EP2971188A1 - Selektiver nachweis von hepatitis a-, b-, c-, d- oder e-viren oder kombinationen davon - Google Patents

Selektiver nachweis von hepatitis a-, b-, c-, d- oder e-viren oder kombinationen davon

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Publication number
EP2971188A1
EP2971188A1 EP14724860.3A EP14724860A EP2971188A1 EP 2971188 A1 EP2971188 A1 EP 2971188A1 EP 14724860 A EP14724860 A EP 14724860A EP 2971188 A1 EP2971188 A1 EP 2971188A1
Authority
EP
European Patent Office
Prior art keywords
seq
hdv
sequence
oligonucleotide
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP14724860.3A
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English (en)
French (fr)
Inventor
Maja KODANI
Tonya MIXSON-HAYDEN
Saleem Kamili
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US Department of Health and Human Services
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US Department of Health and Human Services
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Publication of EP2971188A1 publication Critical patent/EP2971188A1/de
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • This invention relates generally to processes for detection of vims in fluid samples.
  • the instant invention relates to selective detection of hepatitis D virus (HDV) in biological or other fluid media.
  • the invention relates to selective and multiplex detection of hepatitis A, B, C, D, E, or any combination thereof. Processes are described for rapid and sensitive detection of hepatitis viruses in human and animal biological samples and quantification thereof. Diagnostic kits are provided for detection of one or more hepatitis viruses in a clinical, laboratory, or field setting.
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • HDV hepatitis delta virus
  • HAV hepatitis E virus
  • HDV is responsible for severe acute and chronic hepatitis in patients previousl or co-infected with HBV
  • Treatment relies on long-term administration of high-doses of alpha- interferon (IF ) and its efficacy is usually followed by the detection of specific anti-HDV IgM or HDV genome in serum.
  • IF alpha- interferon
  • Individuals with concurrent acute HBV and HDV infection are considered co-infected, whereas patients who are chronically infected with HBV may develop a superinfection with HDV, Co-infection or superinfection with HD can cause more severe hepatitis than HBV infection alone, including more rapid progression to liver disease and higher mortality rates. Due to this risk, it is important for patients who are HBsAg positive to be tested for HDV infection.
  • the small hepatitis D antigen (S-HDAg) is 195 amino acids long (24 kDa), while large hepatitis D antigen (L-HDAg) is 19 amino acids longer on the carboxyl-terminal end (27 kDa), consisting of a stretch of variable, yet genotype-specific, membrane-attaching sequence and serves as the virion assembly signal (Lai 2005). Assembly of the viral particle requires both viral proteins to form the ribonucleoprotein. in the course of viral replication, the ribonucleoprotein buds through the hepatocyte endoplasmic reticulum, and acquires an envelope in which the hepatitis B surface antigens (HBsAg) are embedded.
  • S-HDAg small hepatitis D antigen
  • L-HDAg large hepatitis D antigen
  • Assembly of the viral particle requires both viral proteins to form the ribonucleoprotein. in the course of viral replication, the ribonucleoprotein buds through the hepat
  • Genotype 1 is prevalent world-wide with other genotypes being endemic to different parts 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.
  • N AT Nucleic acid testing
  • FDA Food and Drug Administration
  • NAT -based multiplex assay that can detect all five hepatitis virus in the same format and under identical experimental conditions has not been developed.
  • Reasons for this include the lack of a sufficient detection system specific of HDV as well as the inability to incorporate detection systems for the previously detectable viruses into a single assay suitable for multiplex and simultaneous detection.
  • a first object of the invention is to provide a process for the rapid and specific detection of HDV in simple or complex sample, Processes that address this object include producing an amplification product by amplif ing a hepatitis virus, optionally HDV nucleotide sequence using a forward primer that interacts with an HDV genome at positions 816-834, and a reverse primer, under conditions suitable for a polymerase chain reaction; and measuring the amplification product to defect HDV in said biological sample.
  • the forward primer includes at least two degenerate nucleotide positions.
  • a forward primer consists of the sequence of SEQ ID NO: 1.
  • the reverse primer binds a region of a HDV genome between nucleotides 894 to 908,
  • the reverse primer consists of the sequence of SEQ ID NO: 2.
  • a process optionally used a probe that will bind a region of a HDV genome in the region of nucleotides 853 to 890.
  • a probe optionally includes at least two positions of nucleotide degeneracy.
  • a probe consists of the nucleotide sequence of SEQ ID NO: 3.
  • the use of a probe optionally includes the step of detecting a first detection signal from the probe hybridized to the amplification product.
  • any of the above processes in any combination is used to diagnose HDV infection in a subject.
  • the processes of detecting HDV optionally further include comparing the first detection signal to a second detection signal, where the second detection signal results from detection of a complementary amplification product produced from a sequence of HDV.
  • the second detection signal is generated in parallel with said first detection signal.
  • the first detection signal is compared to a third detection signal from a nucleic acid calibrator extracted in parallel to the biological sample,
  • the nucleic acid calibrator comprises a known amount of HDV and a known amount of a medium similar to the biological sample,
  • the process of any of the above or any combination of the above elements optionally also includes producing a second amplification product by amplifying a nucleotide sequence from, one or more viruses selected from the group consisting of HAV, HBV, HCV, and HEV; and measuring said second amplification product to detect said virus in said biological sample.
  • the one or more viruses is or includes HAV and said step of producing is using one or more nucleotides selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.
  • the one or more viruses is or includes HBV and said step of producing is using one or more nucleotides selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21 .
  • the one or more viruses is or includes HCV and said step of producing is using one or more nucleotides selected from the group consisting of SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.
  • the one or more viruses is or includes HEV and said step of producing is using one or more nucleotides selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27.
  • the hepatitis virus is one or more of the viruses selected from the group consisting of HAV, HBV, HCV, HDV, and HEV.
  • the process detects the presence or absence of HAV, HBV, HCV, HDV, and HDV, wherein the reaction conditions for said step of producing HAV, HBV, HCV, HDV, and HDV amplification products are identical.
  • a forward primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, and combinations of said primers.
  • a reverse primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, and combinations of said primers.
  • said step of measuring is using a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 21 , SEQ ID NO: 24, SEQ ID NO: 27, and combinations of said probes.
  • the detection of one or more viruses in a biological sample optionally diagnoses hepatitis virus infection in a subject.
  • Another object of the invention is to provide a kit for the detection of one or more hepatitis viruses in a biological sample.
  • a kit optionally includes materials suitable for the detection of one or more of HAV, HBV, HCV, HDV, HEV, or any combination thereof.
  • a kit optionally includes a first forward primer, said first forward primer optionally interacts with an HDV genome at positions 816-834,
  • a first forward primer optionally includes at least two degenerate nucleotides.
