Use of 5'-tailed long primers to improve amplification performance when targeting short primer-binding sites

Abstract

Methods for amplifying a template / target nucleic acid, where the original template / target nucleic acid has short primer-binding sites (e.g., ≤ 15 nucleotides), by employing long primers are described. Long forward primers and long reverse primers have two regions: (1) a region for annealing / hybridizing to a region of the template / target nucleic acid; and (2) a region that does not anneal / hybridize to a region of the template / target nucleic acid region. Put another way, the long primers are longer than the length of the primer-binding site (i.e., the site upon which the long primers are to anneal / hybridize). Use of these long primers results overcomes the problems and obstacles of short primer-binding sequences.

Description

[0001] USE OF 5’-TAILED LONG PRIMERS TO IMPROVE AMPLIFICATION PERFORMANCE WHEN TARGETING SHORT PRIMER-BINDING SITES

[0002] SEQUENCE LISTING

[0003] The instant patent application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created July 11, 2024, is named “20240711_P38679- WO Sequence Listing,” and is 6,144 bytes in size.

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to the field of in vitro diagnostics and nucleic acid analysis. Within this field, the present invention concerns: (a) the amplification of a target nucleic acid and / or (b) the amplification and detection of a target nucleic acid that may be present in a sample. In particular, the present invention is directed to overcoming the problem of poor amplification performance of a target nucleic acid, where the target nucleic acid has short primer-binding sites. This problem is overcome by employing longer primers, where the longer primers have one region that anneals / hybridizes to the target nucleic acid, and one region that does not anneal / hybridize to the target nucleic acid.

[0006] BACKGROUND OF THE INVENTION

[0007] Short target primer binding sites (e.g. 15 bases) are challenging to prime off of during polymerase chain reactions (PCR), due to the lower inherent annealing temperature (< 55°C) of these short sequence. This is due to the low potential of primer annealing events to occur during typical thermal-cycling temperatures (> 55°C). Moreover, this poor annealing per cycle impacts final yield of amplification product in an exponential manner, due to the doubling nature per cycle of PCR and multiple rounds of cycling.

[0008] Longer tailed primers with 3’ ends of the primer matching (z.e., complementary to) a short primer binding site of a target nucleic acid, and a 5’ end that does not anneal to (z.e., not complementary to) the initial primer binding site of a target nucleic acid, but lengthens the total primer to typical PCR primer lengths (> 18 bases), and annealing temperatures (> 55°C), can be used to improve PCR yields.

[0009] Starting with an initial template nucleic acid that contains the template of interest flanked on either side by: (a) a short forward primer binding site on one end, and (b) a template reverse primer-binding site on the other side, the initial cycles of PCR will be different than the subsequent cycles. The initial cycle of PCR using longer forward primers will occur while using only the shortened matching primer binding sites of the longer forward primers. For example, in the first cycle, the 3’ end of the long forward primer will anneal to the corresponding portion of the template (z.e., matching), whereas the 5’ end will not anneal to anything (z.e., nonmatching), and the first extension product generated will contain the non-matching 5’ tail of the forward primer, the 3’ matching end of the forward primer, the reverse complement of the initial template, and the short reverse primer-binding site.

[0010] In the second cycle of PCR, the template is the primer extension product generated from the first PCR cycle. That is, the template for the second cycle of PCR will contain the non-matching 5’ tail of the forward primer, the 3’ matching end of the forward primer, the reverse complement of the initial template, and the short reverse primer-binding site. In the second PCR cycle, the 3’ end of the long reverse primer will anneal to the corresponding portion of the template (z.e., matching), whereas the 5’ end will not anneal to anything (z.e., non-matching), and the second extension product generated will contain the longer reverse primer with the nonmatching 5’ tail of the reverse primer, the 3’ matching end of the reverse primer, the reverse complement of the first extension product, and the reverse complement of the longer forward primer, which will serve as the full length primer-binding site of the longer forward primer (> 18 bases long) with a typical PCR annealing temperature (> 55°C). That is, the product of the second PCR cycle is the template nucleic acid flanked by, now, primer-binding sites for: (a) the full-length longer forward primer, and (b) the full-length longer reverse primer.

[0011] In the third cycle of PCR, the template is the primer extension product generated from the 2nd PCR cycle. That is, the template for the third cycle of PCR will contain the template nucleic acids flanked by the primer binding sites for: (a) the full-length longer forward primer, and (b) the full-length longer reverse primer. That is, the template for the third PCR cycle will contain a full-length primer binding site (for example > 18 bases long), with a typical PCR annealing temperature (> 55°C). Thus, in instances where templates have short primer-binding sites, the deficiencies of short-primer binding sites are overcome within two cycles of PCR by employing longer forward and longer reverse primers. In the third PCR cycle, the long forward primer anneals / matches completely to the long forward primer-binding site, and generates a primer extension product that is the template nucleic acid flanked by primer-binding sites for: (a) the full-length longer forward primer, and (b) the full-length longer reverse primer. Like the third PCR cycle, subsequent PCR cycles should also employ templates having primer-binding sites that accommodate / anneal to the longer forward primers and the longer reverse primers, thereby eliminating the problems of short primer-binding sites stemming from the original template.

[0012] Moreover, the use of longer forward primers and longer reverse primers could also allow for additional priming, via extension of the template. That is, with a longer forward primer, having a 5’ end overhang, extension can occur in the two directions (z.e., (a) extension of the longer forward primer, and (b) extension of the template).

[0013] Thus, this method of using long primers with short primer binding sites can be utilized where primer binding sites are generated in synthesized probes or other similar templates where primer binding sites are shorter by design.

[0014] In the field of molecular diagnostics, the amplification and / or detection of nucleic acids is of considerable significance. Such methods can be employed to detect any number of microorganisms, such as viruses and bacteria. The most prominent and widely-used amplification technique is the Polymerase Chain Reaction (PCR). Other amplification techniques include Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and QP-amplification. Automated systems for PCR-based analysis often make use of a real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels. Short target primer binding sites (e.g. 15 bases) are challenging to prime off of during polymerase chain reactions (PCR), due to the lower inherent annealing temperature (< 55°C) of these short sequence. This is due to the low potential of primer annealing events to occur during typical thermal-cycling temperatures (> 55°C). Moreover, this poor annealing per cycle impacts final yield of amplification product in an exponential manner, due to the doubling nature per cycle of PCR and multiple rounds of cycling. The methods disclosed, herein, describe a method for overcoming the problems of short target primer-binding sites, by employing longer forward primers and longer reverse primers.

[0015] SUMMARY OF THE INVENTION

[0016] Certain embodiments in the present disclosure relate to methods for the amplification and / or detection of the presence or absence of a template / target nucleic acid, by a polymerase chain reaction (PCR), using longer forward primers and longer reverse primers. Embodiments include methods of amplification and / or detection of template / target nucleic acids, comprising performing at least one cycling step, which may include an amplifying step and a hybridizing step, using longer forward primers and longer reverse primers. Furthermore, embodiments include primers, probes, and kits that are designed for the amplification and / or detection template / target nucleic acids.

