Digital PCR assay design for multiple hepatitis b virus gene targets and non-extensible blocker oligonucleotides therefor

Abstract

The present invention provides compositions and methods relating to novel digital PCR (dPCR) assay designs (droplet numbers or other systems) for the detection and quantification of various hepatitis B virus (HBV) gene targets. The compositions and methods may further include a non-extensible blocker oligonucleotide for reducing extension between non-specific amplicons.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based upon and claims the benefit of U.S. Provisional Patent Application No. 63 / 435,798, filed on December 28, 2022, the entirety of which is incorporated herein by reference.

[0003] Reference Sequence Listing

[0004] This application contains a sequence listing submitted as an electronic text file entitled "P38057-WO_Seq_Listing," which is 75,424 bytes in size and was created on December 19, 2023. Pursuant to 37 CFR §1.52(e)(5), the information contained in this electronic file is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of in vitro virus diagnosis. Within this field, the present invention relates to the amplification and detection of target nucleic acids that may be present in a sample, and in particular, specific amplification and detection of target nucleic acids including sequence variations and / or individual mutations of hepatitis B virus (HBV), the target nucleic acid being particularly HBV RNA (particularly HBV RNA such as HBV pregenomic RNA (pgRNA) derived from covalently closed circular double-stranded DNA (cccDNA)) and other HBV gene targets, optionally using at least one competitive blocking oligonucleotide to reduce non-specific extension between amplicons. The present invention further provides a method for amplifying and detecting various HBV RNA forms, a reaction mixture, and a kit containing oligonucleotides (such as reverse transcription (RT) primers and competitive blocking oligonucleotides). Background Art

[0006] Hepatitis B is an infectious disease of the liver caused by HBV. HBV can cause acute and / or chronic infection. Many people experience no symptoms during initial infection, while others develop a rapid onset of illness (including vomiting, yellowing of the skin, fatigue, dark urine, and abdominal pain). Those infected around the time of birth are more susceptible to chronic hepatitis B. Most individuals who develop chronic disease also experience no symptoms, but may eventually develop cirrhosis and liver cancer. These complications contribute to the death of 15% to 25% of those who develop chronic disease. HBV is generally transmitted through exposure to infected blood or body fluids, for example, when blood, semen, or other body fluids from an HBV-infected person enter the body of an uninfected person. This can occur through sexual contact; sharing needles, syringes, or other drug injection equipment; or from mother to child during birth. In areas where hepatitis B is endemic, infection around the time of birth or through childhood exposure to another person's blood is the most common way to acquire the disease. In areas where the disease is rare, intravenous drug use and sexual intercourse are the most common routes of infection. Other risk factors include working in a healthcare setting, receiving blood transfusions, receiving dialysis, living with an infected person, traveling to countries with high infection rates, and living in a shelter. HBV infection can be diagnosed 30-60 days after exposure. The diagnosis is then usually confirmed by testing the blood for parts of the hepatitis B virus and anti-HBV antibodies.

[0007] Chronic hepatitis B infection remains a major health burden worldwide, affecting 257 million people. Several therapies are available to control the disease, but cure rates are low. In the absence of a curative therapy, lifelong antiviral therapy is required. Because current therapies cannot directly target the reservoir of episomal HBV genomes in the nucleus of infected cells, withdrawal of therapy often results in a resurgence of HBV viral titers.

[0008] The viral life cycle of HBV 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 produces multiple messenger RNA forms that encode the structural proteins of the virus (core and surface proteins), e antigen, viral polymerase, and X antigen. An mRNA form, called pgRNA, also serves as a template for the RT activity of the viral polymerase, producing new rcDNA copies in encapsidated, secreted viral particles. There is also evidence that a certain proportion of encapsidated pgRNA is released without being reverse transcribed, so that the production of infected cells includes viral particles containing both rcDNA and pgRNA. In addition, there are multiple spliced RNA variants, 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, as this does not produce complete pgRNA molecules; however, it is a common occurrence and may result in host cells producing small, truncated, or fusion mRNAs that contribute to the secretion of surface antigen-containing subviral particles.

[0009] Table 1 below lists the forms of HBV RNA that are believed to be generated from HBV cccDNA.

[0010] Table 1:

[0011]

[0012] Table 2 below lists the forms of HBV RNA that cannot be transcribed from the integrated copy (ie, are derived entirely from cccDNA).

[0013] Table 2:

[0014] Type / Description 3.5kbp gRNA, which is also the mRNA for the core protein and polymerase protein 3.5Kb pre-core mRNA, which is longer than pgRNA at the 5' end and produces HBeAg

[0015] Table 3 below lists some of the HBV RNA forms that can be transcribed from the integrated copies.

[0016] Table 3:

[0017]

[0018]

[0019] Markers of HBV include detection of DNA, e antigen (from pre-core mRNA), core antigen (or a combination of antigens including e antigen and core antigen) and s antigen, as well as antibody production against these antigens by the subject or patient. Suppression of s antigen is a marker of functional cure. However, s antigen can be produced by integrated, non-replicating HBV copies, and therefore quantification of hepatitis B surface antigen (HBsAg) levels is unlikely to accurately reflect the transcriptionally active cccDNA pool. Monitoring DNA titer is a sensitive test for detecting HBV infection, and a decrease in HBV is an indicator of treatment response. However, current nucleoside analog therapy for HBV (which inhibits reverse transcription) does not affect the transcription of pgRNA or other mRNAs, but only affects the production of new rcDNA copies. A decrease in DNA titer in patient blood (plasma or serum sample type) does not always correspond to a decrease in HBV RNA, which may lag behind or even temporarily increase because encapsidated pgRNA (and spliced RNA) can be secreted by infected cells that retain transcriptionally active cccDNA. For this reason, HBV RNA has been explored as a single marker for monitoring HBV disease status and therapy effectiveness. Studies have shown that HBV RNA levels can predict outcomes after discontinuation of treatment, such as e-antigen loss, viral relapse, or "flare" events, and that the biomarker may be crucial for the timing of treatment completion in HBV patients.

[0020] Distinguishing HBV RNA forms is important for understanding disease states and explaining molecular test results. In addition to pgRNA, a variety of mRNAs may also be present in the circulation (see, for example, Stadelmayer et al., J Hepatol. 2020, Vol. 73, pp. 40–51). Their transcription start sites may be different, but due to the close arrangement of the HBV genome, they overlap (see, for example, Altinel et al., J Virol. 2016 Nov 14; 90 (23): 10811-10822). Most proposed HBV RNA detection assays have only one or two targets. The determination of the state of the art uses a two-stage (RT and PCR) RACE method to target poly (A) tails (see, for example, van Bommel et al., Hepatology 2015 61: 66-76; Zhang W et al., Methods Mol Med 2004, Vol. 95, pp. 29-44; Kairat A et al., Intervirology 1999, Vol. 42, pp. 228-237). Poly (A) tail determination will detect pgRNA and other mRNAs that all terminate in the primary polyadenylation site (" full-length " poly (A) tail). These determinations will also detect splice forms. However, in order to distinguish pgRNA or each individual mRNA from other overlapping forms, a quantitative strategy of elimination requiring multiple PCR targets will be needed. For example, pgRNA and pre-core mRNA (slightly longer 3.5kbmRNA) can be distinguished by targeting the genomic region between the transcription start sites of these forms (see, for example, Wang Jie et al., Journal of Hepatology 2016V65:700-710). Similar targeting of the 5' end length difference of mRNA can also be used to distinguish pgRNA from smaller viral mRNAs that produce s antigen and X antigen (see, for example, Butler EK et al., Hepatology.201868 (6):2106-2117). The X gene mRNA transcript is known to be in circulation (see, e.g., Stadelmayer et al., J Hepatology 2020, vol. 73, pp. 40–51), and a target in this region will pick up this transcript as well as the longer mRNA and pgRNA.

[0021] Other assays can target spliced RNA variants, which can be indicators of interferon therapy response (see, e.g., Chen et al., Sci Rep 5, 16459 (2015); Bayliss, J. et al., J Hepatol, 2013, V59, p1022-1028, Preiss, S. et al., Hepatology, 2008, V48, 741-749); targets in the core region may be disrupted by splicing variants, and therefore the ratio of these transcripts in the sample is relevant for accurate quantification. Integrated HBV copies (which can be nearly complete or fragmented in different infected cells) cannot produce active virus, but can produce s antigen-producing transcripts, which interfere with human immune responses and also reduce the predictive power of s antigen monitoring as a marker for HBV virus production. Truncated RNAs from integrated copies of HBV that terminate in a secondary poly(A) site upstream of the full-length site (van Bommel et al., Hepatology 2015 61: 66-76) will not be picked up by the full-length 3' end assay but may be detected by other targets within the S gene.

[0022] In view of the foregoing, there is a need for assays with improved effectiveness in distinguishing between different HBV RNA forms. Summary of the Invention

[0023] The present disclosure overcomes the above challenges by providing assays with improved effectiveness in distinguishing different HBV RNA forms. In one aspect, the present disclosure provides a set of multiple targeted assays for different HBV gene targets, all on the same platform and capable of multiplexing to reduce run-to-run variability.

[0024] Certain aspects of the present disclosure relate to methods for rapidly detecting the presence or absence of HBV RNA in biological or non-biological samples for monitoring HBV disease status and treatment efficacy, such as detecting HBV by polymerase chain reaction (PCR) in a single test tube. Such aspects include methods for detecting HBV, which include performing at least one cycling step, which may include an amplification step and a hybridization step. In addition, aspects include oligonucleotides (including reverse transcription primers (which may also be PCR primers), blocking oligonucleotides, conventional primers, and probes) and kits designed for detecting HBV in a single tube.

