Digital PCR assay design for multiple hepatitis B virus gene targets and non-extendable blocker oligonucleotides for this purpose

Abstract

The present invention provides compositions and methods for novel digital PCR (dPCR) assay designs (droplet digital or other systems) for the detection and quantification of multiple Hepatitis B virus (HBV) gene targets. The compositions and methods can further include a non-extendible blocker oligonucleotide to reduce nonspecific inter-amplicon extension.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on and claims priority to U.S. Provisional Patent Application No. 63 / 435,798, filed December 28, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted as an electronic text file entitled "P38057-WO_Seq_Listing," which is 75,424 bytes in size and created on December 19, 2023. The information contained in this electronic file is hereby incorporated by reference in its entirety in accordance with 37 CFR § 1.52(e)(5).

[0003] FIELD OF THE INVENTION The present disclosure relates to the field of in vitro viral diagnostics. In this field, the present invention relates to the amplification and detection of target nucleic acids that may be present in a sample, particularly target nucleic acids containing sequence variations and / or individual mutations of hepatitis B virus (HBV), particularly HBV RNA (particularly HBV RNA derived from covalently closed circular double-stranded DNA (cccDNA) such as HBV pregenomic RNA (pgRNA)), and other HBV genetic targets, optionally using at least one competitive blocking oligonucleotide to reduce nonspecific inter-amplicon extension. The present invention further provides methods, reaction mixtures including oligonucleotides (such as reverse transcription (RT) primers and competitive blocking oligonucleotides), and kits for the amplification and detection of various HBV RNA forms. [Background technology]

[0004] Background of the Invention Hepatitis B is a liver infection caused by HBV. Notably, HBV can cause both acute and / or chronic infection. While many people are asymptomatic during the initial infection, some exhibit rapid onset of disease (including vomiting, yellowish skin, fatigue, dark urine, and abdominal pain). Chronic hepatitis B preferentially affects those infected around the time of birth. While most of these individuals with chronic disease are also asymptomatic, they may eventually develop cirrhosis and liver cancer. These complications result in 15% to 25% of deaths among those with chronic disease. Generally, HBV is transmitted through exposure to infectious blood or bodily fluids, for example, when blood, semen, or other bodily fluids from an HBV-infected person enters the body of an uninfected person. This can occur through sexual contact, sharing needles, syringes, or other drug injection equipment, or from mother to baby during birth. Infection near birth or through contact with other people's blood during childhood is the most common way of acquiring hepatitis B in areas where the disease is common. In areas where the disease is rare, intravenous drug use and sexual intercourse are the most common routes of infection. Other risk factors include medical procedures, blood transfusions, dialysis, living with an infected person, traveling to a country with high infection rates, and living in an institution. HBV infection can be diagnosed 30 to 60 days after exposure. The diagnosis is then usually confirmed by testing blood for hepatitis B virus fragments and antibodies to HBV.

[0005] Chronic hepatitis B infection, which affects 257 million people worldwide, remains a major health burden. While treatments are available to manage the disease, cure rates are low. In the absence of curative therapy, lifelong adherence to antiviral medications is required. Treatment removal often allows for a rebound in HBV viral titers because current treatments cannot directly target the HBV episomal genome reservoir within the nucleus of infected cells.

[0006] The HBV viral life cycle alternates between DNA and RNA forms. Infectious HBV particles contain a relaxed, circular, incomplete double-stranded DNA genome (rcDNA). In infected cells, HBV DNA replication is completed to form cccDNA in the host cell nucleus. Transcription from this DNA genome generates various messenger RNA forms that encode the viral structural proteins (core and surface proteins), e antigen, viral polymerase, and X antigen. One mRNA form, called pgRNA, also serves as a template for the RT activity of the viral polymerase, which generates new copies of the rcDNA in encapsidated, secreted viral particles. There is also evidence that a proportion of encapsidated pgRNA is released without being reverse transcribed, such that infected cell production contains both rcDNA- and pgRNA-containing viral particles. Furthermore, multiple spliced ​​RNA variants exist, some of which are also reverse transcribed into incomplete forms of HBV DNA and secreted. Integration of the HBV genome into the host chromosome is not part of the replication cycle because complete pgRNA molecules cannot be produced, but this is a common occurrence and can result in host cells producing smaller, truncated, or fused mRNAs that contribute to the secretion of surface antigen-containing subviral particles.

[0007] Table 1 below lists the HBV RNA forms that are thought to be generated from HBV cccDNA. [Table 1]

[0008] Table 2 below lists the forms of HBV RNA that cannot be transcribed from the integrated copy (i.e., originate exclusively from cccDNA). [Table 2]

[0009] Table 3 below shows some of the forms of HBV RNA that can be transcribed from the integrated copy. [Table 3]

[0010] Markers of HBV include DNA, e antigen (derived from precore mRNA), core antigen (or a combination of antigens including e and core), and s antigen, as well as the detection of a subject's or patient's production of antibodies against these antigens. Suppression of s antigen is a marker for functional cure. However, because s antigen can be produced by integrated, non-replicating copies of HBV, quantification of hepatitis B surface antigen (HBsAg) levels is unlikely to accurately reflect the pool of transcriptionally active cccDNA. DNA titer is monitored as a highly sensitive test for detecting HBV infection, and a reduction in HBV titer is an indicator of treatment response. However, current nucleoside analog therapies for HBV (which inhibit reverse transcription) do not affect the transcription of pgRNA or other mRNAs, only the generation of new rcDNA copies. The decrease in DNA titer in a patient's blood (plasma or serum sample types) does not necessarily correspond to a decrease in HBV RNA; this may be delayed or result in a transient increase due to the secretion of encapsidated pgRNA (and spliced ​​RNA) by infected cells harboring transcriptionally active cccDNA. For this reason, HBV RNA is being investigated as a separate marker for monitoring HBV disease status and treatment effectiveness. Studies have shown that HBV RNA levels can predict outcomes such as e antigen loss, viral recurrence, or "relapse" events after treatment cessation, making the biomarker potentially important in timing treatment termination for HBV patients.

[0011] Distinguishing between HBV RNA forms is important for understanding disease status and interpreting molecular test results. Multiple mRNAs may circulate separately from pgRNA (see, e.g., Stadelmayer et al., J Hepatol. 2020 vol. 73 pp. 40-51). These may have different transcription start sites but overlap due to the compact arrangement of the HBV genome (see, e.g., Altinel et al., J Virol. 2016 Nov 14;90(23):10811-10822). Most proposed HBV RNA detection assays have only one or two targets. State-of-the-art assays target poly(A) tails with two-step (RT and PCR) RACE methods (e.g., van Bommel et al., Hepatology 2015 61:66-76; Zhang W et al., Methods Mol Med 2004, Vol 95, p29-44; Kairat A et al., Intervirology 1999, Vol 42, p228-237). Poly(A) tail assays will detect pgRNA and other mRNAs that all end with the primary polyadenylation site ("full-length" poly(A) tail). These assays will also detect spliced ​​forms. However, to distinguish pgRNA or each individual mRNA from other overlapping forms, subtractive quantification strategies requiring multiple PCR targets would be required. For example, pgRNA and pre-core mRNA (a slightly longer 3.5 kb mRNA) could be distinguished by targeting the region of the genome between the transcription start sites of these forms (see, e.g., Wang Jie et al., Journal of Hepatology 2016 V 65:700-710). Similar targeting of differences in the length of the 5' end of mRNAs can also be used to distinguish pgRNA from the smaller viral mRNAs that produce the s and X antigens (see, e.g., Butler EK et al., Hepatology. 2018 68(6):2106-2117).The X gene mRNA transcript is known to circulate (see, e.g., Stadelmayer et al., J Hepatology 2020 vol. 73 pp. 40-51), and targets in this region pick up both this transcript and longer mRNAs and pgRNAs.

[0012] Other assays target spliced ​​RNA variants, which may be indicators of interferon treatment response (see, e.g., Chen et al., Sci Rep 5, 16459 (2015); Bayliss, J. et al., J Hepatol, 2013, V59, pp. 1022-1028; Preiss, S. et al., Hepatology, 2008, V48, pp. 741-749). Because targets in the core region can be disrupted by splice variants, the proportion of these transcripts in a sample is relevant for accurate quantification. While integrated HBV copies (which may be largely intact or fragmented in different infected cells) cannot produce active virus, they can generate S antigen-producing transcripts, which disrupt the human immune response and reduce the predictive ability of antigen monitoring as a marker of HBV viral production. Truncated RNA from integrated copies of HBV that terminate at a secondary poly(A) site upstream of the full-length site (van Bommel et al., Hepatology 2015 61:66-76) is not detected by the full-length 3' end assay but can be detected by other targets within the S gene.

[0013] In light of the above, there is a need for assays with improved effectiveness in distinguishing between different HBV RNA forms. Summary of the Invention

[0014] Summary of the Invention The present disclosure overcomes the aforementioned challenges by providing assays with improved efficacy for distinguishing between different HBV RNA forms. In one aspect, the present disclosure provides a panel of targeted assays for different HBV gene targets, all on the same platform, which can be multiplexed to reduce run-to-run variation.

