Use of dITP for preferential / selective amplification of RNA versus DNA targets based on strand separation temperature

Abstract

A method is described for preferentially and / or selectively detecting and / or quantifying target RNA over DNA in a biological or non-biological sample. The method includes performing an amplification step, a hybridization step, and a detection step. Furthermore, the reaction mixture includes dNTPs, including dITP. Kits, reaction mixtures, and oligonucleotides (e.g., primers and probes) for preferentially and / or selectively amplifying and detecting and / or quantifying target RNA (over DNA) in the presence of dITP are also described. The method, kit, and reaction mixture are for preferentially and / or selectively detecting and / or quantifying RNA over DNA and are particularly useful in samples containing several different types of nucleic acids (e.g., DNA and RNA).

Description

[Technical field]

[0001] The present disclosure relates to the field of nucleic acid detection. In this field, the invention relates to the amplification, detection and / or quantification of target nucleic acids that may be present in a sample, and in particular to the selective and preferential amplification, detection and / or quantification of target ribonucleic acid (RNA) relative to deoxyribonucleic acid (DNA) utilizing 2'-deoxyinosine 5'-triphosphate (dITP) as a deoxynucleoside triphosphate (dNTP) with primers and probes. The invention further provides reaction mixtures and kits containing dITP and primers and probes for the selective and preferential amplification and detection of RNA (e.g., Hepatitis B Virus (HBV) RNA) relative to DNA. [Background technology]

[0002] Many biological samples contain several different types of nucleic acids, including both deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). In certain situations, it may be desirable or advantageous to selectively and preferentially detect only one type of nucleic acid (e.g., RNA rather than DNA, or DNA rather than RNA). For example, it may be desirable or advantageous to detect genomic DNA without detecting potentially interfering RNA that may be present in the same biological sample. Alternatively, it may be desirable or advantageous to detect RNA without detecting any potentially interfering genomic DNA that may be present in the same biological sample.

[0003] There are methods known in the art for selectively and preferentially depleting undesired nucleic acids from biological samples or enriching desired nucleic acids. One example of an existing solution for depleting undesired DNA from biological samples, for example, and thereby improving RNA selectivity in amplification reactions (e.g., polymerase chain reaction (PCR)), is to add deoxyribonuclease (DNase) enzyme to the sample preparation material before PCR cycling. In this case, the DNAse enzyme selectively and preferentially degrades only DNA in the sample, while leaving RNA intact. However, this methodology has drawbacks. For example, this method poses the risk of introducing DNase into the reaction. In addition, this method requires an extra step of removing DNase in the sample before PCR cycling to prevent the degradation of oligonucleotides (e.g., primers, probes and amplicons / amplified products) during PCR by residual DNase. If DNase is not eliminated and introduced into the PCR reaction, oligonucleotides such as primers, probes and amplicons / amplified products will be degraded and PCR will not be successful. Yet another example of an existing solution for preferential and selective amplification of RNA over DNA is to design a PCR assay around the polyA tail region within the RNA. However, since all mRNAs contain a polyA tail, this method is not specific enough to detect a particular RNA target of interest.

[0004] In the field of molecular diagnostics, the amplification and detection of nucleic acids is of considerable importance. Such methods can be used 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 Qβ amplification. Automated systems for PCR-based analysis often utilize real-time detection of product amplification during the PCR process in the same reaction vessel. The key to such methods is the use of modified oligonucleotides bearing reporter groups or labels.

[0005] The present invention represents an improvement over existing technology. The present invention relates to a method for preferentially and / or selectively amplifying and detecting RNA (versus DNA) comprising utilizing 2'-deoxyinosine 5'-triphosphate (dITP) as a deoxynucleoside triphosphate (dNTP) in the amplification reaction. The utilization of dITP results in a shift in the strand separation temperature, thereby allowing for improved RNA selectivity for RNA targets that cannot be easily distinguished from DNA targets or targeted by utilizing obvious biological properties such as polyA tails or intron / exon junctions. In this case, dITP behaves as a deoxyguanosine triphosphate (dGTP) analogue, sharing a similar chemical structure to dGTP, but compared to dGTP, dITP lacks the 2-amino group (see FIG. 1). This difference results in reduced base stacking interactions and hydrogen bonding, resulting in a higher T m Substitution of dITP for dGTP results in a decrease in the temperature of first and second strand complementary DNA (cDNA) synthesis and the subsequent T of the newly formed amplicon. mThe dITP concentration is reduced, which allows for lower temperature thermal cycling since the strand separation temperature is lower than that required for melting natural double-stranded DNA. Thus, incorporating dITP preferentially amplifies RNA by preventing strand separation of double-stranded DNA, thereby preventing polymerase from accessing and amplifying DNA targets. Thus, there remains a need in the art for a rapid, reliable and specific method for preferentially detecting and / or amplifying RNA targets in samples that may contain a mixture of nucleic acid types (e.g., DNA and RNA).

[0006] The present invention provides a method for selective and / or preferential detection and / or amplification of RNA targets by utilizing dITP, which is particularly useful in situations where a biological sample contains many different types of nucleic acid types (e.g., RNA and DNA) and it is desirable to detect only RNA and not potentially interfering DNA. Summary of the Invention

[0007] Certain embodiments of the present disclosure relate to methods for preferentially detecting and / or quantifying target RNA over DNA in biological or non-biological samples. Such methods may be performed in vitro. The embodiments include methods for preferentially detecting and / or quantifying target RNA over DNA, comprising performing at least one cycling step, which may include an amplification step and a hybridization step. Additionally, the embodiments include primers, probes, polymerase(s), dNTPs (including dATP, dTTP, dCTP, dGTP, and dITP), and kits designed to preferentially detect and / or quantify target RNA over DNA.