[0025] One sequence difference between HBV DNA and RNA is the poly(A) tail of pgRNA and other mRNAs; however, methods using oligo d(T) primers can detect non-target RNA or other sequences with poly(A) fragments. "Anchored" poly(A)-containing oligonucleotides can provide some measure of specificity for non-target binding and extension, but this is a trade-off strategy that results in some binding to HBV DNA. As a method for improving the assay performance (sensitivity and specificity) of targeted RNA in the presence of DNA, the methods disclosed herein may include a competitive blocking oligonucleotide that matches the DNA sequence at the target where the RNA sequence has a poly(A) tail attached. Binding of the competitive blocking oligonucleotide to the homologous genomic HBV DNA prevents binding of the primer (e.g., RT primer), thereby reducing the amplification of unwanted homologous genomic HBV DNA. Modified stable bases can be incorporated into the assay oligonucleotide or blocker oligonucleotide to further improve the discrimination ability of the method.

[0026] Primers and probes targeting the poly(A) tail of HBV RNA (particularly HBV RNA transcribed from cccDNA, which has a standard poly(A) tail position for transcripts, such as pgRNA, but also other mRNAs and spliced RNAs) can be provided. Competitive blocking oligonucleotides can be provided that improve specificity for RNA in the presence of HBV DNA. Additional primers and probes can be provided for targeting other poly(A) sites, such as secondary or truncated poly(A) sites of HBV transcripts that can be derived from integrated HBV copies. Competitive blocking oligonucleotides can be provided that improve specificity for RNAs with these specific poly(A) sites in the presence of homologous DNA.

[0027] One aspect of the present invention relates to a method for detecting and quantifying between two and 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, wherein a plurality of forward and reverse primer sets and a plurality of probes are used to amplify and detect each of the HBV target nucleic acids, each probe being labeled with a fluorescent dye that produces a different signal, and measuring the amount of signal generated in each of the partitions to calculate the amount of each HBV target nucleic acid in the sample. In one embodiment, the HBV target nucleic acid is selected from the group consisting of: pre-core-mRNA 5' end (non-pgRNA), core, X gene, truncated RNA 3' end (poly(A) junction), pre-core / core, full-length RNA 3' end (poly(A) junction), selected splice junction, S gene (pre-splice site), S gene (post-splice site), and pg RNA 5' end. In another embodiment, dPCR is measured using forward and reverse primer sets and probes that are specific for HBV target nucleic acids 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 includes reducing undesirable amplification in dPCR by using at least one blocker oligonucleotide having a non-extendable 3' end and a higher melting temperature (Tm) for template nucleic acid relative to the at least two forward and reverse primer sets and the at least two probes used in the dPCR assay. In certain embodiments, undesirable amplification may be caused by the amplification target region being tightly positioned. In another embodiment, undesirable amplification is the presence of a DNA template when RNA is the target nucleic acid. In yet another embodiment, undesirable amplification is the presence of an RNA splice variant when spliced RNA is the target and unspliced RNA is suppressed.

[0028] 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, the method comprising providing a sample; randomly dividing the sample into a plurality of equally sized and independent partitions; performing a PCR assay in each partition with at least two forward and reverse primer sets to amplify each of the HBV target nucleic acids, and performing a PCR assay with at least two probes, each probe labeled with a fluorescent dye that produces a different signal, to detect each of the HBV target nucleic acids; and measuring the amount of signal generated in each of the partitions 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 pre-core-mRNA 5' end (non-pregenomic RNA), core, X gene, truncated RNA 3' end (poly(A) junction), pre-core / core, full-length RNA 3' end (poly(A) junction), selected splice junction, S gene (pre-splice site), S gene (post-splice site), and pre-genomic RNA 5' end. In certain embodiments, the dPCR assay is performed using forward and reverse primer sets and probes specific for HBV target nucleic acids 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-extendable 3' end and a higher melting temperature (Tm) for the template nucleic acid relative to the at least two forward and reverse primer sets and the at least two probes used in the dPCR assay. In some embodiments, undesirable amplification may be caused by tightly positioned amplification target regions. In another embodiment, undesirable amplification is the presence of a DNA template when RNA is the target nucleic acid. In yet another embodiment, undesirable amplification is the presence of an RNA splice variant when spliced RNA is the target and unspliced RNA is suppressed.

[0029] 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 set of primers for producing a first amplification product in the presence of nucleic acid in the sample, a second set of primers for producing a second amplification product in the presence of nucleic acid in the sample, and a blocker oligonucleotide; performing a hybridization step comprising contacting the first amplification product and the second amplification product with at least a first detectable probe and a second detectable probe; and detecting the presence or absence of the first amplification product and the second amplification product, wherein the presence of the first amplification product indicates the presence of the first target in the sample, and wherein the absence of the first amplification product indicates the absence of the first target in the sample, and wherein the presence of the second amplification product indicates the presence of the second target in the sample, and wherein the absence of the second amplification product indicates the absence of the second target in the sample, and wherein the nucleic acid comprises a contiguous sequence comprising the first target, the second target, and an intermediate sequence located between the first target and the second target, and wherein the blocker oligonucleotide is complementary to at least a portion of the intermediate sequence. In some embodiments, the blocker oligonucleotide is not extendable by a 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.

[0030] Another aspect of the invention relates to a method for reducing desired amplification in a multiplex digital PCR (dPCR) assay by using a blocker oligonucleotide having a non-extendable 3' end and a higher melting temperature (Tm) for the template nucleic acid relative to the primers and probes used in the dPCR. In one embodiment, the undesired amplification is caused by the amplification target region being tightly positioned. In another embodiment, the undesired amplification is the presence of a DNA template when RNA is the target. In yet another embodiment, the undesired amplification is the presence of an RNA splice variant when spliced RNA is the target and unspliced RNA is suppressed.

[0031] In another aspect, a kit for selectively detecting at least two targets in a nucleic acid is provided, the kit comprising a first set of primers for generating a first amplification product in the presence of a first portion of the nucleic acid in a sample; a second set of primers for generating a second amplification product in the presence of a second portion of the nucleic acid in the sample; a blocker oligonucleotide complementary to a nucleic acid intermediate the first portion and the second portion; 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 extendable by a DNA polymerase. In some embodiments, the blocker oligonucleotide reduces or eliminates the generation of undesired amplification products comprising the first target and the second target.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0033] Details of one or more embodiments of the present invention are set forth in the accompanying drawings and the following description. Other features, objects, and advantages of the present invention will be apparent from the accompanying drawings and detailed description, as well as from the claims. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of any conflict, the present specification (including definitions) will control. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram showing a digital PCR assay diagram on a linear schematic diagram of HBV RNA transcripts and selected splice variant forms disclosed herein. The elements (horizontal lines, boxes) of the full-length HBV circular genome and multiple example primer pairs (arrow pairs) for amplifying the target region of interest are shown. Example primers are indicated for targets, including pre-core mRNA 5' end (non-pgRNA) 1, core target 2, X gene target 3, truncated RNA 3' end (poly (A) connection) 4, pre-core / core target 5, full-length RNA 3' end (poly (A) connection) 6, selected splicing connection (illustrated example) 7, S gene, pre-splice site 8, S gene, post-splice site 9 and pgRNA+pc-mRNA 5' end 10 of 3.5kb transcript.

[0035] Figure 2 is a schematic diagram illustrating the use of a blocker oligonucleotide to prevent unintended amplification due to two amplification regions being positioned close to each other in a target template.

[0036] Figure 3 is a schematic diagram illustrating the use of blocker oligonucleotides that bind to intron sequences to inhibit DNA-specific amplification without affecting RNA-specific amplification.

[0037] Figure 4 Shown is the use of blocker oligonucleotides designed to inhibit unspliced RNA variants. An example DNA template (top) is shown to illustrate the 3 exons and 2 introns associated with a single target gene.

[0038] Figure 5The use of blocker oligonucleotides designed to inhibit longer forms of fusion RNA is shown. An example DNA template (top) is shown to illustrate two different regions, with the first region (left) comprising two exons and one intron associated with a first target gene, and the second region (right) comprising one exon associated with a second target gene.

[0039] Figure 6 A linear plot of an HBV dPCR assay according to the present disclosure is shown. Primers and probes were designed for six HBV targets, including i) 3' pre-core, ii) 3' poly (A), iii) X gene mRNA, iv) core gene mRNA, v) truncated poly (A), and vi) 5'-pre-core. The logarithm of the measured concentration of each of the six targets in the sample was calculated and plotted on the vertical axis relative to the known (expected) concentration of those samples on the horizontal axis. The linear regression (dashed line) for each series was calculated and plotted. The slope, intercept, and R of each linear regression were plotted. 2 The values are shown in Table 5.

[0040] Figure 7 A box-and-whisker plot of an HBV dPCR assay according to the present disclosure is shown. Primers and probes were designed for six HBV targets, including i) 3' pre-core, ii) 3' poly(A), iii) X gene mRNA, iv) core gene mRNA, v) truncated poly(A), and vi) 5'-pre-core. The logarithm of the measured concentration of each of the six targets in the sample was calculated and plotted on the vertical axis relative to the known (expected) concentration of those samples on the horizontal line.

[0041] Figure 8A and Figure 8B Amplification of 3' pre-core RNA ( Figure 8A ) and 3' pre-core DNA ( Figure 8B ). The fluorescence signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as shown on the lower horizontal axis. The well identifier (well ID) for each sample is shown on the upper horizontal axis, with the data associated with each well ID depicted by (and contained between) vertical dashed lines.

