Compositions and methods for detecting hepatitis delta virus by a dual target assay - Patents.com

Abstract

A method for rapidly detecting the presence or absence of Hepatitis Delta Virus (HDV) in a biological or non-biological sample is described. The method may include performing an amplifying, hybridizing, and detecting step. Additionally, primers, probes, and kits designed for the detection of HDV that target the HDV ribozyme domain and HDV Hepatitis Delta Antigen (HDAg) gene are provided.

Description

[Technical field]

[0001] The present disclosure relates to the field of molecular diagnostics, and more particularly to the detection of Hepatitis Delta Virus (HDV) by a dual-targeted polymerase chain reaction (PCR) assay. [Background technology]

[0002] Hepatitis delta virus (HDV) is a satellite of hepatitis B virus (HBV) for transmission and spread, with a global disease burden estimated at 15-20 million. HDV particles are 35-37 nm in diameter and contain a ribonucleoprotein complex surrounded by the HBV envelope. The ribonucleoprotein is composed of a circular negative single-stranded viral RNA genome, approximately 1700 nucleotides, and two isoforms of HDV protein (small and large). Phylogenetic analysis of the full-length HDV genome has classified HDV into eight genotypes and many sub-genotypes. HDV genotypes show specific geographic divergence and high sequence diversity, with 20%-30% across genotypes and 15% within sub-genotypes.

[0003] Clinically, HBV / HDV dual infection is associated with a higher risk of liver complications and higher mortality compared with HBV infection alone. To manage the higher risk from HDV infection, it is recommended that patients with positive HDV serology undergo active HDV infection evaluation by HDV RNA detection. A score of in-house and commercial reverse transcription polymerase chain reaction assays have been developed to detect and quantify HDV RNA. However, there is a lack of standardization among assays. An international quality control study to evaluate various HDV RNA detection assays showed that only 13 of 28 laboratories (46.3%) across 17 countries demonstrated adequate HDV quantification and good assay performance (Le Gal et al., Hepatology, 2016, Vol. 64, No. 5, p. 1483-1494, which is incorporated herein by reference in its entirety).

[0004] The main challenges associated with robust HDV quantification include (a) the high genetic diversity of the HDV genome, (b) the limited number of sequences with temporally and spatially biased collection, and (c) HDV biology with a high mutation rate combined with editing and recombination. Thus, there is a need in the art for rapid, reliable, specific, and sensitive methods to detect all HDV genotypes and subgenotypes. Summary of the Invention

[0005] The present invention discloses a reverse transcription polymerase chain reaction (RT-PCR) dual target assay for detecting and quantifying HDV in blood, plasma or serum samples. The challenges associated with robust HDV detection as described above have been resolved by detecting two HDV targets, a ribozyme domain and a hepatitis delta antigen (HDAg) gene target region, selected in-silico for their high comprehensiveness on the HDV RNA genome. Compared to current laboratory-developed commercial HDV assays that only detect a single HDV target, the detection of two highly comprehensive targets in the present invention has advanced the field by providing an additional advantage for adequately detecting and quantifying HDV when one target fails to be detected due to rapid viral evolution.

[0006] Certain embodiments of the present disclosure relate to a method for rapid detection of the presence or absence of HDV in a biological or non-biological sample, for example, multiplex detection of HDV by RT-PCR in a single test tube. The embodiments include a method for detection of HDV, comprising performing at least one cycling step, which may include an amplifying step and a hybridizing step. Furthermore, the embodiments include primers, probes and kits designed for detection of HDV in a single tube. The detection method is designed to target a ribozyme domain and the hepatitis delta antigen (HDAg) gene, which allows HDV to be detected in a single test.

[0007] In one aspect, a method for detecting at least two target nucleic acids of HDV in a sample is provided, comprising: (a) providing a sample; (b) performing an amplification step, if the at least two target nucleic acids of HDV are present in the sample, comprising contacting the sample with at least two primer sets to generate amplification products; (c) performing a hybridization step, if the at least two target nucleic acids of HDV are present in the sample, comprising contacting the amplification products with at least two probes; and (d) performing a detection step, comprising detecting the presence or absence of the amplification products, wherein the presence of one amplification product indicates the presence of HDV in the sample and the absence of an amplification product indicates the absence of HDV in the sample, and wherein the at least two primer sets and the at least two probes (i) comprise or are composed of a nucleic acid sequence of SEQ ID NO: 1-4 or any combination of SEQ ID NO: 1-4. and (ii) a first probe or a first probe set comprising or consisting of a nucleic acid sequence of SEQ ID NO: 7-8 or its complement, or a combination of SEQ ID NO: 7-8 or its complement, and (ii) a second primer set comprising a forward primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 9-10 or its combination, and a reverse primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 11-12 or its combination, and a second probe or a second probe set comprising or consisting of a nucleic acid sequence of SEQ ID NO: 13-15 or its complement, or any combination of SEQ ID NO: 13-15 or its complement. In a related embodiment, the sample is a biological sample. In another related embodiment, the biological sample is blood, plasma, or serum.

[0008] In yet another embodiment, the first primer set generates one or more amplification products of a first target nucleic acid that is detected by a first probe or first probe set, and the second primer set generates one or more amplification products of a second target nucleic acid that is detected by a second probe or second probe set. In one embodiment, the first target nucleic acid is an HDV ribozyme domain and the second target nucleic acid is an HDV hepatitis delta antigen (HDAg) gene.