[0008] One embodiment relates to a method for selectively detecting and / or quantifying one or more target RNAs in a sample over DNA, comprising: (a) providing a sample; (b) providing one or more polymerases; (c) providing dNTPs comprising at least dITP; (d) performing an amplification step comprising contacting the sample with one or more set of oligonucleotide primers specific for the target RNA; (e) performing a hybridization step comprising contacting the amplification products with one or more set of oligonucleotide probes if the target RNA is present in the sample; and (f) performing a detection step comprising detecting the presence or absence of the target RNA, wherein the presence of an amplification product indicates the presence of the target RNA in the sample and the absence of an amplification product indicates the absence of the target RNA in the sample, wherein during the amplification step, the one or more polymerases amplify the target RNA, if present in the sample, by incorporating dITP into the amplification product, thereby selectively detecting and / or quantifying the target RNA compared to DNA in the sample. In one embodiment, the dNTPs further comprise dATP, dTTP, dCTP and dGTP. In one embodiment, the ratio of dITP:dGTP is 3:1, 1:1 or 1:3. In one embodiment, the ratio of dITP:dGTP is 3:1. In one embodiment, the dNTPs comprise equal amounts of (i) dATP, (ii) dTTP, (iii) dCTP and (iv) dGTP+dITP. In one embodiment, the ratio of dITP:dGTP is 3:1, 1:1 or 1:3. In one embodiment, the ratio of dITP:dGTP is 3:1. In one embodiment, the sample contains both RNA and DNA. In one embodiment, the sample is a biological sample. In one embodiment, the biological sample is blood, plasma or urine. In one embodiment, the one or more polymerases are DNA polymerases. In one embodiment, the presence of dITP increases the melting temperature (T m) is reduced. In one embodiment, the target RNA is Hepatitis B virus (HBV) RNA. In one embodiment, the one or more sets of oligonucleotide primers and the one or more sets of oligonucleotide probes hybridize to a nucleic acid sequence comprising SEQ ID NO:4. In one embodiment, the one or more sets of oligonucleotide primers comprise oligonucleotide primers having the nucleic acid sequences of SEQ ID NO:1 and SEQ ID NO:2, and the one or more sets of oligonucleotide probes comprise an oligonucleotide probe having the nucleic acid sequence of SEQ ID NO:3. In one embodiment, the HBV RNA is HBV pregenomic RNA (pgRNA). In one embodiment, the HBV pgRNA is a surrogate for HBV covalently closed circular DNA (cccDNA). In one embodiment, the amount of HBV pgRNA quantified is a factor considered for treatment decisions regarding the patient from whom the sample originates.

[0009] One embodiment relates to a kit for selectively detecting and / or quantifying one or more target RNAs over DNA in a sample, the kit comprising: (a) one or more polymerases; (b) dNTPs including at least dITP; (c) one or more sets of oligonucleotide primers specific for the target RNAs; and (d) one or more sets of oligonucleotide probes. In one embodiment, the dNTPs further comprise dATP, dTTP, dCTP, and dGTP. In one embodiment, the ratio of dITP:dGTP is 3:1, 1:1, or 1:3. In one embodiment, the ratio of dITP:dGTP is 3:1. In one embodiment, the dNTPs comprise equal amounts of (i) dATP, (ii) dTTP, (iii) dCTP, and (iv) dGTP+dITP. In one embodiment, the ratio of dITP:dGTP is 3:1, 1:1, or 1:3. In one embodiment, the ratio of dITP:dGTP is 3:1. In one embodiment, the sample contains both RNA and DNA. In one embodiment, the sample is a biological sample. In one embodiment, the biological sample is blood, plasma or urine. In one embodiment, the one or more polymerases are DNA polymerases. In one embodiment, the presence of dITP increases the melting temperature (T m) is reduced. In one embodiment, the target RNA is Hepatitis B virus (HBV) RNA. In one embodiment, the one or more sets of oligonucleotide primers and the one or more sets of oligonucleotide probes hybridize to a nucleic acid sequence comprising SEQ ID NO:4. In one embodiment, the one or more sets of oligonucleotide primers comprise oligonucleotide primers having the nucleic acid sequences of SEQ ID NO:1 and SEQ ID NO:2, and the one or more sets of oligonucleotide probes comprise an oligonucleotide probe having the nucleic acid sequence of SEQ ID NO:3. In one embodiment, the HBV RNA is HBV pregenomic RNA (pgRNA). In one embodiment, the HBV pgRNA is a surrogate for HBV covalently closed circular DNA (cccDNA). In one embodiment, the amount of HBV pgRNA quantified is a factor considered for treatment decisions regarding the patient from whom the sample originates.