  • a forward primer consists of the sequence of SEQ ID NO: 1, SEQ ID NO: 16, SEQ ) ID NO: 19, SEQ ID NO: 22, or SEQ ID NO: 25, or combinations of said forward primers are included.
  • a kit optionally includes a reverse primer.
  • a reverse primer optionally binds a region of a HDV genome between nucleotides 894 to 908.
  • a reverse primer optionally consists of the sequence of SEQ ID NOs: 2, 4, 5, 17, 20, 23, 26 or a combination of primers having at least one of SEQ ID NOs. 2, 4, 5, 17, 20, 23, or 26.
  • a kit optionally further or on its own includes a probe that will bind a region of a HDV genome in the region of nucleotides 853 to 890.
  • a probe optionally comprises the nucleotide sequence SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, or the kit optionally includes any combination of said probes.
  • a kit includes one or more oligonucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or combinations thereof.
  • compositions suitable for the detection of the presence or absence of one or more hepatitis viruses in a sample includes an oligonucleotide that comprises or consists of the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 27.
  • FIG. 1 represents an alignment of the qRT-PCR ampiicon region of HDV using sequences representing ail HDV genotypes
  • FIG. 2 represents HDV qRT-PCR Assay Characteristics where positive control transcript preparation was prepared in 10-fold serial dilutions and A) is the linearity of the assay spans six logs and B) is the standard curve shows the efficiency of over 90%, with the slope of -3.65 and the R 2 value of 0.9907; and
  • FIG. 3 illustrates a Taq an Array Card (TAC) workflow compared to the individual real-time PCR samples for 384 reactions, the number equivalent to one card.
  • TAC Taq an Array Card
  • the above difficulties in detection and quantification of hepatitis viruses in complex biological samples such at plasma, serum, or other are addressed by the present processes and materials.
  • the invention has utility to detect the presence or absence of HDV in a biological sample either alone or in concert with additional methods for detection of other hepatitis viruses including HAV, HBV, HCV, HEV, and new viruses to be discovered. Also provided are materials and processes for the simultaneous detection of HAV, HBV, HCV, HDV, and HEV.
  • the term "variant” defines either a naturally occurring genetic mutant of HDV or a recombinantly prepared variation of HDV, each of which contain one or more mutations in its genome compared to the HDV of HDV genotype 1 (accession no. AF098261).
  • the term “variant” may also refer to either a naturally occurring variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion.
  • analog in the context of a non-pro teinaceous analog defines a second organic or inorganic molecule that possesses a similar or identical function as a first organic or inorganic molecule and is structurally similar to the first organic or inorganic molecule.
  • Structural similarit includes the presence of one or more degenerate nucleotides, one or more nucleotides on either or both ends of the molecule, the presence of a locked nucleic acid, or other similarity.
  • the term "derivative" in the context of a non-proteinaceous derivative defines a second organic or inorganic molecule that is formed based upon the structure of a first organic or inorganic molecule.
  • a derivative of an organic molecule includes, but is not limited to, a molecule modified, e.g., by the addition or deletion of a hydroxy., methyl, ethyl, carboxyl or amine group.
  • An organic molecule may also be esterified, alkylated and/or phosphory!ated.
  • a derivative also defined as a degenerate base mimicking a C/T mix such as that from Glen Research Corporation, Sterling, VA, illustratively LNA-dA or LNA-dT, or other nucleotide modification known in the art or otherwise.
  • mutant defines the presence of mutations in the nucleotide sequence of an organism as compared to a wild-type organism
  • a "purified" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule and is often substantially free of other cellular material, or culture medium such as when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemicall synthesized. I his term is exclusive of a nucleic acid that is a member of a library that has not been purified away from other library clones containing other nucleic acid molecules.
  • hybridizes under stringent conditions describes conditions for hybridization and washing under which nucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to each other typically remain hybridized to each other.
  • hybridization conditions are described in, for example but not limited to, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6.; Basic Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986), pp.
  • a preferred, non-limiting example of stringent hybridization conditions is hybridization in 6x sodium chloride/sodium citrate (SSC), 0.5% SDS at about 68°C followed by one or more washes in 2xSSC, 0.5% SDS at room temperature.
  • Another preferred, non-limiting example of stringent hybridization conditions is hybridization in 6x SSC at about 45°C followed by one or more washes in 0,2x SSC, 0.1% SDS at 50 to 65°C,
  • nucleotide or oligonucleotide sequence is substantiaily free of cellular material or other contaminating proteins from the cell or tissue source from which the nucleotide is derived, or is substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a nucleotide/oligonucleotide in which the nucleotide/oligonucleotide is separated from cellular components of the cells from which it is isola ted or produced.
  • nucleotide/oligonucleotide that is substantiaily free of cellular material includes preparations of the nucleotide having less than about 30%, 20%, 10%, 5%, 2.5%, or 1 % (by dry weight) of contaminating material.
  • nucleotide/oligonucleotide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • nucleotide/oligonucleotide have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the nucleotide/oligonucleotide of interest,
  • the nucleotide/oligonucleotide are isolated or purified.
  • isolated vims or virus-like particle is one which is separated from other organisms which are present in the natural source of the virus, e.g., biological material such as cells, blood, serum, plasma, saliva, urine, stool, sputum, nasopharyngeal aspirates, and so forth.
  • biological material such as cells, blood, serum, plasma, saliva, urine, stool, sputum, nasopharyngeal aspirates, and so forth.
  • the isolated virus or VLP can be used to infect a subject cell,
  • biological sample is defined as sample obtained from a biological organism, a tissue, ceil, cell culture medium, or any medium suitable for mimicking biological conditions, or from the environment.
  • Non-limiting examples include, saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, synovial fluid, blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, seminal fluid, mammary secretions, and vitreai fluid, and nasal secretions, throat or nasal materials
  • viral agents are contained in serum, whole blood, liver tissue, or other biological material believed to have HDV present or absent.
  • the term "medium” refers to any liquid or fluid biological sample in the presence or absence of virus.
  • Non-limiting examples include buffered saline solution, cell culture medium, acetonitrile, trifluoroacetic acid, combinations thereof, or any other fluid recognized in the art as suitable for combination with virus or cells, or for dilution of a biological sample or amplification product for analysis,
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence),
  • the nucleotides at corresponding nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are the same length.
  • the determination of percent identity between two sequences can also be accomplished using a mathema tical algorithm.
  • a preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Kariin and Altsch l, 1990, PNAS 87:2264 2268, modified as in Kariin and Altschul, 1993, PNAS, 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol 215:403.
  • Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402.
  • PSI BLAST is used to perform an iterated search which detects distant relationships between molecules (Id.).