[0017] One embodiment is directed to a method for amplifying a target nucleic acid, wherein the method comprises the following steps: (a) obtaining one or more forward primers and one or more reverse primers, wherein the forward primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid, wherein the reverse primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid; and (b) performing one or more amplification steps, wherein the amplification step comprises contacting the target nucleic acid with the one or more forward primers and one or more reverse primers, in the presence of a polymerase, to produce one or more amplification products. In one embodiment, the region of the forward primer that is complementary to the target nucleic acid is < 20 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is < 15 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is < 10 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is < 20 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is < 15 base pairs long. In one embodiment, the region of the reverse primer that is complementary to the target nucleic acid is < 10 base pairs long. In one embodiment, the forward primer is > 17 base pairs long. In one embodiment, the forward primer is > 20 base pairs long. In one embodiment, the forward primer is > 25 base pairs long. In another embedment, the reverse primer is > 17 base pairs long. In another embodiment, the reverse primer is > 20 base pairs long. In another embodiment, the reverse primer is > 25 base pairs long. In another embodiment, the target nucleic acid is between 110-130 base pairs long. In one embodiment, the target nucleic acid about 120 base pairs long.

[0018] Another embodiment is directed to a method for detecting a target nucleic acid in a sample, wherein the method comprises the following steps: (a) obtaining one or more forward primers and one or more reverse primers, wherein the forward primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid, wherein the reverse primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid; and (b) performing one or more amplification steps, wherein the amplification step comprises contacting the sample with the one or more forward primers and one or more reverse primers, in the presence of a polymerase, to produce one or more amplification products, if the target nucleic acid is present in the sample; (c) performing a hybridization step, wherein the hybridization step comprises contacting one or more probes with the one or more amplification products from step (b); and (d) detecting the presence or absence of the amplification products, wherein the presence of the amplification product is indicative of the presence of the target nucleic acid in the sample, and wherein the absence of the amplification product is indicative of the absence of the target nucleic acid in the sample. In one embodiment, the region of the forward primer that is complementary to the target nucleic acid is < 20 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is < 15 base pairs long. In another embodiment, the region of the forward primer that is complementary to the target nucleic acid is < 10 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is < 20 base pairs long. In another embodiment, the region of the reverse primer that is complementary to the target nucleic acid is < 15 base pairs long. In one embodiment, the region of the reverse primer that is complementary to the target nucleic acid is < 10 base pairs long. In one embodiment, the forward primer is > 17 base pairs long. In one embodiment, the forward primer is > 20 base pairs long. In one embodiment, the forward primer is > 25 base pairs long. In another embedment, the reverse primer is > 17 base pairs long. In another embodiment, the reverse primer is > 20 base pairs long. In another embodiment, the reverse primer is > 25 base pairs long. In another embodiment, the target nucleic acid is between 110-130 base pairs long. In one embodiment, the target nucleic acid about 120 base pairs long.

[0019] Other embodiments provide an oligonucleotide comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs:l-4, or complements thereof, which oligonucleotide has 100 or fewer nucleotides. In another embodiment, the present disclosure provides an oligonucleotide that includes a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs:l-4, or a complement thereof, which oligonucleotide has 100 or fewer nucleotides. Generally, these oligonucleotides may be primer nucleic acids, probe nucleic acids, or the like in these embodiments. In certain of these embodiments, the oligonucleotides have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.). In some embodiments, the oligonucleotides comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability relative to unmodified nucleotides. Optionally, the oligonucleotides comprise at least one label and optionally at least one quencher moiety. In some embodiments, the oligonucleotides include at least one conservatively modified variation. “Conservatively modified variations” or, simply, “conservative variations” of a particular nucleic acid sequence refers to those nucleic acids, which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. One of skill in the art will recognize that individual substitutions, deletions or additions which alter, add or delete a single nucleotide or a small percentage of nucleotides (typically less than 5%, more typically less than 4%, 2% or 1%) in an encoded sequence are “conservatively modified variations” where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.

[0020] In one aspect, amplification can employ a polymerase enzyme having 5’ to 3’ nuclease activity. Thus, the donor fluorescent moiety and the acceptor moiety, e.g., a quencher, may be within no more than 5 to 20 nucleotides (e.g., within 7 or 10 nucleotides) of each other along the length of the probe. In another aspect, the probe includes a nucleic acid sequence that permits secondary structure formation. Such secondary structure formation may result in spatial proximity between the first and second fluorescent moiety. According to this method, the second fluorescent moiety on the probe can be a quencher.

[0021] The present disclosure also provides for methods of detecting the presence or absence of template / target nucleic acids. Such methods generally include performing at least one cycling step, which includes an amplifying step and a dyebinding step. Typically, the amplifying step includes contacting the sample with a plurality of pairs of oligonucleotide primers to produce one or more amplification products if a nucleic acid molecule is present in the sample, and the dye-binding step includes contacting the amplification product with a double-stranded DNA binding dye. Such methods also include detecting the presence or absence of binding of the double-stranded DNA binding dye into the amplification product, wherein the presence of binding is indicative of the presence of template / target nucleic acids in the sample, and wherein the absence of binding is indicative of the absence of template / target nucleic acids in the sample. A representative double-stranded DNA binding dye is ethidium bromide. Other nucleic acid-binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YOYO® and SYBR® Green. In addition, such methods also can include determining the melting temperature between the amplification product and the double-stranded DNA binding dye, wherein the melting temperature confirms the presence or absence of the template / target nucleic acid.

[0022] In a further embodiment, a kit for detecting and / or quantitating one or more nucleic acids of template / target nucleic acids is provided. The kit can include one or more sets of primers specific for amplification of the gene target; and one or more detectable oligonucleotide probes specific for detection of the amplification products.

[0023] In one aspect, the kit can include probes already labeled with donor and corresponding acceptor moieties, e.g., another fluorescent moiety or a dark quencher, or can include fluorophoric moieties for labeling the probes. The kit can also include nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit can also include a package insert and instructions for using the primers, probes, and fluorophoric moieties to detect the presence or absence of the template / target nucleic acid.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0025] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] 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. FIG. 1A shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the short version of the primer-binding site for the long forward primer (SEQ ID NO:2). FIG. IB shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the long version of the primer-binding site for the long forward primer (SEQ ID NO:3).

[0028] FIG. 2A shows alignment of an exemplary long reverse primer (SEQ ID NO:4), and the short version of the primer-binding site for the long reverse primer (SEQ ID NO:5). FIG. 2B shows alignment of an exemplary long forward primer (SEQ ID NO:4), and the long version of the primer-binding site for the long reverse primer (SEQ ID NO:6).

[0029] FIG. 3A shows a first PCR cycle, using a long forward primer (LFP) annealing to a short forward primer-binding site (SFPBS) of a template nucleic acid / insert that also contains a template reverse primer-binding site (TRPBS). Subsequent primer extension of the long forward primer (LRP) results in generation of an oligonucleotide with the long forward primer (having a 5’ unmatched region and a 3’ unmatched region), a reverse complement of the original template nucleic acid / insert, and a short reverse primer-binding site (SRPBS).

[0030] FIG. 3B shows a second PCR cycle, using a long reverse primer (LRP) annealing to a short reverse primer-binding site (SRPBS) of the primer extension product from the first PCR cycle, which also contains a long forward primer (LFP). Subsequent primer extension of the long reverse primer (LRP) results in the generation of an oligonucleotide with the long reverse primer (LRP), the template nucleic acid / insert, and a long forward primer-binding site (LFPBS).