[0010] Another embodiment is a method for selectively detecting and / or quantifying one or more target HBV RNAs in a sample over DNA, comprising: (a) providing a sample; (b) providing one or more polymerases; (c) providing dNTPs, including at least dITP; (d) performing an amplification step, the amplification step comprising contacting the sample with one or more oligonucleotide primers specific for the target HBV RNA, the one or more set of oligonucleotide primers comprising oligonucleotide primers having the nucleic acid sequences of SEQ ID NO:1 and SEQ ID NO:2; (e) performing a hybridization step, the hybridization step comprising contacting the amplification products with one or more set of oligonucleotide probes, if the target HBV RNA is present in the sample, the one or more set of oligonucleotide probes comprising oligonucleotide probes having the nucleic acid sequence of SEQ ID NO:3; and (f) performing a detection step, the detection step comprising detecting the presence or absence of the target HBV RNA, the presence of the amplification product indicating the presence of the target HBV RNA in the sample and the absence of the amplification product indicating the presence of the target HBV RNA in the sample. and performing a detection step indicating the absence of RNA, wherein during the amplification step, one or more polymerases amplify the target HBV RNA, if present in the sample, by incorporating dITP into the amplification product, thereby selectively detecting and / or quantifying the target RNA compared to DNA in the sample. In one embodiment, the dNTPs further comprise dATP, dTTP, dCTP and dGTP. In one embodiment, the ratio of dITP:dGTP is 3:1, 1:1 or 1:3. In one embodiment, the ratio of dITP:dGTP is 3:1. In one embodiment, the dNTPs comprise equal amounts of (i) dATP, (ii) dTTP, (iii) dCTP and (iv) dGTP+dITP. In one embodiment, the ratio of dITP:dGTP is 3:1, 1:1 or 1:3. In one embodiment, the ratio of dITP:dGTP is 3:1. In one embodiment, the sample contains both RNA and DNA. In one embodiment, the sample is a biological sample.In one embodiment, the biological sample is blood, plasma or urine. In one embodiment, the one or more polymerases are DNA polymerases. In one embodiment, the presence of dITP increases the melting temperature (T m ) is reduced. In one embodiment, the one or more sets of oligonucleotide primers and the one or more sets of oligonucleotide probes hybridize to a nucleic acid sequence comprising SEQ ID NO:4. In one embodiment, the HBV RNA is HBV pregenomic RNA (pgRNA). In one embodiment, the HBV pgRNA is a surrogate for HBV covalently closed circular DNA (cccDNA). In one embodiment, the amount of quantified HBV pgRNA is a factor considered for treatment decisions regarding the patient from whom the sample originates.

[0011] In one aspect, the amplification can use a polymerase enzyme with 5' to 3' nuclease activity. Thus, the donor fluorescent moiety and the acceptor moiety, e.g., a quencher, can be within 5 to 20 nucleotides (e.g., 7 or 10 nucleotides) of each other along the length of the probe. In another aspect, the probe includes a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure can result in spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the oligonucleotide probe can be a quencher.

[0012] The present disclosure also provides methods for preferentially and / or selectively detecting and / or quantifying target RNA over DNA in a biological sample from an individual. These methods can be used to detect the presence or absence of target RNA in plasma for use in blood screening and diagnostic testing. Furthermore, the same tests can be used by those skilled in the art to evaluate urine and other sample types to preferentially and / or selectively detect and / or quantify target RNA nucleic acids over DNA nucleic acids. Such methods generally include performing at least one cycling step, including an amplification step and a dye binding step. Typically, the amplification step includes contacting the sample with multiple pairs of oligonucleotide primers that generate one or more amplification products if the nucleic acid molecule is present in the sample, and the dye binding step includes contacting the amplification products 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 to the amplification products, the presence of binding indicating the presence of the target RNA nucleic acid in the sample and the absence of binding indicating the absence of the target RNA nucleic acid 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 YO-YO® and SYBR® Green. Additionally, such methods can also include determining the melting temperature between the amplification product and a double-stranded DNA binding dye, which confirms the presence or absence of the target RNA nucleic acid.

[0013] In a further embodiment, a kit is provided for preferentially and / or selectively detecting and / or quantifying one or more target RNA nucleic acids. The kit may include one or more sets of oligonucleotide primers specific for amplifying a gene target and one or more detectable oligonucleotide probes specific for detecting the amplification products, as well as one or more polymerases and dNTPs, including dATP, dCTP, dTTP, dGTP, and dITP. In one aspect, the kit may include a donor moiety and a corresponding acceptor moiety, e.g., an oligonucleotide probe that is already labeled with another fluorescent moiety or a dark quencher, or may include a fluorophore moiety for labeling the oligonucleotide probe. The kit may also further include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit may also include a package insert and instructions for the use of the oligonucleotide primers, oligonucleotide probes, and fluorescent dye moieties for preferentially and / or selectively detecting and / or quantifying target RNA in a sample.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled 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 are 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.

[0015] 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. [Brief description of the drawings]

[0016] [Figure 1]FIG. 1 shows the molecular differences between dITP and dGTP.

[0017] [Figure 2A] Figure 2A shows the amplification curves and Figure 2B shows the melting curves. These figures show that the Tm of the HBV region 7 amplicon decreases with increasing dITP concentration. These experiments are described in Example 2. [Figure 2B] Same as above.

[0018] [Figure 3A] 3A-3C show PCR growth curves demonstrating that a master mix containing 200 μM dITP restores performance at lower denaturation temperatures. These experiments are described in Example 3. [Figure 3B] Same as above. [Figure 3C] Same as above.

[0019] [Figure 4A] 4A-4C show PCR growth and melting curves demonstrating that 200 μM dITP shows significantly improved performance at lower denaturation temperatures. These experiments are described in Example 3. [Figure 4B] Same as above. [Figure 4C] Same as above.

[0020] [Figure 5A] 5A-5C show PCR growth and melting curves demonstrating that formulations containing 200 μM dITP result in improved performance at lower denaturation temperatures. These experiments are described in Example 3. [Figure 5B] Same as above. [Figure 5C] Same as above.

[0021] [Figure 6A]Figures 6A and 6B show PCR growth curves showing that HBV insert 3 (Figure 6A) and GIC transcripts (Figure 6B) were duplexed with TaqMan probes and tested in a denaturation temperature gradient. Formulations containing 200 μM dITP produced improved performance, especially at lower denaturation temperatures. These experiments are described in Example 4. [Figure 6B] Same as above.