[0009] In another aspect, a method for detecting HDV in a sample includes: (a) performing an amplifying step, comprising contacting the sample with one or more forward primers and one or more reverse primers specific for a HDV ribozyme domain to generate ribozyme domain amplification products if HDV is present in the sample, and with one or more forward primers and one or more reverse primers specific for a HDV hepatitis delta antigen (HDAg) gene to generate HDAg gene amplification products if HDV is present in the sample; (b) performing a hybridizing step, comprising contacting the ribozyme domain amplification products with one or more detectable probes specific for the ribozyme domain, and contacting the HDAg gene amplification products with one or more detectable probes specific for the HDAg gene; and (c) detecting the presence or absence of a ribozyme domain amplification product and / or a HDAg gene amplification product, wherein the presence or absence of either the ribozyme domain amplification product or the HDAg amplification product or both amplification products is detected. the presence of a ribozyme domain amplification product indicates the presence of HDV in the sample, the absence of both a ribozyme domain amplification product and an HDAg amplification product indicates the absence of HDV in the sample, one or more ribozyme domain forward primers comprise or consist of a nucleotide sequence selected from SEQ ID NOs: 1 to 4, or any combination of SEQ ID NOs: 1 to 4, one or more ribozyme domain reverse primers comprise or consist of a nucleotide sequence selected from SEQ ID NOs: 5 to 6, or a combination thereof, one or more detectable ribozyme domain probes comprise or consist of a nucleotide sequence selected from SEQ ID NOs: 7 to 8, or a complement thereof, or a combination of SEQ ID NOs: 7 to 8, or a complement thereof, one or more HDAg gene forward primers comprise or consist of a nucleotide sequence selected from SEQ ID NOs: 9 to 10, or a combination thereof, one or more HDAg gene reverse primers comprise or consist of a nucleotide sequence selected from SEQ ID NOs: 11 to 12, or a combination thereof, one or more detectable HDAg gene probes comprise a nucleotide sequence selected from SEQ ID NOs: 13 to 15, or a complement thereof,or any combination of SEQ ID NOs: 13 to 15 or their complements.

[0010] In one embodiment, the amplification of the ribozyme domain target comprises using all forward primers comprising or consisting of the nucleotide sequences of SEQ ID NOs: 1-4 and both reverse primers comprising or consisting of the nucleotide sequences of SEQ ID NOs: 5-6, and the detection of the ribozyme domain amplification products comprises using both detectable probes comprising or consisting of the nucleotide sequences of SEQ ID NOs: 7-8 or their complements. In another embodiment, the amplification of the HDAg gene target comprises using both forward primers comprising or consisting of the nucleotide sequences of SEQ ID NOs: 9-10 and both reverse primers comprising or consisting of the nucleotide sequences of SEQ ID NOs: 11-12, and the detection of the HDAg gene amplification products comprises using all detectable probes comprising or consisting of the nucleotide sequences of SEQ ID NOs: 13-15 or their complements. In one embodiment, the sample is a biological sample. Specifically, the biological sample is blood, plasma or serum.

[0011] Another aspect provides an oligonucleotide comprising or consisting of a sequence of nucleotides selected from SEQ ID NOs: 1-20 or a complement thereof, having 50 or fewer nucleotides. In another embodiment, the disclosure provides an oligonucleotide comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%, etc.) to one of SEQ ID NOs: 1-20 or a complement thereof, the oligonucleotide having 50 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, etc. in these embodiments. In certain of these embodiments, the oligonucleotide has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. The oligonucleotide optionally comprises at least one label moiety and optionally at least one quencher moiety. In some embodiments, the at least one label moiety and the at least one quencher moiety are fluorescent moieties. In some embodiments, the oligonucleotide comprises at least one conservatively modified variation. A "conservatively modified variation" or simply "conservative variation" of a particular nucleic acid sequence refers to a nucleic acid that encodes the same or essentially identical amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, an essentially identical sequence. Those skilled in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single nucleotide or a small percentage of nucleotides (typically less than 5%, more typically less than 4%, 2%, or 1%) in the encoded sequence are "conservatively modified variations" where the modification results in the deletion of an amino acid, the addition of an amino acid, or the replacement of an amino acid with a chemically similar amino acid.

[0012] In one embodiment, the amplification can use a polymerase enzyme with 5'-3' nuclease activity. Thus, the first and second fluorescent moieties, the label moiety and the quencher moiety, can be within 8 nucleotides of each other along the length of the probe. In another embodiment, the ribozyme domain and / or HDAg gene probe includes a nucleic acid sequence that allows for the formation of a secondary structure. The formation of such a secondary structure generally results in spatial proximity between the first and second fluorescent moieties. According to this method, the second fluorescent moiety on the probe can be a quencher.

[0013] In one embodiment, the ribozyme domain and HDAg gene probe can be labeled with a fluorescent dye that acts as a reporter. The probe can also have a second dye that acts as a quencher. The reporter dye is measured at a defined wavelength, thus allowing detection and discrimination of the amplified HDV ribozyme domain and HDAg gene target. The fluorescent signal of the intact probe is suppressed by the quencher dye. During the amplification step of PCR, hybridization of the probe to a specific single-stranded DNA template results in cleavage by the 5'-3' nuclease activity of DNA polymerase, resulting in separation of the reporter and quencher dyes and generation of a fluorescent signal. With each PCR cycle, the amount of cleaved probe increases, and the cumulative signal of the reporter dye increases accordingly. Optionally, one or more additional probes (e.g., internal reference controls or other targeting probes (e.g., other viral nucleic acids)) can also be labeled with a reporter fluorescent dye that is unique and different from the fluorescent dye label associated with the ribozyme domain and HDAg gene probe. In such cases, a specific reporter dye is measured at a defined wavelength, allowing simultaneous detection and discrimination of the amplified target and one or more additional probes.