[0042] Figure 9 Multiple HBV S gene assays (including one S gene assay and three S gene post-splicing assays (S PSdPCR assay results for a dPCR assay. The fluorescence signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as shown on the lower horizontal axis. The well identifier (and associated assay) for each sample is shown on the upper horizontal axis, with the data associated with each well depicted by (and contained between) the vertical dashed lines.

[0043] Figure 10 is a sensitivity plot showing the Figure 9 S PS Expected (input) concentration (log cps / μL) for assay 2 is compared to measured titer (cps / μL) on the vertical axis. Linear regression identified a strong correlation between input and measured concentrations (R 2 =0.9991).

[0044] Figure 11 Schematic diagram of an HBV 3.5kb RNA assay targeting the pre-core splice site, used to detect pgRNA and pre-core mRNA. The target region of the assay is indicated by a dotted box.

[0045] Figure 12 Shown Figure 11 Detailed view of a schematic diagram of the assay. The target region of the assay is indicated by the dashed box. Two in vitro templates (IVT) containing the target region (IVT 14 and IVT 15) are shown aligned with the corresponding sites on HBV RNA.

[0046] Figure 13 Shown Figure 11 and Figure 12 dPCR assay results for the pgRNA IVT assay are shown. The fluorescence signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as shown on the lower horizontal axis. The well identifier for each sample is shown on the upper horizontal axis, with the data associated with each well depicted by (and contained between) vertical dashed lines.

[0047] Figure 14 Shown in Figure 11 and Figure 12 dPCR assay results for the IVT 14 template tested in the assay presented. The assay demonstrated a high level of sensitivity, being able to detect as little as 10 1 cps / μL of target template (e.g., IVT 14). The fluorescence signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as shown on the lower horizontal axis. The well identifier (and associated input concentration) for each sample is shown on the upper horizontal axis, with the data associated with each well depicted by (and contained between) the vertical dashed lines. For IVT 14, the input concentration is 10 5 and 106 cps / μL, a saturated reaction (in which no negative droplets were detected) was observed, while for each of the other tested concentrations, both positive-reacting droplets and negative-reacting droplets were observed.

[0048] Figure 15 is a linear graph that shows the Figure 14 The expected (input) concentration (log cps / μL) of the 3.5 kb RNA assay is compared to the measured titer (cps / μL) on the vertical axis. Linear regression identified a strong correlation between input and measured concentrations (R 2 =0.9992).

[0049] Figure 16 is a linear graph that shows the Figure 14 Expected (input) concentration of IVT 14 template detected in the 3.5 kb RNA assay (log cps / μL) is compared to the measured titer (cps / μL) on the vertical axis. Linear regression identified a strong correlation between input and measured concentrations (R 2 =0.9999).

[0050] Figure 17 Shown are dPCR assay results for a multiplexed assay targeting the detection of core, X gene, and poly(A) targets in HBV RNA. The assay involved a ratio-based multiplex analysis with 100% core target in channel 1, 100% poly(A) target in channel 2, and 50% X gene target in channel 1 and 50% in channel 2. For each event (detected droplet), the fluorescence signal detected in channel 1 or channel 2 of the dPCR instrument was plotted on the horizontal and vertical axes, respectively. Intercluster rain (corresponding to unexpected and / or undesired amplification products) observed between data clusters corresponding to each of the targets is indicated by rounded rectangular boxes.

[0051] Figure 18 Schematic diagram of an assay for detecting core, gene X, and poly(A) targets in HBV RNA. A non-extendable blocker oligonucleotide is designed to bind to a designated region between gene X and poly(A) to block the potential formation of hybrid amplicons containing both gene X and poly(A). In addition to the binding region of the blocker oligonucleotide, forward (Fwd) and reverse (Rev) primer pairs and corresponding probes are indicated for each of the three targets.

[0052] Figure 19 Shown for Figure 18ddPCR assay results for a multiplex assay including a non-extendable blocker oligonucleotide for detection of core, X gene, and poly(A) targets in HBV RNA are shown. The assay involves a ratio-based multiplex analysis where the core target is 100% in channel 1, 100% poly-A target in channel 2, and 50% X gene target in channel 1 and 50% in channel 2. For each event (detected droplet), the fluorescence signal detected in channel 1 or channel 2 of the dPCR instrument is plotted on the horizontal and vertical axes, respectively. Figure 17 Inter-cluster rain (corresponding to unexpected and / or undesired amplification products) is significantly reduced or eliminated between the data clusters corresponding to each of the targets compared to the assay shown excluding the non-extendable blocker oligonucleotide.

[0053] Figure 20 Schematic diagram of an assay for detecting truncated poly(A) targets in HBV RNA. The non-extendable blocker oligonucleotide TR3_DD is designed to prevent the binding of the truncated poly(A) reverse primer to its 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 also indicated.

[0054] Figure 21 Shown are both with and without the displayed non-extendable blocker oligonucleotides. Figure 20 dPCR assay results for the assay are shown. The fluorescence signal detected in channel 1 of the dPCR instrument is plotted on the vertical axis for each event (detected droplet), as shown on the lower horizontal axis. The well identifier for each sample (and the associated assay condition—i.e., the presence (+) or absence (+) of a non-extendable blocker in the reaction) is shown on the upper horizontal axis, with the data associated with each well depicted by (and contained in) a vertical dashed line. Both positive and negative reaction droplets were observed under each of the conditions tested. DETAILED DESCRIPTION

[0055] Diagnosis of HBV infection by nucleic acid amplification provides a method for detecting and / or quantifying viral infection quickly, accurately, reliably, specifically, and sensitively. This article describes a digital PCR assay for detecting HBV gene targets (e.g., HBV pgRNA and smaller viral mRNA) 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, as well as products or kits comprising such primers, competitive blocking oligonucleotides, and probes, are provided. Compared to other PCR methods, the specificity and sensitivity of digital PCR (dPCR) for quantifying various forms of HBV RNA are increased, making it feasible to implement this technology in clinical laboratories for routine diagnosis and treatment of HBV infection.

[0056] Assay designs for droplet digital PCR (ddPCR) and digital PCR include multiple targets, including 5' and 3' end structures; quantification of overlapping mRNAs 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 (DNA removal is not required for poly(A) targeted designs) and can also detect DNA forms such as incomplete HBV genomes generated by reverse transcribed spliced RNA or integrated copies of DNA released from infected cells. These assays will allow assessment of disease status and the biological effects of antiviral therapy.

[0057] The digital PCR assays disclosed herein include: (i) a polyA-targeted assay targeting the full-length mRNA and the 3' end of the pgRNA; (ii) an X-gene-targeted assay; (iii) a core-targeted assay; (iv) a pre-core assay targeting near the 3' end but omitting the poly(A) tail junction; (v) an assay targeting the 5' end of the pre-core mRNA, a 3.5 kb transcript slightly longer than the pgRNA; (vi) a truncated assay targeting the second poly(A) start site; (vii) an S-gene assay, at locations upstream and downstream of a common splice junction; (viii) an assay targeting the 5' end of the pgRNA+pre-core-mRNA to capture the unspliced 3.5 kb transcript; and (ix) an assay targeting a specific splice junction.

[0058] A linear representation of the dPCR assay showing HBV RNA transcripts and selected splice variant forms is shown in Figure 1These assays can be multiplexed in different combinations to save sample volume. In addition to the capabilities of the platform used for PCR, the main limitation to multiplex analysis is the proximity or overlap of some target primer sets. The assay design includes novel oligonucleotide designs that are incorporated into the master mix as competitive, non-extensible blocking oligonucleotides, thereby improving the assay specificity for the target during multiplex analysis. The blocker oligonucleotide is located between adjacent amplification products of the PCR assay and reduces nonspecific extension between assay oligonucleotide sets across regions between expected targets. These assays are designed for digital PCR platforms, which may include droplet digital systems (such as Bio-Rad's QX200 Droplet Digital PCR System) or systems with other forms of reaction partitioning, including Roche Digital PCR Systems (Digital PCR Systems). ). Further details of digital PCR and the use of blocking oligonucleotide design to improve multiplex analysis are described below.

[0059] The present disclosure includes oligonucleotide primers (including RT primers), competitive blocking oligonucleotides, and fluorescently labeled hydrolysis probes that hybridize to HBV nucleic acid, particularly HBV RNA (particularly HBV RNA transcribed from cccDNA, such as pgRNA), to specifically identify and quantify various forms of HBV RNA.

[0060] The disclosed method may include performing at least one cycling step, which includes using one or more pairs of primers to amplify one or more parts of a nucleic acid molecule gene target from a sample. As used herein, "HBV primers" or "HBVRT primers" refer to oligonucleotide primers that specifically anneal to a nucleic acid sequence found in HBV or HBV RNA (such as HBV pgRNA) under appropriate conditions, and thereby initiate reverse transcription and / or DNA synthesis to produce a corresponding amplified product. Examples of nucleic acid sequences found in HBV suitable for targeting include HBV pgRNA. Each of the HBV primers (including RT primers) discussed anneals to the target so that at least a portion of each amplified product includes a nucleic acid sequence corresponding to the target. If one or more nucleic acids are present in the sample, one or more amplified products are produced, so that the presence of one or more amplified products indicates the presence of HBV and / or HBV RNA (particularly HBV RNA transcribed from cccDNA, such as pgRNA) in the sample. The amplified 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" 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 cycle step includes an amplification step, a hybridization step, and a detection step, wherein the sample is contacted with one or more detectable HBV or HBV RNA probes to detect the presence or absence of HBV and / or HBV RNA (particularly HBV RNA transcribed from cccDNA, such as pgRNA) in the sample. As used herein, the term "blocking oligonucleotide" (or "competitive blocking oligonucleotide" or "blocker") refers to a non-extendable oligonucleotide that specifically anneals to complementary DNA and reduces the occurrence of extension between non-specific amplicons.