[0022] [Figure 7A] 7A-7F show PCR growth curves from six different clinical plasma samples, demonstrating that reactions with dITP demonstrated selective amplification of RNA relative to DNA when compared to reactions without dITP. These experiments are described in Example 5. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above.

[0023] [Figure 8A] 8A-8D show PCR growth curves under various experimental conditions, showing that in the Cy5.5 channel, the GIC minus dITP, minus RT maintenance reactions generate unexpected amplification curves. RT activity may still be present in these reactions even in the absence of RT maintenance. This phenomenon is not observed in the plus dITP, minus RT maintenance reactions. These experiments are described in Example 5. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Methods, kits, and reaction mixtures are described herein for preferentially and / or selectively detecting and / or quantifying target RNA (over DNA). In particular, polymerase(s), dNTPs (including dATP, dTTP, dCTP, dGTP, and dITP), primers, and probes for preferentially and / or selectively detecting and / or quantifying target RNA are provided, as are articles of manufacture or kits that include such reagents. Furthermore, this technology can be used for blood screening and prognosis.

[0025] The term "amplifying" as used herein refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a nucleic acid molecule such as RNA from the HBV genome). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and appropriate buffers and / or cofactors (e.g., MgCl2 and / or KCl) for optimal activity of the polymerase enzyme.

[0026] The term "primer" as used herein is known to those skilled in the art and refers to oligomeric compounds, primarily oligonucleotides, but also modified oligonucleotides that are capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which a further "nucleotide" can be attached by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using a deoxynucleoside triphosphate, thereby releasing pyrophosphate.

[0027] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. "Hybridization conditions" typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0028] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0029] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when exposed to high temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, 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, for example, from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, non-thermostable polymerases can also be used in PCR assays, provided the enzyme is replenished as needed.

[0030] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0031] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a biocatalyst that incorporates a nucleotide, e.g., a polymerase that typically adds a nucleotide to the 3' end of the nucleic acid.

[0032] 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 have the same or a specified percentage of identical nucleotides when compared and aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, 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 et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation," Genome Genet. Res. 7:649-656, which are incorporated herein by reference.

[0033] "Modified nucleotide" in the context of oligonucleotide refers to a change in which at least one nucleotide of an oligonucleotide sequence is replaced by a different nucleotide that provides the oligonucleotide with desired properties.Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, t-butylbenzyl, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU ... Examples of modified nucleotides include 7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxy-xanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, 5-propynyl-dU, 5-propynyl-dC, 7-deaza-deoxyguanosine (deazaG(u-deaza)), and the like. Many other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or known in the art. In certain embodiments, modified nucleotide substitutions result in a lower melting temperature (T) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. m ) to modify the nucleic acid sequence. To further illustrate, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of the intended target amplicon, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference. Other modified nucleotide substitutions can alter the stability of the oligonucleotide or provide other desirable characteristics.

[0034] The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that allows it to hybridize, by design or selection, specifically (i.e., preferentially and / or selectively) under a defined, given stringency to a "target nucleic acid," in this case an RNA (target) nucleic acid. A "probe" may also be referred to as a "detection probe," which means that it detects a target nucleic acid.

[0035] 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, such as a fluorescent dye, and a corresponding acceptor moiety, such as a quencher.

[0036] The design of oligonucleotides used as probes can be done in a similar manner to the design of primers. Embodiments can use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probe(s) used can include at least one label and / or at least one quencher moiety. Like 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 compromised during synthesis. Oligonucleotide probes are generally 15-40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0037] Polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as initiation points for nucleic acid synthesis within the described RNA target nucleic acid sequence. Primers can be purified from restriction digests by conventional methods or can be produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, but primers may also 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.

[0038] 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 achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating the nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration, as well as the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90° C. to about 105° C., depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0039] When the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is usually about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient to cause extension from the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range 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 seconds to about 5 minutes (e.g., from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0040] PCR assays can use nucleic acids such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified and can be a minor fraction of a complex mixture such as the target RNA contained in human cells. The target RNA nucleic acid molecule can be extracted from a biological sample by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, including plasmids or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0041] The oligonucleotide primers are combined with PCR reagents under reaction conditions conducive to primer extension. Such PCR reagents include, but are not limited to, one or more polymerases and dNTPs, including dATP, dTTP, dCTP, dGTP, and dITP. For example, a chain extension reaction typically includes 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, and dITP, or one or more analogs thereof.

[0042] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to generate a desired amount of amplification product corresponding to the target RNA nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, 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 enough for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0043] Fluorescence Resonance Energy Transfer (FRET) FRET technology (see, e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance of each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light irradiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light irradiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0044] 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) (BHQ-2, etc.)) that may or may not be fluorescent and 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 the fluorescent emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in 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) (e.g., BHQ2) (Biosearch Technologies, Inc., Novato, CA), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, IA), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, MI).

[0045] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target RNA target nucleic acid sequence. When the oligonucleotide probe hybridizes to the amplification product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0046] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor a particular range of fluorescence emission), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed using 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 of the desired range.

[0047] As used herein with respect to a donor moiety and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety, so that efficient non-irradiative energy transfer can take place between them.

[0048] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift in emission as far as possible into the red portion of the visible spectrum (>600 nm); and (d) a shift in emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0049] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0050] 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 since it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Typically, the linker arm is from about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods of attaching the linker arm to a particular nucleotide base and attaching the fluorescent moiety to the linker arm.

[0051] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.) to generate, for example, LC Red 640-labeled oligonucleotides. Linkers commonly used to attach donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from Fluorescein-NHS-esters, such as CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires attachment of a Fluorescein-NHS-ester after oligonucleotide synthesis.