[0031] FIG. 3C shows a third PCR cycle, using a long forward primer (LFP) annealing to a long forward primer-binding site (LFPBS) of the primer extension product from the second PCR cycle, which also contains a long reverse primer (LRP). Subsequent primer extension of the long forward primer (LFP) results in the generation of an oligonucleotide with the long forward primer (LFP), the reverse complement of the template nucleic acid / insert, and a long reverse primer-binding site (LRPBS).

[0032] FIG. 4 shows additional priming that may occur when using long forward primers (LFP) and / or long reverse primers (LRP). In such cases, the non- hybridizing / unmatched region of the long forward primer can act as a template, and the template / PCR insert can act as a primer. Naturally, in such instances, the long forward primer (LFP) also acts as a primer and extends (as shown in FIG. 3A). Thus, FIG. 6 shows simultaneous priming and extension reactions in two directions.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] Described herein, is a method for amplifying a template / target nucleic acid, by using long forward primers and long reverse primers. Use of these long forward primers and long reverse primers overcome the situation or problem of when the primer-binding region is short (e.g., around 15 bases). In such cases, short primerbinding sites then result in lower inherent annealing temperatures (< 55°C), which make it challenging for conventional primers to prime. After one or more primer extension events with long forward primers and long reverse primers, the primerbinding site is increased and is no longer short, which results in higher annealing temperatures (> 55°C), which improves PCR yields.

[0035] In particular, the long forward primers and the long reverse primers have a region that is complementary to the target nucleic acid (z.e., matched), and a region that is not complementary to the target nucleic acid (ie., unmatched). Therefore, the long forward primers and the long reverse primers are considered to be “long” or “longer,” by virtue of having regions that do not hybridize / anneal to the target nucleic acid, which appear as single-stranded overhang regions. During the first few cycles of PCR, where the full length of the long forward primers and the long reverse primers do not anneal completely with the template / target nucleic acid, the melting temperature (Tm) is lower. During later cycles of PCR, the template / target nucleic acid will lengthen, due to addition of the unmatched regions of the long forward primer and the long reverse primer. Accordingly, during later cycles of PCR, the length of the primer-binding region matches the length of the long forward / reverse primers, and the Tmis higher, allowing for more efficient cycling and PCR.

[0036] In particular, this method is useful for amplification of template / target nucleic acid and / or for detecting a template / target nucleic acid in a sample by nucleic acid amplification, thereby providing a method for rapidly, accurately, reliably, specifically, and sensitively detecting and / or quantitating a template / target nucleic acid. Primers (where the oligonucleotide primers are long forward primers and long reverse primers) and / or probes for amplification and / or detecting of a template / target nucleic acid are provided, as are articles of manufacture or kits containing such primers (where the oligonucleotide primers are long forward primers and long reverse primers) and / or probes.

[0037] The present disclosure includes oligonucleotide primers (where the oligonucleotide primers are long forward primers and long reverse primers) and / or fluorescent labeled hydrolysis probes that hybridize to template / target nucleic acid.

[0038] The disclosed methods may include performing at least one cycling step that includes amplifying one or more portions of the nucleic acid molecule gene target / template using one or more pairs of long forward primers and long reverse primers (where the oligonucleotide primers are long forward primers and long reverse primers). Each of the discussed long primers anneals to a template / target nucleic acid, such that at least a portion of each amplification product contains nucleic acid sequence corresponding to the template / target nucleic acid. Each cycling step includes an amplification step, and a hybridization step.

[0039] As used herein, the term “amplifying” refers to the process of synthesizing nucleic acid molecules that are complementary to one or both strands of a template nucleic acid molecule (e.g., nucleic acid molecules from the Plasmodium genome). Amplifying a nucleic acid molecule typically includes denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature that is below the melting temperatures of the primers, and enzymatically elongating from the primers to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate buffer and / or co-factors for optimal activity of the polymerase enzyme (e.g., MgCh and / or KC1).

[0040] The term “primer” as used herein is known to those skilled in the art and refers to oligomeric compounds, primarily to oligonucleotides but also to modified oligonucleotides that are able to “prime” DNA synthesis by a template-dependent DNA polymerase, z.e., the 3’-end of the, e.g., oligonucleotide provides a free 3’-OH group where further "nucleotides" may be attached by a template-dependent DNA polymerase establishing 3’ to 5’ phosphodiester linkage whereby deoxynucleoside triphosphates are used and whereby pyrophosphate is released.

[0041] The term “hybridizing” refers to the annealing of one or more probes to an amplification product. “Hybridization conditions” typically include a temperature that is below the melting temperature of the probes but that avoids non-specific hybridization of the probes.

[0042] The term “5’ to 3’ nuclease activity” refers to an activity of a nucleic acid polymerase, typically associated with the nucleic acid strand synthesis, whereby nucleotides are removed from the 5’ end of nucleic acid strand.

[0043] The term “thermostable polymerase” refers to a polymerase enzyme that is heat stable, z.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of doublestranded template nucleic acids. Generally, the synthesis is initiated at the 3’ end of each primer and proceeds in the 5’ to 3’ direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished, if necessary.

[0044] The term “complement thereof’ refers to nucleic acid that is both the same length as, and exactly complementary to, a given nucleic acid.

[0045] The term “extension” or “elongation” when used with respect to nucleic acids refers to when additional nucleotides (or other analogous molecules) are incorporated into the nucleic acids. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase that typically adds nucleotides at the 3’ terminal end of a nucleic acid.

[0046] The terms “identical” or percent “identity” in the context of two or more nucleic acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet. 3:266-272, Madden et al. (1996) “Applications of network BLAST server” Meth. Enzymol. 266: 131-141, Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res. 25:3389-3402, and Zhang etal. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation” Genome Res. 7:649-656, which are each incorporated herein by reference.

[0047] A “modified nucleotide” in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, e.g., a t-butyl benzyl, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5- propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a 7- deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2’-0-methyl ribo-U, 2’-0-methyl ribo-C, an N4-ethyl-dC, an N6- methyl-dA, a 5-propynyl dU, a 5-propynyl dC, 7-deaza-deoxy guanosine (deaza G (u-deaza)) and the like. Many other modified nucleotides that can be substituted in the oligonucleotides are referred to herein or are otherwise known in the art. In certain embodiments, modified nucleotide substitutions modify melting temperatures (Tm) of the oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides. To further illustrate, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation or the like), increase the yield of an intended target amplicon, and / or the like in some embodiments. Examples of these types of nucleic acid modifications are described in, e.g., U.S. Patent No. 6,001,611, which is incorporated herein by reference. Other modified nucleotide substitutions may alter the stability of the oligonucleotide, or provide other desirable features. Amplification and / or Detection of Template / Tar et Nucleic Acid

[0048] The present disclosure provides methods to amplify a template / target nucleic acid. Specifically, long primers and / or long primers and probes to amplify and detect and / or quantitate template and / or target nucleic acids are provided by the embodiments in the present disclosure.

[0049] For amplification and / or detection and / or quantitation template / target nucleic acids, long primers and / or long primers and probes to amplify and detect / quantitate the template / target nucleic acids are provided. Long primer nucleic acids other than those exemplified herein can also be used to amplify template / target nucleic acid. For example, functional variants can be evaluated for specificity and / or sensitivity by those of skill in the art using routine methods. Representative functional variants can include, e.g., one or more deletions, insertions, and / or substitutions in the long primer nucleic acids disclosed herein.