  • BLAST Gapped BLAST
  • PSI Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBI website).
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABI ' OS 4: 11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.
  • the MEGA5 software is used to align sequences. Tamura K, et al, Molecular Biology and Evolution, 201 1 ; 28:2731-2739.
  • inventive processes involve the use of real-time polymerase chain reactions (qRT-PCR).
  • qRT-PCR is an accurate and sensitive assay of quantifying viral genomes, with the major advantage of avoiding post-PCR handling that can be source of DNA carryover.
  • primers and probes for HDV detection are incapable of use in a multiplex or simultaneous reaction with other primers and probes for detection of other hepatitis virus types.
  • These problems are fully addressed by the presently provided primers and probes that: 1) do not suffer from negative secondary structure formation; 2) are used for PAN detection of all HDV genotypes; 3) are suitably matched in size, gc content, and melting temperature to be used as a component of a broader assay to detect all five hepatitis viral types; and 4) and are readily tailorabie to additional detection of future HDV types.
  • the primers and probes also are capable of working with other assays for hepatitis virus infection due to their ability to function with samples extracted with a common extraction technique, function with the same PGR chemistry; and function under common cycling parameters. While HBV is a DNA virus, the rest are RNA viruses, so a universal nucleic acid extraction needs to be performed in order to amplify any of them from an infected individual
  • the primers and probes of the invention are specifically identified and produced to satisfy all these needs.
  • Some embodiments of the instant inventive process provide rapid, specific, and sensitive assay process for detection of HDV in biological samples by amplifying one or more nucleotide sequences with greater specificity to strains of HDV than HCV or other viral agents.
  • a process optionally involves qRT-PCR methods.
  • General principles of real time quantitative RT PGR are known in the Art and are for instance described in Poitras et al.. Reviews in Biology and Biotechnology, 2002; 2: 1 -1 1 and Gibson et al ., Genome Research, 1996; 6: 995-1001.
  • Real-time PG based on the TaqMan® technology allows DNA or cDNA quantification over a large dynamic range (10 to 10 7 copies), and is therefore well adapted to the quantification of viral genomes.
  • the possibility of handling numerous samples and the absence of post-PC R handling makes it a safe and convenient approach for clinical diagnosis.
  • An oligonucleotide forward primer with a nucleotide sequence complementary to a unique sequence in an HDV nucleotide sequence illustratively in the region upstream from the HDAg coding region is hybridized to its complementary sequence and extended.
  • a reverse oligonucleotide primer complementary to a second strand of HDV nucleotide sequence in the same or an a!teniate HDV region is hybridized and extended. This system allows for amplification of specific gene sequences and is suitable for simultaneous or sequential detection systems.
  • the present invention relates to the use of the sequence information of HDV for diagnostic process.
  • the present invention provides a process for detecting the presence or absence of nucleic acid molecules of HDV, natural or artificial variants, analogs, or derivatives thereof, in a biological sample.
  • the process involves obtaining a biological sample from various sources and contacting the sample with a compound or an agent capable of detecting a nucleic acid sequence of HDV, natural or artificial variants, analogs, or derivatives thereof, such that the presence of HDV, natural or artificial variants, analogs, or derivatives thereof, is detected in the sample.
  • the presence of HDV, natural or artificial variants, analogs, or derivatives thereof * is detected in the sample by a quantitative real time polymerase chain reaction (qRT-PCR) using the primers that are constructed based on a partial nucleotide sequence of a fit of all known HDV vims genome sequences.
  • qRT-PCR quantitative real time polymerase chain reaction
  • an amplicon is ideally less than 150 nucleotides, optionally from 75 to 150 nucleotides or any value or range therebetween. In some embodiments, an amplicon should not exceed 150 nucleotides.
  • a forward primer should be no longer than 19 nucleotides and be used to bind in the region of the HDV in nucleotides 816 to 834 using the numbering of Wang, et a!., Nature, 1986; 323: 508-514; corrigendum 328:456 and as found in the NCBI database at GenBank accession number X04451.1.
  • a forward primer for HDV detection should include at least two degenerate nucleotide positions, but may include more than 2 such as 3, 4, or 5.
  • a reverse primer for detection of HDV optionally has 32 nucleotides or fewer and optionally includes a core region that is suitable to bind HDV in the region of nucleotides 894 to 908 using the numbering of Wang, et al., Nature, 1986; 323: 508-514; corrigendum 328:456 and as found in the NCBI database at GenBank accession number X044 1.1 , optionally without a mutation or degeneracy. No extensions are permissible beyond the 5 ' end of a primer sequence including nucleotides TCCTC at the 5' end.
  • A. probe for detection of HDV optionall extends between nucleotides 853 and 890 using the numbering of Wang, et al, Nature, 1986; 323: 508-514; corrigendum 328:456 and as found in the NCBI database at GenBank accession number X04451.1.
  • a probe optionally includes at least a core sequence of nucleotides 858-876 and include at least two degenerate nucleotides within those positions.
  • a forward primer to be used in a qRT-PCR. process includes or consists of 5'- TCTCCCTTWGCCATCMG G -3' (SEQ ID NO: 1) where W represents A. or T, and M represents A or C,
  • a reverse primer optionally includes or consists of 5'- TCCTCTTCGGGTCGG -3' (SEQ ID NO: 2),
  • the primers used in the process are SEQ ID NOS: 1 and 2.
  • An agent for detecting HDV nucleic acid sequences is a labeled nucleic acid probe capable of hybridizing to an amplification product produced by a forward and reverse primer pair.
  • the nucleic acid probe is a nucleic acid molecule comprising or consisting of the nucleic acid sequence of 5 '-CYCGCGGTCCGWCCTGGGC-3 ' (SEQ ID NO: 3), where Y represents C or T, and W represents A or T.
  • a nucleotide primer or probe optionally includes one or more locked nucleic acids.
  • LNA denotes a locked nucleic acid. LNAs were first detailed in Koshkin et al., Tetrahedron 54, 3607-3630 (1998).
  • a simultaneous or sequential detection of the presence or absence of nucleic acid molecules of HAV, HBV, HCV, HDV, and HEV individually or in any combination, natural or artificial variants, analogs, or derivatives thereof, in a biological sample involves obtaining a biological sample from various sources and contacting the sample with a compound or an agent capable of detecting a nucleic acid sequence of HAV, HBV, HCV, HDV, or HEV, natural or artificial variants, analogs, or derivatives thereof, such that the presence of HAV, HBV, HCV, HDV, or HEV, natural or artificial variants, analogs, or derivatives thereof, is detected in the sample.