[0061] As used herein, the term "amplification" refers to the process of synthesizing a nucleic acid molecule that is 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 the primers to the template nucleic acid at a temperature below the melting temperature of the primers, and enzymatically extending from the primers to produce an amplified product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase (e.g., Taq) and an appropriate buffer and / or cofactors (e.g., MgCl2 and / or KCl) for optimizing polymerase activity.

[0062] The term "primer" as used herein is known to those skilled in the art and refers to an oligomeric compound, primarily an oligonucleotide, but also refers to a modified oligonucleotide capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., a free 3'-OH group is provided at the 3' end of the oligonucleotide, wherein further "nucleotides" can be connected by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using deoxynucleoside triphosphates and thereby releasing pyrophosphate. In some embodiments, the primer is also a reverse transcription (RT) primer (RT primer). Several types of RT primers are known in the art, including oligo(dT)N primers, anchored oligo(dT)N primers, random hexamer primers, and sequence-specific primers. In some embodiments, the RT primer will anneal to RNA (e.g., HBV RNA) and extend to produce a DNA complementary sequence (i.e., reverse transcription of the target). In some embodiments, the RT primer targets HBV RNA containing poly(A), and therefore the RT primer is an oligonucleotide containing polyT.

[0063] The term "hybridization" refers to the annealing of one or more probes to an amplification product. "Hybridization conditions" generally include a temperature below the melting point of the probes but which avoids nonspecific hybridization of the probes.

[0064] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase, typically associated with nucleic acid strand synthesis, whereby nucleotides are removed from the 5' end of the nucleic acid strand.

[0065] The term "thermostable polymerase" refers to a thermostable polymerase that catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when in elevated temperature experiences realizing the time required for denaturation of double-stranded template nucleic acid. Typically, synthesis is initiated at the 3' end of each primer and advances in a 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. However, non-thermostable polymerases may also be used in PCR assays if the enzyme is supplemented (if necessary).

[0066] The term "its complement" refers to a nucleic acid that has the same length as a given nucleic acid and is completely complementary thereto.

[0067] When applied to nucleic acids, the term "extension" or "elongation" refers to when additional nucleotides (or other similar molecules) are incorporated into the nucleic acid. For example, the nucleic acid is optionally extended by a biocatalyst that incorporates nucleotides, such as a polymerase that typically adds nucleotides to the 3' end of the nucleic acid.

[0068] As used herein, the terms "identical" or "percentage identity" refer to two or more nucleic acid sequences that are identical or have a specified percentage of nucleotides that are the same when compared and aligned for maximum correspondence (e.g., using a sequence comparison algorithm available to the skilled artisan or as measured by visual inspection). An exemplary algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST program, which is described, for example, in 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.

[0069] "Modified nucleotides" in the context of oligonucleotides refer to changes in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide to provide the desired properties to the oligonucleotide. Exemplary modified nucleotides that may be substituted in the oligonucleotides described herein include, for example, tert-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-alk ...U, C7-propynyl-dG, C5-propargylamino-dC, C5-alkynyl-dU, C7-propynyl-d Propylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthine nucleoside, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-O-methylribose-U, 2'-O-methylribose-C, N4-ethyl-dC, N6-methyl-dA, 5-propynyl dU, 5-propynyl dC and N6 benzyl-dA etc. Some oligonucleotides described herein contain modified bases to increase stability or for the improvement of other performances. An example is 5-propynyl-dU (modified uracil), which can replace T (thymine). In the oligonucleotide sequences provided, pdU, T and U nucleotide names will be considered to be interchangeable because assays may contain modified or unmodified versions of specific oligonucleotides. Another example of modified nucleotides includes locked nucleic acid (LNA). LNA (also known as inaccessible RNA) is a modified RNA nucleotide in which the ribose moiety is modified by an additional bridge connecting the 2' oxygen and the 4' carbon. The bridge locks the ribose in a 3' inner (North) conformation, which is typically found in A-type duplexes. The effect of LNA is that the locked ribose conformation enhances base stacking and backbone pre-organization, which significantly improves the hybridization characteristics (melting temperature) of the oligonucleotide. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or are otherwise known in the art. In certain embodiments, the modified nucleotide substitution changes the melting temperature (T) of the oligonucleotide relative to the melting temperature of the corresponding unmodified oligonucleotide. m). To further illustrate, in some embodiments, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimizing the formation of primer dimers, etc.), increase the yield of expected target amplicon, etc. The examples of these types of nucleic acid modifications are described in, for example, U.S. Patent No. 6,001,611, which is incorporated herein by reference. Other modified nucleotide substitutions can change the stability of the oligonucleotide, or provide other desired features. For example, some modifications can make the oligonucleotide non-extendable, which is useful for probes and competitive blocking oligonucleotides. In addition to phosphate, the non-extendable end can also be promoted by a C3 spacer, a dideoxynucleotide, connecting the 3' end of the second oligonucleotide to the 3' end of the oligonucleotide, etc.

[0070] For example, computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) can be used to design the oligonucleotides for the nucleic acid molecules (for example, encoding the nucleic acid of HBV alternative parts) of amplification coding HBV targets, including the oligonucleotides and oligonucleotide analogs of modification. When designing the oligonucleotides to be used as amplification primers, important features include but are not limited to an amplification product of appropriate size for ease of detection (for example, by electrophoresis), the similar melting temperature of the member of a pair of primers and the length of each primer (that is, primers need to be long enough to anneal with sequence specificity and initiate synthesis, but can not be too long so that fidelity is reduced during oligonucleotide synthesis). Typically, the length of oligonucleotide primers is 8 to 50 nucleotides (for example, a length of 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 nucleotides).

[0071] In the assay, "competitive blocking oligonucleotide," "competitive blocking nucleotide," "competitive blocking nucleic acid," "blocking oligonucleotide," "blocking nucleotide," "blocker" and / or "blocking nucleic acid" are used and are terms that refer to competitive blocking oligonucleotides that bind to regions in HBV DNA or RNA.

[0072] Forward primer sets for detecting the presence or absence of HBV nucleic acids, such as HBV RNA, and other gene targets include sequences of SEQ ID NOs: 20, 23, 24, 210, 213, 214, 387, and 389. Reverse transcription primer sets that can function as reverse primers (e.g., 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), include sequences of SEQ ID NOs: 16, 18, 19, 25-30, 33-190, 206, 208, 209, 215-220, and 223-380. Competitive blocking oligonucleotide sets for improving 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), include sequences of SEQ ID NOs: 1-15, 21, 22, 191-205, 211, and 212. The probe set for detecting the presence or absence of HBV nucleic acid, such as HBV RNA (e.g., HBV derived from cccDNA, such as pgRNA) includes sequences of SEQ ID NOs: 17, 31, 32, 207, 221, 222, 381-386, 388, and 390-392.

[0073] In addition to a set of primers and competitive blocking oligonucleotides, the method can also use one or more probes to detect the presence or absence of HBV nucleic acids such as HBV RNA (e.g., HBV derived from cccDNA, such as pgRNA). The term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA) that is designed or selected to contain a specific nucleotide sequence that allows them to specifically (i.e., preferentially) hybridize with a "target nucleic acid" under a specified predetermined stringency, in this case HBV nucleic acid (including HBV RNA, for example, HBV RNA transcribed from cccDNA, such as pgRNA) (target) nucleic acid. A "probe" can be referred to as a "detection probe," meaning that it detects a target nucleic acid.

[0074] In some embodiments, the HBV nucleic acid probe (including a probe for HBV RNA) may be labeled with at least one fluorescent tag. In one embodiment, the HBV nucleic acid probe (including a probe for HBV RNA) may 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.

[0075] The oligonucleotide designed as a probe can be carried out in a manner similar to primer design. Embodiments can use a single probe or a pair of probes to detect amplified products. According to an embodiment, the probe used may include at least one label and / or at least one quencher moiety. Like primers, probes generally have a melting temperature that is suitable for the thermal cycle parameters of amplification methods, and the length of each probe must be sufficient to allow sequence-specific hybridization to occur, but not too long to reduce fidelity during synthesis. The length of the oligonucleotide probe is generally 15 to 40 (e.g., 16, 18, 20, 21, 22, 23, 24 or 25) nucleotides.

[0076] The construct may include a vector, each vector containing a primer, a competitive blocking oligonucleotide, and one or more sequences of a probe nucleic acid molecule for HBV (e.g., SEQ ID NO: 1-392). The construct may be used as, for example, a control template nucleic acid molecule. Applicable vectors are commercially available and / or produced by conventional recombinant nucleic acid technology methods in the art. HBV nucleic acid molecules may be obtained, for example, by chemical synthesis, direct cloning from HBV, or by nucleic acid amplification.

[0077] In addition to HBV nucleic acid molecules (e.g., nucleic acid molecules comprising one or more SEQ ID NOs: 1-392 sequences), constructs suitable for the methods generally include sequences encoding selectable markers (e.g., antibiotic resistance genes) for selecting desired constructs and / or transformants, as well as an origin of replication. The choice of vector system generally depends on several factors, including but not limited to host cell selection, replication efficiency, selectivity, inducibility, and ease of recovery.