[0050] More specifically, embodiments of the oligonucleotides, a substantially identical variant thereof in which the variant has at least, e.g., 80%, 90%, or 95% sequence identity to one of the sequences of long primers.

[0051] In one embodiment, the above-described sets of long primers and / or long primers and probes are used in order to provide for amplification and / or detection of a template / target nucleic acid. In one embodiment, the above described sets of long primers and / or long primers and probes are used in order to provide for amplification and / or detection of a template / target nucleic acid.

[0052] In one embodiment, variants of the long primers and / or the long primers and probe may be employed. The variants may vary from sequences by one or more nucleotide additions, deletions or substitutions such as one or more nucleotide additions, deletions or substitutions at the 5’ end and / or the 3’ end of a sequence. As detailed above, a long primer and / or probe may be chemically modified, z.e., a long primer and / or probe may comprise a modified nucleotide or a non-nucleotide compound. A probe (or a long primer) is then a modified oligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differ from a natural “nucleotide” by some modification but still consist of a base or base-like compound, a pentofuranosyl sugar or a pentofuranosyl sugar-like compound, a phosphate portion or phosphate-like portion, or combinations thereof. For example, a “label” may be attached to the base portion of a “nucleotide” whereby a “modified nucleotide” is obtained. A natural base in a “nucleotide” may also be replaced by, e.g., a 7- desazapurine whereby a “modified nucleotide” is obtained as well. The terms “modified nucleotide” or “nucleotide analog” are used interchangeably in the present application. A “modified nucleoside” (or “nucleoside analog”) differs from a natural nucleoside by some modification in the manner as outlined above for a “modified nucleotide” (or a “nucleotide analog”).

[0053] Oligonucleotides including modified oligonucleotides and oligonucleotide analogs that detect and amplify a given template / target nucleic acid can be designed using, for example, a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features when designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (z.e., the primers need to be long enough to anneal with sequence-specificity and to initiate synthesis but not so long that fidelity is reduced during oligonucleotide synthesis).

[0054] In addition to a set of long primers, the methods may use one or more probes in order to detect and amplify a template / target nucleic acid. The term “probe” refers to synthetically or biologically produced nucleic acids (DNA or RNA), which by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies specifically (z.e., preferentially) to “target nucleic acids”, in the present case to a Plasmodium (target) nucleic acid. A “probe” can be referred to as a “detection probe” meaning that it detects the target nucleic acid.

[0055] In some embodiments, the described probes can be labeled with at least one fluorescent label. In one embodiment, the probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher.

[0056] Designing oligonucleotides to be used as probes can be performed in a manner similar to the design of primers. Embodiments may use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probe(s) use may comprise at least one label and / or at least one quencher moiety. As with the primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15 to 40 (e.g., 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, or 40) nucleotides in length.

[0057] Constructs can include vectors each containing one of long primers and / or probes nucleic acid molecules. Constructs can be used, for example, as control template nucleic acid molecules. Vectors suitable for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art.

[0058] Constructs suitable for use in the methods typically include long primers and / or probes for amplification and / or detection of a template / target nucleic acid, sequences encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting desired constructs and / or transformants, and an origin of replication. The choice of vector systems usually depends upon several factors, including, but not limited to, the choice of host cells, replication efficiency, selectability, inducibility, and the ease of recovery.

[0059] Constructs containing long primers and / or probes for amplification and / or detection of a template / target nucleic acid, can be propagated in a host cell. As used herein, the term host cell is meant to include prokaryotes and eukaryotes such as yeast, plant and animal cells. Prokaryotic hosts may include E. coh. Salmonella typhimurium, Serratia marcescens. and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae. S. pombe. Pichia pasloris. mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. A construct can be introduced into a host cell using any of the techniques commonly known to those of ordinary skill in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral -mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be delivered directly to cells (see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466). Polymerase Chain Reaction (PCR)

[0060] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides capable of acting as points of initiation of nucleic acid synthesis. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer is preferably single-stranded for maximum efficiency in amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating.

[0061] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90°C to about 105°C for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5 min).

[0062] If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence. The temperature for annealing is usually from about 35°C to about 65°C (e.g., about 40°C to about 60°C; about 45°C to about 50°C). Annealing times can be from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primer to generate products complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40°C to about 80°C (e.g., about 50°C to about 70°C; about 60°C). Extension times can be from about 10 sec to about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about 3 min; about 1 min 30 sec to about 2 min).

[0063] The genome of a retrovirus or RNA virus is comprised of a ribonucleic acid, i.e., RNA. In such case, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) via the action of the enzyme reverse transcriptase. Reverse transcriptases use an RNA template and a short primer complementary to the 3’ end of the RNA to direct synthesis of the first strand cDNA, which can then be used directly as a template for polymerase chain reaction.

[0064] PCR assays can employ Plasmodium nucleic acid, and / or primers / probes that amplify and / or detect Plasmodium, such as RNA or DNA (cDNA). The template nucleic acid need not be purified; it may be a minor fraction of a complex mixture, such as Plasmodium nucleic acid contained in human cells. Plasmodium nucleic acid molecules, and / or primers / probes that amplify and / or detect Plasmodium may be extracted from a biological sample by routine techniques such as those described in Diagnostic Molecular Microbiology. Principles and Applications (Persing, el al. (eds), 1993, American Society for Microbiology, Washington D.C.). Nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0065] The oligonucleotide long primers are combined with PCR reagents under reaction conditions that induce primer extension. For example, chain extension reactions generally include 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 15 mM MgCh, 0.001% (w / v) gelatin, 0.5-1.0 pg denatured template DNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO). The reactions usually contain 150 to 320 pM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0066] The newly-synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the template / target nucleic acid molecules. The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (z.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficient for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.

[0067] Fluorescence Resonance Energy Transfer (FRET)

[0068] FRET technology (see, for example, U.S. Patent Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on a concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer takes place between the two fluorescent moieties that can be visualized or otherwise detected and / or quantitated. The donor typically transfers the energy to the acceptor when the donor is excited by light radiation with a suitable wavelength. The acceptor typically re-emits the transferred energy in the form of light radiation with a different wavelength. In certain systems, non-fluorescent energy can be transferred between donor and acceptor moieties, by way of biomolecules that include substantially non-fluorescent donor moieties (see, for example, US Patent. No. 7,741,467).

[0069] In one example, an oligonucleotide probe can contain a donor fluorescent moiety or dye (e.g., HEX or FAM) and a corresponding quencher (e.g, BlackHole Quencher™ (BHQ) (such as BHQ-2)), which may or not be fluorescent, and which dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties such that fluorescent emission from the donor fluorescent moiety is quenched the acceptor moiety. During an extension step of a polymerase chain reaction, a probe bound to an amplification product is cleaved by the 5’ to 3’ nuclease activity of, e.g., a Taq Polymerase such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described in, e.g., U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quencher™ (BHQ) (such as BHQ2), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™, (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ- 650), (Berry & Assoc., Dexter, Mich.).

[0070] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to an amplification product at particular positions determined by the complementarity of the oligonucleotide probes to the Plasmodium target nucleic acid sequence. Upon hybridization of the oligonucleotide probes to the amplification product nucleic acid at the appropriate positions, a FRET signal is generated. Hybridization temperatures can range from about 35°C. to about 65°C. for about 10 sec to about 1 min.