[0014] The present disclosure provides methods for detecting the presence or absence of HDV or HDV nucleic acid in a biological sample from an individual. These methods can be used to detect the presence or absence of HDV or HDV nucleic acid in biological samples such as serum, plasma, whole blood, liver tissue or other biological material in which HDV is believed to be present, for use in diagnostic testing. Furthermore, the same tests may be used by those skilled in the art to evaluate other sample types to detect HDV or HDV nucleic acid. Such methods generally include performing a reverse transcription step and at least one cycling step including an amplifying step and either a detectable probe binding step or a dye binding step. Typically, the amplifying step includes contacting the sample with multiple pairs of oligonucleotide primers to generate one or more amplification products if a nucleic acid molecule is present in the sample, the probe binding step includes contacting the amplification product with one or more detectable probes specific to the amplification product, and the dye binding step includes contacting the amplification product with a double-stranded DNA binding dye. Such methods may also include detecting the presence or absence of binding of a double-stranded DNA binding dye to the amplification product, the presence of binding indicating the presence of HDV or HDV nucleic acid in the sample, and the absence of binding indicating the absence of HDV or HDV nucleic acid in the sample. An exemplary double-stranded DNA binding dye is ethidium bromide. Other nucleic acid binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green. Furthermore, such methods may also include determining the melting temperature between the amplification product and the double-stranded DNA binding dye, which confirms the presence or absence of HDV or HDV nucleic acid.

[0015] In a further aspect, a kit for detecting one or more target nucleic acids of HDV is provided. In one embodiment, a kit for detecting a first target nucleic acid of HDV and a second target nucleic acid of HDV in a sample is provided, the kit comprising: (a) a DNA polymerase having 5'-3' nuclease activity; (b) a nucleotide monomer; and (c) a first primer set and a first probe or a first probe set for detecting the first target nucleic acid of HDV, the first primer set and the first probe or the first probe set comprising at least a forward primer comprising or consisting of a nucleic acid sequence of SEQ ID NO: 1 to 4 and any combination of SEQ ID NO: 1 to 4, and a reverse primer comprising at least a nucleic acid sequence of SEQ ID NO: 5 to 6 or a combination thereof, and the first probe or the first probe set comprising a nucleic acid sequence of SEQ ID NO: 7 to 8 or a complement thereof, or a combination of SEQ ID NO: 7 to 8 or a complement thereof. The amplification reagent includes a first primer set and a first probe or a first probe set for detecting a target nucleic acid, and (d) a second primer set and a second probe or a second probe set for detecting a second target nucleic acid of HDV, the second primer set and the second probe or the second probe set including at least a forward primer including or consisting of a nucleic acid sequence of SEQ ID NO: 9 to 10 or a combination thereof, and a reverse primer including or consisting of a nucleic acid sequence of SEQ ID NO: 11 to 12 or a combination thereof, and the second probe or the second probe set including a nucleic acid sequence of SEQ ID NO: 13 to 15 or a complement thereof, or any combination of SEQ ID NO: 13 to 15 or a complement thereof. In one embodiment, the first target nucleic acid is an HDV ribozyme domain, and the second target nucleic acid is an HDV hepatitis delta antigen (HDAg) gene. In a further embodiment, a kit for detecting one or more target nucleic acids of HDV is provided.The kit may include multiple sets of ribozyme domain and / or HDAg gene primers specific for amplifying ribozyme domain targets and / or HDAg gene targets, and one or more detectable ribozyme domain and / or HDAg gene probes specific for detecting the respective amplification products. The kit may include probes already labeled with donor fluorescent moieties and corresponding acceptor fluorescent moieties, or may include fluorescent moieties for labeling the probes. The kit may also include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit may also include a package insert and instructions for using the primers, probes, and fluorophore moieties to detect the presence or absence of HDV in a sample.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0017] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, as well as the claims. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows the relative positions of the primers and probes used for amplification and detection of the HDV ribozyme domain target with respect to GenBank Accession No. AF 098261 (HDV genotype 1). [Diagram 2]FIG. 2 shows the relative positions of the primers and probes used for amplification and detection of the HDV HDAg gene target, relative to GenBank Accession No. AF098261. [Diagram 3] FIG. 3 shows the results of the experiment described in Example 4 to determine assay sensitivity using 24 replicates of the WHO HDV NAT at 25, 10, 5, 2, 1 and 0.5 IU / mL levels, respectively. [Figure 4] FIG. 4 shows the results of the experiment described in Example 4 to determine assay linearity in which HDV armored RNA (arRNA) was tested in eight replicates at concentrations ranging from 100 copies / mL to 10 copies / mL. [Diagram 5] FIG. 5 shows the results of the experiment described in Example 4 to determine assay linearity in which HDV RNA from positive plasma samples was tested in triplicate at 10-fold dilutions between 2.3E+00 IU / mL and 2.3E+05 IU / mL. [Figure 6] 6 shows the results of the experiment described in Example 4 to determine the comprehensiveness of the dual target assay using in vitro transcribed HDV genotype 1-8 sequences for the ribozyme domain and HDAg gene as described in Example 3. Each genotype was tested over a 4 log concentration range from 103 copies to 107 copies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Diagnosis of HDV infection by nucleic acid amplification provides a method for rapid and accurate detection of viral infection. A real-time reverse transcription polymerase chain reaction (RT-PCR) assay for detecting HDV in a sample is described herein. Primers and probes for detecting HDV are provided, as are articles of manufacture or kits containing such primers and probes. The increased sensitivity of real-time PCR for detection of HDV compared to other methods, and the improved features of real-time PCR, including sample containment and real-time detection of amplification products, make the implementation of this technology feasible for routine diagnosis of HDV infection in clinical laboratories.

[0020] Hepatitis delta virus (HDV) is an infectious agent that depends on hepatitis B virus (HBV) for the formation of viral particles. The HDV genome is a small, single-stranded RNA, approximately 1700 nucleotides long, that is circular in conformation. The genomic RNA can fold and form an unbranched rod-like structure using approximately 74% base pairing. Replication of the HDV genome occurs via a symmetric rolling circle mechanism involving an RNA intermediate, leading to the accumulation of new genomes and complementary RNA species, known as antigens. In classical HDV infection, up to 300,000 copies of genome and 100,000 copies of antigenome accumulate per infected cell during HDV genome replication. The genomic and antigenome RNA circles are thought to act as templates for the generation of bipolar multimeric chains longer than 1700 nucleotide unit lengths. These are processed into unit-length RNAs due to the presence of site-specific ribozyme sequences in both the genome and antigenome. After ribozyme cleavage, the unit-length RNAs are ligated to form new circular RNA species. HDV does not encode its own replicase and can replicate autonomously within its host, redirecting one or more host RNA polymerases for its replication (Taylor and Pelchat, Future Microbiol 5:393-402, 2010).