[0078] The construct containing HBV nucleic acid molecules can be bred in a host cell. As used herein, the term host cell is intended to include prokaryotes and eukaryotes, such as yeast, plants and animal cells. Prokaryotic hosts can include Escherichia coli (E.coli), Salmonella typhimurium (Salmonella typhimurium), Serratia marcescens (Serratiamarcescens) and Bacillus subtilis (Bacillus subtilis). Eukaryotic hosts include yeast (such as Saccharomyces cerevisiae (S.cerevisiae), Schizosaccharomyces pombe (S.pombe), Pichia pastoris (Pichia pastoris)), mammalian cells (such as COS cells or Chinese hamster ovary (CHO) cells), insect cells and plant cells (such as Arabidopsis thaliana (Arabidopsis thaliana) and Nicotiana tabacum (Nicotiana tabacum)). Any technique known to those of ordinary skill in the art can be used to introduce the construct into a host cell. 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 cells. In addition, naked DNA can be delivered directly to cells (see, for example, U.S. Patent numbers 5,580,859 and 5,589,466).

[0079] The construct (plasmid vector) can be used to generate RNA molecules (by in vitro transcription or other processes) to generate RNA templates, which may also contain binding sites for primers and probes. RNA template molecules can also be produced synthetically. One type of RNA template that can be produced as a control material is armored RNA (an RNA molecule encapsulated in a protein coating), which involves the production of RNA and a coating protein (such as a viral capsid protein) by a construct (for example, in a bacterial host) and an assembly of the coating protein that encapsulates the RNA molecule. DNA molecules can also be encapsulated in a protein coating for use as a control material.

[0080] polymerase chain reaction (PCR)

[0081] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers used in some embodiments include oligonucleotides that can serve as starting points for nucleic acid synthesis within the HBV nucleic acid sequence (e.g., SEQ ID NOs: 15, 18-20, 23-30, 33-190, 206, 208-210, 213-220, and 223-380). In certain embodiments, the primer is a reverse transcription (RT) primer (RT primer). The primer can be purified from a restriction digest by conventional methods, or it can be synthesized. For maximum efficiency in amplification, the primer is preferably single-stranded, but the primer can be double-stranded. First, the double-stranded primer is denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0082] If the template nucleic acid is double-stranded, the two strands must be separated before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturation method, including physical, chemical, or enzymatic methods. One method for separating nucleic acid chains involves heating the nucleic acid until most of it is denatured (e.g., denaturation is greater than 50%, 60%, 70%, 80%, 90%, or 95%). The heating conditions necessary for denaturing the template nucleic acid will depend, for example, on the buffer salt concentration and the length and nucleotide composition of the denatured nucleic acid, 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 performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0083] If the double-stranded template nucleic acid is denatured by heating, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence. The annealing temperature is typically about 35°C to about 65°C (e.g., about 40°C to about 60°C; about 45°C to about 50°C). The annealing time may be 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 at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient to allow extension to occur from the annealed primers to generate products complementary to the template nucleic acid. The temperature should be sufficient to synthesize extension products from each primer annealed to the nucleic acid template, but should not be so high as to denature the extension products from their complementary templates (e.g., temperatures for extension typically range from about 40°C to about 80°C (e.g., about 50°C to about 70°C; about 60°C). The extension time may be about 10 seconds to about 5 minutes (e.g., about 30 seconds to about 4 minutes; about 1 minute to about 3 minutes; about 1 minute 30 seconds to about 2 minutes).

[0084] The genome of a retrovirus or RNA virus is composed of ribonucleic acid, or RNA. HBV is a pararetrovirus, a non-retroviral virus that still uses reverse transcription during its replication process and requires RNA produced by host enzymes for viral replication. In this case, the template nucleic acid RNA must first be transcribed into complementary DNA (cDNA) by the action of reverse transcriptase. Reverse transcriptase uses the RNA template and a short primer complementary to the 3' end of the RNA to guide the synthesis of the first-strand cDNA, which can then be used directly as a template for the polymerase chain reaction. For general preparation of RNA, primers can also be random or assay / target specific, depending on the method.

[0085] PCR assays can be performed using HBV nucleic acids, such as RNA (e.g., HBV pgRNA) or DNA (cDNA). The template nucleic acid does not need to be purified; it can be a small portion of a complex mixture, such as HBV nucleic acid contained in human cells. HBV nucleic acid molecules can be extracted from biological samples by conventional 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 many sources, such as plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms, such as plants or animals.

[0086] Oligonucleotide primers (e.g., forward primers comprising SEQ ID NOs: 20, 23, 24, 210, 213, 214, 387, and 389; and RT / reverse primers comprising 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 that induce primer extension. For example, the chain extension reaction typically includes 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U Taq polymerase, and 10% DMSO. Reactions typically contain 150 to 320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0087] The newly synthesized chain forms a double-stranded molecule and can be used for the subsequent steps of the reaction. The steps of chain separation, annealing and extension can be repeated as many times as needed to produce the desired number of amplification products corresponding to the target HBV nucleic acid molecules (including HBVRNA, such as HBVpgRNA). The limiting factor in the reaction is the amount of primers, thermostable enzymes and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing and extension) are preferably repeated at least once. For use in detection, the number of cycling steps will depend on, for example, the properties of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be needed to amplify the target sequence sufficient for detection. Typically, the cycling steps are repeated at least about 20 times, but can be repeated up to 40, 60 or even 100 times.

[0088] Fluorescence resonance energy transfer (FRET)

[0089] FRET technology (e.g., see U.S. Patent 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 located within a certain distance of each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. When the donor is excited by light radiation having a suitable wavelength, the donor typically transfers energy to the acceptor. The acceptor typically re-emits the transferred energy in the form of light radiation having a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties by biomolecules comprising a substantially non-fluorescent donor moiety (see, e.g., U.S. Patent No. 7,741,467).

[0090] In one example, an oligonucleotide probe can comprise a donor fluorescent moiety (e.g., FAM) and a corresponding quencher (e.g., Black Hole Quenchers TM (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 fluorescence 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 by the 5' to 3' nuclease activity of, for example, Taq polymerase, such that the fluorescence 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 BlackHoleQuenchers TM(BHQ) (e.g., BHQ2) (Biosearch Technologies, Inc., Novato, Calif.), Iowa Black TM (IntegratedDNATech., Inc., Coralville, Iowa) and BlackBerry TM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0091] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplified product at a specific location, determined by the complementarity of the oligonucleotide probes to the HBV RNA target nucleic acid sequence (including HBV RNA, such as HBV RNA transcribed from cccDNA, such as pgRNA). After the oligonucleotide probes hybridize to the amplified product nucleic acid at the appropriate location, a FRET signal is generated. The hybridization temperature can be in the range of about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0092] Fluorescence analysis can be performed using, for example, a photon counting epifluorescence microscope system (containing appropriate dichroic mirrors and filters for monitoring fluorescence emission in a specific range), a photon counting photomultiplier tube system, or a fluorimeter. Excitation 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 that is appropriately filtered to excite in the desired range to initiate energy transfer or allow direct detection of fluorophores.

[0093] As used herein, "corresponding" with respect to a donor and a corresponding acceptor moiety means that the acceptor fluorescent moiety or dark quencher has an absorption spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The maximum wavelength of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the maximum wavelength of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-radiative energy transfer can occur between them.

[0094] The fluorescent donor and the corresponding acceptor moiety are generally selected for (a) efficient Foerster energy transfer; (b) a large final Stokes shift (>100 nm); (c) shifting the emission as far into the red portion of the visible spectrum (>600 nm) as possible; and (d) shifting the emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has an excitation maximum near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0095] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET techniques include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridinium isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-stilbene-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, depending on the donor fluorescent moiety used, 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). Donor and acceptor fluorescent moieties can be obtained from, for example, Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0096] The donor and acceptor fluorescent moieties can be linked 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 is measured in angstroms. The distance from the nucleotide base to the fluorescent moiety is measured in units of . Typically, the linker arm is approximately to about The linker arm may be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching a linker arm to a specific nucleotide base, and for attaching a fluorescent moiety to a linker arm.

[0097] Acceptor fluorescent moieties, such as LCRed640, can be combined with oligonucleotides containing amino linkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, VA) to produce, for example, LCRed640-labeled oligonucleotides. Frequently used linkers for coupling donor fluorescent moieties, such as fluorescein, to oligonucleotides include thiourea linkers (FITC-derived, such as fluorescein-CPG's from Glen Research or ChemGene (Ashland, Mass.)), amide linkers (fluorescein-NHS-ester derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, Calif.), or 3'-amino-CPGs that require coupling to fluorescein-NHS-ester after oligonucleotide synthesis.

[0098] In each thermal cycler operation, a control sample can also be circulated. Positive control samples can use, for example, control primers and control probes to amplify target nucleic acid control templates (different from the amplified product of the target gene). Positive control samples can also be amplified, for example, containing a plasmid construct of a target nucleic acid molecule. Such plasmid controls can be amplified internally (for example, within a sample), or amplified using the same primers and probes used to detect the expected target in a separate sample run side by side with a patient sample. Such controls are indicators of the success or failure of amplification, hybridization, and / or FRET reactions. Each thermal cycler operation can also include a negative control, for example, lacking target template DNA. Negative controls can measure contamination. This ensures that system and reagents do not produce false positive signals. Therefore, control reactions can easily determine, for example, the ability of primers to anneal and initiate extension with sequence specificity, and the ability of probes to hybridize with sequence specificity and FRET to occur.

[0099] In one embodiment, the method includes steps to avoid contamination. For example, U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 describe an enzymatic method using uracil-DNA glycosylase to reduce or eliminate contamination between one thermal cycler run and the next.