[0015] Certain embodiments of the present disclosure relate to methods for rapid detection of the presence or absence of HBV RNA in biological or non-biological samples, e.g., for detecting HBV by polymerase chain reaction (PCR) in a single test tube, to monitor HBV disease status and treatment effectiveness. Such embodiments include methods for detecting HBV that involve performing at least one cycling step, which may include an amplifying step and a hybridizing step. Additionally, embodiments include oligonucleotides (including reverse transcription primers (which may be PCR primers), blocking oligonucleotides, conventional primers and probes), and kits designed for detecting HBV in a single tube.

[0016] Although one sequence difference between HBV DNA and RNA is the poly(A) tail of pgRNA and other mRNAs, methods using oligo-d(T) primers can detect non-target RNA or other sequences with poly(A) extensions. While "fixed" poly(T)-containing oligonucleotides may provide some measure of specificity for non-target binding and extension, this is a trade-off strategy that results in some binding to HBV DNA. The methods disclosed herein can include competitive blocking oligonucleotides whose RNA sequence matches the DNA sequence in targets with poly(A) tail junctions as a way to improve the performance (sensitivity and specificity) of assays targeting RNA in the presence of DNA. Binding of the competitive blocking oligonucleotide to homologous genomic HBV DNA prevents binding of primers (e.g., RT primers), thereby reducing undesired amplification of homologous genomic HBV DNA. To further improve the discrimination of this method, modified stabilizing bases can be incorporated into the assay oligonucleotide or blocker oligonucleotide.

[0017] Primers and probes can be provided that target the poly(A) tail of HBV RNA (particularly HBV RNA transcribed from cccDNA, which has the standard poly(A) tail position for transcripts such as pgRNA, but also includes other mRNAs and spliced ​​RNAs). Competitive blocking oligonucleotides can be provided that increase specificity for RNA in the presence of HBV DNA. Additional primers and probes can be provided that target other poly(A) sites, such as secondary or truncated poly(A) sites of HBV transcripts that may originate from integrated HBV copies. Competitive blocking oligonucleotides can be provided that increase specificity for RNA with these specific poly(A) sites in the presence of homologous DNA.

[0018] One aspect of the present invention relates to a method for detecting and quantifying two to six different Hepatitis B virus (HBV) target nucleic acids in a sample by digital PCR (dPCR), the method comprising: providing a sample; randomly dividing the sample into a plurality of equally sized, independent partitions; performing a dPCR assay in each partition using a plurality of sets of forward and reverse primers and a plurality of probes, each probe labeled with a fluorescent dye that generates a different signal for amplifying and detecting each of the HBV target nucleic acids; and measuring the amount of signal generated in each partition to calculate the amount of each HBV target nucleic acid in the sample. In one embodiment, the HBV target nucleic acids are selected from the group consisting of precore-mRNA 5' end (non-pgRNA), core, X gene, truncated RNA 3' end (poly(A) junction), precore / core, full-length RNA 3' end (poly(A) junction), alternative splice junction, S gene (pre-splice site), S gene (post-splice site), and pgRNA 5' end. In another embodiment, the dPCR assay is performed using a set of forward and reverse primers and a probe specific to the HBV target nucleic acid selected from the oligonucleotides listed in Tables 4 and 6. In one embodiment, the fluorescent dye on the probe is selected from the group consisting of Atto-425, FAM, HEX, Texas Red, Cy5, and Cy5.5. In one embodiment, the method further comprises reducing undesired amplification in the dPCR assay by using at least one blocker oligonucleotide with a non-extendible 3' end and a higher melting temperature (Tm) for the template nucleic acid compared to the set of at least two forward and reverse primers and at least two probes used in the dPCR assay. In some embodiments, undesired amplification can be due to the close proximity of amplification target regions. In another embodiment, undesired amplification is due to the presence of a DNA template where RNA is the target nucleic acid.In yet another embodiment, the unwanted amplification is the presence of RNA splice variants in situations where spliced ​​RNA is targeted and unspliced ​​RNA is inhibited.

[0019] In another aspect, a method for detecting and quantifying at least two different hepatitis B virus (HBV) target nucleic acids in a sample by polymerase chain reaction (PCR) is provided, comprising: randomly dividing the sample into a plurality of equally sized independent partitions; performing a PCR assay in each partition using at least two sets of forward and reverse primers for amplifying each of the HBV target nucleic acids and at least two probes labeled with fluorescent dyes that generate different signals for detecting each of the HBV target nucleic acids; and measuring the amount of signal generated in each partition to calculate the amount of each of the at least two different HBV target nucleic acids in the sample. In some embodiments, the PCR assay performed in each partition is a digital PCR (dPCR) assay. In some embodiments, the at least two different HBV target nucleic acids are selected from the group consisting of precore-mRNA 5' end (non-pregenomic RNA), core, X gene, truncated RNA 3' end (poly(A) junction), precore / core, full-length RNA 3' end (poly(A) junction), selected splice junction, S gene (pre-splice site), S gene (post-splice site), and pregenomic RNA 5' end. In certain embodiments, the dPCR assay is performed using a set of forward and reverse primers and a probe specific for the HBV target nucleic acid selected from the oligonucleotides listed in Tables 4 and 6. In some embodiments, the fluorescent dye on the labeled probe is selected from the group consisting of Atto-425, FAM, HEX, Texas Red, Cy5, and Cy5.5. In some embodiments, the method further comprises reducing undesired amplification in the dPCR assay by using at least one blocker oligonucleotide having a non-extendible 3' end and a higher melting temperature (Tm) relative to the template nucleic acid compared to the set of at least two forward and reverse primers and at least two probes used in the dPCR assay.In some embodiments, undesired amplification can be caused by the close location of amplification target regions.In another embodiment, undesired amplification is the presence of DNA template where RNA is the target nucleic acid.In yet another embodiment, undesired amplification is the presence of RNA splice variants where spliced ​​RNA is the target and unspliced ​​RNA is inhibited.

[0020] Another aspect of the present invention relates to a method for selectively detecting at least two targets in a sample, the method comprising: performing an amplification step comprising contacting the sample with a first primer set to produce a first amplification product if nucleic acid is present in the sample, a second primer set to produce a second amplification product if nucleic acid is present in the sample, and a blocker oligonucleotide; and performing a hybridization step comprising contacting the first and second amplification products with at least a first detectable probe and a second detectable probe. and detecting the presence or absence of first and second amplification products, wherein the presence of the first amplification product indicates the presence of the first target in the sample, the absence of the first amplification product indicates the absence of the first target in the sample, the presence of the second amplification product indicates the presence of the second target in the sample, and the absence of the second amplification product indicates the absence of the second target in the sample, wherein the nucleic acid comprises a contiguous sequence including the first target, the second target, and an intermediate sequence located between the first target and the second target, and the blocker oligonucleotide is complementary to at least a portion of the intermediate sequence. In some embodiments, the blocker oligonucleotide is not extendible by DNA polymerase. In some embodiments, the blocker oligonucleotide reduces or eliminates the production of undesired amplification products comprising the first target and the second target.

[0021] Another aspect of the present invention relates to a method for reducing undesired amplification in a multiplex digital PCR (dPCR) assay by using a blocker oligonucleotide that has a non-extendible 3' end and a higher melting temperature (Tm) for the template nucleic acid compared to the primers and probes used in dPCR. In one embodiment, the undesired amplification is due to the close location of amplification target regions. In another embodiment, the undesired amplification is due to the presence of a DNA template that targets RNA. In yet another embodiment, the undesired amplification is due to the presence of an RNA splice variant that targets spliced ​​RNA and inhibits unspliced ​​RNA.

[0022] In another aspect, a kit for selectively detecting at least two targets in a nucleic acid is provided, the kit comprising: a first primer set for generating a first amplification product when a first portion of the nucleic acid is present in a sample; a second primer set for generating a second amplification product when a second portion of the nucleic acid is present in the sample; a blocker oligonucleotide complementary to a nucleic acid intermediate between the first and second portions; a first detectable probe complementary to the first amplification product; and a second detectable probe complementary to the second amplification product. In some embodiments, the blocker oligonucleotide is not extendible by DNA polymerase. In some embodiments, the blocker oligonucleotide reduces or eliminates the production of undesired amplification products comprising the first and second targets.

[0023] 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.

