WO2001064959A1 - Detection d'arn du virus de l'hepatite b - Google Patents

Detection d'arn du virus de l'hepatite b Download PDF

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Publication number
WO2001064959A1
WO2001064959A1 PCT/EP2001/002143 EP0102143W WO0164959A1 WO 2001064959 A1 WO2001064959 A1 WO 2001064959A1 EP 0102143 W EP0102143 W EP 0102143W WO 0164959 A1 WO0164959 A1 WO 0164959A1
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hbv
rna
dna
amplification
nucleic acid
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PCT/EP2001/002143
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English (en)
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Jaap Goudsmit
Sol Carl Yates
Maarten Tjerk Penning
Lambertus Henricus Maria Weijer Van De
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Akzo Nobel N.V.
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Priority to AU2001254670A priority Critical patent/AU2001254670A1/en
Publication of WO2001064959A1 publication Critical patent/WO2001064959A1/fr

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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Definitions

  • the present invention is concerned with oligonucleotides that can be used as in the detection of Hepatitis B mRNA. Furthermore a method for the diagnosis of Hepatitis B infection is provided.
  • Hepatitis B virus (HBV) infection in humans is widespread.
  • the hepatitis infection is transmitted by three general mechanisms: (1 ) by parenteral inoculation of infected blood or body fluids, either in large amounts as in blood transfusions or in minute amounts as through an accidental skinprick; (2) by close family or sexual contact; and (3) by some mothers, who transmit the virus to their new-born children.
  • HBV is not highly contagious. Transmission by inhalation occurs rarely, if ever. The transmission route through contaminated blood or blood products is a major threat to the human health.
  • HBV infection elicits a spectrum of disease entities ranging from the most severe form of chronic active hepatitis to less severe chronic persistent hepatitis to the asymptomatic carrier state.
  • An array of diagnostic assays have recently been developed to aid the clinician in differentiating hepatitis B virus infections from other forms of viral hepatitis (i.e., hepatitis A, hepatitis C or hepatitis E).
  • hepatitis A, hepatitis C or hepatitis E forms of viral hepatitis
  • the ability to distinguish between an acute hepatitis B infection and symptomatic chronic hepatitis B infection is still problematic. This is especially true since chronic active hepatitis and chronic persistent hepatitis patients often demonstrate a cyclic pattern of hepatitis characterised by acute exacerbation of liver injury alternating with normal liver function.
  • HBsAg hepatitis B surface antigen
  • HBeAg hepatitis B e antigen
  • the appearance of antibody to HBsAg is usually not observed until approximately two months following disappearance of circulating HBsAg.
  • the viral particles present in the serum are known to shed their surface coat exposing the nucleocapsid, known as the core antigen (HBcAg).
  • Antibody production of HBcAg occurs early in the course of the acute phase of HBV infection and can persist for many years, and chronically infected patients can produce high titers of anti-HBc antibodies.
  • HBsAg is established as the most important marker of acute or chronic hepatitis B infection, detectable in serum of infected individuals. HBsAg screening of donor blood for example, is essential to avoid transmission of hepatitis B. It is clear that sensitivity is of utmost importance in diagnostic HBV assays.
  • HBV is the smallest DNA virus known, and its genome shows a highly compacted organization.
  • a unique aspect in the replication cycle of HBV is that a pregenomic mRNA serves as a template for the synthesis of the first viral DNA strand ⁇ -4.
  • the RNAse H activity of the HBV DNA polymerase subsequently removes the mRNA and the complementary DNA strand is then synthesized, generating a partially double stranded DNA molecule for packaging in virions. Upon successful virus entry, the partially double stranded DNA molecule is converted into a fully double stranded DNA molecule.
  • HBV DNA can be detected in the blood of infected hosts who are HBsAg and HBeAg positive in more than 90% of cases.
  • the state of the art method of measuring the quantity of infectious particles is by measuring the quantity of viral DNA in serum or plasma, because it most reliably reflects the amount of replicating virus.
  • assays are available for this purpose, such as the branched DNA (bDNA) assay 5 , DNA hybridization assays and quantitative PCR '7 Most of these assays, however, have only limited sensitivity. K ⁇ ck et al. ° found that a considerable number of HBV virus particles circulating in the serum may contain viral mRNA rather than DNA.