[0052] Detection and / or quantification of target RNA amplification products (amplicons) The present disclosure provides a method for preferentially and / or selectively detecting and / or quantifying target RNA (over DNA) in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes performing at least one cycling step including preferentially and / or selectively amplifying a portion of the target RNA from the sample using one or more pairs of target RNA primers, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed to preferentially and / or selectively detect and / or quantify target RNA (over DNA) using target RNA primers and probes, where the presence of the target RNA and the detection of the target RNA indicates the presence of the target RNA in the sample.

[0053] As described herein, the amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of the amplification products and thus the presence or absence of the target RNA. TaqMan® technology utilizes one single-stranded hybridization probe that is labeled with, for example, 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 the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or dark quencher according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification products) and is degraded during the subsequent extension step, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent and quencher moieties are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein to preferentially and / or selectively detect and / or quantitate target RNA (over DNA) in a sample.

[0054] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. 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 a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). The secondary structure formed within the probe results in both fluorescent sites being in spatial proximity when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0055] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., 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 occur when the fluorescent moieties are in direct local proximity and 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 to the number of original target DNA / RNA molecules (e.g., the number of target RNA molecules). When amplification of the target RNA occurs and an amplification product is produced, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0056] Generally, the presence of FRET indicates the presence of target RNA in the sample, and the absence of FRET indicates the absence of target RNA in the sample. However, insufficient specimen collection, transport delays, improper transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.

[0057] Representative biological samples that can be used to carry out the method include, but are not limited to, whole blood, respiratory specimens, urine, fecal specimens, blood specimens, plasma, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release nucleic acids (such as target RNA), or in some cases, the biological sample can be directly contacted with PCR reaction components and appropriate oligonucleotides. In some examples, the biological sample is whole blood. When whole blood is typically collected, it is often collected in a container that contains an anticoagulant, such as heparin, citrate, or EDTA, which allows the whole blood to be stored at an appropriate temperature. However, under such conditions, the nucleic acids in whole blood undergo a significant amount of degradation. Therefore, it may be advantageous to collect the blood in a reagent that dissolves, denatures, and stabilizes whole blood components, including nucleic acids, such as a nucleic acid stabilizing solution. In such cases, the nucleic acids may be better preserved and stabilized for subsequent isolation and analysis by nucleic acid tests such as PCR, etc. Such nucleic acid stabilizing solutions are well known in the art and include, but are not limited to, cobas® PCR medium containing 4.2 M guanidinium salt (GuHCl) and 50 mM Tris at pH 7.5.

[0058] The sample can be collected by any method or device designed to adequately hold and preserve the sample before analysis. Such methods and devices are well known in the art. When the sample is a biological sample such as whole blood, the method or device can include a blood collection tube. Such blood collection tubes are well known in the art and can include, for example, a blood collection tube. In many cases, it may be advantageous to use a blood collection tube, where the blood collection tube is under pressure in the space intended for sample intake, such as a blood tube with a vacuum chamber, such as a vacutainer blood collection tube. Such blood collection tubes with vacuum chambers, such as vacutainer blood collection tubes, are well known in the art. It may be further advantageous to collect blood into a blood collection tube with or without a vacuum chamber, such that the whole blood drawn is immediately in contact with the nucleic acid stabilizing solution in the blood collection tube, with or without a vacuum chamber, containing therein a solution that dissolves, denatures, and stabilizes whole blood components, including nucleic acids, such as a nucleic acid stabilizing solution.

[0059] Melting curve analysis is an additional step that can be included in the cycle profile. Melting curve analysis is performed to determine the melting temperature (T), which is defined as the temperature at which one half of a nucleic acid duplex separates into single strands. m It is based on the fact that DNA / RNA melts at a characteristic temperature called the Tm. The melting temperature of DNA / RNA depends mainly on its nucleotide composition. Thus, a nucleic acid molecule rich in G and C nucleotides has a higher Tm than a nucleic acid molecule rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the target RNA probe from the target RNA amplification product can confirm the presence or absence of the target RNA in the sample.

[0060] During each thermocycler run, control samples can be cycled as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., in the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect 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, for example, a negative control lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reaction can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, as well as the ability of probes to hybridize with sequence specificity and FRET to occur.

[0061] In one embodiment, the method includes a step to avoid contamination, for example, the enzymatic method utilizing uracil-DNA glycosylase described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycling run and the next.

[0062] This method can be carried out using conventional PCR combined with FRET technology. In one embodiment, the LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0063] The LightCycler® can be operated using a PC workstation and utilizes a Windows NT operating system. Signals from samples are obtained as the machine sequentially positions the capillaries over the optical unit. The software can display the fluorescent signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The data generated can be stored for further analysis.

[0064] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® GREEN or SYBR® GOLD (Molecular Probes)) can be used to detect the amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Also, double-stranded DNA binding dyes such as nucleic acid intercalating dyes can be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of the amplification product.

[0065] Those skilled in the art will appreciate that other nucleic acid or signal amplification methods may also be used, including, but not limited to, branched DNA signal amplification, loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3 SR), strand displacement amplification (SDA), or smart amplification process version 2 (SMAP 2).

[0066] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0067] Manufactured products / kits An embodiment of the present disclosure further provides an article of manufacture or kit for preferentially and / or selectively detecting and / or quantifying target RNA (over DNA). The article of manufacture may include primers and probes used to preferentially and / or selectively detect and / or quantify target RNA with suitable packaging materials including dNTPs (including dATP, dCTP, dTTP, dGTP, and dITP). Exemplary primers and probes for preferentially and / or selectively detecting and / or quantifying target RNA (over DNA) can hybridize to target RNA molecules. Furthermore, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that preferentially and / or selectively amplify and hybridize to RNA target molecules are provided.