[0021] A third HDV RNA species of approximately 900 nucleotides in length and antigenomic polarity is also produced in classical HDVHBV infection at approximately 500 copies per infected cell. The open reading frame of this RNA encodes a 195 amino acid long protein, referred to in this disclosure as small delta antigen (S-HDAg), or simply HDAg. During replication, adenosine deaminase acting on dsRNA converts the adenosine in the stop codon of HDAg to inosine. This amino acid conversion leads to the production of an mRNA whose stop codon codes for tryptophan, resulting in the production of a second viral protein species with a C-terminal 19 amino acids longer, referred to as large delta antigen (L-HDAg) (Taylor and Pelchat, Future Microbiol 5:393-402, 2010).

[0022] Extensive sequence analysis of a large number of isolates has led to the classification of HDV in at least eight different clades with different geographic distributions. Genotype 1 is prevalent worldwide, while the other genotypes are endemic to various regions of the world. Genotype 2 is found in Southeast Asia, Taiwan, China, and Japan. Genotype 3 is endemic to the Amazon basin. Genotype 4 is found in Taiwan and Japan. Finally, genotypes 5 to 8 are prevalent in Africa.

[0023] The disclosed method may include performing at least one cycling step that includes amplifying one or more portions of HDV ribozyme domain nucleic acid targets and HDV HDAg gene nucleic acid targets from a sample using one or more pairs of ribozyme domain primers and / or one or more pairs of HDAg gene primers. As used herein, "ribozyme domain primer" or "HDAg primer" refers to an oligonucleotide primer that specifically anneals to a ribozyme domain and HDAg gene nucleic acid sequence, respectively, and initiates DNA synthesis therefrom under appropriate conditions. Each of the contemplated ribozyme domain or HDAg gene primers anneals to a target within or adjacent to a respective target nucleic acid molecule such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. Provided that one or more ribozyme domain nucleic acid and / or HDAg gene nucleic acid are present in the sample, one or more ribozyme domain amplification products and / or HDAg gene amplification products are generated, and thus the presence of one or more of these amplification products indicates the presence of HDV in the sample. The amplification products must contain nucleic acid sequences complementary to one or more detectable probes for the ribozyme domain or HDAg gene. Each cycling step includes an amplification step, a hybridization step, and a detection step, in which the sample is contacted with one or more detectable probes for the ribozyme domain or HDAg gene to detect the presence or absence of HDV in the sample.

[0024] As used herein, the term "amplifying" 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., HDV ribozyme domain or HDV HDAg gene). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically involves the use of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq) and appropriate buffers and / or cofactors for optimal activity of the polymerase enzyme (e.g., MgCl 2 and / or KCl) is required.

[0025] The term "primer" as used herein is known to those skilled in the art and refers to an oligomeric compound, primarily oligonucleotides, but also modified oligonucleotides that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group, whereby a deoxynucleoside triphosphate is used, whereby a further "nucleotide" can be attached by the template-dependent DNA polymerase to establish a 3' to 5' phosphodiester bond, whereby pyrophosphate is released. Thus, there is no fundamental difference between a "primer", an "oligonucleotide", or a "probe", except perhaps in the intended function.

[0026] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0027] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0028] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, catalyzes the formation of primer extension products complementary to a template, and does not irreversibly denature when exposed to high temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated, for example, from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, non-thermostable polymerases can also be used in PCR assays, provided the enzyme is replenished.

[0029] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0030] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase, which typically adds nucleotides to the 3' end of the nucleic acid.

[0031] The terms "identical" or "percent identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that are identical or have a particular percentage of identical nucleotides, when compared or aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Genet. Res. 7:649-656, each of which is incorporated herein by reference.

[0032] A "modified nucleotide" in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of an oligonucleotide sequence is replaced with a different nucleotide that provides desired properties to the oligonucleotide. Exemplary modified nucleotides that may be substituted in the oligonucleotides described herein include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-O-methylriboU, 2'-O-methylriboC, N4-ethyl-dC, N6-methyl-dA, etc. Many other modified nucleotides that may be substituted in oligonucleotides are mentioned herein or known in the art. In certain embodiments, modified nucleotide substitutions modify the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. To further explain, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase the yield of intended target amplicon, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference.

[0033] The present disclosure provides a method for detecting hepatitis delta virus (HDV), for example, by amplifying a portion of the HDV ribozyme domain nucleic acid sequence and / or the HDV hepatitis delta antigen (HDAg) gene nucleic acid sequence. Nucleic acid sequences of various genotypes and subgenotypes of HDV are available (e.g., GenBank accession numbers AF098261 for genotype 1, AF104264 for genotype 2, AB037948 for genotype 3, AB118820 for genotype 4, AM183326 for genotype 5, AM183332 for genotype 6, AM183333 for genotype 7, and AM183330 for genotype 8). In particular, primers and probes for amplifying and detecting the ribozyme domain and HDAg gene nucleic acid molecular targets are provided by embodiments of the present disclosure.

[0034] For the detection of HDV, primers and probes are provided for amplifying ribozyme domain and / or HDAg gene. HDV nucleic acids other than those exemplified herein can also be used to detect HDV in a sample. For example, functional variants can be evaluated for specificity and / or sensitivity by those skilled in the art using routine methods. Representative functional variants can include, for example, one or more deletions, insertions and / or substitutions in the HDV nucleic acids disclosed herein.