[0100] Principles of digital PCR

[0101] Digital PCR (dPCR), sometimes called droplet digital PCR, is a PCR-based method for quantifying DNA or RNA targets. In digital PCR, a reaction mixture containing target nucleic acids, primers, probes, and other reagents is randomly partitioned into thousands of independent partitions of equal size, and end-point PCR is performed. TaqMan hydrolysis probes are typically used to detect amplification of the target, and the fluorescence signal of each partition is measured at the end. Partitions without target nucleic acid will have relatively low fluorescence and are therefore negative, while partitions that begin with one or more target nucleic acid molecules will have high fluorescence and are therefore positive. For each reaction, the proportion of negative partitions provides the basis for absolute quantification using Poisson statistics.

[0102] Methods involving dPCR provide a fairly new method for nucleic acid detection and quantification, providing an alternative to conventional real-time quantitative PCR for absolute quantification of nucleic acids and detection of rare alleles. The dPCR assay works by partitioning a nucleic acid sample into many separate, parallel PCR reactions; some of these reactions contain target molecules (positive), while others do not (negative). After PCR analysis, the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample. One of the main advantages of dPCR over real-time PCR is its excellent quantitative accuracy. This advantage relies on the inherent properties of dPCR, as quantification only requires the correct counting of positive partitions and an understanding of the theoretical partition volume (the number of counts is not very sensitive to PCR efficiency). No quantification standard is required. This eliminates potential quantification errors caused by the standard itself.

[0103] As described in detail above, dPCR samples are partitioned so that the individual nucleic acid molecules within the sample are positioned and concentrated in many separate areas (reaction areas). The partitioning of the sample allows the quantity of nucleic acid to be estimated by assuming that the molecular population follows a Poisson distribution. Therefore, each part will contain a negative reaction or a positive reaction ("0" or "1" respectively). After PCR amplification, the nucleic acid can be quantified by counting the areas containing the positive reactions of the PCR final product. In traditional quantitative PCR, the quantitative result may 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, eliminates the reliance on uncertain index data to quantify the target nucleic acid, and therefore provides absolute quantification.

[0104] Next, in each reaction zone of the reaction zone array, sample is carried out dPCR. In dPCR, the nucleic acid in question is amplified and detected, and many independent molecules are isolated in independent reaction zones. Each reaction zone (hole, chamber, pearl, emulsion, etc.) will have a negative result for amplification and detection when the starting molecule does not exist, or has a positive result when the target starting molecule exists. This is a kind of technology, wherein across many independent PCR reactions, sample is carried out limiting dilution so that the partial reaction does not have template molecule, and negative amplification result is given. When the quantity of the positive PCR reaction at the reaction end point is counted, the single template molecule present in the original sample is counted one by one. PCR-based technology has an additional advantage, namely only amplifiable molecules (such as molecules related to the large-scale parallel PCR step in the sequencing workflow) are counted. In the method based on digital PCR, nucleic acid to be analyzed is assigned to many different reaction zones (such as chambers in holes, pearls, emulsions, gel spots, microfluidic devices, etc.). Importantly, some reaction zones (but not all) contain at least one molecule. Usually, each reaction zone will contain one or zero molecule. In fact, molecules will be randomly distributed in reaction areas (such as holes). When the percentage of reaction areas (e.g., 80%) is positive, many areas will contain one or more molecules (e.g., 2.2 molecules per hole on average). Statistical methods can be used to calculate the expected total number of molecules in the sample based on the number of different reaction areas and the number of positives. This will result in the calculated amount or concentration of nucleic acid in the parts applied to different reaction areas. Many statistical methods based on sampling and probability can be used to obtain this concentration. Dube et al., arXiv:0809.1460v2 "Computation of Maximal Resolution of Copy Number Variation on a Nanofluidic Device using Digital PCR (2008)" provide an example of such analysis, see arxiv.org, citing arXiv:0809.1460v2[q-bio.GN], first uploaded on September 8, 2008. This publication provides a series of equations that can be used to estimate the concentration and statistical confidence interval of molecules based on the number of reaction areas used in the digital PCR array and the number of positive results. Another example of this type of calculation can be found in US patent application US 2009 / 0239308 A1.

[0105] Typically, a Poisson distribution is used to predict digital schemes in which only a single DNA amplicon will appear in a reactor of random discrete volume, 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 discrete reactor volumes. Quantification is then achieved by counting how many digital reactors emit amplified fluorescent signals corresponding to an intercalating dye or a specific DNA polymerase probe sequence. Since each reactor volume is limited to no more than a single DNA chain in the digital scheme, one can correctly assume that 100% of its amplified fluorescent signals come only from this one DNA chain and the corresponding primer and probe set. However, very low concentration schemes are generally disadvantageous in terms of imprecise results.

[0106] There are various methods for dPCR. For example, emulsion PCR has been used to prepare beads with cloned amplified DNA, in fact, each bead contains the amplicon of one type of dPCR. Fluorescent probe-based technology can be carried out "in situ" (i.e., in the same well) on the PCR product, which is particularly suitable for this application. U.S. Patent No. 6,440,705 contains a more detailed description of this amplification procedure. These amplifications can be carried out in emulsions or gels, on beads or in multi-well plates. dPCR also includes microfluidic-based technology, in which channels and pumps are used to transport molecules to many reaction zones. Suitable microsomal devices are known in the art.

[0107] dPCR is carried out basically like conventional PCR. Nucleic acid (reference or interested) in a suitable medium is contacted with primers, probes and a thermostable polymerase (e.g., Taq polymerase), and thermal cycling (repeated heating and cooling reaction cycles) is performed to separate chains and enzymatic replication. The culture medium usually contains deoxynucleotides, a buffer solution and ions (e.g., Mg2+). The selectivity of PCR comes from using primers complementary to the region for amplification under specific thermal cycling conditions. The resulting amplified product is detected using a suitable probe, which is usually labeled, such as a fluorescent label. For PCR based on mRNA, the RNA sample is first reverse transcribed into complementary DNA (cDNA) with a reverse transcriptase.

[0108] The PCR process typically consists of a series of temperature changes repeated 25 to 50 times. These cycles typically consist of three phases: the first phase, at around 95°C, allows the double strands of the nucleic acid to separate; the second phase, at temperatures around 50°C to 60°C, allows the primers to bind to the DNA template; and the third phase, at between 68°C and 72°C, promotes polymerization by the DNA polymerase. Due to the small size of the fragments, the final step is often omitted in this type of PCR, as the enzyme is able to increase their number during the transition between the alignment and denaturation phases. Furthermore, signals, such as fluorescence, are measured at a temperature of, for example, 80°C to reduce signals caused by the presence of primer dimers when using nonspecific dyes. The temperature and time used depend on various parameters, such as the enzyme used to synthesize the DNA, the concentrations of divalent ions and deoxyribonucleotides (dNTPs) in the reaction, and the binding temperature of the primers.

[0109] The dPCR method has achieved the unique ability to identify a large number of fluorescent probe sequences (such as TaqMan probe sequences) by encoding each unique probe sequence using a variety of colors, time and intensity combinations. In addition, cheaper non-TaqMan probe real-time PCR amplification indicators such as SYBR- or PicoGreen can be used to implement multiple dPCR based on individual time clues, individual intensity clues or a combination of intensity and time clues, thereby distinguishing primer pairs to a greater extent, and significantly reducing costs. If necessary, these can also be used to enhance control and standardize results to obtain higher accuracy. Using fluorescent reporter genes, the typical multiple analysis limit of typical 5-plex qPCR can be increased to 100-plex dPCR, and the spectral band is limited.

[0110] There are many available dPCR systems that can be used in the present invention. Commercial digital PCR platforms include the microwell chip-based PCR system from Fluidigm. dPCR, through-hole-based QuantStudio 12kflex dPCR and 3D dPCR from Life Technologies, and Droplet-based ddPCR (ddPCR) QX100 and QX200 and from RainDrop. Microfluidic chip-based dPCR can have up to hundreds of reaction areas per panel. Droplet-based dPCR typically has about 20,000 partitioned droplets and can have up to 10,000,000 per reaction. Digital PCR analysis was performed on a plate containing 64 reaction areas per subarray and 48 subarrays in total, equivalent to a total of 3072 reaction areas per array.

[0111] Droplet dPCR (ddPCR) is based on water-oil emulsion droplet technology. The sample is divided into multiple droplets (e.g., about 20,000), and PCR amplification of the template molecule occurs in each individual droplet. The reagents and workflow used in ddPCR technology are similar to those used in most standard assays based on TaqMan probes (including droplet formation chemistry). In addition, intercalating dyes such as Evagreen can be used. A large number of sample reaction partitions are a key aspect of ddPCR technology. Non-spherical partitions (e.g., nanopores) actually have a larger area per sample volume than the same number of spherical partitions.

[0112] In general, the accuracy and, more importantly, the precision of the determination by dPCR can be improved by using a larger number of reaction areas. One can use about 100 to 200, 200 to 300, 300 to 400, 700 or more reaction areas for determining the amount or concentration in question by PCR. In a preferred embodiment of the method of the present invention, dPCR is performed identically in at least 100 reaction areas, in particular at least 1,000 reaction areas, and in particular at least 5,000 reaction areas. In a preferred embodiment of the method of the present invention, dPCR is performed identically in at least 10,000 reaction areas, in particular at least 50,000 reaction areas, and in particular at least 100,000 reaction areas.