  • the presence of HAV, HBV, HCV, HDV, or HEV, natural or artificial variants, analogs, or derivatives thereof is detected in the sample by a quantitative real time polymerase chain reaction (qRT-PCR) optionally using the primers that are constructed based on a partial nucleotide sequence of a fit of ail known HDV vims genome sequences.
  • qRT-PCR quantitative real time polymerase chain reaction
  • a process is used to detect the presence or absence of HDV and HAV.
  • a process is used to detect the presence or absence of HDV and HBV.
  • a process is used to detect the presence or absence of HDV and HCV.
  • a process is used to detect the presence or absence of HDV and HEV.
  • a process is used to detect the presence or absence of HAV and HBV.
  • a process is used to detect the presence or absence of HAV and HCV.
  • a process is used to detect the presence or absence of HAV and HEV.
  • a process is used to detect the presence or absence of HBV and HCV.
  • a process is used to detect the presence or absence of HBV and HEV.
  • a process is used to detect the presence or absence of HCV and HEV.
  • a process is used to detect the presence or absence of three of HAV, HBV, HCV, HDV, or HEV in any combination.
  • a process is used to detect the presence or absence of four of HAV, HBV, HCV, HDV, or HEV.
  • a process is used to detect the presence or absence of ail of HAV, HBV, HCV, HDV, and HEV.
  • the processes of the present invention can involve a real-time quantitative PGR assay.
  • the quantitative PCR used in the present invention is a TaqMan assay (Holland et al., PNAS 88(16): 7276 (1991)). It is appreciated that the current invention is amenable to performance on other RT-PCR systems and protocols that use alternative reagents illustratively including, but not limited to Molecular Beacons probes, Scorpion probes, multiple reporters for multiplex PCR, combinations thereof, or other DNA detection systems.
  • the assays are performed on an instrument designed to perform such assays, for example those available from Applied Biosystems (Foster City, CA).
  • the present invention provides a real-time quantitative PCR assay to detect the presence of any species of hepatitis virus selected from HAV, HBV, HCV, HDV, or HEV, natural or artificial variants, analogs, derivatives thereof, or any combination thereof, in a biological sample by subjecting the hepatitis virus nucleic acid from the sample to PCR reactions using specific primers, and detecting the amplified product using a probe.
  • HXV HAV; HBV; HCV; HDV; HEV; HDV and
  • the probe is a TaqMan probe which consists of an oligonucleotide with a 5'-reporter dye and a 3 '-quencher dye.
  • a fluorescent reporter dye such as FAM dye (illustratively 6-car oxyfluorescein)
  • FAM dye is covalently linked to the 5' end of the oligonucleotide probe.
  • Other dyes illustratively include TAMRA, AlexaFluor dyes such as AlexaFluor 495 or 590, Cascade Blue, Marina Blue, Pacific Blue, Oregon Green, Rhodamine, Fluorescein, TET, HEX, Cy5, Cy3, and Tetramethylrhodamine.
  • Each of the reporters is quenched by a dye at the 3' end or other non-fluorescent quencher.
  • Quenching molecules are suitably matched to the fluorescence maximum of the dye.
  • Any suitable fluorescent probe for use in real-time PGR (RT-PCR) detection systems is illustratively operable in the instant invention.
  • any quenching molecule for use in RT-PCR systems is illustratively operable.
  • a 6-carboxyfluorescein reporter dye is present at the 5 '-end and matched to Black Hole Quencher (BHQl, Biosearch Technologies, Inc., Novate, CA). The fluorescence signals from these reactions are captured at the end of extensi on steps as PCR product is generated over a range of the thermal cycles, thereby allowing the quantitative determination of the viral load in the sample based on an amplification plot.
  • the HXV virus nucleic acid sequences are optionally amplified before being detected.
  • amplified defines the process of making multiple copies of the nucleic acid in either RNA or cDNA form from a single or lower copy number of nucleic acid sequence molecule.
  • the amplification of nucleic acid sequences is carried out in vitro by biochemical processes known to those of skill in the art.
  • the amplification agent may be any compound or system that will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, E. coll DNA polymerase ⁇ , Taq polymerase, Klenow fragment of E.
  • c li DNA polymerase I T4 DNA polymerase, AmpliTaq Gold DNA Polymerase from Applied Biosystems, other available DNA polymerases, reverse transcriptase (preferably iScript RNase H+ reverse transcriptase), ligase, and other enzymes, including heat-stable enzymes (i.e., those enzymes that perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation).
  • reverse transcriptase preferably iScript RNase H+ reverse transcriptase
  • ligase i.e., those enzymes that perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation.
  • heat-stable enzymes i.e., those enzymes that perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation.
  • the AgPath-ID One Step Kit by Applied Biosystems is used, Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each mutant nucleotide
  • the synthesis is initiated at the 3 '-end of each primer and proceed in the S'-direction along the template strand, until synthesis terminates, producing molecules of different lengths.
  • One process of in vitro amplification may include the polymerase chain reaction (PCR) such as those described in U.S. Patent Nos, 4,683,202 and 4,683, 195.
  • PCR polymerase chain reaction
  • the term "polymerase chain reaction” refers to a process for amplifying a DNA base sequence using a heat-stable DNA polymerase and two oligonucleotide primers, one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (-)-strand at the other end.
  • RNA is subjected to cycling parameters in a thermocycler optionally as follows: 45°C for 10 rnin, 94°C for 10 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 1 rnin.
  • RNA is subjected to a polymerase chain in a thermocycler using parameters of denaturing at 94°C for 10 rnin followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 1 min with a final extension step of 72°C for 10 min at a denaturing temperature of 95°C for 30 sec, followed by varying annealing temperatures ranging from 54 to 58°C for 1 min, an extension step at 70°C for 1 min, with a final extension step at 70°C for 5 min.
  • the primers for use in amplifying the mRNA or genomic RNA of HXV may be prepared using any suitable process, such as conventional phosphotriester and phosphodiester processes or automa ted embodiments thereof so long as th e primers are capable of hybridizing to the nucleic acid sequences of interest,
  • Any suitable process for synthesizing oligonucleotides on a modified solid support is described in U.S. Patent No. 4,458,066.
  • the exact length of primer will depend on man factors, including desired melting temperature, buffer, and nucleotide composition.
  • the primer must prime the synthesis of extension products in the presence of the inducing agent for amplification.
  • Primers used according to the process of the invention are complementary to each strand of nucleotide sequence to be amplified.
  • the term "complementary" means that the primers must hybridize with their respective strands under conditions that allow the agent for polymerization to function.
  • the primers that are complementary to the flanking sequences hybridize with the flanking sequences and permit amplification of the nucleotide sequence.
  • the 3' terminus of the primer that is extended is perfectly base paired with the complementary flanking strand, in some embodiments, probes possess nucleotide sequences complementary to one or more strands of the 5 '-NCR of HDV.