[0024] 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. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. [Brief explanation of the drawings]

[0025] [Figure 1] Figure 1 is a schematic diagram showing a diagram of the digital PCR assay disclosed herein in a linear schematic representation of HBV RNA transcripts and selected splice variant forms. Elements of the full-length HBV circular genome (horizontal lines, boxes) are shown along with multiple exemplary primer pairs (pairs of arrows) for amplifying target regions of interest. Exemplary primers are shown for targets including the pre-core mRNA 5' end (non-pgRNA) 1, core target 2, X gene target 3, truncated RNA 3' end (poly(A) junction) 4, pre-core / core target 5, full-length RNA 3' end (poly(A) junction) 6, selected splice junction (example shown) 7, S gene, pre-splice site 8, S gene, post-splice site 9, and pgRNA + pc-mRNA 5' end of the 3.5 kb transcript 10. [Figure 2] FIG. 2 is a schematic diagram illustrating the use of blocker oligonucleotides to prevent unexpected amplification as a result of two amplification regions located close to each other in a target template. [Figure 3] FIG. 3 is a schematic diagram illustrating the use of blocker oligonucleotides that bind to intronic sequences to inhibit DNA-specific amplification without affecting RNA-specific amplification. [Figure 4] Figure 4 illustrates the use of blocker oligonucleotides designed to inhibit unspliced ​​RNA variants. An exemplary DNA template (top) is shown to depict three exons and two introns associated with a single target gene. [Figure 5]Figure 5 illustrates the use of blocker oligonucleotides designed to inhibit longer forms of fusion RNA. An exemplary DNA template (top) is shown showing two distinct regions: the first region (left) contains two exons and one intron associated with a first target gene, and the second region (right) contains one exon associated with a second target gene. [Figure 6] Figure 6 shows the linearity plot of the HBV dPCR assay according to the present disclosure. Primers and probes were designed for six HBV targets, including i) 3' precore, ii) 3' poly(A), iii) X gene mRNA, iv) core gene mRNA, v) truncated poly(A), and vi) 5' precore. The logarithm of the measured concentration of each of the six targets in the samples was calculated and plotted on the vertical axis against the known (expected) concentration of those samples on the horizontal axis. The linear regression of each series was calculated and plotted (dashed lines). The slope, intercept, and R2 value of each linear regression are shown in Table 5. [Figure 7] Figure 7 shows a boxplot of an HBV dPCR assay according to the present disclosure. Primers and probes were designed for six HBV targets, including i) 3' precore, ii) 3' poly(A), iii) X gene mRNA, iv) core gene mRNA, v) truncated poly(A), and vi) 5' precore. The logarithm of the measured concentration of each of the six targets in the samples was calculated and plotted on the vertical axis against the known (expected) concentrations of those samples on the horizontal axis. [Figure 8A] Figure 8A and Figure 8B show a comparison of dPCR assay results for the amplification of 3' precore RNA (Figure 8A) and 3' precore DNA (Figure 8B) using either a control reverse primer complementary to precore HBV (excluding the poly(A) tail) or a reverse primer complementary to precore HBV (including the poly(A) tail) containing an 8 bp poly(T). The fluorescent signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as indicated on the bottom horizontal axis. The well identifier (well ID) for each sample is indicated on the top horizontal axis, and data associated with each well ID are depicted by (and included between) vertical dashed lines. [Figure 8B] Figure 8A and Figure 8B show a comparison of dPCR assay results for the amplification of 3' precore RNA (Figure 8A) and 3' precore DNA (Figure 8B) using either a control reverse primer complementary to precore HBV (excluding the poly(A) tail) or a reverse primer complementary to precore HBV (including the poly(A) tail) containing an 8 bp poly(T). The fluorescent signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as indicated on the bottom horizontal axis. The well identifier (well ID) for each sample is indicated on the top horizontal axis, and data associated with each well ID are depicted by (and included between) vertical dashed lines. [Figure 9] Figure 9 shows dPCR assay results for multiple HBV S gene assays, including one S gene (S) assay and three S gene post-splicing (SPS) assays. The fluorescent signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as indicated on the bottom horizontal axis. The well identifier (and associated assay) for each sample is indicated on the top horizontal axis, and the data associated with each well is depicted by (and contained between) vertical dashed lines. [Figure 10] Figure 10 is a sensitivity plot showing measured titer (cps / μL) on the vertical axis compared to expected (input) concentration (log cps / μL) for SPS Assay 2 in Figure 9. A strong correlation between input and measured concentrations was determined by linear regression (R = 0.9991). [Figure 11] Figure 11 is a schematic diagram of an HBV 3.5 kb RNA assay targeting the pre-splice site of the core region to detect both pgRNA and pre-core mRNA. The target region of the assay is indicated by the dashed box. [Figure 12] Figure 12 shows a detailed view of the schematic diagram in Figure 11. The target regions of the assay are indicated by dashed boxes. Two in vitro templates (IVTs) containing the target regions (IVT14 and IVT15) are shown aligned with the corresponding sites on HBV RNA. [Figure 13] Figure 13 shows the dPCR assay results for the pgRNA IVT assay shown in Figures 11 and 12. The fluorescent signal detected in Channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as indicated on the bottom horizontal axis. The well identifier for each sample is indicated on the top horizontal axis, and the data associated with each well is depicted by (and contained between) vertical dashed lines. [Figure 14] Figure 14 shows dPCR assay results for the IVT14 template as detected in the assay illustrated in Figures 11 and 12. This assay demonstrates a high level of sensitivity, capable of detecting target templates (e.g., IVT14) down to 101 cps / µL. The fluorescent signal detected in Channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as shown on the bottom horizontal axis. The well identifier (and associated input concentration) for each sample is shown on the top horizontal axis, and the data associated with each well is depicted by (and contained between) vertical dashed lines. Saturating responses (no negative droplets detected) were observed at IVT14 input concentrations of 105 cps / µL and 106 cps / µL, while both positive and negative reaction droplets were observed at each of the other concentrations tested. [Figure 15] Figure 15 is a linearity plot showing measured titer (cps / μL) on the vertical axis compared to expected (input) concentration (log cps / μL) for the 3.5 kb RNA assay in Figure 14. A strong correlation between input and measured concentrations was determined by linear regression (R2=0.9992). [Figure 16] Figure 16 is a linearity plot showing measured titer (cps / μL) on the vertical axis compared to expected (input) concentration (log cps / μL) for the IVT14 template detected in the 3.5 kb RNA assay of Figure 14. A strong correlation between input and measured concentrations was determined by linear regression (R2=0.9999). [Figure 17]Figure 17 shows dPCR assay results for a multiplex assay targeting the detection of core, X gene, and poly(A) targets in HBV RNA. The assay involves ratio-based multiplexing with 100% core target in channel 1, 100% poly(A) target in channel 2, and 50% X gene target in channel 1 and 50% X gene target in channel 2. The fluorescent signal detected in either channel 1 or channel 2 of the dPCR instrument is plotted on the horizontal and vertical axes, respectively, for each event (detected droplet). Inter-cluster variations (corresponding to unexpected and / or unwanted amplification products) observed between clusters of data corresponding to each of the targets are indicated by rounded rectangular boxes. [Figure 18] Figure 18 is a schematic diagram of an assay directed to the detection of core, X gene, and poly(A) targets in HBV RNA. A non-extendable blocker oligonucleotide was designed to bind to the indicated region between the X gene and poly(A) to block the potential formation of a hybrid amplicon containing the X gene and poly(A). In addition to the binding region of the blocker oligonucleotide, forward (Fwd) and reverse (Rev) primer pairs and corresponding probes are shown for each of the three targets. [Figure 19] Figure 19 shows ddPCR assay results for a multiplex assay including non-extendible blocker oligonucleotides directed at detecting HBV RNA core, X gene, and poly(A) targets, as shown in Figure 18. The assay involves ratio-based multiplexing with 100% core target in channel 1, 100% poly(A) target in channel 2, and 50% X gene target in channel 1 and 50% X gene target in channel 2. The fluorescent signal detected in either channel 1 or channel 2 of the dPCR instrument is plotted on the horizontal and vertical axes, respectively, for each event (detected droplet). Inter-cluster rain (corresponding to unexpected and / or undesired amplification products) was significantly reduced or eliminated between clusters of data corresponding to each of the targets, compared to assays that excluded the non-extendible blocker oligonucleotides, as shown in Figure 17. [Figure 20]Figure 20 is a schematic diagram of an assay directed to the detection of a truncated poly(A) target in HBV RNA. The non-extendable blocker oligonucleotide TR3_DD was designed to prevent binding of the truncated poly(A) reverse primer to the corresponding target region in the HBV RNA template. In addition to the blocker oligonucleotide TR3_DD, the forward and reverse primers and corresponding probes are shown. [Figure 21] Figure 21 shows the dPCR assay results for the assay shown in Figure 20, both with and without the non-extension blocker oligonucleotide shown. The fluorescent signal detected in Channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as indicated on the bottom horizontal axis. The well identifier for each sample (and associated assay condition—i.e., whether non-extension blocker was present in the reaction (+) or not (-)) is indicated on the top horizontal axis, with data associated with each well indicated by the vertical dashed lines (included between). Both positive and negative reaction droplets were observed for each of the conditions tested. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description of the Invention Diagnosing HBV infection by nucleic acid amplification provides a rapid, accurate, reliable, specific, and sensitive method for detecting and / or quantifying viral infection. Described herein are digital PCR assays for detecting HBV gene targets (e.g., HBV pgRNA and smaller viral mRNAs) in the presence of homologous HBV DNA in non-biological or biological samples. Primers (including RT primers), competitive blocking oligonucleotides, and probes for detecting and quantifying HBV are provided, as are articles of manufacture or kits containing such primers, competitive blocking oligonucleotides, and probes. Compared with other PCR methods, the increased specificity and sensitivity of digital PCR (dPCR) for quantifying various forms of HBV RNA makes it feasible to implement this technology for routine diagnosis and therapeutic efficacy of HBV infection in clinical laboratories.

[0027] Droplet digital PCR (ddPCR) and digital PCR assay designs include multiple targets, including 5'- and 3'-end structures, overlapping mRNA formation quantification by targeting regions before and after the transcription start site, splice junction targeting assays, and assays for integrated copy transcripts. The assays can be used as RT-PCR assays after DNA removal (though poly(A)) and can also detect DNA forms such as incomplete HBV genomes resulting from reverse-transcribed spliced ​​RNA or integrated copies of DNA released from infected cells. These assays enable the evaluation of disease states and the biological effects of antiviral therapy.