  • HBV mRNA may be packaged in virion particles, its nature, quantity and clinical significance remain unknown.
  • K ⁇ ck et al. ° describe an assay for the detection of this HBV RNA in in vitro infected cells and in patient's serum, however the publication is silent about the clinical and diagnostic relevance of this assay.
  • the monitoring of antiviral therapy plays an important role in the management of HBV patient care.
  • a close monitoring of the efficacy of the therapy is necessary in order to adjust antiviral therapy to the needs of the patient Typical problems that are encountered during the course of therapy are the emergence of resistant virus strains and undesired co-effects of the drugs used.
  • the HBV RNA assay as presented here has several advantages over methods known in the art such as conventional HBV DNA assays.
  • the HBV RNA assay is able to detect HBV RNA earlier than HBV DNA in the course of HBV infection which would allow an earlier start of the treatment as well as an earlier exclusion of such patients from giving blood for transfusion.
  • the HBV RNA assay can distinguish between patients that will go into a chronic course of the disease and those with a transient course.
  • HBV RNA is detectable in higher quantities than HBV DNA after therapy is stopped and therefore is a more sensitive marker of recurrent disease.
  • PCR polymerase chain reaction
  • the strands of the double stranded DNA are separated from each other by thermal denaturation and each strand serves as a template for primer annealing and subsequent elongation in a following cycle.
  • the PCR method has been described in Saiki et al., Science 230, 135, 1985 and in European Patents no. EP 200362 and EP 201184.
  • TAS transcription based amplification system
  • NASBA NASBA process
  • NASBA includes the use of T7 RNA polymerase to transcribe multiple copies of RNA from a template including a T7 promoter.
  • RNA and DNA are known in the art. Many use a primer set and suitable amplification reagents for that purpose, however, amplification methods employing only one primer are known in the art. The skilled person would know how to select an amplification technique particularly suitable for the desired purpose.
  • HBV RNA can be amplified by RT- PCR from culture medium of in vitro HBV infected cells (Table 1 , example 1 ). This assay is highly specific for RNA since samples containing only DNA gave negative test results (Example 1 ).
  • HBV RNA results of RT PCR for HBV RNA. HBV RNA could be detected in duplicate experiments in Culture medium from HBV infected cells not in water and also not when no reverse transcription (RT) step was performed when using culture medium. See Example 1 for details of the procedure.
  • HBV mRNA Amplification of HBV mRNA on 5 serum samples and 1 plasma sample, tested positive for HBV with serological markers (HbsAg, HbcAg) and bDNA.
  • RT-PCR was performed in duplicate.
  • POS/POS both duplicates positve
  • POS/NEG one of duplicates negative the other positive
  • NEG/NEG both duplicates negative.
  • HBV RNA amplification assay performed on a serum sample when using different primers: HBV 340, HBV 1050, HBV 1160, HBV 2537, HBV 2674 which are situated 1650, 950, 850, 750 and 640 nucleotides away respectively from the poly-A strectch of the HBV mRNA.
  • RNA isolated form human serum could not withstand incubation in plasma in its isolated form. It is hypothesized that the RNA becomes rapidly degraded when exposed to the RNAses which naturally occur in serum and plasma. The same phenomenon was observed in culture medium, which is known to contain a relatively high concentration of RNAses originating from one of its constituents, the fetal calf serum.
  • RNAse A was added to serum from HBV patients and culture medium from HBV infected cells. Even this extra RNAse A was not able to degrade the RNA while it was present in its natural environment, whereas the equivalent amount of RNAse A added to naked nucleic acid rapidly degraded the HBV RNA.
  • HBV RNA isolated from culture medium is 10 times enriched in the pellet fraction upon ultracentrifugation while no longer detectable in the supernatant (table 7).
  • Table 8B Semi-quantitative analysis of the sensitivity of the HBV DNA assay.
  • the P1 primers contained a T7 RNA promoter sequence (Table 11 ). In addition to that, they contained an initiation transcription site with the sequence AGAAGG. Primer P1.4 detected only RNA whereas P1.11 detected both RNA and DNA (Example 6).
  • the invention relates to a method for monitoring anti-viral therapy wherein HBV nucleic acid is detected, said method comprising the following steps: a) obtaining a sample from an individual suspected of HBV infection and optionally extracting nucleic acid from that sample b) amplifying a target nucleic acid sequence in that sample using at least one oligonucleotide primer and suitable amplification reagents c) detecting the amplified sequence.
