-well plate were then added WarmStart LAMP 2× Master Mix (NEB, E1700) to 1× and 10 mU/ul RNase H2 (IDT, catalog #Nov. 3, 2002-02) dilute to 0.26 mU/ul or RNase H2 dilution buffer were incubated at 65° C. on a StepOnePlus Real Timer PCR System and fluorescence of SYBR was collected at each cycle for 75 cycles (cycle/minute). Fluorescence was plotted against cycle numbers in the example (
LAMP assay was designed to amplify SARS-CoV-2 N gene with 6 primers. The standard primer reaction used all-DNA oligonucleotides: F3: TGG CTA CTA CCG AAG AGC T (SEQ ID NO: 8); B3: TGC AGC ATT GTT AGC AGG AT (SEQ ID NO:2); FIP: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO:9); BIP: AGA CGG CAT CAT ATG GGT TGC ACG GGT GCC AAT GTG ATC T (SEQ ID NO:11); LF: GGA CTG AGA TCT TTC ATT TTA CCG T (SEQ ID NO:10); LB: ACT GAG GGA GCC TTG AAT ACA (SEQ ID NO:1). For the reaction with ribo-primers, one ribonucleotide was incorporated into each of three of the primers, B3, BIP and LB (B3: TGC AGC ATT GrUT AGC AGG+AT (SEQ ID NO:6); BIP: AGA CGG CAT rCAT ATG GGT TGC ACG GGT GCC AAT GTG AT+C T (SEQ ID NO:7); LB: ACT GAG GGA GCrC TTG AAT A+CA (SEQ ID NO:3)), while the other three, F3, FIP and LF, were standard primers (F3: TGG CTA CTA CCG AAG AGC T (SEQ ID NO:8); FIP: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO:9); LF: GGA CTG AGA TCT TTC ATT TTA CCG T (SEQ ID NO:10)). Primer concentrations in the final reaction were 400 nM for F3 and B3, 1.6 μM FIP and BIP, and 400 nM LF and LB. The primers were pre-annealed with 2000 copies of Covid-19 N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Catalog #10006625) with 1×SYBR in a volume of 5.85 ul by heating up to 95° C. and cooling to 20° C. on a StepOnePlus Real Timer PCR System. The primers and plasmid mixtures in reaction wells of a 96-well plate were then added WarmStart Colorimetric LAMP 2× Master Mix (NEB, M1800) to 1×, 10 mU/ul RNase H2 (IDT, catalog #Nov. 3, 2002-02; NEB, catalog #M0288) dilutes to 0.26 mU/reaction and 3.2 U/ul Bsu DNA Polymerase, Large Fragment (NEB, M0330) to 2 U/reaction (total volume 9.15 ul) were incubated at 37° C. for 30 cycles of 37° C./15 sec and 37° C./1 min, 40 cycles of 65° C./15 sec and 65° C./1 min on a StepOnePlus Real Timer PCR System and fluorescence of SYBR was collected at each cycle. Fluorescence was plotted against cycle numbers in the example (
R-primers were used to amplify the N gene cloned in Covid-19 N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Catalog #10006625). Each of the forward primer and reverse primer contained a ribonucleotide in the sequences (Forward r-primer: ACC AAT AGC AGT CCA GAT GAC rCAA ATT GGC TAC TAC CGA AGA GCT (SEQ ID NO:12); Reverse r-primer: GTT CCT TGA GGA AGT TGT AGC ArCG ATT GCA GCA TTG TTA GCA GGA T (SEQ ID NO:13)). The amplification was compared to corresponding standard primers (Forward primer: TGG CTA CTA CCG AAG AGC T (SEQ ID NO:8); Reverse primer: TGC AGC ATT GTT AGC AGG AT (SEQ ID NO:2)). 500 nM of each of the r-primers or standard primers were used to amplify 2000 copies of N gene plasmid in a 10 ul reaction with a strand displacement DNA polymerase, SD Polymerase (Boca Scientific, Catalog #1089100) at 0.3 U/ul in 1×SD DNA polymerase reaction buffer supplemented with 0.375 mM dNTPs, 3.5 mM MgCl2, 1×SYBR and 2.6 mU/ul RNase H2 (IDT, catalog #Nov. 3, 2002-02). The reactions were thermocycled with the protocols of 93 C/3 min, 40 cycle of 60 C/1 min and 65 C/1 min on a StepOnePlus Real Time PCR System (Thermo Fisher Scientific, Catalog #4376600) and the amplification was monitored at SYBR fluorescence channel (