[0028] The digital PCR assays disclosed in this invention include: (i) a polyA-targeting assay of full-length mRNA and the 3' end of pgRNA; (ii) an X gene-targeting assay; (iii) a core-targeting assay; (iv) a precore assay that targets near the 3' end but excludes the poly(A) tail junction; (v) an assay of the 5' end of precore mRNA, a 3.5 kb transcript slightly longer than pgRNA; (vi) a truncated assay that targets a secondary poly(A) start site; (vii) an S gene assay at positions upstream and downstream of the common splice junction; (viii) an assay of the 5' end of pgRNA plus precore-mRNA to capture the unspliced ​​3.5 kb transcript; and (ix) an assay for specific splice junctions.

[0029] A diagram of the dPCR assay, showing a linear schematic of HBV RNA transcripts and selected splice variant forms, is shown in Figure 1. These assays can be multiplexed in different combinations to conserve sample volume. Aside from the capabilities of the platform used for PCR, the main limitation of multiplexing is the proximity or overlap of several target primer sets. The assay design includes a novel oligonucleotide design incorporated into the master mix as a competitive, non-extendible blocking oligonucleotide, which improves assay specificity for targets when multiplexed. This blocker oligonucleotide is placed between adjacent amplification products in the PCR assay, reducing nonspecific extension between assay oligonucleotide sets across the region between the intended targets. These assays are designed for digital PCR platforms, which may include droplet digital systems (such as the QX200 Droplet Digital PCR System from Bio-Rad) or systems with other forms of reaction partitioning, including the Roche digital PCR system (Digital LightCycler®). Further details of digital PCR and the use of blocking oligonucleotide designs to improve multiplexing are described below.

[0030] The present disclosure includes oligonucleotide primers (including RT primers), competitive blocking oligonucleotides, and fluorescently labeled hydrolysis probes that hybridize to HBV nucleic acids, particularly HBV RNA (particularly HBV RNA transcribed from cccDNA, e.g., pgRNA), to specifically identify and quantify various forms of HBV RNA.

[0031] The disclosed methods may include performing at least one cycling step, which involves amplifying one or more portions of a nucleic acid molecule gene target from a sample using one or more primer pairs. As used herein, "HBV primer(s)" or "HBV RT primer(s)" refers to oligonucleotide primers that specifically anneal to a nucleic acid sequence found in HBV or HBV RNA (e.g., HBV pgRNA) and initiate reverse transcription and / or DNA synthesis therefrom under appropriate conditions to produce a respective amplification product. An example of a nucleic acid sequence found in HBV suitable for targeting includes HBV pgRNA. Each of the HBV primers (including RT primers) discussed anneals to a target such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. One or more amplification products are produced when one or more nucleic acids are present in the sample, and therefore the presence of one or more amplification products indicates the presence of HBV and / or HBV RNA (particularly HBV RNA transcribed from cccDNA, such as pgRNA) in the sample. The amplification product should contain a nucleic acid sequence complementary to one or more detectable probes for HBV and / or HBV RNA. As used herein, "HBV probe(s)" refers to an oligonucleotide probe that specifically anneals to a nucleic acid sequence found in an HBV target nucleic acid (e.g., HBV RNA). Each cycling step includes an amplification step, a hybridization step, and a detection step, in which the sample is contacted with one or more detectable HBV or HBV RNA (particularly HBV RNA transcribed from cccDNA such as pgRNA) probes to detect the presence or absence of HBV and / or HBV RNA in the sample. As used herein, a "blocking oligonucleotide" ("competitive blocking oligonucleotide" or "blocker") refers to a non-extendable oligonucleotide that specifically anneals to complementary DNA and reduces the occurrence of nonspecific inter-amplicon extension.

[0032] As used herein, the term "amplifying" 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 from HBV and / or HBV RNA). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature below the melting temperature of the primers, and enzymatically extending 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 cofactors for optimal activity of the polymerase enzyme (e.g., MgCl and / or KCl).

[0033] As used herein, the term "primer" is known to those skilled in the art and refers to an oligomeric compound, primarily an oligonucleotide, but also a modified oligonucleotide capable of "priming" DNA synthesis by a template-dependent DNA polymerase; i.e., 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 and releasing pyrophosphate. In some embodiments, a primer is also a reverse transcription (RT) primer (RT primer). There are several types of RT primers known in the art, including oligo(dT)N primers, fixed oligo(dT)N primers, random hexamer primers, and sequence-specific primers. In some embodiments, the RT primer anneals to RNA (e.g., HBV RNA) and extends to generate a DNA complement (i.e., reverse transcription of the target). In some embodiments, the RT primer targets poly(A)-containing HBV RNA, and thus the RT primer is a poly(T)-containing oligonucleotide.

[0034] 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.

[0035] 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.

[0036] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to elevated 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. Nevertheless, non-thermostable polymerases can also be employed in PCR assays, provided the enzyme is replenished as needed.

[0037] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0038] The terms "extension" or "lengthening" when used with respect to nucleic acids refer to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase, which typically adds nucleotides to the 3' end of the nucleic acid.

[0039] 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 the same 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 that are 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 Res. 7:649-656, each of which is incorporated herein by reference.

[0040] " Modified nucleotide " in the context of oligonucleotide refers to the change in which at least one nucleotide in the oligonucleotide sequence is replaced with a different nucleotide that provides the oligonucleotide with desired properties.The exemplary modified nucleotide that can be substituted in the oligonucleotide described herein includes, 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 ... Examples of modified nucleotides include 2'-O-methylribo-dU, 2'-O-methylribo-C, N4-ethyl-dC, N6-methyl-dA, 5-propynyl-dU, 5-propynyl-dC, and N6-benzyl-dA. Some oligonucleotides described herein contain modified bases for increased stability or other performance improvements. One example is 5-propynyl-dU (modified uracil), which can replace T (thymine). In the provided oligonucleotide sequences, the pdU, T, and U nucleotide designations are considered interchangeable, as assays can contain either modified or unmodified versions of a particular oligonucleotide. Other examples of modified nucleotides include locked nucleic acids (LNAs). LNA (also known as inaccessible RNA) is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. This bridge locks the ribose in the 3'-endo (North) conformation, which is often found in A-type duplexes. The effect of LNA is that the locked ribose conformation enhances base stacking and backbone preorganization, which significantly increases the hybridization properties (melting temperature) of oligonucleotides. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art.In certain embodiments, the modified nucleotide substitutions increase the melting temperature (T) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. m ) to modify the nucleotide sequence. To further illustrate, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimizing 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. Pat. No. 6,001,611, incorporated herein by reference. Other modified nucleotide substitutions can alter the stability of the oligonucleotide or provide other desirable characteristics. For example, some modifications can render the oligonucleotide non-extendable, which is useful for probes and competitive blocking oligonucleotides. Non-extendable ends can be promoted by adding phosphates, C3 spacers, dideoxynucleotides, or by attaching the 3' end of a second oligonucleotide to the 3' end of the oligonucleotide, etc.

[0041] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding HBV targets, such as nucleic acids encoding other portions of HBV, can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, appropriate size amplification products for ease of detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (i.e., primers must be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0042] The assays use the terms "competitive blocking oligonucleotide," "competitive blocking nucleotide," "competitive blocking nucleic acid," "blocking oligonucleotide," "blocking nucleotide," "blocker," and / or "blocking nucleic acid," which refer to the binding of a competitive blocking oligonucleotide to a region of HBV DNA or RNA.

[0043] A set of forward primers for detecting the presence or absence of HBV nucleic acids, such as HBV RNA and other gene targets, includes the sequences of SEQ ID NOs: 20, 23, 24, 210, 213, 214, 387, and 389. A set of RT / reverse primers for detecting the presence or absence of HBV nucleic acids, such as HBV RNA (e.g., HBV derived from cccDNA, such as pgRNA), includes the sequences of SEQ ID NOs: 16, 18, 19, 25-30, 33-190, 206, 208, 209, 215-220, and 223-380. A set of competitive blocking oligonucleotides for increasing the specificity of detecting the presence or absence of HBV nucleic acids, such as HBV RNA (e.g., HBV derived from cccDNA, such as pgRNA), includes the sequences of SEQ ID NOs: 1-15, 21, 22, 191-205, 211, and 212. A set of probes for detecting the presence or absence of HBV nucleic acid, such as HBV RNA (eg, HBV derived from cccDNA, such as pgRNA) includes the sequences of SEQ ID NOs: 17, 31, 32, 207, 221, 222, 381-386, 388, and 390-392.

[0044] In addition to a set of primers and competitive blocking oligonucleotides, the present method may use one or more probes to detect the presence or absence of HBV nucleic acid, such as HBV RNA (e.g., HBV derived from cccDNA, such as pgRNA). The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that enables it to specifically (i.e., preferentially) hybridize to a "target nucleic acid," in this case, HBV nucleic acid (HBV RNA, e.g., HBV RNA transcribed from cccDNA, such as pgRNA) (target) nucleic acid, under a defined, predetermined stringency. The "probe" may also be referred to as a "detection probe," which means that it detects the target nucleic acid.