  • a preferred embodiment is a method according to the invention wherein the nucleic acid is amplified using the NASBA amplification method.
  • This anchor sequence should consist of 4 to 10 nucleotides hybridisable to the sequence of the HBV mRNA adjacent to the poly-A tail which is 5' GCA TGGACA TTG ACC CTT ATA AAG AAT TTG GAG CT polyA 3'.
  • the anchor sequence therefore consists of at least nucleotides AGCT and at most of nucleotides AGCTCCAAAT (SEQ ID NO 1 ).
  • Example 7 shows a comparison of primers with and without such an anchor sequence and the results shown in Figure 3 and 4 clearly indicate that a primer with an anchor sequence has an improved sensitivity.
  • P1 primers with less than 4 nucleotides did not show this improved performance, whereas P1 primers with more than 10 nucleotides of anchor sequence were found to amplify DNA as well.
  • the invention therefore relates to an oligonucleotide primer comprising a T7 RNA promoter sequence followed by a transcription initiation site followed by an oligo dT stretch followed by an anchor sequence consisting of at least the first 4 nucleotides of SEQ ID NO 1 optionally followed by the remaining nucleotides of SEQ ID NO 1 in consecutive order.
  • the invention also relates to a pair of oligonucleotide primers for the amplification of HBV RNA comprising a first oligonucleotide according to the invention and a second oligonucleotide capable of hybidising to HBV RNA at such a position that amplification of the region between the first and the second oligonucleotide may occur.
  • a possible commercial embodiment of the invention is a test kit for the diagnosis of HBV infection comprising: a) one or more oligonucleotides according to the invention or a pair of oligonucleotides according to the invention b) a suitable probe optionally provided with a detectable label, c) optionally suitable amplification reagents
  • kits would have the advantage that they provide quality controlled reagents suitable for use together as well as controls for their performance
  • FIG. 1 CsCI gradient analysis of HBV-positive cell line sample. • designates the density as determined by refractometry, o HBsAg titer, v HBV DNA level, and
  • HBV mRNA level HBV DNA and mRNA are both found to peak at 1.36 g/ml, where nude core particles representing the majority of HBV DNA containing particles are located. Note that there is another NA peak at the density expected to contain the HBV Dane particle population (1.26 g/ml) and the peak of HBsAg at lower density (around 1.23 g/ml) representing empty HBV envelope (sAg) particles.
  • FIG. 1 CsCI gradient analysis of HBV-positive cell line sample. • designates the density as determined by refractometry, 0 HBsAg titer, v HBV DNA level, and
  • HBV mRNA level HBV DNA and mRNA are both found to peak at 1.28 g/ml, where Dane particles, representing the majority of HBV DNA particles, are located. Note the peak of HBsAg at lower densities (around 1.23 g/ml) representing empty HBV envelope (sAg) particles.
  • Figure 3 and 4 NASBA amplifications with two different P1 primers.
  • FIG. 8 Samples of an HBV infected individual analysed for HBV RNA and DNA markers at several time points during the infection and treatment thereof. Treatment regimes are indicated by the dotted areas. Definitions
  • oligonucleotide refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides such as primers and probes.
  • primer refers to an oligonucleotide either naturally occurring (e.g. as a restriction fragment) or produced synthetically, which is capable of acting as a point of initiation of synthesis of a primer extension product which is complementary to a nucleic acid strand (template or target sequence) when placed under suitable conditions (e.g. buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization, such as DNA dependent or RNA dependent polymerase.
  • suitable conditions e.g. buffer, salt, temperature and pH
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of an agent for polymerization.
  • a typical primer contains at least about 10 nucleotides in length of a sequence substantially complementary (P1) or homologous (P2) to the target sequence, but somewhat longer primers are preferred.
  • primers contain about 15-26 nucleotides but longer primers may also be employed.
  • a set of primers will consist of at least two primers, one 'upstream' and one 'downstream' primer which together define the amplificate (the sequence that will be amplified using said primers).
  • the oligonucleotides according to the invention may also be linked to a promoter sequence.
  • promoter sequence defines a region of a nucleic acid sequence that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the process of transcription by which an RNA transcript is produced.