[0071] Fluorescent analysis can be carried out using, for example, a photon counting epifluorescent microscope system (containing the appropriate dichroic mirror and filters for monitoring fluorescent emission at the particular range), a photon counting photomultiplier system, or a fluorimeter. Excitation to initiate energy transfer, or to allow direct detection of a fluorophore, can be carried out with an argon ion laser, a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation in the desired range.

[0072] As used herein with respect to donor and corresponding acceptor moieties “corresponding” refers to an acceptor fluorescent moiety or a dark quencher having an absorbance spectrum that overlaps the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Accordingly, efficient non-radiative energy transfer can be produced therebetween.

[0073] Fluorescent donor and corresponding acceptor moieties are generally chosen for (a) high efficiency Foerster energy transfer; (b) a large final Stokes shift (>100 nm); (c) shift of the emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) shift of the emission to a higher wavelength than the Raman water fluorescent emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be chosen that has its excitation maximum near a laser line (for example, helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, a high quantum yield, and a good overlap of its fluorescent emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be chosen that has a high extinction coefficient, a high quantum yield, a good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red part of the visible spectrum (>600 nm).

[0074] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, Lucifer Yellow VS, 4- acetamido-4’-isothio-cyanatostilbene-2, 2’ -disulfonic acid, 7-diethylamino-3-(4’- isothiocyanatophenyl)-4-methylcoumarin, succinimdyl 1 -pyrenebutyrate, and 4- acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid derivatives. Representative acceptor fluorescent moieties, depending upon the donor fluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of Lanthanide ions (e.g., Europium, or Terbium). Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0075] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important, as the linker arms will affect the distance between the donor and acceptor fluorescent moieties. The length of a linker arm can be the distance in Angstroms (A) from the nucleotide base to the fluorescent moiety. In general, a linker arm is from about 10 A to about 25 A. The linker arm may be of the kind described in International Patent Publication No. WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching linker arms to a particular nucleotide base, and also for attaching fluorescent moieties to a linker arm. An acceptor fluorescent moiety, such as an LC Red 640, can be combined with an oligonucleotide that contains an amino linker (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, VA)) to produce, for example, LC Red 640-labeled oligonucleotide. Frequently used linkers to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, for example, fluorescein- CPG's from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3’-amino-CPGs that require coupling of a fluorescein-NHS-ester after oligonucleotide synthesis.

[0076] Detection of Amplified Product (Amplicon)

[0077] The present disclosure provides methods for amplifying and detecting the presence or absence of template / target nucleic acid. Methods provided avoid problems of sample contamination, false negatives, and false positives. The methods include performing at least one cycling step that includes amplifying a portion of target nucleic acid molecules using one or more pairs of long primers, and a FRET detecting step. Multiple cycling steps are performed, preferably in a thermocycler. Methods can be performed using the long primers and probes to amplify and / or detect a template / target nucleic acid.

[0078] As described herein, amplification products can be detected using labeled hybridization probes that take advantage of FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of an amplification product, and hence, the presence or absence of the template / target nucleic acid. TaqMan® technology utilizes one single- stranded hybridization probe labeled with, e.g., one fluorescent moiety or dye (e.g., HEX or FAM) and one quencher (e.g., BHQ-2), which may or may not be fluorescent. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety or a dark quencher according to the principles of FRET. The second moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (z.e., the amplification product) and is degraded by the 5’ to 3’ nuclease activity of, e.g., the Taq Polymerase during the subsequent elongation phase. As a result, the fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected. By way of example, an ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology, and is suitable for performing the methods described herein for amplifying and / or detecting a template / target nucleic acid.

[0079] Molecular beacons in conjunction with FRET can also be used to detect the presence of an amplification product using the real-time PCR methods. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g., a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the target nucleic acids (i.e., amplification products), the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

[0080] Another common format of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and are generally designed to hybridize in close proximity to each other in a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, for example, fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, the fluorescein transfers its energy to an acceptor fluorescent moiety such as LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of Plasmodium genomes). If amplification of Plasmodium target nucleic acid occurs and an amplification product is produced, the step of hybridizing results in a detectable signal based upon FRET between the members of the pair of probes.

[0081] Generally, the presence of FRET indicates the presence of Plasmodium in the sample, and the absence of FRET indicates the absence of Plasmodium in the sample. Inadequate specimen collection, transportation delays, inappropriate transportation conditions, or use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and / or accuracy of a test result, however.

[0082] Representative biological samples that can be used in practicing the methods include, but are not limited to whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs, wound swabs, blood cultures, skin, and soft tissue infections. Collection and storage methods of biological samples are known to those of skill in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release Plasmodium nucleic acid or in some cases, the biological sample can be contacted directly with the PCR reaction components and the appropriate oligonucleotides. In some instances, the biological sample is whole blood. When whole blood is typically collected, it is often collected in vessels containing anticoagulants, such as heparin, citrate, or EDTA, which enables the whole blood to be stored at suitable temperatures. However, under such conditions, the nucleic acids within the whole blood undergo considerable amount of degradation. Therefore, it may be advantageous to collect the blood in a reagent that will lyse, denature, and stabilize whole blood components, including nucleic acids, such as a nucleic acidstabilizing solution. In such cases, the nucleic acids can be better preserved and stabilized for subsequent isolation and analysis, such as by nucleic acid test, such as PCR. Such nucleic acid-stabilizing solution are well known in the art, including, but not limited to, cobas PCR media, which contains 4.2 M guanadinium salt (GuHCl) and 50 mM Tris, at a pH of 7.5.

[0083] The sample can be collected by any method or device designed to adequately hold and store the sample prior to analysis. Such methods and devices are well known in the art. In the case that the sample is a biological sample, such as whole blood, the method or device may include a blood collection vessel. Such a blood collection vessel is well known in the art, and may include, for example, a blood collection tube. In many cases, it may be advantageous to use a blood collection tube wherein the blood collection vessel is under pressure in the space intended for sample uptake, such as a blood vessel with an evacuated chamber, such as a vacutainer blood collection tube. Such blood collection tubes with an evacuated chamber, such as a vacutainer blood collection tube are well known in the art. It may further be advantageous to collect the blood in a blood collection vessel, with or without an evacuated chamber, that contains within it, a solution that will lyse, denature, and stabilize whole blood components, including nucleic acids, such as a nucleic acid-stabilizing solution, such that the whole blood being drawn immediately contacts the nucleic acid-stabilizing solution in the blood collection vessel.

[0084] Melting curve analysis is an additional step that can be included in a cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA duplexes have separated into single strands. The melting temperature of a DNA depends primarily upon its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than those having an abundance of A and T nucleotides. By detecting the temperature at which signal is lost, the melting temperature of probes can be determined. Similarly, by detecting the temperature at which signal is generated, the annealing temperature of probes can be determined. The melting temperature(s) of the Plasmodium probes from the Plasmodium amplification products can confirm the presence or absence of Plasmodium in the sample.

[0085] Within each thermocycler run, control samples can be cycled as well. Positive control samples can amplify target nucleic acid control template (other than described amplification products of target genes) using, for example, control primers and control probes. Positive control samples can also amplify, for example, a plasmid construct containing the target nucleic acid molecules. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run side-by-side with the patients' samples using the same primers and probe as used for detection of the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run can also include a negative control that, for example, lacks target template DNA. Negative control can measure contamination. This ensures that the system and reagents would not give rise to a false positive signal. Therefore, control reactions can readily determine, for example, the ability of primers to anneal with sequencespecificity and to initiate elongation, as well as the ability of probes to hybridize with sequence-specificity and for FRET to occur.