[0035] More specifically, each of the oligonucleotide embodiments includes a nucleic acid having a sequence selected from SEQ ID NOs: 1-20, a substantially identical variant thereof having at least, e.g., 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1-20, or the complement of SEQ ID NOs: 1-20 and variants thereof.

[0036] [Table 1]

[0037] [Table 2]

[0038] In one embodiment, the above-mentioned set of HDV ribozyme domain and HDAg gene primers and probes are used to provide detection of HDV in biological samples suspected of containing HDV. The primer and probe set may comprise or consist of primers and probes specific for the ribozyme domain or HDAg gene nucleic acid sequences comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-20. In another embodiment, the primers and probes for the ribozyme domain and HDAg gene targets comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOs: 1-15.

[0039] Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-20 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-20 relate to primers and / or probes that provide similar or higher specificity and sensitivity compared to the respective sequences of SEQ ID NOs: 1-20 in the described methods or kits.

[0040] A variant may vary from the sequence of SEQ ID NO: 1-20 by, for example, the addition, deletion or substitution of one or more nucleotides, such as the addition, deletion or substitution of one or more nucleotides at the 5'-end and / or 3'-end of each of the sequences of SEQ ID NO: 1-20. As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may comprise modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide". A natural base in a "nucleotide" may also be replaced, for example, by 7-desazapurine, thereby obtaining a "modified nucleotide" as well. The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a naturally occurring nucleoside by some modification in the manner outlined above for "modified nucleotides" (or "nucleotide analogs").

[0041] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs that amplify nucleic acid molecules from ribozyme domains or HDAg gene nucleic acid sequences, can be designed using computer programs such as, for example, OLIGO (Molecular Biology Insights Inc., Cascade, CO). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the pair of primers, and the length of each primer (i.e., primers must be long enough to anneal with sequence specificity and initiate synthesis, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 nucleotides long (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides long). For example, oligonucleotide primers can be up to 30, 35, or 40 nucleotides long.

[0042] In addition to a set of primers, the method may use one or more probes to detect the presence or absence of HDV. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to HDV ribozyme domain (target) nucleic acid and / or HDV HDAg gene (target) nucleic acid under a defined, predetermined stringency by design or selection. "Probe" can also be called "detection probe", which means to detect target nucleic acid.

[0043] In some embodiments, the ribozyme domains and HDAg gene probes described can be labeled with at least one fluorescent label. In one embodiment, the ribozyme domains and HDAg gene probes can be labeled with a donor fluorescent moiety, such as a fluorescent dye, and a corresponding acceptor fluorescent moiety, such as 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: 7, 8, 13, 14, and 15.

[0044] The design of oligonucleotides used as probes can be done in a similar manner to the design of primers. Embodiments can use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probe(s) used can include at least one label and / or at least one quencher moiety. Like primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15-30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length. In some examples, oligonucleotide probes can be up to 30, 35, or 40 nucleotides in length.

[0045] Constructs containing HDV nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant and animal cells. Prokaryotic hosts can include E. coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into the host cell using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and viral-mediated nucleic acid transfer are common methods for introducing nucleic acid into the host cell. In addition, naked DNA can be delivered directly into the cell (e.g., U.S. Patent Nos. 5,580,859 and 5,589,466).

[0046] Conventional PCR techniques are disclosed in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as initiation points for nucleic acid synthesis within the described HDV ribozyme domain nucleic acid sequences (e.g., SEQ ID NOs: 1-6) and HDV HDAg gene nucleic acid sequences (e.g., SEQ ID NOs: 9-12). Primers can be purified from restriction digests by conventional methods or produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, although primers may be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0047] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating the nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration, as well as the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0048] When the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the described ribozyme domain and HDAg gene nucleic acid molecule. 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 can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient for extension to occur from the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). Extension times can be from about 10 seconds to about 5 minutes (e.g., from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0049] The PCR assay can use HDV nucleic acid, e.g., RNA or DNA (cDNA). The template nucleic acid does not need to be purified and can be a minor fraction of a complex mixture, such as HDV nucleic acid contained in human cells. HDV nucleic acid molecules can be extracted from biological samples by routine techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, including plasmids or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0050] Oligonucleotide primers (e.g., SEQ ID NOs: 1-6 and 9-12) are combined with PCR reagents under reaction conditions conducive to primer extension. For example, chain extension reactions typically contain 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl 2 The reaction mixture typically contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more of their analogs.

[0051] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing and extension can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target ribozyme domain and / or HDAg gene nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence enough for detection. Generally, the cycling steps are repeated at least about 20 times, but can be repeated 40, 60, or even 100 times.

[0052] FRET technology (e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are placed within a certain distance of each other, energy transfer occurs between two fluorescent moieties that can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light irradiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light irradiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0053] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent, that dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, resulting in quenching of the fluorescence emission from the donor fluorescent moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, such that the 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 BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0054] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the HDV target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product at the appropriate position, it generates a FRET signal. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0055] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission in a particular range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation of the desired range.

[0056] As used herein with respect to a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, "corresponding" refers to an acceptor fluorescent moiety that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-irradiative energy transfer can occur between them.

[0057] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) high efficiency Forster energy transfer, (b) large final Stokes shift (>100 nm), (c) emission shift as far as possible to the red portion of the visible spectrum (>600 nm), and (d) emission shift to a wavelength higher than the Raman water fluorescence emission caused by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0058] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0059] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Typically, the linker arm is from about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods of attaching the linker arm to a particular nucleotide base and attaching the fluorescent moiety to the linker arm.

[0060] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to produce, for example, LC Red 640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from fluorescein-NHS-ester, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0061] Hepatitis Delta Virus Detection The present disclosure provides a method for detecting the presence or absence of Hepatitis Delta Virus (HDV) in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes performing at least one cycling step including amplifying a portion of a HDV ribozyme domain and / or HDV Hepatitis Delta Antigen (HDAg) gene target nucleic acid molecule from a sample using multiple pairs of ribozyme domain and / or HDV Hepatitis Delta Antigen (HDAg) gene primers, and a FRET detecting step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed to detect the presence of HDV using ribozyme domain and / or HDAg gene primers and probes, and detection of the HDV ribozyme domain and / or HDV HDAg gene indicates the presence of HDV in the sample.