  • any promoter sequence may be employed for which there is a known and available polymerase that is capable of recognizing the initiation sequence.
  • Known and useful promoters are those that are recognized by certain bacteriophage RNA polymerases such as bacteriophage T3, T7 or SP6. It is understood that oligonucleotides consisting of the sequences of the present invention may contain minor deletions, additions and/or substitutions of nucleic acid bases, to the extent that such alterations do not negatively affect the yield or product obtained to a significant degree.
  • An oligonucleotide sequence used as detection-probe may be labeled with a detectable moiety.
  • a detectable moiety may, for example, either be a radioactive compound, a detectable enzyme (e.g. horse radish peroxidase (HRP)) or any other moiety capable of generating a detectable signal such as a colorimetric, fluorescent, chemiluminescent or electrochemiluminescent signal.
  • Preferred analysis systems wherein said labels are used are electrochemiluminescence (ECL) based analysis or enzyme linked gel assay (ELGA) based analysis.
  • the cDNA was PCR amplified with the following protocol: 2.5 ⁇ l cDNA, 5.0 ⁇ l 10x PCR buffer, 0.75 ⁇ l MgCI2 (1.5 mM), 0.25 ⁇ l Taq DNA polymerase (2.5 U), 0.4 ⁇ l dNTPs (0.2 mM each), 0.5 ⁇ l primer R0 (50 ng, sequence 5' AA GGA TCC GTC GAC ATC 3'), 0.5 ⁇ l primer HBV2659+ (50 ng, sequence 5' GCT GCT AGG CTG TGC TGC CAA 3') and 40.1 ⁇ l H2O were added together in an eppendorf tube and incubated 3 minutes at 95°C in PE9700 thermocycler.
  • the PCR was performed by applying 40 cycles of 30 seconds 95°C, 20 seconds 53°C and 45 seconds 72°C. The last cycle was followed by a 10 minutes 72°C incubation. The amplification products of this first PCR amplification were further amplified in a subsequent PCR reaction, i.e. the nested PCR.
  • PCR product was mixed with 5.0 ⁇ l 10x PCR buffer, 0.5 ⁇ l R1 (50 ng, sequence 5' GAC ATC GAT AAT ACG AC 3'), 0.5 ⁇ l primer HBV2674+ (50 ng, sequence 5' TGC CAA CTG GAT CCT GCG CGG 3'), 0.4 ⁇ l dNTPs (50 ⁇ mol each dNTP), 1.0 ⁇ l MgCI2 (2.0 mM), 0.25 ⁇ l Taq DNA polymerase (2.5 U) and 41.4 ⁇ l H2O.
  • the reaction mix was incubated 3 minutes at 95°C in PE9700 thermocycler.
  • the nested PCR was performed by applying 30 cycles of 30 seconds 95°C, 20 seconds 53°C and 45 seconds 72°C. The last cycle was followed by a 10 minutes 72°C incubation.
  • the HBV specific primers used in this protocol have been described by Sallie et al. 14
  • the primers are designed such that the chance of DNA amplification is minimal due to the application of a poly A stretch in the downstream primer.
  • the specific RNA amplification is confirmed by the fact that without the use of reverse transcriptase in the method there is no amplification (see table 1 ).
  • RNA molecule that is amplified by the nested RT-PCR was investigated by the subsequent use of 5' sense primers that were each time located further away from the 3' anti-sense primer containing the poly A stretch using the protocol described in example 1.
  • the primers used in this study are shown in table 1. Only sequences smaller than approximately 800 bp were amplifiable by both HBV-positive plasma and HBV-positive cell culture medium. This suggests that the RNA has a size of 800 nucleotides (see table 4). In HBV- positive cells and plasmid-derived nucleic acids, all primer combinations gave positive results.
  • a different antisense primer was used (Rcpro) in a normal DNA PCR, because the HBV plasmid construct does not contain an poly-A tail.
  • HBV nucleic acids purified from a plasma containing a high titer (10 9 copies per ml) of HBV was added to HBV-negative cell line culture medium or HBV-negative human plasma, no HBV mRNA could be detected due the presence of high RNase concentration in the medium (as part of the fetal calf serum) and plasma.