[0068] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or may label the probes provided with the kit. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the target RNA probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0069] The article of manufacture may also include a package insert or packaging label with instructions for using the target RNA primers and probes to detect target RNA in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0070] Embodiments of the present disclosure also provide a set of primers and one or more detectable probes for preferentially and / or selectively detecting and / or quantifying a target RNA in a sample.

[0071] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. EXAMPLES

[0072] The following examples and figures are provided to aid in 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 to the procedures described without departing from the scope of the invention.In all of the following examples, the polymerase used was Z05D polymerase, which is the D580G mutant of Z05 polymerase described in, for example, U.S. Patent No. 8,962,293, U.S. Patent No. 9,102,924 and U.S. Patent No. 9,738,876.

[0073] Example 1: Approach to Test the Effect of dITP in Hepatitis B Virus (HBV) Assays HBV is a partially double-stranded DNA virus that packages a single-stranded RNA pregenome (pgRNA) that is reverse transcribed. Current treatments for chronic HBV infection include nucleoside analogs (NAs) that disrupt reverse transcription of pgRNA and inhibit DNA synthesis. HBV pgRNA is a surrogate for cccDNA (covalently closed circular DNA) and has been shown to be detectable in plasma even when HBV DNA production is suppressed. Due to severe side effects and long-term benefits seen in less than half of treated patients, monitoring patients for treatment discontinuation is important. Current monitoring methods include HBV DNA quantification or HBV antigen detection, both of which are believed to have limitations. Because NA therapy does not directly eliminate the cccDNA reservoir, these markers may be insufficient to reflect cccDNA activity. Thus, discontinuing NA therapy may result in rebound HBV infection. Because the continued presence of HBV RNA particles may indicate active transcription of cccDNA, HBV pgRNA may be a potential biomarker for identifying long-term virally suppressed patients who may be candidates for treatment discontinuation.

[0074] Therefore, an HBV pgRNA quantitative assay is useful for monitoring patient treatment discontinuation. However, one of the key challenges in developing an HBV pgRNA assay is to distinguish between HBV RNA and viral DNA amplification. Elimination of HBV DNA is difficult to achieve due to the limited number of sequence and structural differences with HBV pgRNA that can be targeted. Therefore, the development of an HBV quantitative assay provided a platform to evaluate the utility of dITP to ensure RNA selectivity in a useful manner.

[0075] To evaluate the effect of dITP, an HBV assay was developed with HBV region 7. The HBV region 7 amplicon is called insert 3. In order to avoid divalent metal ion adjustment (Mn(OAc)2), the total dNTP concentration of 2.0 mM remained constant, and only the ratio of dGTP and dITP was varied to determine the optimal concentration for HBV region 7 and the generic internal control (GIC) assay. The GIC assay was evaluated together with the HBV region 7 assay, because the GIC assay detects the generic internal control, which serves as a full process control (FPC), internal control (IC), and internal quantification standard (IQS) in all cobas® 6800 / 8800 assays. The optimal dGTP:dITP ratio depends on the GC content of the primers, probe, and amplicon. The determination of the ideal denaturation temperature for selective RNA amplification depends on the amount of dIMP incorporated and the length of the amplicon. It is noted that the dITP derivative, dIMP, is the monomeric form of dITP that is incorporated into DNA. HBV region 7 and GIC assay sequence, T m The temperature (° C.) and GC content are listed in Tables 1 and 2 below, respectively.

[0076] [Table 1]

[0077] [Table 2]

[0078] Perform an initial experiment with SYTO 9 intercalating dye to identify lysates and amplicons m The effect of dITP on HBV RNA was observed. SYTO 9 experiments were performed on HBV Insert 3 transcripts, HBV Insert 3 DNA gBlock (a double-stranded DNA fragment identical to the sequence of the HBV transcript), and GIC transcripts. HBV and GIC duplex reactions using TaqMan probes were then evaluated at various dGTP:dITP ratios across a denaturing and annealing temperature gradient to determine optimal dITP concentrations and thermocycling parameters. Performance evaluation of HBV Insert 3 transcripts spiked with HBV Insert 3 DNA gBlock was performed to examine the amplification efficiency and specificity of HBV RNA targets compared to HBV DNA gBlock. After finalizing the dITP concentrations and thermocycling temperatures, clinical HBV plasma samples with various viral loads extracted by cobas® 6800 / 8800 were evaluated with the optimized dITP conditions.

[0079] Example 2: Use of dITP m This results in a decrease in Therefore, the HBV region 7 assay was performed using dITP titration with SYTO 9. HBV insert 3 amplicon T m The effect of dITP on was assessed utilizing SYTO 9 dye (1 μM / reaction). To optimize the assay, the HBV region 7 assay was tested with different ratios of dGTP:dITP. The final dNTP concentration in the master mix was 2.0 mM, consisting of 400 μM dATP, 400 μM dCTP, 800 μM dUTP, and 400 uM (dGTP+dITP). The following five formulations with various dGTP and dITP ratios were tested in the backbone cobas® 6800 / 8800 master mix and are shown in Table 3 below: [Table 3]

[0080] Titrations of HBV insert 3 transcript were tested at 0.1, 0.01, and 0.001 ng / reaction. A generic cobas® 6800 / 8800 thermal profile (including denaturation temperatures of 95° C. and 91° C.) was tested in the experiments listed in Table 4 below.