[0113] 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 or as a molecular beacon or as a hydrolysis probe. dPCR may involve the use of one or more fluorescent probes to detect nucleic acids of interest and / or reference nucleic acids, particularly in combination with a quencher or as a molecular beacon or as a hydrolysis probe. Representative donor and acceptor fluorescent moieties in FRET technology have been described above. In order to detect and quantify more than one target in the same reaction, primer and probe sets for each target can be combined to perform multiplex analysis. Roche Digital The dPCR system has six optical channels, allowing multiplexing of up to six targets in a single reaction, using a differently labeled TaqMan hydrolysis probe for each target. Other systems with fewer optical channels may require more complex multiplexing strategies, for example, using a combination of dyes for a single probe.

[0114] Use of blocker oligonucleotides to reduce unwanted amplification

[0115] The biggest challenge in optimizing multiplex assays is ensuring that there are no significant oligonucleotide interactions and assay interference. One possible scenario is that multiple primer and probe sets overlap in the amplifiable sequence and thus produce multiplex amplifications that include both intended and unintended multiplex amplifications. Figure 2 An example of an HBV RNA assay design is shown, in which two amplifications are close to each other (within about 2 kb) in the target template. In addition to the expected amplification of a single primer / probe set by design, there may be additional unexpected amplification events from one primer of each assay and two probe cleavage events caused by one primer. Due to the amplicon length and primer-probe distance, these multiple amplification and probe cleavage events may have different efficiencies and therefore result in reduced endpoint fluorescence ("rain" phenotype) in some partitions, and even inaccurate quantification. Blocker oligonucleotides with higher Tm (65°C to 90°C, achieved by Tm enhancer modification such as LNA) and non-extendable 3'-ends (including 3-C spacer or phosphorylation) designed for the middle sequence can effectively bind to the template with high affinity and inhibit primer extension to undesirable regions. When blocker oligonucleotides are added to the reaction, the "rain" phenotype in the dPCR reaction will be greatly reduced, and the quantitative results will be improved.

[0116] General blocker oligonucleotide applications for non-HBV assays as well as HBV targets may include closely located amplified targets of interest for multiplex analysis. Two targets cannot share the same primer set because the resulting amplicons are outside the optimal size range for dPCR and there may be sensitivity issues with sample fragmentation. This utility is applicable to both DNA and RNA assays (see Figure 2 ).

[0117] Applications of general blocker oligonucleotides 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. Although probes can be designed for exon junctions to avoid cleavage from DNA amplification, this inevitably depletes the primers and reduces the efficiency of amplification on the target. Blocker oligonucleotides can be designed to bind to intronic sequences that inhibit DNA-specific amplification without affecting RNA amplification (see Figure 3 )

[0118] General blocker oligonucleotide applications for non-HBV assays as well as HBV targets can further include samples that are non-homogeneous in template sequence. Such samples may include splice variants and fusion products. Regarding splice variants, some splice variants may produce small amplicons that compete with longer amplicons from non-spliced RNA species. In this case, blocker oligonucleotides can be designed to inhibit longer amplifications, and additional priming after the blocker can be better multiplexed in the reaction (see Figure 4). Regarding the detection of multiple fusion products, when multiple fusion products have overlapping sequences, a set of primer pairs can produce amplicons with different lengths and, therefore, different PCR efficiencies. Long amplicon sizes exceeding 300 bp are not optimal for digital PCR. In this case, a blocker oligonucleotide can be designed to inhibit longer amplification. Additional priming after the blocker oligonucleotide can produce amplicons of similar size that are compatible with smaller fusion products, and can be distinguished using a second probe of a different color (see Figure 5 ).

[0119] Products / Kits

[0120] Embodiments of the present disclosure further provide products or kits for detecting HBV RNA and other gene targets. Products may include primers and probes for detecting HBV RNA targets, and suitable packaging materials. Representative primers and probes for detecting HBV RNA (including HBV RNA transcribed from cccDNA, such as HBV pgRNA) are capable of hybridizing with HBV target nucleic acid molecules. In addition, the kit may also include reagents and materials required for DNA fixation, hybridization and detection, such as solid supports, buffers, enzymes and DNA standards, which are appropriately packaged. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes for amplifying and hybridizing HBV target nucleic acid molecules are provided.

[0121] The article may also include one or more fluorescent moieties for labeling a probe, or alternatively, may label a probe provided with the kit. For example, the article may include donor and / or acceptor fluorescent moieties for labeling an HBV probe (which may include a probe targeting HBV RNA). Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0122] The article of manufacture may also contain a package insert or package 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 for practicing the methods disclosed herein (e.g., buffers, polymerases, cofactors, or agents to prevent contamination). Such reagents may be specifically designed for use with one of the commercially available instruments described herein.

[0123] 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 for targeting other poly (A) sites, such as secondary or truncated poly (A) sites of HBV transcripts that may be derived from integrated HBV copies.

[0124] Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0125] Examples

[0126] The following examples and figures are provided to aid the understanding of the present invention, the true scope of the subject matter being set forth in the appended claims.It should be understood that modifications can be made to the procedures set forth without departing from the spirit of the invention.

[0127] Example 1:

[0128] This example demonstrates oligonucleotide sequences used in HBV dPCR assays. Table 4 lists the nucleotide sequences and descriptions of primers (forward and reverse), probes, and blocker oligonucleotides used in dPCR assays for detection and quantification of various HBV RNA forms and gene targets.

[0129] Table 4:

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140] Example 2:

[0141] This example demonstrates 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, such as Figure 1As shown, the sequence includes 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 a 3.5 kb transcript 10. In this example, primers and probes were designed for the following six HBV targets: i) 3' pre-core, ii) 3' poly(A), iii) X gene mRNA, iv) core gene mRNA, v) truncated poly(A), and vi) 5'-pre-core. A dilution series of known concentrations was prepared for each of the six targets, and a ddPCR assay containing the primers and probes in Table 4 was performed to detect each of the six targets. The measured concentrations of each of the six targets in the samples were calculated and plotted against the known (expected) concentrations for these samples, as shown in Figure 5. Figure 6 Linear graph and Figure 7 As shown in the box-and-whisker plot of . As confirmed by the linear regression data shown in Table 5, the expected concentrations were highly correlated with the measured concentrations.

[0142] Table 5

[0143] HBV targets Slope intercept <![CDATA[R 2 ]]> 3' pre-core 0.9451 0.2902 0.9961 3'poly(A) 1.0008 0.0840 0.9898 X gene (XIVT) 1.0388 0.0312 0.9900 Core Gene (Core IVT) 1.0563 0.1825 0.9947 Truncated poly(A) 0.9556 0.0687 0.9954 5' front core 0.9222 0.2341 0.9664

[0144] Steering Figure 8A and Figure 8B , shows the amplification of 3' pre-core RNA ( using a control reverse primer complementary to pre-core HBV (excluding the poly(A) tail) or a reverse primer containing 8 bp poly(T) complementary to pre-core HBV (including the poly(A) tail) Figure 8A ) and 3' pre-core DNA plasmid ( Figure 8B ) comparison of dPCR assay results. Figure 8A As shown, experiments using control and poly(T) primers in samples containing 3' pre-core HBV RNA successfully generated a detectable fluorescent signal of approximately 10,0000 units above a baseline signal amplitude of approximately 2,500 units. As expected, experiments using samples containing 3' pre-core HBV DNA generated a positive signal of approximately 12,000 units, compared to a baseline of approximately 3,500 units for the control primer alone ( Figure 8B ), because the 3' pre-core HBV DNA template does not contain a poly(A) tail complementary to the poly(T) amplification primer.

[0145] Example 3:

[0146] This example shows the oligonucleotide sequences used in the HBV dPCR assay for the S gene. Two S gene assays were designed to accommodate the possible effects of splicing: the S gene assay (S) and the S gene assay post-splicing (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 a probe (HBV_S600_PR-1-1_FZI (SEQ ID NO: 123)) were used. Three different combinations of primers and probes were used for the SPS assay: i) SPS assay-1 (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-2-1 (SEQ ID NO: 129)). Note that the universal probe HBV_S1000_PR-1-1_FZI (SEQ ID NO: 118) was used for all three SPS assays.

[0147] The template for these assays was HBV pgRNA in vitro transcripts (IVT pgRNA). The first experiments focused on preliminary testing with the S gene assay, with the specific aim of implementing two assay designs that could accommodate the potential impact of splicing. Figure 9 Both the S gene assay and the S gene post-splicing (SPS) assay successfully detected the intended target region. While SPS assay 1 exhibited a second positive droplet band at a lower channel 1 amplitude, SPS assays 1 and 3, along with the S assay, demonstrated optimal performance, exhibiting only a single positive droplet band with minimal background signal. In summary, the S gene assay of the present invention is effective for specifically detecting two adjacent regions within the S gene sequence.

[0148] Figure 10 The results show the results of the sensitivity test of SPS assay 2. The previous test pattern persisted, which involved a concentration of 10 6 and 10 5 The reaction of pgRNA IVT with 10 copies / μL (cps / μL) reaches saturation. 3One of the IVT repeats showed lower fluorescence at 1 cps / μL and was therefore omitted from the titer calculation. The assay demonstrated sensitivity to detect template even at concentrations as low as 1 cps / μL.

[0149] Example 4:

[0150] This example demonstrates an HBV assay targeting the pre-splice site of the core region while avoiding the major downstream splicing intron.

[0151] The HBV 3.5kb RNA assay targets the pre-splice site of the core region and strategically avoids the major downstream splicing intron. This design allows the detection of pgRNA and slightly longer pre-core mRNA, such as Figure 11 Reference Figure 12 , the assay was designed and tested with two similar templates (IVT 14 and IVT 15), each containing at least a portion of the 5' pre-core region representing the pre-core RNA as well as pgRNAIVT. IVT 14, IVT 15, and pgRNA( Figure 13 and Figure 14 ).