  • one or more primers are complementary to HDV genetic sequences encompassing positions 800-910,
  • primers are used in pairs wherein a pair contain the nucleotide sequences of SEQ ID NOs: 1 and 2; 16 and 17; 19 and 20; 22 and 23; 25 and 26; or any combination of the primer pairs.
  • the complement of SEQ ID NOs: 1 , 2, 16, 17, 19, 20, 22, 23, 25, or 26 are similarly suitable for use in the instant invention.
  • oligonucleotide sequences that hybridize with SEQ ID NO; 1, 2, 16, 17, 19, 20, 22, 23, 25, or 26 are also similarly suitable.
  • the primers used in the present invention are highly restricted both positionaily and compositionally so as to provide the proper melting temperature, ability to recognize all known HDV genotypes or all of HAV, HBV, HCV, HDV, and HEV, refrain from formation of secondary structure, and have a specific ability to bind with sufficient hybridization to the genome so as to be useful in the invention,
  • nucleic acid sequences detected in the process of the invention are optionally further evaluated, detected, cloned, sequenced, and the Like, either in solution or after binding to a solid support, by any process usually applied to the detection of a specific nucleic acid sequence such as another polymerase chain reaction, oligomer restriction (Saiki et al., BioTechnology 3:1008 1012 (1985)), allele-specific oligonucleotide (ASO) probe analysis (Conner et al., PNAS 80: 278 (1983)), oligonucleotide ligation assays (OLAs) (Landegren et al., Science 241:1077 ( 1988)), R ase Protection Assay and the like.
  • oligomer restriction Saiki et al., BioTechnology 3:1008 1012 (1985)
  • ASO allele-specific oligonucleotide
  • OVAs oligonucleotide ligation assays
  • the reaction product may be detected by Southern blot analysis, without using radioactive probes.
  • a small sample of DNA containing the nucleic acid sequence obtained from the tissue or subject is amplified, and analyzed via a Southern blotting technique.
  • the use of non-radioactive probes or labels is facilitated by the high level of the amplified signal.
  • one nucleoside triphosphate is radioactivelv labeled, thereby allowing direct visualization of the amplification product by autoradiography.
  • amplification primers are fluoreseently labeled and run through an electrophoresis system. Visualization of amplified products is by laser detection followed by computer assisted graphic display, without a radioactive signal.
  • labeled with regard to the probe is intended to encompass direct labeling of the probe by coupling (i.e., physically linking) a detectable substance to the probe, as well as mdirect labeling of the probe by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a probe using a fluorescently labeled antibody and end-labeling or centrally labeling of a DNA probe with biotirt such that it can be detected with fluorescently labeled streptavidin.
  • the detection method of the invention can be used to detect RNA (particularly mRNA) or genomic nucleic acid in a sample in vitro as well as in vivo.
  • in vitro techniques for detection of nucleic acid include northern hybridizations, in situ hybridizations, RT- PCR, real-time RT-PCR, and DNase protection.
  • in vivo techniques for detection of HXV include introducing into a subject organism a labeled antibody directed against a capsid or polypeptide component or directed against a particular nucleic acid sequence of HXV,
  • the antibody can be labeled with a radioactive marker whose presence and location in the subject organism can be detected by standard imaging techniques, including autoradiography.
  • HXV is at least 5, and often 10, 15, 20, 25, or 30 nucleotides in length.
  • a primer is 10- 30 nucleotides in length or any value or range therebetween.
  • the GC ratio should be above 30%, 35%, 40%, 45%, 50%, 55%, or 60% so as to prevent hair-pin structure on the primer.
  • the amplicon should be sufficiently long enough to be detected by standard molecular biology methodologies. Although it is not desired in the invention, techniques for modifying the T TO of either primer are operable herein.
  • An illustrative forward primer contains LNA-dA and L A-dT (Glen Research Corporation) so as to match T m with a corresponding alternate primer.
  • An inventive process uses a polymerization reaction which employs a nucleic acid polymerizing enzyme, illustratively a DNA polymerase, RNA polymerase, reverse transcriptase, or mixtures thereof, it is further appreciated that accessory proteins or molecules are present to form the replication machinery.
  • the polymerizing enzyme is a thermostable polymerase or thermodegradable polymerase.
  • thermostable polymerases Use of thermostable polymerases is well known in the art such as Taq polymerase available from Invitrogen Corporation. Thermostable polymerases allow a polymerization reaction to be initiated or shut down by changing the temperature other condition in the reaction mixture without destroying activity of the polymerase.
  • thermostable polymerases illustratively include those isolated from Thermits aquaticus, Thermus thermophihi , Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis and Thermotoga maritima.
  • Themiodegradable polymerases illustratively include E, coii UNA polymerase, the lenow fragment of E. coli DNA polymerase, T4 DNA polymerase, T7 DNA polymerase and other examples known in the art, It is recognized in the art that other polymerizing enzymes are similarly suitable illustratively including E. coli, T7, T3, SP6 RNA polymerases and AMY, M-MLV, and HIV reverse transcriptases.
  • the polymerases are optionally bound to the primer.
  • the HXV is a single- stranded RNA molecule due to heat denaturing the polymerase is bound at the primed end of the single-stranded nucleic acid at a origin of replication.
  • a binding site for a suitable polymerase is optionally created by an accessory protein or by any primed single-stranded nucleic acid.
  • PCR amplification products are generated using complementary forward and reverse oligonucleotide primers.
  • HDV genetic sequences or fragments thereof are amplified by the primer pair SEQ ID NOS: 1 and 2 that amplify a conserved sequence in the HDV 5 '-NCR encompassing nucleotides 816-908.
  • the resulting amplification product is processed and prepared for detection by processes known in the art, It is appreciated that the complements of SEQ ID NOS: 1 and 2 are similarly suitable for use in the instant invention. It is further appreciated that oligonucleotide sequences that hybridize with SEQ ID NO: 1 or 2 are also similarly suitable.
  • multiple amplification products are simultaneously produced in a PCR reaction that is then available for simultaneous detection and quantification.
  • multiple detection signals are inherently produced or emitted that are separately and uniquely detected in one or more detection systems. It is appreciated that multiple detection signals are optionally produced in parallel.
  • a single biological sample is subjected to analysis for the simultaneous or sequential detection of HXV genetic sequences. It is appreciated that three or more independent or overlapping sequences are simultaneousl or sequentiall measured in the instant inventive process.
  • Oligonucleotide matched primers are simultaneously or sequentially added and the biological sample is subjected to proper thermocycling reaction parameters.
  • the processes further involve obtaining a control sample from a control subject, contacting the control sample with a compound or agent capable of detecting the presence of HXV nucleic acid in the sample, and comparing the presence of mRNA or genomic RNA in the control sample with the presence of mRNA or genomic RNA in the test sample.