[0062] As described herein, the amplification product can be detected using a labeled hybridization probe that utilizes FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of the amplification product, and thus the presence or absence of HDV. TaqMan® technology utilizes, for example, a single-stranded hybridization probe labeled with a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded during the subsequent extension stage, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein for detecting the presence or absence of HDV in a sample.

[0063] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). The secondary structure formed within the probe results in both fluorescent moieties being in spatial proximity when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0064] Another common form of FRET technology utilizes two hybridization probes. Each probe may be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of HDV genomes). When amplification of the HDV target nucleic acid occurs and an amplification product is generated, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0065] Generally, the presence of FRET indicates the presence of HDV in the sample, and the absence of FRET indicates the absence of HDV in the sample.However, insufficient specimen collection, delayed transportation, improper transportation conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results.Using the method disclosed herein, for example, detection of FRET within 45 cycling steps indicates infection with HDV.

[0066] Representative biological samples that can be used to carry out the method include, but are not limited to, blood, plasma, serum, liver samples, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release HDV nucleic acid, or in some cases, the biological sample can be directly contacted with PCR reaction components and appropriate oligonucleotides.

[0067] Melting curve analysis is an additional step that can be included in the cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called melting temperature (Tm), which is defined as the temperature at which half of a DNA double strand separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the ribozyme domain and HDAg gene probe from each amplification product can confirm the presence or absence of HDV in the sample.

[0068] During each thermocycler run, control samples may be cycled as well. A positive control sample may amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and a control probe. A positive control sample may also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control may be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such controls are indicative of the success or failure of the amplification, hybridization and / or FRET reaction. Each thermocycler run may also include a negative control, for example, lacking target template DNA. The negative control may measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reaction may easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, as well as the ability of probes to hybridize with sequence specificity and FRET to occur.

[0069] In one embodiment, the method includes a step to avoid contamination, for example, the enzymatic method utilizing uracil-DNA glycosylase described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycling run and the next.

[0070] The method can be implemented using conventional PCR methods combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0071] The LightCycler® may be operated using a PC workstation and may utilize the Window NT operating system. Signals from samples are obtained as the machine sequentially positions the capillaries over the optical unit. The software can display the fluorescent signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The data generated may be stored for further analysis.

[0072] As an alternative to FRET, the amplification products can be detected using double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Also, double-stranded DNA binding dyes such as nucleic acid intercalating dyes can be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of the amplification products.

[0073] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0074] Manufactured products / kits The embodiments of the present disclosure further provide an article of manufacture, composition or kit for detecting HDV. The article of manufacture may include primers and probes used to detect HDV, together with suitable packaging materials. Representative primers and probes for detecting HDV can hybridize to HDV target nucleic acid molecules (e.g., HDV ribozyme domain and / or HDV HDAg gene). In addition, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to HDV target nucleic acid molecules are provided.

[0075] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or the probes provided with the kit may be labeled. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the HDV ribozyme domain and / or HDV HDAg gene probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0076] The article of manufacture may also include a package insert or packaging label with instructions for using the HDV ribozyme domain and / or HDV HDAg gene primers and probes to detect HDV in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific for one of the commercially available instruments described herein.

[0077] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. EXAMPLES

[0078] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0079] Example 1 HDV gene targets Figure 1 shows the relative positions of primers and probes used for amplification and detection of HDV ribozyme domain targets relative to GenBank Accession No. AF098261 (HDV genotype 1). Figure 2 shows the relative positions of primers and probes used for amplification and detection of HDV HDAg gene targets relative to GenBank Accession No. AF098261.

[0080] Example 2 PCR experimental conditions Real-time PCR detection of ribozyme domain targets or HDAg genes was performed using the cobas® 6800 / 8800 system (Roche Molecular Systems, Inc., Pleasanton, Calif.). The final concentrations of amplification reagents were as follows: [Table 3]

[0081] The following table shows a typical thermal profile used in a PCR amplification reaction: [Table 4]

[0082] The Pre-PCR program included incubations at 55°C, 60°C and 65°C for initial denaturation and reverse transcription of the RNA template. The three temperature incubations have the advantageous effect that at the lower temperatures even slightly mismatched target sequences (such as genetic variants of an organism) are transcribed, while at the higher temperatures the formation of RNA secondary structures is suppressed, thus resulting in more efficient transcription. The PCR cycling is divided into two runs, both of which apply a one-stage setup (combining annealing and extension). The first five cycles at 55°C allow for increased inclusiveness by preamplifying the slightly mismatched target sequences, whereas the 45 cycles of the second run increase specificity by using an annealing / extension temperature of 58°C.

[0083] Example 3 Materials and Methods Samples: HDV armored RNA (arRNA), commercially available HDV positive plasma and serum samples, WHO HDV IS standard (PEI code number 7657 / 12), custom collected human volunteer donor HBV / HCV / HIV negative pooled plasma, and human volunteer donor HBV / HCV / HIV negative pooled serum (SeraCare) were used in the study. HDV positive serum and plasma samples were obtained from Boca Biologics.

[0084] HDV RNA Assay: Ribozyme and HDAg target regions on HDV RNA genome were selected for assay design (Figure 1 and Figure 2). HDV sequence was downloaded from Genbank, aligned, and genotype-generic primers and probes were designed. Sample volume of 0.5 mL was extracted with cobas® 6800 System (Roche Molecular Systems, Inc.). Extracted sample was eluted in 50 μL. 25 μL sample was used for amplification using generic RNA master mix and generic thermal profile on cobas® 6800 System. Data was analyzed using Algorithm Testing Framework (ATF) software with custom optimized parameters for HDV assay.