[0081] [Table 4]

[0082] The results are shown in Figures 2A and 2B. Amplification curves are shown in Figure 2A. The melting curves shown in Figure 2B show that across all formulations, amplification curves were generated except for the master mix in the absence of dGTP. This indicates that at least some dGTP is required to amplify the target under the thermocycling conditions tested. The Tm of the amplicon decreases as the concentration of dITP in the reaction increases. The average amplicon Tm of the minus dITP reaction is 87.2°C. The amplicon Tm of the formulation containing 300 μM dITP decreased significantly to 74.1°C. From this experiment, the effect of dITP on the HBV insert 3 amplicon was demonstrated by the resulting decrease in Tm of 13°C. Thus, these studies demonstrate that as the concentration of dITP increases, the Tm of the amplicon decreases.

[0083] Example 3: dITP performance evaluation for HBV region 7 and gBlock and general internal control (GIC) transcripts gBlock double-stranded DNA fragments with sequence identity to HBV region 7 transcripts were evaluated. Because viral HBV RNA and HBV DNA have very similar sequences, evaluation of HBV gBlock and HBV transcript target pools was of interest to determine HBV RNA amplification efficiency and specificity. Initial experiments with SYTO 9 dye were tested with HBV region 7 transcripts, HBV region 7 DNA gBlocks and GIC transcripts to determine amplicon melting curves and T mTwo master mix formulations containing 100 μM and 200 μM dITP were evaluated with all three targets over a denaturation temperature gradient ranging from 79.0 to 87.0° C. in a Roche LightCycler® 96. SYTO 9 dye was tested at 1 μM and a melting step was included at the end of the thermal profile. All targets were tested in singleplex since SYTO 9 intercalating dye was present in the reaction. HBV Insert 3 transcript, gBlock, and GIC transcript were tested at one concentration.

[0084] The results show that the master mix containing 200 μM dITP (and 200 μM dGTP) (FIG. 3B) shows significantly improved performance across all three targets at lower denaturation temperatures compared to the master mix containing only 100 μM dITP (and 300 μM dGTP) (FIG. 3A). The master mix containing 200 μM dITP (and 200 μM dGTP) reliably amplifies HBV region 7 transcripts at denaturation temperatures down to 81.1° C., which is at least 10° C. lower than the denaturation temperature of the typical cobas® 6800 / 8800 thermal profile shown in Table 3 above, as shown in FIGS. 3A-C. As shown in FIGS. 4A-C and 5A-C, a singleplex reaction of HBV region 7 gBlock can also be amplified, likely due to its short length (318 bp), and the GIC transcript can be reliably detected at denaturation temperatures down to about 79.0° C. Example 4: GIC dual performance evaluation using HBV region 7 and dITP

[0085] The majority of the evaluation was performed using SYTO 9 dye to examine the effect of dITP on Tm. In this experiment, HBV and GIC assays were duplexed with TaqMan probes and evaluated over a denaturation temperature gradient of 79 to 84.0 °C. Two master mix formulations containing 100 μM dITP and 200 μM dITP were tested against target pools of HBV insert 3 (Figure 6A) and GIC transcripts (Figure 6B). Both HEX and Cy5.5 TaqMan probes were present in this evaluation.

[0086] The results show that there is a substantial performance difference between the two formulations across the denaturation temperature gradient, as shown in Figures 6A and 6B. A dITP concentration of 200 μM restored performance at lower denaturation temperatures, allowing reliable detection of HBV RNA targets at 79.0°C, up to 16°C lower than the denaturation temperatures utilized in the typical cobas® 6800 / 8800 thermal profile (Figure 6A). A similar trend was observed for the GIC transcript in channel Cy5.5 (Figure 6B). Example 5: Performance of dITP on clinical HBV plasma samples

[0087] After further titration and temperature gradient experiments, the optimal nucleotide concentration and thermocycling parameters were determined to be 300 μM dITP (and 100 μM dGTP), maintaining denaturation (during PCR cycles) and UNG inactivation at 78° C., and maintaining the annealing temperature at 55° C. (optimization data not shown). The general cobas® 6800 / 8800 thermal profile remained the same except for the temperature changes specified. The final dITP thermocycling profile is listed in Table 5 below.

[0088] [Table 5]

[0089] The reverse transcription (RT) PCR step in the thermal cycling profile includes three temperature holds of 55, 60 and 65°C after the UNG inactivation step as listed in Table 4. RT-PCR occurs during the RT step where the synthesis of complementary DNA is created from RNA. The purpose of evaluating with and without RT hold is to compare the amplification of DNA (minus RT hold) and RNA (plus RT hold). The thermal profiles in Table 4 were tested with and without RT hold to compare the RNA and DNA amplification in clinical HBV plasma eluates described herein. Six clinical HBV plasma samples were extracted by cobas® 6800 / 8800 and Roche High Pure Viral Nucleic Acid Kit. The clinical samples were collected across treated and untreated patients. The HBV viral load in these clinical samples ranged from a very high titer of 5.7E9 to 4.8E4 as shown in Table 6 below.

[0090] [Table 6]

[0091] All samples were evaluated with ±dITP (optimized concentration of 300 μM) and ±RT hold to assess RNA and DNA amplification in all nucleic acid purified samples. The dITP optimized thermal profile (including 78°C denaturation / UNG inactivation and 55°C annealing temperature) was tested in the plus dITP reaction. For comparison, the minus dITP reaction was tested using the generic cobas® 6800 / 8800 generic thermal profile (including 95°C and 91°C denaturation hold).

[0092] [Table 7]

[0093] The results, as shown in Figures 7A-7F, indicate that across all clinical plasma samples tested (six samples total), the plus dITP reaction produced significantly higher fluorescent signals and the ΔCp between the ±RT-maintained reactions was significantly larger. Table 6 shows the average Cps for the clinical HBV plasma samples. Table 6 reveals that across both extraction methods, the plus dITP reaction produced average ΔCps of 6.2 and 5.7, which is significantly greater compared to the minus dITP reaction (average ΔCp of 1.9), as shown in Table 7.