[0045] In some embodiments, the described HBV nucleic acid probes (including probes for HBV RNA) can be labeled with at least one fluorescent label. In one embodiment, the HBV nucleic acid probes (including probes for HBV RNA) can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor moiety, e.g., a quencher. In one embodiment, the probe comprises or consists of a fluorescent moiety, and the nucleic acid sequence comprises or consists of SEQ ID NOs: 17, 31, 32, 207, 221, 222, 381-386, 388, and 390-392.

[0046] The design of oligonucleotides used as probes can be performed similarly to the design of primers. In embodiments, a single probe or a pair of probes may be used to detect amplification products. Depending on the embodiment, the probe(s) used may contain at least one label and / or at least one quencher moiety. Like primers, probes typically have a melting temperature appropriate for the thermal cycling parameters of the amplification method, and the length of each probe must be sufficient to allow sequence-specific hybridization to occur, but not so long that it reduces fidelity during synthesis. Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0047] The construct may comprise a vector containing one or more of the sequences of a primer for HBV, a competitive blocking oligonucleotide, and a probe nucleic acid molecule (e.g., SEQ ID NOs: 1-392). The construct may be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. HBV nucleic acid molecules may be obtained, for example, by chemical synthesis, direct cloning from HBV, or nucleic acid amplification.

[0048] Constructs suitable for use in the present methods typically include, in addition to an HBV nucleic acid molecule (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOs: 1-392), a sequence encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The choice of vector system usually depends on several factors, including, but not limited to, host cell choice, replication efficiency, selectability, inducibility, and ease of recovery.

[0049] Constructs containing HBV nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant, and animal cells. Prokaryotic hosts can include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris; mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells; insect cells; and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. Construct can be introduced into host cell by any technique commonly known to those skilled in the art.For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and virus-mediated nucleic acid transfer are common methods for introducing nucleic acid into host cell.In addition, naked DNA can be directly delivered into cell (see, for example, U.S. Patent No. 5,580,859 and U.S. Patent No. 5,589,466).

[0050] Constructs (plasmid vectors) can be used to generate RNA molecules through in vitro transcription or other processes to generate RNA templates that may also contain primer and probe binding sites. RNA template molecules can also be produced synthetically. One type of RNA template that can be created as a control material is an armored RNA (an RNA molecule enclosed within a protein coat), which involves the production of RNA and coat proteins (e.g., viral capsid proteins) by a construct (e.g., in a bacterial host) and the assembly of the coat proteins to encapsulate the RNA molecule. DNA molecules can also be enclosed in a protein coat for use as a control material.

[0051] 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). In some embodiments, useful primers include oligonucleotides that can act as initiation points for nucleic acid synthesis within the described HBV nucleic acid sequences (e.g., SEQ ID NOS: 15, 18-20, 23-30, 33-190, 206, 208-210, 213-220, and 223-380). In some embodiments, the primers are reverse transcription (RT) primers (RT primers). Primers can be purified from restriction digests by conventional methods or produced synthetically. While primers are preferably single-stranded for maximum efficiency in amplification, 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.

[0052] 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 for separating nucleic acid strands involves heating the nucleic acid until it 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 and the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C for a period of time, depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0053] Once 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. Annealing temperatures are typically 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 range from about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). If necessary, the reaction mixture is adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient for extension of the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize extension products from each primer annealed to the nucleic acid template, but not so high as to denature the extension products from their complementary templates (e.g., extension temperatures generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). The extension time can be from about 10 seconds to about 5 minutes (eg, 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).

[0054] The genome of retroviruses, or RNA viruses, is composed of ribonucleic acid, or RNA. HBV is a pararetrovirus, a non-retrovirus that still uses reverse transcription in its replication process, requiring RNA produced by host enzymes for viral replication. In such cases, the template nucleic acid, RNA, must first be transcribed into complementary DNA (cDNA) through the action of the enzyme reverse transcriptase. Using the RNA template and a short primer complementary to the 3' end of the RNA, reverse transcriptase directs the synthesis of a first-strand cDNA, which can then be used directly as a template for polymerase chain reaction. For general preparation of RNA, primers can also be random or assay / target-specific, depending on the method.

[0055] PCR assays can use HBV nucleic acids, such as RNA (e.g., HBV pgRNA) or DNA (cDNA). The template nucleic acid need not be purified and can be a minor fraction of a complex mixture, such as HBV nucleic acids contained in human cells. HBV nucleic acid molecules can be extracted from biological samples 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.

[0056] Oligonucleotide primers (e.g., forward primers containing SEQ ID NOs: 20, 23, 24, 210, 213, 214, 387, and 389, and RT / reverse primers containing SEQ ID NOs: 16, 18, 19, 25-30, 33-190, 206, 208, 209, 215-220, and 223-380) are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl, 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, or one or more analogs thereof.

[0057] The newly synthesized strand forms a double-stranded molecule that can be used in subsequent steps of the reaction. The strand separation, annealing, and extension steps can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target HBV nucleic acid molecule (including HBV RNA, e.g., HBV pgRNA). The limiting factor in the reaction is 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 may depend, 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 sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0058] 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 from each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, when the donor is excited by light radiation of a suitable wavelength, it transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule containing a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0059] In one example, an oligonucleotide probe can contain a donor fluorescent moiety (e.g., FAM) and a corresponding quencher (e.g., BlackHole Quencher™ (BHQ) (e.g., BHQ2)), which 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, so 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 Quenchers™ (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).

[0060] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to an amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the HBV RNA target nucleic acid sequence (HBV RNA, e.g., HBV RNA transcribed from cccDNA, such as pgRNA). 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.

[0061] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission in a specific range), 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.

[0062] As used herein with respect to a donor and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher having 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. This allows for efficient non-irradiative energy transfer between them.

[0063] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) highly efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift in emission as far as possible toward the red portion of the visible spectrum (>600 nm), and (d) a shift in emission to a wavelength higher than the Raman water fluorescent 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 fluorescent 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).

[0064] 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, succinimidyl 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, diethylenetriamine pentaacetate, 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, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0065] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the angstrom (Å) distance from the nucleotide base to the fluorescent moiety. Typically, the linker arm is 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 for attaching the linker arm to a specific nucleotide base and for attaching the fluorescent moiety to the linker arm.

[0066] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)) to generate, for example, LC Red 640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, MA)), amide linkers (derived from fluorescein-NHS-esters, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0067] Control samples can be cycled during each thermocycler run as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the listed target gene) using, for example, control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within 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 a negative control, for example, lacking target template DNA. The negative control can measure contamination, ensuring that the system and reagents do not produce false-positive signals. Thus, control reactions can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate elongation, and the ability of probes to hybridize with sequence specificity and allow FRET to occur.

[0068] In one embodiment, the method includes a step to avoid contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase to reduce or eliminate contamination between thermocycler runs are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313.

[0069] Principles of digital PCR Digital PCR (dPCR), sometimes referred to as droplet digital PCR, is a PCR-based method for quantifying DNA or RNA targets. In digital PCR, a reaction mixture containing target nucleic acid, primers, probes, and other reagents is randomly distributed into thousands of equally sized, independent partitions, followed by end-point PCR. TaqMan hydrolysis probes are commonly used to detect target amplification, and the fluorescent signal of each partition is measured at the end. Partitions that do not contain target nucleic acid have relatively low fluorescence and are therefore negative, while partitions primed with one or more target nucleic acid molecules have high fluorescence and are therefore positive. For each reaction, the proportion of negative partitions provides the basis for absolute quantification using Poisson statistics.

[0070] Methods involving dPCR offer a fairly novel approach to nucleic acid detection and quantification, offering an alternative to traditional real-time quantitative PCR for absolute quantification of nucleic acids and rare allele detection. dPCR assays work by partitioning a sample of nucleic acid into many individual, parallel PCR reactions, some of which contain the target molecule (positive) and others not (negative). Following PCR analysis, the proportion of negative reactions is used to generate an absolute count of the number of target molecules in the sample. One key advantage of dPCR over real-time PCR is its superior quantitative accuracy. This advantage relies on the inherent properties of dPCR, as quantification requires only an accurate count of the positive partitions and knowledge of the theoretical partition volume (counts are less dependent on PCR efficiency). Quantitation standards are not required, eliminating potential quantitation errors caused by the standards themselves.

[0071] As detailed above, dPCR samples are partitioned so that individual nucleic acid molecules within the sample are localized and concentrated within many distinct regions (reaction regions). Partitioning the sample allows for estimation of the number of nucleic acids by assuming that the molecular population follows a Poisson distribution. As a result, each region contains either a negative or positive reaction ("0" or "1", respectively). After PCR amplification, nucleic acids may be quantified by counting the regions containing the positive PCR end product. In conventional quantitative PCR, quantitative results can depend on the amplification efficiency of the PCR process. However, dPCR does not rely on the number of amplification cycles to determine the initial sample amount, eliminating reliance on uncertain exponential data to quantify target nucleic acids and therefore providing absolute quantification.