  • HBV- transfected cell line (2.2.15) culture medium 60 ml HBV- transfected cell line (2.2.15) culture medium was centrifuged in a table-top centrifuge for 10 min. at 2800 RPM to pellet cell debris, and the resulting supernatant was then passaged through a 0.45 ⁇ M filter. Subsequently, the sample was ultracentrifuged using a SW41TI rotor for 20 hours at 30K and room temperature. The resulting supernatant was collected for analysis and the corresponding pellet was resuspended in 1 ml PBS for further analysis. Analysis was performed by extraction (as described in example 1 ) followed by RT-PCR. These data (see table 7) show that the HBV mRNA is probably associated with a particle, possibly the HBV Dane particle.
  • HBV mRNA was found to peak at the same density as HBV DNA in both cell culture medium (see figure 1 ) and plasma sample (see figure 2), at 1.36 g/ml and 1.28 g/ml, respectively. These densities correspond to the expected densities at which the majority of HBV DNA containing particles can be found. For cell culture medium, these are the nude core particles, while for plasma they are the Dane particles. This will be confirmed by electron microscopy analyses of these fractions. Taken together, these data strongly suggest that the HBV mRNA is co-packaged with the HBV DNA into the same particle.
  • sensitivity of these assays were determined by usage of an in vitro generated HBV mRNA (640 bp) and a plasmid containing the full genome of HBV in tandem (pHBTIII-6). Detection was by visualization on an ethidium bromide agarose gel following (nested RT-) PCR. The results are shown in table 8A and 8B.
  • RNA/DNA ratio was about 1x1 ⁇ 5 in cells and 10 ⁇ in culture medium.
  • RNA/DNA ratio was on average about 10 ⁇ , comparable to culture medium.
  • nucleic acid extracted from the serum of an HBV infected individual was amplified with NASBA using different primer sets for HBV DNA and RNA amplification.
  • the nucleic acid was extracted and purified from 6 aliquots of 200 ⁇ l serum of the same patient using the "Boom" method (Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J, 1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol; 28(3):495-503). After the extraction all isolated nucleic acid was pooled and subsequently aliquoted into 3 batches of 100 ⁇ l each.
  • the 3 nucleic acid batches were treated with RNase A (addition of 10 ⁇ l RnaseA [50 mg/ml], 1 hour incubation at 37°C and subsequently the addition of 1 ⁇ l Proteinase K [20 mg/ml] followed by an incubation of 15 minutes at 37°C) or DNase I (addition of 3 ⁇ l DNasel [10 U/ ⁇ l, Pharmacia] and 2 ⁇ l MgCI 2 [0.5 M] and incubated for 1 hour at 37°C followed by a 10 minutes incubation at 70°C to inactivate the DNase I).
  • RNase A addition of 10 ⁇ l RnaseA [50 mg/ml]
  • 1 hour incubation at 37°C 1 hour incubation at 37°C
  • DNase I addition of 3 ⁇ l DNasel [10 U/ ⁇ l, Pharmacia] and 2 ⁇ l MgCI 2 [0.5 M] and incubated for 1 hour at 37°C followed by a 10 minutes incubation at
  • nucleic acid samples were re-extracted with the Boom method (Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J, 1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol; 28(3):495-503) the nucleic acid extracted in 50 ⁇ l water.
  • Boom method Bit R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J, 1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol; 28(3):495-503
  • the nucleic acid extracted in 50 ⁇ l water For amplification by NASBA 5 ⁇ l of this nucleic acid solution were used directly and 5 ⁇ l of a ten-fold dilution were used as input for the amplification reactions.
  • P1 primer PolA P1.4 is specific for the amplification of HBV RNA, while P1 primer PolA P1.11 amplifies both HBV DNA and RNA (see table 11 for sequences).
  • the molecular beacon probe that is used in this experiment (MB WT1 , see table 11 ) is labeled with fluoresceine (the label) at its 5' end and DABCYL (the quencher) at its 3' end.
  • P1 primer PolA P1.4 is only capable of amplifying HBV RNA since no signal could be observed after RNase A treatment, whereas signals obtained after DNAse I treatment were identical with those obtained with untreated sample.
  • P1 primer PolA P1.11 can amplify both RNA and DNA indicated by the positive results after both RNase A and DNase I treatment, showing amplification of DNA and RNA, respectively.
  • nucleic acid extracted from the serum of an HBV infected individual was amplified with NASBA using different primer sets for HBV RNA amplification.
  • the nucleic acid was in vitro generated by transcription using a plasmid as template.