[0086] In an embodiment, the methods include steps to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patent Nos. 5,035,996, 5,683,896 and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.

[0087] Conventional PCR methods in conjunction with FRET technology can be used to practice the methods. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR as used in the LightCycler® technology: International Patent Publication Nos. WO 97 / 46707, WO 97 / 46714, and WO 97 / 46712.

[0088] The LightCycler® can be operated using a PC workstation and can utilize a Windows NT operating system. Signals from the samples are obtained as the machine positions the capillaries sequentially over the optical unit. The software can display the fluorescence signals in real-time immediately after each measurement. Fluorescent acquisition time is 10-100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence vs. cycle number can be continually updated for all samples. The data generated can be stored for further analysis.

[0089] As an alternative to FRET, an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A doublestranded DNA binding dye such as a nucleic acid intercalating dye also can be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product. One of skill in the art would appreciate that other nucleic acid- or signalamplification methods may also be employed. Examples of such methods include, without limitation, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), selfsustained sequence replication (3 SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP 2).

[0090] It is understood that the embodiments of the present disclosure are not limited by the configuration of one or more commercially available instruments.

[0091] Articles of Manufacture / Kits

[0092] Embodiments of the present disclosure further provide for articles of manufacture or kits to amplifying and / or detecting template / target nucleic acid. An article of manufacture can include long primers and / or long primers and probes used to amplify and / or detect a template / target nucleic acid, together with suitable packaging materials. Representative long primers and probes for amplification and / or detection of template / target nucleic acid are capable of hybridizing to template / target nucleic acid. In addition, the kits may also include suitably packaged reagents and materials needed for DNA immobilization, hybridization, and detection, such solid supports, buffers, enzymes, and DNA standards. Methods of designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to template / target nucleic acid.

[0093] Articles of manufacture can also include one or more fluorescent moieties for labeling the probes or, alternatively, the probes supplied with the kit can be labeled. For example, an article of manufacture may include a donor and / or an acceptor fluorescent moiety for labeling the Plasmodium probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0094] Articles of manufacture can also contain a package insert or package label having instructions thereon for using the long primers and / or the long primers and probes to amplify and / or detect a template / target nucleic acid. Articles of manufacture may additionally include reagents for carrying out the methods disclosed herein (e.g., buffers, polymerase enzymes, co-factors, or agents to prevent contamination). Such reagents may be specific for one of the commercially available instruments described herein.

[0095] Embodiments of the present disclosure also provide for a set of long primers and one or more detectable probes for the amplification and / or detection of template / target nucleic acids.

[0096] Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0097] EXAMPLES

[0098] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0099] Example 1 : Design of Long Forward Primers and Long Reverse Primers

[0100] This disclosure provides for long forward primers and long reverse primers for amplification of a template / target nucleic acid, where the template / target nucleic acid has a short primer-binding region. Long forward primers and long reverse primers were designed to have two regions: (1) a region for annealing / hybri dizing to a region of the template / target nucleic acid; and (2) a region that does not anneal / hybridize to a region of the template / target nucleic acid region. Put another way, the long primers are longer than the length of the primer-binding site (z.e., the site upon which the long primers are to anneal / hybridize). Table 1: Exemplary long primers and the, relatively, shorter primer-binding sites.

[0101] Table 1, above, shows the sequences of exemplary long forward primers (SEQ ID NO: 1), the correspondingly (short) binding-region for the long forward primer (SEQ ID NO:2), long reverse primers (SEQ ID NO:3), and the correspondingly (short) binding-region for the long reverse primer (SEQ ID NON).

[0102] FIG. 1A shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the short version of the primer-binding site for the long forward primer (SEQ ID NO:2). As can be seen in FIG. 1A, the 3 ’ region of the long forward primer hybridizes / aligns with the entirety of the short version of the primer-binding site for the long forward primer (SEQ ID NO:2), and the 5’ region of the long forward primer is not hybridized and not aligned, and therefore appears as a single-stranded tail (of 5 bases, ACTCG). During the first few PCR cycles alignment of the long forward primer will contain matched and mismatched regions, as depicted in FIG. 1A, and the calculated Tmwith this alignment is 51.1 °C. By contrast, FIG. IB shows alignment of an exemplary long forward primer (SEQ ID NO: 1), and the long version of the primer-binding site for the long forward primer (SEQ ID NON). As can be seen in FIG. IB, the long forward primer (SEQ ID NO: 1) hybridizes / aligns entirely with the long version of the primer-binding site for the long forward primer (SEQ ID NON) (z.e., the singled-stranded 5’ tail from FIG. 1A no longer exists). During the later cycles of PCR, alignment of the long forward primer (SEQ ID NON) will be across the entirety of the long primer-binding region (SEQ ID NON), as depicted in FIG. IB, and the calculated Tmwith this alignment is 61.1°C. Therefore, over time, use of the long primers in PCR cycles results in increased Tm, which results in better annealing and improved PCR yields.

[0103] FIG. 2A shows alignment of an exemplary long reverse primer (SEQ ID NON), and the short version of the primer-binding site for the long reverse primer (SEQ ID NON). As can be seen in FIG. 2A, the 3’ region of the long reverse primer hybridizes / aligns with the entirety of the short version of the primer-binding site for the long reverse primer (SEQ ID NON), and the 5’ region of the long reverse primer is not hybridized and not aligned, and therefore appears as a single-stranded tail (of 5 bases, ATGCC). During the first few PCR cycles alignment of the long reverse primer will contain matched and mismatched regions, as depicted in FIG. 2A, and the calculated Tmwith this alignment is 46.9°C. By contrast, FIG. 2B shows alignment of an exemplary long reverse primer (SEQ ID NO:4), and the long version of the primer-binding site for the long forward primer (SEQ ID NO:6). As can be seen in FIG. 2B, the long reverse primer (SEQ ID NO: 1) hybridizes / aligns entirely with the long version of the primer-binding site for the long reverse primer (SEQ ID NO:6) (z.e., the single-stranded 5’ tail from FIG. 2A no longer exists). During the later cycles of PCR, alignment of the long reverse primer (SEQ ID NO: 1) will be across the entirety of the long primer-binding region (SEQ ID NO:3), as depicted in FIG. 2B, and the calculated Tmwith this alignment is 58.4°C. Therefore, over time, use of the long primers in PCR cycles results in increased Tm, which results in better annealing and improved PCR yields.

[0104] Thus, these exemplary sequences for long forward primers (SEQ ID NO: 1) and long reverse primers (SEQ ID NO:4) are, over time, effective at increasing Tm, exhibit better annealing, and improve overall PCR yields.

[0105] Example 2: PCR Cycles of Long Forward Primers and Long Reverse Primers

[0106] This disclosure provides for long forward primers and long reverse primers for amplification of a tempi ate / target nucleic acid, where the template / target nucleic acid has a short primer-binding region. Long forward primers and long reverse primers were designed to have two regions: (1) a region for annealing / hybri dizing to a region of the template / target nucleic acid; and (2) a region that does not anneal / hybridize to a region of the template / target nucleic acid region. Put another way, the long primers are longer than the length of the primer-binding site (z.e., the site upon which the long primers are to anneal / hybridize).