[0072] In the next step, dPCR is performed using the sample in each reaction area of ​​the series of reaction areas. In dPCR, the nucleic acid of interest is amplified and detected, and a large number of individual molecules are isolated in each separate reaction area. Each reaction area (well, chamber, bead, emulsion, etc.) has either a negative result if the starting molecule is absent or a positive result for amplification and detection if the target starting molecule is present. This technique involves limiting dilution of the sample across a large number of separate PCR reactions, so that a portion of the reactions lack template molecules, resulting in a negative amplification result. When counting the number of positive PCR reactions at the end of the reaction, each individual template molecule present in the original sample is counted. PCR-based techniques have the additional advantage of counting only molecules that can be amplified, such as those associated with a massively parallel PCR step in a sequencing workflow. In digital PCR-based methods, the nucleic acid to be analyzed is distributed across several different reaction areas (e.g., wells, beads, emulsions, gel spots, chambers, etc. in a microfluidic device). It is important that some, but not all, reaction areas contain at least one molecule. Typically, each reaction area contains one or zero molecules. In reality, there is a random distribution of molecules to reaction regions, such as wells. If a percentage of reaction regions (e.g., 80%) are positive, some regions contain one or more molecules (e.g., an average of 2.2 molecules per well). Statistical methods can be used to calculate the expected total number of molecules in a sample based on the number of different reaction regions and the number of positives. This provides a calculated amount or concentration of nucleic acid in the portion applied to the different reaction regions. Several statistical methods based on sampling and probability can be used to arrive at this concentration.An example of such an analysis is Dube et al., arXiv:0809.1460v2 "Computation of Maximal Resolution of Copy Number Variation on a Nanofluidic Device using Digital PCR (2008)," found at arxiv.org, citation arXiv:0809.1460v2 [q-bio.GN], originally uploaded on September 8, 2008. This publication provides a set of formulas that can be used to estimate the concentration and statistical confidence interval of a molecule based on the number of reaction regions used in the digital PCR array and the number of positive results. Another example of this type of calculation can be found in U.S. Patent Application Publication No. 2009 / 0239308.

[0073] Typically, a Poisson distribution is used to predict the digital regime, ensuring that only a single DNA amplicon occurs within randomly discretized volumetric reactors, favoring only one DNA amplicon of interest per reaction volume. In this way, the PCR amplification signal (e.g., fluorescence) emitted by each reactor volume is the product of only one amplicon and is isolated from all other separate reactor volumes. Quantification is then achieved by counting the number of digital reactors that emit an amplified fluorescent signal corresponding to an intercalating dye or specific DNA polymerase probe sequence. Because each reactor volume is limited to no more than a single DNA strand in the digital regime, it can be accurately assumed that 100% of that amplified fluorescent signal originates from only one DNA strand and the corresponding primer and probe set. However, very low concentration regimes are usually unfavorable due to the inaccuracy of results.

[0074] Several methodologies exist for dPCR. For example, emulsion PCR has been used to prepare small beads with clonally amplified DNA; essentially, each bead contains one dPCR amplicon. Fluorescent probe-based techniques that can be performed on PCR products "in situ" (i.e., in the same well) are particularly well suited for this application. U.S. Patent No. 6,440,705 contains a more detailed description of this amplification procedure. These amplifications can be performed in emulsions or gels, on beads, or in multiwell plates. dPCR also includes microfluidic-based techniques that use channels and pumps to deliver molecules to several reaction regions. Suitable microfluidic devices are known in the art.

[0075] dPCR is performed essentially as conventional PCR. Nucleic acids (reference or target) in an appropriate medium are contacted with primers, probes, and a thermostable polymerase (e.g., Taq polymerase) and thermocycling (repeated heating and cooling cycles of the reaction for strand separation and enzymatic replication). The medium typically contains deoxynucleotides, buffer, and ions (e.g., Mg2+). The selectivity of PCR arises from the use of primers complementary to the region targeted for amplification under specific thermocycling conditions. The resulting amplification products are detected by using an appropriate probe, which is usually labeled, e.g., fluorescently labeled. In mRNA-based PCR, the RNA sample is first reverse-transcribed into complementary DNA (cDNA) using reverse transcriptase.

[0076] Typically, the PCR process consists of a series of temperature changes repeated 25–50 times. These cycles usually consist of three steps: first, at approximately 95°C, to allow separation of nucleic acid duplexes; second, at approximately 50–60°C, to allow binding of primers to the DNA template; and third, at temperatures between 68–72°C, to promote polymerization by DNA polymerase. Because the fragments are small, the enzyme can increase their number during the transition between the alignment and denaturation steps. Furthermore, to reduce the signal resulting from the presence of primer dimers when nonspecific dyes are used, signals, such as fluorescence, are measured at temperatures of, for example, 80°C. The temperatures and timing used depend on a variety of parameters, including the enzyme used to synthesize DNA, the concentrations of divalent ions and deoxyribonucleotides (dNTPs) in the reaction, and the temperature at which primers bind.

[0077] The dPCR method allows for the inherent ability to identify a larger number of fluorescent probe sequences (e.g., TaqMan probe sequences) by using multiple color, time, and intensity combinations to encode each unique probe sequence. Furthermore, using less expensive non-TaqMan probe real-time PCR amplification indicators, such as SYBR or PicoGreen, multiplexed dPCR based on temporal cues alone, intensity cues alone, or combined intensity and temporal cues can be achieved, thus enabling greater discrimination between primer pairs at significantly reduced cost. These can also be used to enhance control and, if necessary, normalize results with greater precision. Typical multiplexing limits from typical 5-plex qPCR can be increased to 100-plex dPCR with limited spectral bands using fluorescent reporters.

[0078] There are many available dPCR systems that can be used in the present invention. Commercially available digital PCR platforms include Fluidigm's microwell chip-based BioMark® dPCR, the hole-based QuantStudio12k flex dPCR and Life Technologies' 3D dPCR, as well as Bio-Rad®'s droplet-based ddPCR (ddPCR) QX100 and QX200, and RainDance®'s RainDrop. Microfluidic chip-based dPCR can have up to several hundred reaction regions per panel. Droplet-based dPCR typically has approximately 20,000 partitioned droplets, with up to 10,000,000 per reaction. The QuantStudio 12k dPCR performs digital PCR analysis on OpenArray® plates, which contain 64 reaction regions per subarray and a total of 48 subarrays, equivalent to a total of 3,072 reaction regions per array.

[0079] Droplet dPCR (ddPCR) is based on water-oil emulsion droplet technology. A sample is partitioned into a large number of droplets (e.g., approximately 20,000), and PCR amplification of template molecules is performed in each individual droplet. ddPCR technology uses reagents and workflows similar to those used in most standard TaqMan probe-based assays, including droplet formation chemistry. Intercalating dyes such as Evagreen may also be used. Partitioning of large sample reactions is a key aspect of ddPCR technology. Non-spherical partitions (e.g., nanowells) actually provide a larger area per sample volume than the same number of spherical partitions.

[0080] Typically, the use of more reaction regions can improve the accuracy, and more importantly, the precision, of dPCR determinations. Approximately 100-200, 200-300, 300-400, 700, or more reaction regions can be used to determine the quantity or concentration of interest by PCR. In a preferred embodiment of the method of the present invention, dPCR is performed identically in at least 100 reaction regions, particularly at least 1,000 reaction regions, and particularly at least 5,000 reaction regions. In a preferred embodiment of the method of the present invention, dPCR is performed identically in at least 10,000 reaction regions, particularly at least 50,000 reaction regions, and particularly at least 100,000 reaction regions.

[0081] Preferably, dPCR involves the use of one or more fluorescent dPCR probes to detect one or more nucleic acids of interest, particularly in combination with a quencher, as a molecular beacon, or as a hydrolysis probe. dPCR may also involve the use of one or more fluorescent probes to detect the nucleic acid of interest and / or a reference nucleic acid, particularly in combination with a quencher, as a molecular beacon, or as a hydrolysis probe. Representative donor and acceptor fluorescent moieties in FRET technology are described above. To detect and quantify two or more targets in the same reaction, primer sets and probe sets for each target can be combined for multiplexing. The Roche Digital LightCycler® dPCR system has six optical channels, allowing multiplexing of up to six targets in a single reaction using differently labeled TaqMan hydrolysis probes for each target. Other systems with fewer optical channels may require more complex strategies for multiplexing, such as using a combination of dyes in a single probe.

[0082] Use of blocker oligonucleotides to reduce unexpected amplification The greatest challenge in optimizing a multiplex assay is ensuring the absence of significant oligonucleotide interactions and assay interference. One possible scenario is when multiple primer and probe sets overlap in the amplifiable sequence, resulting in multiple amplifications, both expected and unexpected. Figure 2 shows an example of an HBV RNA assay design in which two amplifications are located close to each other (within approximately 2 kb) within the target template. In addition to the expected amplification for which each primer / probe set was designed, there may be an additional unexpected amplification event from one primer in each assay and two probe cleavage events from one primer. These multiple amplification and probe cleavage events may occur with different efficiencies due to amplicon length and primer-probe distance, resulting in reduced endpoint fluorescence in some partitions (the "rain" phenotype) and inaccurate quantification. Blocker oligonucleotides designed with intermediate sequences with higher Tm (65°C-90°C, possible with Tm enhancer modifications such as LNA) and non-extendable 3' termini (containing a 3-C spacer or phosphorylation) can effectively bind to the template with high affinity and inhibit primer extension into undesired regions. Adding blocker oligonucleotides to the reaction significantly reduces the "rhine" phenotype in dPCR reactions and improves quantitative results.

[0083] A general application of blocker oligonucleotides for non-HBV assays as well as HBV targets may involve closely spaced amplification targets that are amenable to multiplexing. The two targets cannot share the same primer set because the resulting amplicons would fall outside the optimal size range for dPCR and would have sensitivity issues associated with sample fragmentation. This utility applies to both DNA and RNA assays (see Figure 2).