  • mass spectrometry has several advantages over RT-PCR or qRT-PCR systems in that it similarly can be used to simultaneously detect the presence of HXV and decipher mutations in target nucleic acid sequences allowing identification and monitoring of emerging strains. Further, mass spectrometers are prevalent in the clinical laboratory. Similar to fluorescence based detection systems mass spectrometry is capable of simultaneously detecting multiple amplification products for a multiplexed and controlled approach to accurately quantifying components of biological or environmental samples.
  • Mass spectrometry platforms are suitable for use in the instant invention illustratively including matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI), electrospray mass spectrometry, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-F ' TICR), multi-stage mass spectrometry fragmentation analysis (MS/MS), mass spectrometry coupled with liquid chromatography such as high performance liquid chromatography mass spectrometry (HPLC) and ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/M S/MS), variations thereof, or combinations thereof.
  • MALDI matrix assisted laser desorption ionization time of flight mass spectrometry
  • ESD-F ' TICR electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry
  • MS/MS multi-stage mass spectrometry fragmentation analysis
  • HPLC high performance liquid
  • TaqMan Array Cards (TAC, Life Technologies, Grand Island,
  • TAC TaqMan Low Density Array
  • TAC-ready assays provide a potential for building modular diagnostic platforms for acute and chronic viral hepatitis, jaundice syndromic surveillance, blood-borne pathogens screening of blood or solid organ donors and recipients, and testing at-risk populations, including intravenous drug users and hemodialysis patients.
  • kits for detecting the presence of HXV viral nucleic acids in a test sample includes a labeled compound or agent capable of detecting a nucleic acid molecule in a test sample and, in certain embodiments, for determining the titer in the sample.
  • the kit includes for example: (1) an oligonucleotide, e.g., a detectably labeled probe, which hybridizes to a nucleic acid sequence of the HXV vims and/or (2) a pair of primers (one forward and one reverse) useful for amplifying a nucleic acid molecule containing the HXV viral sequence.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent.
  • the kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples which is assayed and compared to the test sample contained.
  • Each component of the kit is usually enclosed within an individual container and ail of the various containers are usually enclosed within a single package along with instructions for use.
  • a kit optionally includes a primer including a nucleotide sequence of SEQ ID NO: 1, 2, 16, 17, 19, 20, 22, 23, 25, or 26, or combinations of the primers.
  • a kit optionally includes a probe of SEQ I D NO: 3 , 18, 21, 24, or 27, or combinations of the probes.
  • HXV infection in a subject insects, and any inclusive other organism capable of infection or transfection by or with HXV.
  • a purified and titered HXV solution is optionally used as a biological sample.
  • the level of HXV in the unknown biological sample is determined.
  • the purified and titered HXV solution is analyzed in parallel with the unknown biological sample to reduce inter assay error or to serve as a standard curve for quantitation of unknown HXV in the biological sample.
  • Using purified and titered HXV solution provides for a similar complete genetic base RNA strand for amplification.
  • a subgenomic fragment is cloned into a plasmid for amplification, purification, and use as a quantitative comparator or nucleic acid calibrator.
  • a RNA subgenomic fragment of HDV is optionally amplified from a positive plasma sample using primers bracketing the qRT-PCR target regions in the 5 '-NCR of HDV, It is appreciated that other sequences are similarly suitable for use as a quantitative control.
  • the known concentration of the subgenomic fragment is used to create a standard curve for quantitative determinations and to access amplification efficiency.
  • kits for detecting HXV infection that contains reagents for the amplification, or direct detection of HXV or portions thereof.
  • An exemplary kit illustratively includes a forward and reverse primer pair, and a probe.
  • the forward and reverse primers have the oligonucleotide sequence SEQ ID NOS: 1 and 2 and a nondegenerate probe of the sequence SEQ ID NO: 3.
  • a kit includes one or more primers or probes as listed in Table 1A and IB.
  • a diagnostic kit may optionally contain primers and probes that are the complements of SEQ ID NOS: 1-3 or that hybridize with oligonucleotides SEQ ID NOS: 1-3, or a complements or hybridize with, any other probe or primer from Table 1A and IB, It is further appreciated that a diagnostic kit optionally includes ancillary reagents such as buffers, solvents, thermostable polymerases, nucleotides, and other reagents necessary and recognized in the art for amplification and detection of HXV in a biological sample.
  • ancillary reagents such as buffers, solvents, thermostable polymerases, nucleotides, and other reagents necessary and recognized in the art for amplification and detection of HXV in a biological sample.
  • the invention provides a host cell containing a nucleic acid sequences according to the invention as an alternative to synthetic primer sequence generation.
  • Plasmids containing the polymerase components of the HXV virus are generated in prokaryotie ceils for the expression of the components in relevant cell types (bacteria, insect cells, eukaryotic cells).
  • the cell line is a primate ceil line.
  • These cell lines may be cultured and maintained using known cell culture techniques such as described in Cells, Julio, ed., 1994, Cell Biology Laboratory Handbook, Academic Press, NY.
  • Various culturing conditions for these cells including media formulations with regard to specific nutrients, oxygen, tension, carbon dioxide and reduced serum levels, can be selected and optimized by one of skill in the art.
  • the preferred cell line of the present invention is a eukaryotic cell line, preferably an insect cell line, such as Si9 per, transiently or stably capable of expressing one or more full-length or partial HXV proteins.
  • Such cells can be made by transfection (proteins or nucleic acid vectors), infection (viral vectors) or transduction (viral vectors).
  • the cell lines for use in the present invention are cloned using known cell culture techniques familiar to one skilled in the art. The cells are cultured and expanded from a single cell using commercially available culture media under known conditions suitable for propagating cells.
  • a host ceil is a cell derived from a mammal, insect, yeast, bacteria, or any other single or multicellular organism recognized in the art.
  • Host cells are optionally primary cells or immortalized derivative ceils.
  • Immortalized ceils are those which can be maintained in-vitro for several replication passages.
  • an HXV antigen such as an amino acid sequence representative of a capsid protein is used as a control for a PCR based assay for the detection and measurement of the presence of HXV in a biological sample.
  • the process of detecting HXV antibodies in a biological sample is optionally performed in parallel with the same or control biological samples that are used to detect HXV genetic sequences.
  • kits for detection of HXV infection in a patient optionally contains reagents for
  • kits are any of the reagents described abo ve or other necessary and non-necessary reagents known in the art for solubilization, detection, washing, storage, or other need for in a diagnostic assay kit.
  • the provided inventive processes and compositions can be used as a quantitative one-step RT-PCR assay or other assay for HXV RNA detection and quantitation.