[0094] This demonstrates improved HBV RNA specificity, indicating preferential and / or selective amplification of RNA targets versus DNA in the presence of dITP. The minus dITP reaction produced the smallest Cp delay between ±RT maintenance, suggesting that the amplification efficiency of HBV DNA was comparable or close to that of the HBV RNA target.

[0095] In the Cy5.5 channel, reactions with plus dITP generally delayed the GIC amplification curve and generated a low fluorescent signal. The use of dITP may require adjustment of the GIC target concentration. As expected, the GIC transcript plus dITP / -RT maintenance does not generate any Cp calls, as shown in Figure 8D. The minus dITP / minus RT maintenance reactions generate an amplification curve and a Cp call. This may be due to the presence of RT activity in these reactions even in the absence of RT maintenance. Thermal cycling at higher temperatures could potentially contribute to this result.

[0096] Taken together, these examples showed that preferential and / or selective amplification of HBV RNA targets instead of HBV DNA was demonstrated in the HBV region 7 assay by integrating dITP chemistry and limiting variations in the cobas® 6800 / 8800 master mix formulation to alter the thermal cycling profile. These examples demonstrate the feasibility of applying this method to selective RNA amplification of RNA targets that cannot be easily distinguished from natural DNA targets or targeted by utilizing polyA tails or intron / exon junctions.

[0097] Although the foregoing invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, all of the techniques and devices described above may 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 was individually indicated to be incorporated by reference for all purposes.

Claims

1. 1. A method for selectively detecting and / or quantifying one or more target Hepatitis B virus pregenomic RNAs (HBV pgRNAs) over Hepatitis B virus (HBV) DNA in a sample, comprising: (a) providing a sample; (b) providing one or more polymerases; (c) providing dNTPs including dITP; (d) performing an amplification step comprising denaturing a template nucleic acid, comprising contacting the sample with one or more sets of primers specific for the target HBV pgRNA; (e) performing a hybridization step comprising contacting the amplification products with one or more labeled probes if the target HBV pgRNA is present in the sample; (f) performing a detection step, the detection step comprising detecting the presence or absence of the target HBV pgRNA, the presence of the amplification product indicating the presence of the target HBV pgRNA in the sample and the absence of the amplification product indicating the absence of the target HBV pgRNA in the sample; the HBV pgRNA is a surrogate for HBV covalently closed circular DNA (cccDNA); During the amplification step, the one or more polymerases amplify the target HBV pgRNA if present in the sample by incorporating the dITP into the amplification product derived from the target HBV pgRNA, thereby lowering the melting temperature (Tm) of the amplification product derived from the target HBV pgRNA and allowing the amplification product derived from the target HBV pgRNA to be denatured during the amplification step at a temperature lower than that required for strand separation of double-stranded HBV DNA, thereby preventing the polymerase from accessing and amplifying the double-stranded HBV DNA target, and selectively detecting and / or quantifying the target HBV pgRNA relative to the HBV DNA in the sample. The method.

2. The method of claim 1 , wherein the dNTPs further comprise dATP, dTTP, dCTP and dGTP.

3. 3. The method of claim 2, wherein the ratio of dITP:dGTP is 3:1, 1:1 or 1:

3.

4. 3. The method of claim 2, wherein the dNTPs are in equal amounts (i) dATP, (ii) dTTP; and (iii) dCTP; and (iv) dGTP+dITP.

5. The method of claim 1 , wherein the one or more polymerases is a DNA polymerase.

6. 6. The method of claim 1 , wherein the one or more sets of primers and the one or more sets of probes hybridize to a nucleic acid sequence comprising SEQ ID NO:

4.

7. 7. The method of claim 6, wherein the one or more sets of primers comprise primers having the nucleic acid sequences of SEQ ID NO:1 and SEQ ID NO:2, and the one or more sets of probes comprise a probe having the nucleic acid sequence of SEQ ID NO:

3.

8. The method of claim 1, wherein the detection step (f) further comprises quantifying the one or more target HBV pgRNAs.

9. The method of claim 8, wherein the amount of quantified HBV pgRNA is a factor considered for treatment decisions regarding the patient from which the sample originated.

10. 1. A kit for selectively detecting and / or quantifying one or more target Hepatitis B virus pregenomic RNAs (HBV pgRNAs) in a sample over HBV DNA, comprising: (a) one or more polymerases; (b) dNTPs including dITP; (c) one or more sets of primers specific for the target HBV pgRNA; and (d) one or more labeled probes, A kit comprising a pair of primers and a labeled probe that hybridize to a nucleic acid sequence comprising SEQ ID NO:

4.

11. The kit of claim 10 , wherein the dNTPs further comprise dATP, dTTP, dCTP and dGTP.

12. The kit of claim 11, wherein the ratio of dITP:dGTP is 3:1, 1:1 or 1:

3.

13. 12. The kit of claim 11, wherein the dNTPs are in equal amounts (i) dATP, (ii) dTTP; and (iii) dCTP; and (iv) dGTP+dITP.

14. The kit of any one of claims 10 to 13, wherein the one or more polymerases are DNA polymerases.

15. 15. The kit of any one of claims 10 to 14, wherein the one or more sets of primers comprise primers having the nucleic acid sequences of SEQ ID NO:1 and SEQ ID NO:2, and the one or more sets of probes comprise a probe having the nucleic acid sequence of SEQ ID NO:3.

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