[0084] General blocker oligonucleotide applications for non-HBV assays as well as HBV targets may also include RNA assays that may cross-react with DNA templates present in the sample. Even if probes can be designed at exon junctions to avoid cleavage from DNA amplification, they still inevitably deplete primers and reduce on-target amplification efficiency. Blocker oligonucleotides can be designed to bind to intronic sequences that inhibit DNA-specific amplification without affecting RNA amplification (see Figure 3).

[0085] General applications of blocker oligonucleotides for HBV targets as well as non-HBV assays may include samples with heterogeneous template sequences. Such samples may contain splice variants and fusion products. Regarding splice variants, some splice variants may generate small amplicons that compete with longer amplification from unspliced ​​RNA species. In this case, blocker oligonucleotides can be designed to inhibit longer amplification and additional priming after the blocker, allowing for better multiplexing in this reaction (see Figure 4). Regarding detection of multiple fusion products, if multiple fusion products have overlapping sequences, a single primer pair may generate amplicons with varying lengths and therefore different PCR efficiencies. Amplicon sizes longer than 300 bp are not optimal for digital PCR. In this case, blocker oligonucleotides can be designed to inhibit longer amplification. Additional priming after the blocker oligonucleotide may generate amplicons of similar size compatible with the smaller fusion products, which could be distinguished using a second probe of a different color (see Figure 5).

[0086] Manufactured Products / Kits Embodiments of the present disclosure further provide articles of manufacture or kits for detecting HBV RNA and other gene targets. The articles of manufacture can include primers and probes used to detect HBV RNA targets, along with appropriate packaging materials. Representative primers and probes for detecting HBV RNA, including HBV RNA transcribed from cccDNA, such as HBV pgRNA, can hybridize to HBV target nucleic acid molecules. Furthermore, the kits can 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 amplify and hybridize to HBV target nucleic acid molecules are provided.

[0087] The article of manufacture may also include one or more fluorescent moieties for labeling the probes, or the probes provided with the kit may be labeled. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling HBV probes (which may include probes targeting HBV RNA). Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0088] The article of manufacture may also include a package insert or packaging label with instructions for using the primers and probes to detect HBV (including HBV 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.

[0089] Embodiments of the present disclosure also provide a set of primers and one or more detectable probes for detecting HBV RNA, including HBV RNA, in a sample. Additional primers and probes can be provided that target other poly(A) sites, such as secondary or cleaved poly(A) sites in HBV transcripts that may originate from integrated HBV copies.

[0090] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. [Example]

[0091] 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.

[0092] Example 1: This example shows the sequences of oligonucleotides used in HBV dPCR assays. The nucleotide sequences and descriptions of the primers (forward and reverse), probes, and blocking oligonucleotides used to perform dPCR assays for the detection and quantification of various HBV RNA forms and gene targets are listed in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0093] Example 2: This example illustrates an HBV dPCR assay according to the present disclosure. An exemplary digital PCR assay for detecting HBV RNA can be designed to detect the presence or absence of one or more HBV targets, including the precore mRNA 5' end (non-pgRNA) 1, core target 2, X gene target 3, truncated RNA 3' end (poly(A) junction) 4, precore / core target 5, full-length RNA 3' end (poly(A) junction) 6, selected splice junction (example shown) 7, S gene, pre-splice site 8, S gene, post-splice site 9, and pgRNA + pc-mRNA 5' end of the 3.5 kb transcript 10, as shown in Figure 1. In this example, primers and probes were designed for six HBV targets: i) 3' precore, ii) 3' poly(A), iii) X gene mRNA, iv) core gene mRNA, v) truncated poly(A), and vi) 5' precore. A dilution series of known concentrations of each of the six targets was prepared, and a ddPCR assay containing the primers and probes in Table 4 was performed to detect each of the six targets. The measured concentrations for each of the six targets in the samples were calculated and plotted against the known (expected) concentrations in those samples, as shown in the linear plot in Figure 6 and the box-and-whisker plot in Figure 7. The expected concentrations correlated strongly with the measured concentrations, as evidenced by the linear regression data shown in Table 5. [Table 5]

[0094] Referring to Figures 8A and 8B, a comparison of dPCR assay results is shown for the amplification of 3' precore RNA (Figure 8A) and 3' precore DNA plasmid (Figure 8B) using either a control reverse primer complementary to precore HBV (excluding the poly(A) tail) or an 8-bp poly(T)-containing reverse primer complementary to precore HBV (including the poly(A) tail). As shown in Figure 8A, experiments using both the control and poly(T) primers in samples containing 3' precore HBV RNA successfully generated a detectable fluorescent signal approximately 10,0000 units above a baseline signal amplitude of approximately 2,500 units. As expected, experiments using samples containing 3' precore HBV DNA yielded a positive signal of approximately 12,000 units over a baseline of approximately 3,500 units only for the control primer (Figure 8B), as the 3' precore HBV DNA template does not contain a poly(A) tail complementary to the poly(T) amplification primer.

[0095] Example 3: This example presents the sequences of oligonucleotides used in HBV dPCR assays for the S gene. To address possible splicing effects, two S gene assays were designed: the S gene assay (S) and the spliced ​​S gene assay (SPS). For the S gene assay, specific primers (HBV_S600_FP-1-1_7G2 (SEQ ID NO: 121) and HBV_S600_RP-1-1_7G2 (SEQ ID NO: 125)) and probe (HBV_S600_PR-1-1_FZI (SEQ ID NO: 123)) were used. For the SPS assay, three different combinations of primers and probes were performed: i) SPS assay-1 (HBV_S1000_FP-1-1_7G2) and probe-2 (HBV_S600_RP-1-1_7G2) were used. HBV_S1000_FP-1-1 (SEQ ID NO: 117) and HBV_S1000_RP-1-1 (SEQ ID NO: 120)), ii) SPS assay-2 (HBV_S1000_FP-1-1 (SEQ ID NO: 117) and HBV_S1000_RP-2-1 (SEQ ID NO: 129)), and iii) SPS assay-3 (HBV_S1000_FP-1-1 (SEQ ID NO: 117) and HBV_S1000_RP-3-1 (SEQ ID NO: 130)). Of note, a common probe, HBV_S1000_PR-1-1_FZI (SEQ ID NO: 118), was used in all three SPS assays.

[0096] The template for these assays was HBV pgRNA in vitro transcript (pgRNA IVT). The first experiment focused on conducting preliminary tests using the S gene assay, with the specific goal of implementing two assay designs that could address the potential effects of splicing. Referring to Figure 9, both the S gene (S) assay and the S gene post-spliced ​​(SPS) assay successfully detected the target region as intended. While SPS assay 1 showed a second positive droplet band at lower channel 1 amplitude, SPS assays 1 and 3, as well as the S assay, demonstrated optimal performance, exhibiting only a single positive droplet band with minimal background signal. In conclusion, the present S gene assay was effective in specifically detecting two adjacent regions within the S gene sequence.

[0097] The results in Figure 10 show the results of the sensitivity test of SPS Assay 2. The previous test pattern persisted, with 10 6 and 10 5 Reactions containing pgRNA IVT at a concentration of 10 copies / microliter (cps / µL) reached saturation. 3 One of the replicates from the cps / μL IVT showed lower fluorescence and was therefore omitted from the titer calculation. The assay demonstrated sensitivity in detecting template even at concentrations as low as 1 cps / μL.

[0098] Example 4: This example demonstrates an HBV assay directed at the pre-splicing site of the core region while avoiding the major downstream spliced ​​intron.

[0099] The HBV 3.5 kb RNA assay targets the pre-splice site of the core region and strategically avoids the major downstream spliced ​​intron. This design allows for the detection of both pgRNA and the slightly longer pre-core mRNA, as shown in Figure 11. Referring to Figure 12, the assay was designed and tested using two similar templates (IVT14 and IVT15), each of which contains at least a portion of the 5' pre-core region representing the pre-core RNA as well as the pgRNA IVT. Successful detection of IVT14, IVT15, and pgRNA was achieved by ddPCR (Figures 13 and 14).

[0100] Referring to Figure 14, further sensitivity testing revealed that the 3.5 kb RNA assay of this example did not exhibit significant background noise or rain using the IVT14 template. 5 At copies / µL or higher, ddPCR reactions using the IVT14 template were observed to be saturated. As expected, no significant positive droplets were observed with the template representing the 3' end of HBV mRNA due to the absence of the reverse primer binding region. Similar results were observed with the IVT15 template (not shown). Linearity studies, shown in Figures 15 and 16, indicate that the assay was stable at 104 Copies / µL to 10 1 pgRNA and up to 10 copies / µL 3 Copies / µL to 10 1 The assay was sensitive enough to detect IVT14 down to 10 copies / µL, with relatively low standard deviations within these concentrations. 4 pgRNA and 10 copies / µL 3 Reactions tested with more than 14 copies / µL of IVT resulted in saturation and no negative reaction droplets were observed.