  • the processes use a simple one-step qRT-PCR set or reactions that can be run on various real-time PCR instruments from as little as 200 ⁇ of serum. While most published HDV RNA assays target the delta antigen sequence, the inventive assay for HDV targets the adjacent region located upstream from the delta antigen, in view of its higher level of conservation among all eight delta genotypes. Another advantage of our assay is that the inventive positive control transcript controls for every step of the qRT-PCR reaction, including the reverse transcription step. We have also described the preparation of HXV transcripts for use as a positive control. Both the qRT-PCR and transcript preparation are useful for detection and quantitation of HXV RNA for diagnostic and surveillance purposes.
  • Example 1 HDV Primer and probe design.
  • PAN-HDVF and PAN-HDVR and probe PA -HDVP demonstrating significantly better analytical performance (Table 1).
  • These primers amplify a sequence located upstream of the L-HDAg ORF (FIG. 1). All primers and probes were analyzed for melting temperature, primer-dimer formation, self-annealing, and secondary structure formation using the Oligoanalyzer software. All primer and probe sequences were analyzed by BLAST to verify sequence uniqueness of the chosen regions.
  • Example 2 Obtaining viral strains and clinical specimens.
  • the evaluation panel included 132 HBsAg/anti-HDV positive sera previously referred to our laboratory that had been stored at -70°C. These sera originated from the United States, Moldova, Romania and Venezuela. Qualitative confirmation of the HBsAg-positive status was determined using the VITROS ECi Immunodiagnostic System (Ortho-Clinical Diagnostics, Inc., Rochester, NY). Total anti-HDV was detected using the EIA-AB DELTAK-2 Kit (DiaSorin, Stillwater, MN); positive samples were tested for HDV RNA in two independent runs by the qRT-PCR assay.
  • Total nucleic acid was extracted from 200 ⁇ of each serum specimen using the MagNA Pure LC 2.0 Instrument and the MagNA Pure Total Nucleic Acid Isolation Kit (Roche Diagnostics, Indianapolis, IN). All extractions were performed using 200 ⁇ of serum or plasma according to the manufacturer's protocols; TNAs were eluted in 50 ⁇ of the Elution buffer. TNAs were aliquoted and stored at -8Q°C until use.
  • Example 4 One- Step qRT-PCR.
  • qRT-PCR reactions were performed on the ABI 7500 real-time PCR instrument using the AgPath-ID One-Step Kit (Applied Biosystems, Foster City, CA) following the manufacturer- recommended protocols. All reactions were performed in 96-weli plates in 25 ⁇ reaction volume, containing I X RT-PCR enzyme mix, l x RT-PCR buffer, 600 nM each primer and 200nM probe, and 5 ⁇ of TNA extract. The following cycling conditions were used: 45°C for 10 min, 94°C for 10 min, followed by 45 cycles of 95°C for 30 sec and 60°C for 1 min.
  • the primers of SEQ ID Nos: 1 and 2 and the probe of SEQ ID NO:3 were chosen for particular additional exemplary use, Utilizing 600 iiM of each primer and 200 nM of probe in the qRT-PCR reaction was determined to achieve the best detection sensitivity: HDV RNA could be detected down to the 10 "5 dilution of the serum. Specificity was determined using 48 anti-HDV-negative human sera (SeraCare, Milford, MA); HDV RNA was not detected in any of the specimens. When applied to test the HBV DNA Genotype Performance Panel PHD201 (M) (SeraCare), no positive reactions were observed, suggesting no interference from the presence of HBV DNA.
  • M HBV DNA Genotype Performance Panel PHD201
  • Example 5 Detection and Quantitation of HDV RNA in Clinical Samples.
  • Example 1 Of the 132 specimens of Example 1 that tested positive for anti-HDV, 41 (31.06%) were positive for HDV RNA by the TaqMan qRT-PCR assay, We also determined the HDV RNA viral titers in all HDV RNA positive samples. To generate a standard curve, serial dilutions of the transcript preparation were made, ranging from 10 8 to 10 1 transcript copies per reaction. The assay exhibited a 6-log linear range with a lower detection limit of 7.5 x 10 2 copies/ml and an upper detection limit of 7.5 x 10 copies/ml. The slope of -3.65 suggested the qRT-PCR efficiency of over 90% ( Figure 2).
  • a 10-fold dilution series of the positive control transcript was used to determine the HDV RNA titers of the 41 serum specimens that tested positive for HDV RNA.
  • the mean viral titer of the 41 HDV RNA positive sera was 1.3 10 s copies/ml (range 45.2 - 1.79 x 10 9 ).
  • the 257-base pair amplicon used for genotyping is located in the L-HDAg ORF, The first step of the nested PCR reaction was performed using the AgPath-ID buffer as described above and the DESP and DEAP primers. If no amplicon was obtained, the DEAP-alt reverse primer was used in an otherwise identical reaction, The nested reaction was performed using the DISP and DIAP primers and the PerfeCTa i M 8YBR ® Green SuperMix, Low ROX (Quanta Biosciences, Gaithersburg, MD).
  • the cycling conditions for the second PCR reaction using nested primers DISP and DIAP, were as follows: 94°C for 10 min followed by 35 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 1 min with a final extension step of 72°C for 10 min.
  • the amp ikons from the second nested PCR reaction were run on a 2% agarose gel to confirm the amplification status and the size of the amplicon.
  • the nested amplicons were sequenced directly from the second nested PCR reaction using each internal nested primer in a separate reaction for bidirectional sequencing, the BigDye Terminator v3, l Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, and the 3130x1 Genetic Analyzer (Applied Biosystems, Foster City, CA).
  • HDV R A-positive samples yielded HDV-specific sequences and could be genotyped; 15 of 41 belonged to genotype 1 and four belonged to genotype 3.
  • the genotype 3 samples originated from Venezuela.
  • Example 6 Detection of HDV amplicoes via mass spectroscopy.
  • Detection of amplification products obtained as in Example 4 will be performed essentially as described by Blyn, L, et al. J. Clin. Microbiol. 2008; 46(2):644-651. Following amplification each PCR mixture will be desalted and purified using a weak anion-exchange protocol based on the method of Jiang and Hofstadier (Jiang, Y., and S. A. Hofstadler. Anal. Biochem. 2003; 316:50-57). ESI-TOF will be used to obtain accurate-mass ( ⁇ 1 ppm), high-resolution ( ⁇ / ⁇ , >10,000 full width half maximum) mass spectra.
  • a quantitative RT-PCR assay was desiged tor detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA, hereafter referred to as HABCDE-NAs, using TAG compatible chemistry and identical cycling conditions.