[0107] In the first PCR cycle, depicted in FIG. 3A, the template for primer extension is the original template / insert (shown in black), and including a template reverse primer-binding site (TRPBS) on its 5’ end (shown in purple) and a short forward primer-binding site (SFPBS) on its 3’ end (shown in light blue). During the first PCR cycle, the long forward primer (LFP) (shown in light blue and dark blue) acts as the primer, upon which extension will occur, and the long forward primer (LFP) contains the following 2 regions: (1) a region for annealing / hybridizing to a primer- binding site (depicted in light blue), and (2) a region that does not anneal / hybridize to a primer-binding site (depicted in dark blue). The long forward primer (LFP) anneals, in part, to the short forward primer-binding site (SFPBS) (shown in light blue), which is < 15 nucleotides, as shown in FIG. 3A. A region of the 5’ end of the long forward primer (LFP) does not anneal / hybridize to any portion of the short forward primer-binding site (SFPBS), and appears as a single-stranded 5’ tail (shown in dark blue), as shown in FIG. 3A. Here, the Tmof annealing is lower, but primer concentration is high, which allows for annealing to take place (albeit, with lower efficiency for this cycle). Primer extension is then initiated and completed, generating a first primer extension product, depicted as a dashed line (black and purple), as shown in FIG. 3A. The first primer extension product includes the long forward primer (LFP), as well as the reverse complement of the original template / insert (shown as a dashed black line), and a short reverse primer-binding site (SRPBS) (shown as a dashed purple line), as shown in FIG. 3A. That is, during the first primer extension reaction, the template / insert is copied into its reverse complement (depicted as a dashed black line), and the template reverse primerbinding site (TRPBS) is copied into the short reverse primer-binding site (SRPBS) (depicted as a dashed purple line), as shown in FIG. 3A. Due to the short forward primer-binding site (SFPBS), the Tmof annealing is lower, and annealing / priming occurs at a lower efficiency for this first cycle.

[0108] In the second PCR cycle, depicted in FIG. 3B, the template for primer extension is first primer extension product, which was generated during the first PCR cycle, which is shown in FIG. 3A. That is, the template for primer extension in the second PCR cycle includes the reverse complement of the original template / insert (shown as a dashed black line), and including a long forward primer (LFP) on its 5’ end (shown in dark blue and light blue) and a short reverse primer-binding site (SRPBS) on its 3’ end (shown as a dashed purple line). During the second PCR cycle, the long reverse primer (LRP) (shown in purple and red) acts as the primer, upon which extension will occur, and the long reverse primer (LRP) contains the following 2 regions: (1) a region for annealing / hybridizing to a primer-binding site (depicted in purple), and (2) a region that does not anneal / hybridize to a primerbinding site (depicted in red). During the second PCR cycle, the long reverse primer (LRP) anneals, in part, to the short reverse primer-binding site (SRPBS), which is < 15 nucleotides, and shown as a dashed purple line, as shown in FIG. 3B. A region of the 5’ end of the long reverse primer (LRP) does not anneal / hybridize to any portion of the short reverse primer-binding site (SRPBS), and appears as a singlestranded 5’ tail (shown in red), as shown in FIG. 3B. Here, the Tmof annealing is lower, but primer concentration is high, which allows for annealing to take place (albeit, with lower efficiency for this second cycle). Primer extension is then initiated and completed, generating a second primer extension product, depicted as a dotted line (black, light blue, and dark blue), as shown in FIG. 3B. The second primer extension product includes the long reverse primer (LRP), as well as the reverse complement of the template / insert (shown as a dotted black line), and a long forward primer-binding site (LFPBS) (shown as a dotted light blue and dark blue line), as shown in FIG. 3B. That is, during the second primer extension reaction, the template / insert is copied into its reverse complement (depicted as a dotted black line), and the long forward primer-binding site (LFPBS) is generated from the long forward primer (LFP) in the template (depicted as a dotted light and dark blue line), as shown in FIG. 3B. Due to the short reverse primer-binding site (SRPBS), the Tmof annealing is lower, and annealing / priming occurs at a lower efficiency for this second cycle. The primer extension product generated after the second PCR cycle yields a template having an extended version of both of the original short forward primer-binding site (SFPBS) and the original short reverse primer-binding site (SRPBS). That is, the primer extension product generated after the second PCR contains, now, longer primer-binding sites for the long forward primer (LFP) and the long reverse primer (LRP), thereby eliminating the short primer-binding sites (short forward primer-binding site (SFPBS) and short reverse primer-binding site (SRPBS)). The short primer-binding sites (short forward primer-binding site (SFPBS) and short reverse primer-binding site (SRPBS)) have been replaced by long primer-binding sites (long forward primer-binding site (LFPBS) and long reverse primer-binding site (LRPBS)). Thus, the problem of short-primer binding sites is eliminated by the conclusion of the second PCR cycle, as can be seen in FIG. 3B.

[0109] In the third PCR cycle, depicted in FIG. 3C, the template for primer extension is second primer extension product, which was generated during the second PCR cycle, which is shown in FIG. 3B. That is, the template for primer extension in the third PCR cycle includes the template / insert (shown as a dotted black line), and including a long reverse primer (LRP) on its 5’ end (shown in red and purple) and a long forward primer-binding site (LFPBS) on its 3’ end (shown as a dotted light blue and dark blue line). During the third PCR cycle, the long forward primer (LFP) (shown in dark blue and light blue) acts as the primer, upon which extension will occur, and the long forward primer (LFP), and the entire length of the long forward primer (LFP) will anneal / hybridize to a primer-binding site (z.e., the long forward primer-binding site (LFPBS). During the third PCR cycle, the long forward primer (LFP) anneals / hybridizes completely to the long forward primerbinding site (LFPBS), which is < 15 nucleotides, and shown as a dotted dark blue and light blue line, as shown in FIG. 3C. Because the entire region of the long forward primer (LFP) anneals / hybridizes to the entire region of the long forward primer-binding site (LFPBS), the Tmof annealing is high, and annealing / priming will occur at a higher efficiency for the third cycle, and the subsequent remaining cycles. Primer extension is then initiated and completed, generating a third primer extension product, depicted as a dashed line (black, purple, and red), as shown in FIG. 3C. The third primer extension product includes the long forward primer (LFP), as well as the reverse complement of the template / insert (shown as a dashed black line), and a long reverse primer-binding site (LRPBS) (shown as a dashed purple and red line), as shown in FIG. 3C. That is, during the third primer extension reaction, the template / insert is copied into its reverse complement (depicted as a dashed black line), and the long reverse primer-binding site (LRPBS) is generated from the long reverse primer (LRP) in the template (depicted as a dashed purple and red line), as shown in FIG. 3C. Due to the long forward primer-binding site (LFPBS), the Tmof annealing is higher, and annealing / priming occurs at a higher efficiency for this third cycle, as well as all later cycles (ie., fourth, fifth, sixth, etc.).

[0110] Taken together, FIG. 3A, FIG. 3B, and FIG. 3C show the first few cycles of PCR, where there are short primer-binding sites, flanking a template, and employing long primers. As can be seen in FIG. 3A, FIG. 3B, and FIG. 3C, at the conclusion of the second PCR cycle using long primers, the short primer-binding sites have been replaced by long primer-binding sites. That is, by the conclusion of the second PCR cycle, the short primer-binding sites are gone, replaced by long primer-binding sites, allowing for higher Tm, better annealing and priming, and improved PCR yields of amplicons.