[0152] Go to Figure 14 Further sensitivity testing showed that the 3.5 kb RNA assay in this example demonstrated no significant background noise or rain under the IVT14 template. 5 Saturation of the ddPCR reaction was observed with the IVT14 template at 10 copies / μL and higher assay input concentrations. As expected, no significant positive droplets were observed with the template representing the 3' end of HBV mRNA due to the lack of a reverse primer binding region. Similar results were observed with the IVT15 template (not shown). Figure 15 and Figure 16 The linearity test depicted in the Figure 2 shows that the assay is sensitive enough to detect 10 4 Copies / μL as low as 10 1 copies / μL of pgRNA and 10 3 Copies / μL as low as 10 1 copies / μL of IVT14 with relatively low standard deviations within these concentrations. 4 copies / μL of pgRNA and >10 3 The reaction tested at IVT 14) resulted in saturation, where no negative reaction droplets were observed.

[0153] Example 5:

[0154] This example demonstrates a HBV triplex assay including a blocker according to the present disclosure. Initial ddPCR multiplex assays directed at detecting at least core, X gene, and poly(A) targets resulted in the observation of inter-cluster rain ( Figure 17 ). Upon closer inspection, this intercluster rain was determined to be localized to clusters containing both the gene X and the poly(A) target. To reduce or eliminate the observed intercluster rain, oligonucleotides that are incapable of extending DNA polymerase were designed to bind to the region between the gene X and the poly(A) region, with the goal of i) preventing the formation of dual-target amplicons (e.g., amplicons that include both the gene X and the poly(A) region), and ii) promoting the generation of single-target amplicons, such as Figure 18 As shown. A non-extendable blocker oligonucleotide was strategically designed to bind to the region between gene X and poly(A), blocking the potential formation of hybrid amplicons involving gene X and poly(A). The initial ddPCR multiplex assay targeting core, gene X, and poly(A) was repeated by adding blocker oligonucleotide at the same concentration as the primers. Figure 19 The ddPCR assay data depicted in demonstrate that the presence of blocker oligonucleotides results in a significant reduction in inter-cluster rain.

[0155] Example 6:

[0156] This example demonstrates an HBV assay for detecting truncated poly(A) according to the present disclosure. Figure 20 , a truncated poly(A) assay was developed to detect truncated HBV RNA species characterized by an earlier poly(A) region. The assay includes the forward primer HBV_TR_A_FMIX1-N (SEQ ID NO: 57), the reverse primer HBV_TRPA_7HS (SEQ ID NO: 60), the probe HBV_TRPA_FL_FZIB_PR (SEQ ID NO: 58), and the blocker oligonucleotide TR3_DD (SEQ ID NO: 62). Similar to Figure 4 and Figure 18 The blocker oligonucleotide TR3_DD is used to prevent the truncated poly(A) reverse primer from binding to the corresponding target region in the untruncated HBV RNA and / or HBV DNA. The results of the ddPCR assay comprising the above primers, probes and blockers showed that the 3' poly(A) ( Figure 21 ) was effectively inhibited, whereas amplification was successfully achieved for in vitro transcripts containing a truncated 3'poly(A) region.

[0157] Example 7:

[0158] This example demonstrates additional oligonucleotide sequences used in HBV dPCR assays. Table 6 lists the nucleotide sequences and descriptions of primers (forward and reverse), probes, and blocker oligonucleotides used in dPCR assays for detection and quantification of various HBV RNA forms and gene targets.

[0159] Table 6:

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] In Table 6,<D_LNA_T> Refers to D-locked nucleic acid thymine,<D_LNA_G> Refers to D-locked nucleic acid guanine,<BHQ_2> Refers to black hole quencher 2,<Spc_C3> Refers to the 3-carbon spacer region, <cy5>Refers to Cyanine5 fluorescent dye,<CY5.5> <5_TEX_615> refers to Cyanine5 fluorescent dye variant, <5TEX_615> refers to Texas Red fluorescent dye, <5_FAM_ABD> refers to fluorescein dye, HEG refers to hexaethylene glycol spacer, and <5_HEX_ABD> refers to hexachloro-fluorescein dye.Should be understood that although specific dye, spacer and quencher molecule are assigned to the nucleotide sequence in Table 6 (and the disclosure in full), it should be understood that replacement is possible.For example, a kind of dye molecule can replace another molecule, and does not affect the effectiveness of corresponding nucleotide sequence substantially.

[0166] Although the above-mentioned invention has been described in considerable detail for the purpose of clarity and understanding, it will be clear to those skilled in the art upon reading this disclosure that various changes may be made in form and detail without departing from the true scope of the invention. For example, all of the above-mentioned techniques and equipment 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 were individually indicated as being incorporated by reference for all purposes.

Claims

1. A method for detecting and quantifying between two and six different hepatitis B virus (HBV) target nucleic acids in a sample by digital PCR (dPCR), the method comprising: - providing said sample; - randomly allocating the sample into a plurality of equally sized and independent partitions; - performing a dPCR assay in each partition, wherein between two and six sets of forward and reverse primers are used to amplify each of the HBV target nucleic acids, and between two and six probes are used to detect each of the HBV target nucleic acids, each probe being labeled with a fluorescent dye that produces a different signal; as well as - measuring the amount of signal generated in each of the partitions to calculate the quantity of each of the between two and six different HBV target nucleic acids in the sample.

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

3. The method according to any one of claims 1 to 2, wherein the dPCR assay is performed using the forward and reverse primer sets and probes specific for the HBV target nucleic acid selected from the oligonucleotides listed in Tables 4 and 6.

4. The method according to 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. 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), the method comprising: - providing said sample; - randomly allocating the sample into a plurality of equally sized and independent partitions; - performing a PCR assay in each partition, wherein at least two forward and reverse primer sets are used to amplify each of the HBV target nucleic acids, and at least two probes are used to detect each of the HBV target nucleic acids, each probe being labeled with a fluorescent dye that produces a different signal; as well as - measuring the amount of signal generated in each of said partitions to calculate the amount of each of said at least two different HBV target nucleic acids in said sample. The method of claim 5 , wherein the PCR assay performed in each partition is a digital PCR (dPCR) assay.

7. The method according to any one of claims 5 to 6, wherein the at least two different HBV target nucleic acids are selected from the group consisting of pre-core-mRNA 5' end (non-pregenomic RNA), core, X gene, truncated RNA 3' end (poly(A) linkage), pre-core / core, full-length RNA 3' end (poly(A) linkage), selected splice junction, S gene (front splice site), S gene (back splice site) and pregenomic RNA 5' end.

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

9. The method according to 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 according to 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-extendable 3' end and a higher melting temperature (Tm) for the template nucleic acid relative to the at least two forward and reverse primer sets and the at least two probes used in the dPCR assay. The method of claim 10 , wherein the undesired amplification is caused by proximity of amplification target regions.

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

13. The method of claim 10, wherein the undesired amplification is the presence of RNA splice variants in which spliced RNA is targeted and unspliced RNA is inhibited.

14. A method for selectively detecting at least two targets in a sample, the method comprising: - performing an amplification step, said amplification step comprising contacting said sample with: - a first set of primers for generating a first amplification product in the presence of nucleic acid in said sample, - a second set of primers for producing a second amplification product in the presence of nucleic acid in said sample, and -Blocker oligonucleotide; - performing a hybridization step comprising contacting the first amplification product and the second amplification product with at least a first detectable probe and a second detectable probe; and - detecting the presence or absence of the first amplification product and the second amplification product, wherein the presence of the first amplification product indicates the presence of the first target in the sample, and wherein the absence of the first amplification product indicates the absence of the first target in the sample, and wherein the presence of the second amplification product indicates the presence of the second target in the sample, and wherein the absence of the second amplification product indicates the absence of the second target in the sample, and wherein the nucleic acid comprises a contiguous sequence comprising the first target, the second target, and an intermediate sequence located between the first target and the second target, and wherein the blocker oligonucleotide is complementary to at least a portion of the intermediate sequence.

15. The method of claim 14, wherein the blocker oligonucleotide is non-extendable by a DNA polymerase.

16. The method of any one of claims 14 to 15, wherein the blocker oligonucleotide reduces or eliminates the production of an undesired amplification product comprising the first target and the second target.

17. A method for reducing unwanted amplification in a multiplex digital PCR (dPCR) assay by using a blocker oligonucleotide having a non-extendable 3' end and a higher melting temperature (Tm) for a template nucleic acid relative to primers and probes used in dPCR. The method of claim 17 , wherein the undesired amplification is caused by proximity of amplification target regions.

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

20. The method of claim 17, wherein the undesired amplification is the presence of RNA splice variants in which spliced RNA is targeted and unspliced RNA is inhibited.

21. A kit for selectively detecting at least two targets in a nucleic acid, the kit comprising: - a first set of primers for producing a first amplification product in the presence of a first portion of nucleic acid in said sample; - a second set of primers for producing a second amplification product in the presence of a second portion of said nucleic acid in said sample; - a blocker oligonucleotide complementary to the nucleic acid between said first portion and said second portion; - a first detectable probe complementary to said first amplification product; and - a second detectable probe complementary to said second amplification product.

22. The kit of claim 21, wherein the blocker oligonucleotide is non-extendable by a DNA polymerase.

23. The kit of any one of claims 21 to 22, wherein the blocker oligonucleotide reduces or eliminates the production of an undesired amplification product comprising the first target and the second target.

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