[0085] In-vitro RNA transcription: HDV sequences (genotypes 1-8) were downloaded from Genbank. Sequences spanning the ribozyme and HDAg targets were cloned into the pSP6-polyA vector (Integrated DNA technologies, Inc.). The cloned sequences were verified by DNA sequencing. Plasmids carrying the cloned sequences were used in in-vitro transcription reactions using the SP6 promoter-based MegaScript amplification kit (ThermoFisher). In-vitro RNA transcripts were purified by MegaClear kit (ThermoFisher) and copy numbers were determined by HDV droplet digital PCR assay.

[0086] HDV Assay Performance Study: Assay linearity was determined using HDV arRNA and HDV positive plasma samples. Linearity was reported in IU / mL using WHO HDV NAT standards as calibrators. Linearity data was analyzed by polynomial regression. Assay sensitivity in terms of limit of detection (LOD) was determined using WHO HDV NAT standards with 24 replicates each at 25, 10, 5, 2, 1 and 0.5 IU / mL levels. HBV / HCV / HIV negative pooled plasma was used as a control. LOD data was analyzed by probit. Assay specificity was determined using multiple replicates (n=92) of HBV / HCV / HIV negative pooled plasma and negative pooled serum.

[0087] In-silico inclusion and exclusivity: HDV sequences detected in-silico by the HDV dual-target assay were downloaded from Genbank, aligned, and a phylogenetic analysis (bootstrap=100) was constructed using Geneious bioinformatics software (genious biologics). For exclusivity, HDV dual-target assay primers and probes were aligned for cross-reactivity with HAV, HBV, HCV, HEV, and HIV sequences in local databases.

[0088] Plasma Separation Cards (PSC): Whole blood was collected in EDTA sample collection tubes. Samples such as HDV Armored Control, HDV Plasma, and HDV WHO IS were spiked into whole blood at the designated concentrations. To prepare PSC samples, 140 mL of whole blood (with or without spike-in HDV) was spotted onto a 1 cm circle within the spotting area. After sample spotting, the PSC was left at room temperature for 4 hours and then kept in a ziplock bag containing 4 g of desiccant. All bags containing PSC were stored at various temperatures (ambient, -20°C, 45°C) prior to analysis.

[0089] Example 4 Experimental Results The performance of the dual target assay was evaluated by several means. Determination of assay sensitivity was performed using the WHO HDV NAT standard described in Gal et al., Hepatology, 2016, Vol. 64, No. 5, p. 1483-1494 (24 replicates at 25, 10, 5, 2, 1 and 0.5 IU / mL levels, respectively). Figure 3 shows the results of this experiment with the hit rate and the limit of detection (LOD) determined from the probit curve fit to be 1.11 IU / mL.

[0090] Assay linearity was determined using two types of samples. First, HDV armored RNA (arRNA) was diluted to 100 copies / mL and then diluted to 10 10 The results are shown in Figure 4. The average Ct value was 10 10 The mean Ct values ​​ranged from 9.0 at 100 copies / mL to 34.96 at 100 copies / mL, and accurate quantification could be observed over a wide range of more than 8 logs. HDV RNA from positive plasma samples was then tested in triplicates at every 10-fold dilution between 2.3E+00 IU / mL and 2.3E+05 IU / mL. As can be seen in Figure 5, the mean Ct values ​​ranged between 20.38 and 36.49, and accurate quantification of HDV with clinical HDV samples could be observed over a wide range (>5 logs).

[0091] Experimental determination of the comprehensiveness of the dual target assay was performed using in vitro transcribed HDV genotypes 1-8 sequences for the ribozyme domain and HDAg gene as described in Example 3. Each genotype was analyzed using 10 3 Copy ~10 7 The results are shown in Figure 6 and demonstrate that, unlike many laboratory-developed HDV tests, the dual target assay of the present invention was able to detect all eight HDV genotypes. In silico comprehensiveness and exclusivity testing was performed and the results showed that the dual target assay of the present invention can detect all HDV genotypes (1-8), with no cross-reactivity expected with HAV, HBV, HCV, HEV and HIV (data not shown).

[0092] Although the foregoing invention has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the invention. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.

Claims

1. 1. A method for detecting at least two target nucleic acids of Hepatitis Delta Virus (HDV) in a sample, the method comprising: (a) providing a sample; (b) performing an amplification step, if the at least two target nucleic acids of HDV are present in the sample, comprising contacting the sample with at least two primer sets to generate amplification products; (c) if the at least two target nucleic acids of HDV are present in the sample, performing a hybridization step comprising contacting the amplification products with at least two probes; (d) performing a detection step, comprising detecting the presence or absence of said amplification products, wherein the presence of one of said amplification products indicates the presence of HDV in said sample, and the absence of said amplification product indicates the absence of HDV in said sample; where The at least two primer sets and the at least two probes (i) a first primer set comprising at least one forward primer comprising a nucleic acid sequence of SEQ ID NO: 1-4 or any combination of SEQ ID NO: 1-4 and at least one reverse primer comprising a nucleic acid sequence of SEQ ID NO: 5-6 or a combination thereof, and a first probe or a first probe set comprising a nucleic acid sequence of SEQ ID NO: 7-8 or a complement thereof, or a combination of SEQ ID NO: 7-8 or a complement thereof; (ii) a second primer set comprising at least one forward primer comprising a nucleic acid sequence of SEQ ID NO: 9-10 or a combination thereof and at least one reverse primer comprising a nucleic acid sequence of SEQ ID NO: 11-12 or a combination thereof, and a second probe or a second probe set comprising a nucleic acid sequence of SEQ ID NO: 13-15 or a complement thereof, or any combination of SEQ ID NO: 13-15 or a complement thereof; 1. A method for detecting at least two target nucleic acids of Hepatitis Delta Virus (HDV) in a sample, comprising:

2. the hybridizing step comprises contacting the amplification product with two or more probes, each labeled with a donor fluorescent moiety and a corresponding acceptor moiety; 2. The method of claim 1, wherein the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, the presence or absence of fluorescence indicating the presence or absence of HDV in the sample.