[0111] Example 3 : Additional Priming (Bi-Directional Priming)

[0112] This disclosure provides for long forward primers and long reverse primers for amplification of a tempi ate / target nucleic acid, where the template / target nucleic acid has a short primer-binding region. Long forward primers and long reverse primers were designed to have two regions: (1) a region for annealing / hybri dizing to a region of the template / target nucleic acid; and (2) a region that does not anneal / hybridize to a region of the template / target nucleic acid region. Put another way, the long primers are longer than the length of the primer-binding site (z.e., the site upon which the long primers are to anneal / hybridize).

[0113] As described in Example 2, and as shown in FIG. 3A, FIG. 3B, and FIG. 3C, when using a template that has short primer-binding sites, it is demonstrated that priming and extending off of the long forward primers and the long reverse primers eventually results in extension of the once-short primer-binding sites to become long primer-binding sites (z.e., primer-binding sites that match the length of the long primers).

[0114] Additionally, it is possible that the long primers can act as templates, and the template (having short primer-binding sites) acts as a primer. This is depicted in FIG. 4. That is, by employing long primers, it is possible to have priming and primer extension in two directions, simultaneously (simultaneous bi-directional primer extension).

[0115] Priming and Extending Off the Long Primer

[0116] In FIG. 4, the long forward primer (LFP) (shown in dark blue and light blue) anneals to the short forward primer-binding site (SFPBS) (shown in light blue). In this case, the Tmof annealing is lower, but primer concentration is high, and the annealing / priming occurs at lower efficiency. As discussed previously, the long forward primer (LFP) anneals, in part, to the short forward primer-binding site (SFPBS) (shown in light blue), which is < 15 nucleotides, as shown in FIG. 3A, and also FIG. 4. A region of the 5’ end of the long forward primer (LFP) does not anneal / hybridize to any portion of the short forward primer-binding site (SFPBS), and appears as a single-stranded 5’ tail (shown in dark blue), as shown in FIG. 3A, and also FIG. 4. Here, the Tmof annealing is lower, but primer concentration is high, which allows for annealing to take place (albeit, with lower efficiency for this cycle). Primer extension is then initiated and completed, generating a first primer extension product, depicted as a dashed line (black and purple), as shown in FIG. 3A, and also FIG. 4

[0117] Priming and Extending Off the Short Primer-Binding Site

[0118] Additionally, if the 3’ end of the targeted template is extendable (z.e., 3 ’-OH), then priming occurs from the 3’ end of the targeted template, while using the long forward primer (LFP) as a template, as shown in FIG. 4 (depicted by the short forward primer-binding site, in light blue, having an arrowhead at its 3’ end). In this case, the long forward primer (LFP) acts as a template, and the short forward primerbinding site (SFPBS) acts as a primer, and extension can occur off it, resulting in synthesis of the reverse complement of the 5’ tail of the long forward primer (depicted by a dark blue dotted line), as seen in FIG. 4. In such case, the 3’ end of the template extends on the long forward primer (LFP), thereby generating a long forward primer-binding site (LFPBS) as an extension product, as shown in FIG. 4. As a result, subsequent priming with the long forward primer (LFP) on this product will be high efficiency. This type of priming and extension off the short primerbinding site can occur with the long reverse primer (LRP) and the short reverse primer-binding site (SRPBS) as well (not shown).

[0119] Thus, these studies demonstrate that long primers can function as primers, from which extension can originate, and can function as templates themselves.

[0120] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED:

1. A method for amplifying a target nucleic acid, wherein the method comprises the following steps:(a) obtaining one or more forward primers and one or more reverse primers, wherein the forward primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid, wherein the reverse primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid; and(b) performing one or more amplification steps, wherein the amplification step comprises contacting the target nucleic acid with the one or more forward primers and one or more reverse primers, in the presence of a polymerase, to produce one or more amplification products.

2. The method of claim 1, wherein the region of the forward primer that is complementary to the target nucleic acid is < 20 base pairs long.

3. The method of claim 2, wherein the region of the forward primer that is complementary to the target nucleic acid is < 15 base pairs long.

4. The method of claim 3, wherein the region of the forward primer that is complementary to the target nucleic acid is < 10 base pairs long.

5. The method of claim 1, wherein the region of the reverse primer that is complementary to the target nucleic acid is < 20 base pairs long.

6. The method of claim 2, wherein the region of the reverse primer that is complementary to the target nucleic acid is < 15 base pairs long.

7. The method of claim 3, wherein the region of the reverse primer that is complementary to the target nucleic acid is < 10 base pairs long.

8. The method of claim 1, wherein the forward primer is > 17 base pairs long.

9. The method of claim 8, wherein the forward primer is > 20 base pairs long.

10. The method of claim 9, wherein the forward primer is > 25 base pairs long.

11. The method of claim 1, wherein the reverse primer is > 17 base pairs long.

12. The method of claim 11, wherein the reverse primer is > 20 base pairs long.

13. The method of claim 12, wherein the reverse primer is > 25 base pairs long.

14. The method of claim 1, wherein the target nucleic acid is between 110-130 base pairs long.

15. The method of claim 14, wherein the target nucleic acid about 120 base pairs long.

16. A method for detecting a target nucleic acid in a sample, wherein the method comprises the following steps:(a) obtaining one or more forward primers and one or more reverse primers, wherein the forward primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid, wherein the reverse primer comprises: (i) a region that is complementary to the target nucleic acid, and (ii) a region that is not complementary to the target nucleic acid;(b) performing one or more amplification steps, wherein the amplification step comprises contacting the sample with the one or more forward primers and one or more reverse primers, in the presence of a polymerase, to produce one or more amplification products, if the target nucleic acid is present in the sample;(c) performing a hybridization step, wherein the hybridization step comprises contacting one or more probes with the one or more amplification products from step (b); and(d) detecting the presence or absence of the amplification products, wherein the presence of the amplification product is indicative of the presence of the target nucleic acid in the sample, and wherein the absence of the amplification product is indicative of the absence of the target nucleic acid in the sample.

17. The method of claim 16, wherein the region of the forward primer that is complementary to the target nucleic acid is < 20 base pairs long.

18. The method of claim 17, wherein the region of the forward primer that is complementary to the target nucleic acid is < 15 base pairs long.

19. The method of claim 18, wherein the region of the forward primer that is complementary to the target nucleic acid is < 10 base pairs long.

20. The method of claim 16, wherein the region of the reverse primer that is complementary to the target nucleic acid is < 20 base pairs long.

21. The method of claim 20, wherein the region of the reverse primer that is complementary to the target nucleic acid is < 15 base pairs long.

22. The method of claim 21 , wherein the region of the reverse primer that is complementary to the target nucleic acid is < 10 base pairs long.

23. The method of claim 16, wherein the forward primer is > 17 base pairs long.

24. The method of claim 23, wherein the forward primer is > 20 base pairs long.

25. The method of claim 24, wherein the forward primer is > 25 base pairs long.

26. The method of claim 16, wherein the reverse primer is > 17 base pairs27. The method of claim 26, wherein the reverse primer is > 20 base pairs long.

28. The method of claim 27, wherein the reverse primer is > 25 base pairs long.

29. The method of claim 16, wherein the target nucleic acid is between110-130 base pairs long30. The method of claim 29, wherein the target nucleic acid about 120 base pairs long.

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