Real time amplification of DNA was detected by probes that consisted of ribonucleotides without the need of 5′-nuclease activities. In a 10 ul reaction, 800 copies of SARS-CoV-2 N gene in Covid-19 N gene plasmid (IDT, 2019-nCoV_N_Positive Control, Catalog #10006625) was amplified with a pair of primers that targeted the N gene (Fw primer: TCT GGC CCA GTT CCT AGG TAG TCC AGA CGA ATT CGT GGT GG (SEQ ID NO:9); Rv primer: AGA CGG CAT CAT ATG GGT TGC ACG GGT GCC AAT GTG ATC T (SEQ ID NO:11)) at 400 nM each in 1×Q5 Hot Start High-Fidelity Master Mix (NEB, Catalog #M0494). Real time amplification was monitored by 400 nM probe (/56-FAM/AGC TGG ACT TCrC rCTA TGG TGC TAA CAA/3BHQ_1/, IDT (SEQ ID NO:14)) in the presence of 5.2 mU/ul RNase H2 (IDT, catalog #Nov. 3, 2002-02) on a StepOnePlus Real Time PCR System. The thermocycle condition was 92° C./2 min, 40 cycles of 95° C./15 sec, 60° C./15 sec and 70° C./1 min. Amplification of the target DNA was detected by the ribo-probe DNA in the presence of RNase H2 using a polymerase that lacks 5′-nuclease activity (
SDCR was used to amplify and detect SARS-CoV-2 E gene and compared to standard primers. Forward primer and reverse primer were selected to target the E gene. Standard primers (Fw primer: GCT TTC GTG GTA TTC TTG CTA GTT (SEQ ID NO:15); Rv primer: GTT AAC AAT ATT GCA GCA GTA CGC A (SEQ ID NO:16)) contained all DNA bases while ribo-primers had the same 3′ half and a 5′ half of similar Tm separated by a ribonucleotide (R-Fw primer: CGT TAA TAG TTA ATA GCG TAC TTC TTT TTC TTIG CTT TCG TGG TAT TCT TGC TAG TT (SEQ ID NO:17); R-Rv primer: CGT AAA AAG AAG GTT TTA CAA GAC TCA CrGT TAA CAA TAT TGC AGC AGT ACG CA (SEQ ID NO:18)). A ribo-probe was selected from the internal sequence between the forward and reverse primers and consisted of a ribonucleotide and labeled with a 5′ fluorophore and 3′ quencher (R-Probe:/5HEX/ACA CTA GCC ATC rCTT ACT GCG CTT CG/3BHQ_1/(SEQ ID NO:19)). To compare SDCR to standard PCR, either each of the forward and reverse was switched with the r-ribo primer or both with ribo primers. The same probe was used for all the reactions. In each 10 ul reaction, 2000 copies of E gene plasmid (IDT, 2019-nCoV_E Positive Control, catalog #10006896) were amplified with 500 nM primers, 250 nM probe, 3 U SD polymerase (Boca Scientific, catalog #108910) and 2.7 mU RNase H2 (IDT, catalog #Nov. 3, 2002-02) in 60 mM Tris-HCl buffer (pH 8.5) supplemented with 625 nM ROX (Lumiprobe, ROX reference dye for qPCR, catalog #31110), 45 mM KCl, 4 mM MgCl2, 0.01% Triton X-100, 100 ng/ul BSA and 0.4 mM dNTPs. The thermocyling conditions were 92° C./2 min, 40 cycles of 60° C./1 min. The amplification was carried out on a StepOnePlus Real Time PCR System (Thermo Fisher Scientific) and monitored by fluorescence increase at VIC channel. Logarithm of ROX normalized HEX fluorescence intensities were plotted with cycle number. As shown in
SDCR assays for SARS-CoV-2 E gene were compared to standard primers (
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
The steps shown in the figures, explained in the specification and recited in the claims are for illustration purposes only. It is understood that these steps do not necessarily need to be performed sequentially according to the illustrated order unless specifically required or dictated by the context.
Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/060598 | 1/12/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63298890 | Jan 2022 | US |