3. The method of claim 1 or 2, wherein the amplifying step uses a polymerase enzyme having 5'-3' nuclease activity.

4. The method of claim 1 , wherein the sample is a biological sample.

5. The method of claim 4, wherein the biological sample is blood, plasma or serum.

6. 2. The method of claim 1, wherein the first primer set generates one or more amplification products of a first target nucleic acid that is detected by the first probe or the first probe set, and / or the second primer set generates one or more amplification products of a second target nucleic acid that is detected by the second probe or the second probe set.

7. 1. A method for detecting Hepatitis D Virus (HDV) in a sample, the method comprising: (a) performing an amplifying step, the amplifying step comprising contacting the sample with one or more forward primers and one or more reverse primers specific for a HDV ribozyme domain to generate an amplification product of the ribozyme domain if HDV is present in the sample, and contacting the sample with one or more forward primers and one or more reverse primers specific for a HDV hepatitis delta antigen (HDAg) gene to generate an amplification product of the HDAg gene if HDV is present in the sample; (b) performing a hybridizing step, the hybridizing step comprising contacting the ribozyme domain amplification product with one or more detectable probes specific for the ribozyme domain, and contacting the HDAg gene amplification product with one or more detectable probes specific for the HDAg gene; (c) detecting the presence or absence of the ribozyme domain amplification product and / or the HDAg gene amplification product, wherein the presence of either or both of the ribozyme domain amplification product or the HDAg amplification product indicates the presence of HDV in the sample, and the absence of both the ribozyme domain amplification product and the HDAg amplification product indicates the absence of HDV in the sample; and where the one or more ribozyme domain forward primers comprise a nucleotide sequence selected from SEQ ID NOs: 1-4, or any combination of SEQ ID NOs: 1-4; the one or more ribozyme domain reverse primers comprise a nucleotide sequence selected from SEQ ID NOs: 5-6, or a combination thereof; and the one or more detectable ribozyme domain probes comprise a nucleotide sequence selected from SEQ ID NOs: 7-8, or a complement thereof, or a combination of SEQ ID NOs: 7-8, or a complement thereof; A method for detecting Hepatitis D virus (HDV) in a sample, wherein the one or more HDAg gene forward primers comprise a nucleotide sequence selected from SEQ ID NOs: 9-10 or a combination thereof, the one or more HDAg gene reverse primers comprise a nucleotide sequence selected from SEQ ID NOs: 11-12, or a combination thereof, and the one or more detectable HDAg gene probes comprise a nucleotide sequence selected from SEQ ID NOs: 13-15 or a complement thereof, or any combination of SEQ ID NOs: 13-15 or a complement thereof.

8. The method of claim 7, wherein the one or more ribozyme domain forward primers comprise all four nucleotide sequences of SEQ ID NOs: 1-4, the one or more ribozyme domain reverse primers comprise both nucleotide sequences of SEQ ID NOs: 5-6, and the one or more detectable ribozyme domain probes comprise both nucleotide sequences of SEQ ID NOs: 7-8 or complements thereof.

9. The method of claim 7 or 8, wherein the one or more HDAg gene forward primers comprise the nucleotide sequences of both SEQ ID NOs: 9-10, the one or more HDAg gene reverse primers comprise the nucleotide sequences of both SEQ ID NOs: 11-12, and the one or more detectable HDAg gene probes comprise all three nucleotide sequences of SEQ ID NOs: 13-15 or complements thereof.

10. the hybridizing step comprises contacting the amplification products with detectable probes, each labeled with a donor fluorescent moiety and a corresponding acceptor moiety; 8. The method of claim 7, wherein the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the detectable probe, and the presence or absence of fluorescence indicates the presence or absence of HDV in the sample.

11. The method of claim 7, wherein the amplifying step uses a polymerase enzyme having 5'-3' nuclease activity.

12. The method of claim 7, wherein the sample is a biological sample.

13. 13. The method of claim 12, wherein the biological sample is blood, plasma or serum.

14. 1. A kit for detecting a first target nucleic acid of HDV and a second target nucleic acid of HDV in a sample, the kit comprising an amplification reagent, the amplification reagent comprising: (a) a DNA polymerase having 5'-3' nuclease activity; (b) a nucleotide monomer; and (c) a first primer set and a first probe or a first probe set for detecting the first target nucleic acid of HDV, The first primer set and the first probe or the first probe set include at least a forward primer including a nucleic acid sequence of SEQ ID NO: 1 to 4 and any combination of SEQ ID NO: 1 to 4, and at least a reverse primer including a nucleic acid sequence of SEQ ID NO: 5 to 6 or a combination thereof, and the first probe or the first probe set includes a nucleic acid sequence of SEQ ID NO: 7 to 8 or a complement thereof, or a combination of SEQ ID NO: 7 to 8 or a complement thereof; a first primer set and a first probe or a first probe set for detecting the first target nucleic acid of HDV; (d) a second primer set and a second probe or a second probe set for detecting the second target nucleic acid of HDV, The second primer set and the second probe or the second probe set include at least a forward primer including a nucleic acid sequence of SEQ ID NO: 9 to 10 or a combination thereof, and at least a reverse primer including a nucleic acid sequence of SEQ ID NO: 11 to 12 or a combination thereof, and the second probe or the second probe set includes a nucleic acid sequence of SEQ ID NO: 13 to 15 or a complement thereof, or any combination of SEQ ID NO: 13 to 15 or a complement thereof; a second primer set and a second probe or a second probe set for detecting the second target nucleic acid of HDV; 23. A kit for detecting a first target nucleic acid of HDV and a second target nucleic acid of HDV in a sample, comprising:

15. 15. The kit of claim 14, wherein the first and second probes and the first and second probe sets are labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

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