  • primers and probes routinely used in our laboratory for nucleic acid testing (NAT) of HAV, HBV, HC V and HEV were transferred into the TAC -compatible buffer system and PCR cyclmg conditions, and compared to the original assay for their performance characteristics.
  • TAC- chemistry compatible assay reagents for detection of HDV RNA are presented in Table 1.
  • Real-time RT-PCR reactions were performed on the ABI 7500 real-time PCR instrument using the AgPath-ID One-Step kit (Life Technologies) following the manufacturer- recommended protocols. All reactions were performed in 96-well plates in 25 ⁇ 1 reaction volume, containing lx RT-PCR enzyme mix, 1 ⁇ RT-PCR buffer, variable concentrations of each primer and probe (Table 1), and 5 ul of TNA extract. The following cycling conditions were used: 45°C for 10 min, 94°C for 10 min, followed by 45 cycles of 95 °C for 30 sec and 60°C for 1 min.
  • HABCDE-NA assays were run on TAC in triplicate wells.
  • two experimental controls were used in duplicate: 1) an internal positive control - IPC2, for checking the card performance and 2) ribo-nuclear protein 3 (RNP3) control, for nucleic acids extraction.
  • RNP3 control ribo-nuclear protein 3
  • Each card has eight different ports, which were utilized to run six specimens in addition to positive and negative controls.
  • A. positive control transcript was developed to encompass detection of HABCDE- NAs as described previously. Briefly, a synthetic gene was designed containing the forward primer sequence, the probe sequence, and the reverse complement of the reverse primer sequence for HAV UNA, HBV D ' NA, HCV R A, HDV RNA, HEV KNA and RNP3 targets respectively, followed by the reverse complement of the SP6 promoter sequence. This compound sequence was synthesized and cloned into pIDTSmart Amp vector (IDT, Coralville, lA). For use as a control, transcripts were generated from the plasmid using the SP6 MEGAscript kit according to the manufacturer's instructions (Life Technologies).
  • the transcript preparation was serially diluted from the 10 s copies/ml dilution in IxTE buffer containing 50 ng ml of carrier RNA, aliquoted and stored at -70°C until use.
  • TAG assays were run on the ViiA7 instrument (Life Technologies) using the AgPath- ID One-Step Kit (Life Technologies). All reactions contained l x RT-PCR enzyme mix, l x RT- PCR buffer, and 40 ⁇ of TNA extract. Each sample was set up in 100 ⁇ final reaction and loaded entirely into the TAG port, The universal workflow for setting up PCR reactions, as described above, was followed with set up of TAG reactions. Upon loading, the cards were centrifuged twice at 336 x g for 1 min each time, sealed and loaded into the thermal cycler. The cycling conditions described above were used to run the cards.
  • the reproducibility of the cards was determined based on the a verage cycle threshold (Ct) values for 31 cards, which included 93 data points for HAV, HBV, HCV, HDV, and HEV NAT assays each, and 62 data points for two controls, RNP3 and IPC2 assays each.
  • the limit of detection of hepatitis TAG assay was determined using serial dilutions of standards for HAV, HBV, HCV, and HEV and the quantified transcript for HDV.
  • the T AG-compatible HAV RNA PCR assay exhibited a slope of -3,36, and a linear range of 5 logs, with the limit of detection of 125 ge/ml.
  • the new HAV RNA assay was further evaluated using 17 HAV RNA positive and 33 negative human sera, previously tested by a nested RT-PCR (Nianan, et ai., Clinical microbiology reviews 19:63-79). Comparing TAC-compatible HAV qRT-PCR to the HAY nested PGR (Id), clinical sensitivity of the HAV qRT-PCR assay was determined to be 100% and the specificity was 88% [95% CI: 77-99].
  • the individual 25 ⁇ assay using primers and probes from Tables 1 A and B, for detection of HBV DNA Using SEQ ID os; 1 , 2, and 3) exhibited a slope of -3.229, and a linear range of 7 togs, with the limit of detection of 100 lU/mt; the individual 25 ⁇ assay for detection of HCV RNA exhibited a slope of -3.327, and a linear range of 7 logs, with the limit of detection of 100 IU/ml; the individual 25 ⁇ assay for detection of HDV RNA exhibited a slope of -3.65, and a linear range of 6 logs, with the limit of detection of 1000 ge/nil; finally, the individual 25 ⁇ assay for detection of HEV RNA exhibited a slope of -3.238, and a linear range of 7 logs, with the limit of detection of 250 IU/ml.
  • the HAV TAC assay was half a logic less sensitive than its individual assay- equivalent; the HBV and HCV TAC assays were one fifth of a logto less sensitive than their individual assay equivalents: the HDV TAC assay was as sensitive as its individual assay equivalent; and the HEV TAC assay was 1 logio less sensitive than its individual assay equivalent.
  • the overall limit of detection was established at 250 ge/ml for HAV TAC, 500 IU/ml for HBV TAC, 500 IU/ml for HCV TAC, 1000 ge/ml for HDV TAC, and 2500 IU/ml for HEV TAC.
  • the HAY TAC exhibited 94% [95% CI: 86-100] sensitivity and 93% [95% CI: 85-100] specificity. Compared to an in-house NAT test for HBV DMA, the HBV TAC exhibited 92% [95% CI: 84-100] sensitivity and 100% specificity.
  • the HCV TAC assay was compared to in vitro diagnostic COBAS platform for HCV RNA detection as the standard, The clinical sensitivity and specificity were 100%.
  • the individual 25 ⁇ assay identical to the TAC assay was used as the standard, since this is the assay developed specifically for HDV RNA as described above, Both clinical specificity and clinical sensitivity for HDV TAC were 100%.
  • the HEV TAC assay was compared to the HEV qRT-PCR assay used in our laboratory for detection of HEV RNA as the standard. This assay shares identical primers and probes with the HEV TAC assay, but they differ in chemistry and PGR running conditions. According to this comparison, the HEV TAC assay exhibited 100% specificity and sensiti vity.
  • the sensiti vity of TAC assays was reasonably high, ranging from 92% (HBV) to 100% (HCV, HDV and HE V).
  • the specificity of the TAC assays was high, ranging from 93% (HAV) to 100% (H BV, HCV, HDV, and HEV) (Table 6).
  • Delta agent association of delta antigen with hepatitis B surface antigen and R A in serum of delta-infected chimpanzees. Proc Natl Acad Sci US A 77, 6124-6128,
  • TaqMan Array Card (TAG) for the simultaneous detection of multiple respiratory viruses in children with acute respiratory infection. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 57:254-260.

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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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EP14724860.3A 2013-03-15 2014-03-14 Selektiver nachweis von hepatitis a-, b-, c-, d- oder e-viren oder kombinationen davon Ceased EP2971188A1 (de)

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