[0101] Example 5: This example describes an HBV triplex assay containing blockers according to the present disclosure. Initial ddPCR multiplex assays targeting at least the core, X, and poly(A) targets observed intercluster rain (Figure 17). Upon closer inspection, this intercluster rain was determined to be localized to the cluster containing the X gene and poly(A) target. To reduce or eliminate the observed intercluster rain, a DNA polymerase-nonextendable oligonucleotide was designed to bind to the region between the X gene and poly(A), as shown in Figure 18, with the objective of i) preventing the formation of a double-target amplicon (e.g., an amplicon containing both the X gene and poly(A) region) and ii) promoting the production of a single-target amplicon. A non-extendable blocker oligonucleotide was strategically designed to bind to the region between the X gene and poly(A) and block the potential formation of a hybrid amplicon containing the X gene and poly(A). The initial ddPCR multiplex assay targeting the core, X, and poly(A) was repeated with the addition of blocker oligonucleotides present at the same concentration as the primers. The ddPCR assay data shown in Figure 19 indicated that the presence of the blocker oligonucleotide resulted in a significant reduction in inter-cluster rain.

[0102] Example 6: This example demonstrates an HBV assay for detecting truncated poly(A) according to the present disclosure. Referring to Figure 20, a truncated poly(A) assay was developed to detect truncated HBV RNA species characterized by a former poly(A) region. The assay included forward primer HBV_TR_A_FMIX1-N (SEQ ID NO: 57), reverse primer HBV_TRPA_7HS (SEQ ID NO: 60), probe HBV_TRPA_FL_FZIB_PR (SEQ ID NO: 58), and blocker oligonucleotide TR3_DD (SEQ ID NO: 62). Similar to the applications described in Figures 4 and 18, the blocker oligonucleotide TR3_DD served to prevent binding of the truncated poly(A) reverse primer to its corresponding target region in uncleaved HBV RNA and / or HBV DNA. The results of ddPCR assays containing the primers, probes, and blockers described above showed that amplification of in vitro transcripts with uncleaved 3' poly(A) (Figure 21) was effectively inhibited, whereas amplification was successfully achieved for in vitro transcripts containing the truncated 3' poly(A) region.

[0103] Example 7: This example provides additional sequences of oligonucleotides used in HBV dPCR assays. The nucleotide sequences and descriptions of primers (forward and reverse), probes, and blocking oligonucleotides used to perform dPCR assays for the detection and quantitation of various HBV RNA forms and gene targets are listed in Table 6. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0104] In Table 6,<D_LNA_T> refers to D-locked nucleic acid thymine,<D_LNA_G> refers to the D-locked nucleic acid guanine,<BHQ_2> refers to Black Hole Quencher 2,<Spc_C3> refers to a three-carbon spacer, <cy5>refers to the cyanine 5 fluorescent dye,<CY5.5> <5_HEX_ABD> refers to a cyanine 5 fluorescent dye variant, <5TEX_615> refers to a Texas Red fluorescent dye, <5_FAM_ABD> refers to a fluorescein dye, HEG refers to a hexaethylene glycol spacer, and <5_HEX_ABD> refers to a hexachloro-fluorescein dye. While specific dye, spacer, and quencher molecules are assigned to nucleic acid sequences in Table 6 (and throughout this disclosure), it will be understood that substitutions may be possible. For example, one dye molecule may be substituted for another without substantially affecting the utility of the corresponding nucleic acid sequence.

[0105] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail may be made therein 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 detecting and quantifying two to six different Hepatitis B virus (HBV) target nucleic acids in a sample by digital PCR (dPCR), comprising: - providing said sample; - randomly distributing the sample into a plurality of equally sized independent partitions; - in each partition, performing a dPCR assay using a set of 2 to 6 forward and reverse primers for amplifying each of the HBV target nucleic acids and 2 to 6 probes, each probe labeled with a fluorescent dye that generates a different signal for detecting each of the HBV target nucleic acids; - measuring the amount of signal generated in each of said partitions to calculate the amount of each of said 2 to 6 different HBV target nucleic acids in said sample; A method comprising:

2. 2. The method of claim 1, wherein the two to six different HBV target nucleic acids are selected from the group consisting of precore-mRNA 5' end (non-pregenomic RNA), core, X gene, truncated RNA 3' end (poly(A) junction), precore / core, full-length RNA 3' end (poly(A) junction), alternative splice junction, S gene (pre-splice site), S gene (post-splice site), and pregenomic RNA 5' end.

3. 3. The method of claim 1 or 2, wherein the dPCR assay is performed using the set of forward and reverse primers and a probe specific for the HBV target nucleic acid selected from the oligonucleotides listed in Table 4 and Table 6.

4. The method of any one of claims 1 to 3, wherein the fluorescent dye on the labeled probe is selected from the group consisting of Atto-425, FAM, HEX, Texas Red, Cy5 and Cy5.

5.

5. 1. A method for detecting and quantifying at least two different Hepatitis B virus (HBV) target nucleic acids in a sample by polymerase chain reaction (PCR), comprising: - providing said sample; - randomly distributing the sample into a plurality of equally sized independent partitions; - performing a PCR assay in each partition using at least two sets of forward and reverse primers for amplifying each of the HBV target nucleic acids and at least two probes, each probe labeled with a fluorescent dye that generates a different signal for detecting each of the HBV target nucleic acids; - measuring the amount of signal generated in each of said partitions in order to calculate the amount of each of said at least two different HBV target nucleic acids in said sample; A method comprising:

6. 6. The method of claim 5, wherein the PCR assay performed in each partition is a digital PCR (dPCR) assay.

7. 7. The method of claim 5 or 6, wherein the at least two different HBV target nucleic acids are selected from the group consisting of precore-mRNA 5' end (non-pregenomic RNA), core, X gene, truncated RNA 3' end (poly(A) junction), precore / core, full-length RNA 3' end (poly(A) junction), alternative splice junction, S gene (pre-splice site), S gene (post-splice site), and pregenomic RNA 5' end.

8. 8. The method of any one of claims 5 to 7, wherein the dPCR assay is performed using a set of forward and reverse primers and a probe specific for the HBV target nucleic acid selected from the oligonucleotides listed in Tables 4 and 6.

9. The method of any one of claims 5 to 8, wherein the fluorescent dye on the labeled probe is selected from the group consisting of Atto-425, FAM, HEX, Texas Red, Cy5 and Cy5.

5.

10. The method of any one of claims 1 to 4 and 6 to 9, further comprising reducing undesired amplification in the dPCR assay by using at least one blocker oligonucleotide having a non-extendible 3' end and a higher melting temperature (Tm) for the template nucleic acid compared to the set of at least two forward and reverse primers and the at least two probes used in the dPCR assay.

11. The method of claim 10, wherein the unwanted amplification is due to the closely spaced location of the amplification target regions.

12. 11. The method of claim 10, wherein the undesired amplification is the presence of a DNA template in which RNA is the target nucleic acid.

13. 11. The method of claim 10, wherein the unwanted amplification is the presence of an RNA splice variant in a situation where spliced ​​RNA is the target and unspliced ​​RNA is inhibited.

14. 1. A method for selectively detecting at least two targets in a sample, said method comprising: - said sample, a first primer set for producing a first amplification product if nucleic acid is present in said sample, - a second primer set for producing a second amplification product if said nucleic acid is present in said sample, and - carrying out an amplification step, which includes contacting with a blocker oligonucleotide; - carrying out a hybridization step comprising contacting said first and second amplification products with at least a first detectable probe and a second detectable probe; and - detecting the presence or absence of said first and second amplification products; Including, the presence of the first amplification product indicates the presence of the first target in the sample, and the absence of the first amplification product indicates the absence of the first target in the sample; the presence of the second amplification product indicates the presence of a second target in the sample, and the absence of the second amplification product indicates the absence of the second target in the sample; wherein the nucleic acid comprises a contiguous sequence including the first target, the second target, and an intermediate sequence located between the first target and the second target, and the blocker oligonucleotide is complementary to at least a portion of the intermediate sequence.

15. 15. The method of claim 14, wherein the blocker oligonucleotide is not extendible by a DNA polymerase.

16. 16. The method of claim 14 or 15, wherein the blocker oligonucleotide reduces or eliminates production of undesired amplification products comprising the first target and the second target.

17. A method for reducing undesired amplification in multiplex digital PCR (dPCR) assays through the use of blocker oligonucleotides that have non-extendible 3' ends and a higher melting temperature (Tm) for template nucleic acids compared to the primers and probes used in dPCR.

18. 18. The method of claim 17, wherein the unwanted amplification is due to the closely spaced location of the amplification target regions.

19. 18. The method of claim 17, wherein the undesired amplification is the presence of a DNA template where RNA is the target nucleic acid.

20. 18. The method of claim 17, wherein the unwanted amplification is the presence of an RNA splice variant in a situation where spliced ​​RNA is targeted and unspliced ​​RNA is inhibited.

21. 1. A kit for selectively detecting at least two targets in a nucleic acid, comprising: a first set of primers for producing a first amplification product if a first portion of nucleic acid is present in said sample; a second set of primers for producing a second amplification product if a second portion of the nucleic acid is present in the sample; a blocker oligonucleotide complementary to said nucleic acid intermediate said first and second portions; a first detectable probe complementary to said first amplification product; a second detectable probe complementary to said second amplification product; Includes a kit.

22. 22. The kit of claim 21, wherein the blocker oligonucleotide is not extendible by a DNA polymerase.

23. 23. The kit of claim 21 or 22, wherein the blocker oligonucleotide reduces or eliminates production of undesired amplification products comprising the first target and the second target.

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