  • NASBA 5 ⁇ l of this RNA solution or the appropriate dilution were used.
  • the T7 RNA promoter sequence (including the first 3 G residues of the RNA that is being made) that is part of the P1 primers is shown in italics, the inserted transcription initiation site is shown underlined.
  • the detection probe WT-1d was labeled at the NH2 group with an electrochemiluminescence (ECL) label for detection pruposes.
  • ECL electrochemiluminescence
  • the enzyme mix was added (BSA 2.1 mg, RNase H 0.01 units, T7 RNA Polymerase 37 units, AMV-RT 7.5 units) and after gentle mixing by tapping the reactions were incubated at 41 °C in a waterbath for 90 minutes. After the amplification the amplicons were hybridized with the capture and detection probe and bound to magnetic particles covered with streptavidin (that binds the biotin group of the capture probe). After hybridization the ECL signals were measured in an Origin ECL reader (Igen Inc), the signal strength corresponds with the amount of amplicons that were made during the amplification. The results of the experiment are shown in figures 3 and 4.
  • HBV RNA is the first nucleic acid marker that can be measured in the course of a HBV infection. Therefore HBV RNA can be used as a marker for early diagnosis of HBV infection.
  • HBV RNA remains detectable in patient samples after the HBV DNA detection has become negative. This persistent HBV RNA positivity is a marker for disease development and a chronic course of the infection.
  • HBV RNA remains undetectable in patient samples after the HBV DNA detection has become positive.
  • HBV RNA negativity is a marker for absence of future disease development and a transient course of the infection.
  • RNA is a better marker for measuring the effect of stopping of therapy on the replication of the HBV virus. This result also indicates that RNA probably is also a better marker for measuring the breakthrough to therapy of resistant viruses because HBV RNA is a better market for active replication than HBV DNA.

Abstract

L'invention a trait à un procédé de surveillance de traitement antiviral qui comporte une détection d'ARN du virus de l'hépatite B, et à des oligonucléotides pouvant être utilisés dans la détection d'ARN d'hépatite B. Les outils actuels permettant de surveiller un traitement contre le virus de l'hépatite B sont basés sur des marqueurs immunologiques ou sur la détection d'ADN du virus de l'hépatite B dans des particules virales. Cependant, les traitements les plus récents tendent de plus en plus à cibler la polymérase virale, ces traitements influençant directement la réplication du virus. Il existe en conséquence un besoin en outils permettant de surveiller l'efficacité du traitement. Ces outils de surveillance devraient permettre d'obtenir directement une mesure réelle de la réplication du virus et d'indiquer immédiatement l'apparition de souches résistantes de virus. L'invention concerne un tel outil. Elle a trait à un procédé de détection d'ARN du virus de l'hépatite B, et fournit des moyens d'interprétation des résultats permettant de prédire l'issue du traitement. Un traitement plus efficace peut ensuite être prescrit au patient concerné.
PCT/EP2001/002143 2000-03-02 2001-02-26 Detection d'arn du virus de l'hepatite b WO2001064959A1 (fr)

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Cited By (3)

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EP1513958A1 (fr) * 2002-06-14 2005-03-16 Gen-Probe Incorporated Compositions et methodes de detection du virus de l'hepatite b
WO2005071117A2 (fr) 2004-01-23 2005-08-04 Biomerieux, Inc. Amorces et sondes configurees pour permettre une amplification et une detection efficaces de la region non traduisante 3' du vhc
CN107475444A (zh) * 2017-08-04 2017-12-15 广州市第八人民医院 一种从乙型肝炎病毒总核酸中选择性扩增rna的pcr引物、试剂盒和方法

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US7785844B2 (en) 2002-06-14 2010-08-31 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
EP2292801A3 (fr) * 2002-06-14 2011-09-21 Gen-Probe Incorporated Compositions et procédé pour la détection du virus de l'hépatite B
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WO2005071117A2 (fr) 2004-01-23 2005-08-04 Biomerieux, Inc. Amorces et sondes configurees pour permettre une amplification et une detection efficaces de la region non traduisante 3' du vhc
WO2005071117A3 (fr) * 2004-01-23 2006-04-20 Bio Merieux Inc Amorces et sondes configurees pour permettre une amplification et une detection efficaces de la region non traduisante 3' du vhc
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