WO2009087685A2 - Procédé de détection d'acide nucléique de l'hépatite et ses utilisations - Google Patents

Procédé de détection d'acide nucléique de l'hépatite et ses utilisations Download PDF

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WO2009087685A2
WO2009087685A2 PCT/IN2009/000013 IN2009000013W WO2009087685A2 WO 2009087685 A2 WO2009087685 A2 WO 2009087685A2 IN 2009000013 W IN2009000013 W IN 2009000013W WO 2009087685 A2 WO2009087685 A2 WO 2009087685A2
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seq
oligonucleotides
hepatitis
sample
virus
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PCT/IN2009/000013
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WO2009087685A3 (fr
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Rajeev Soni
Nupur Mehrotra
Prabuddha Kumar Kundu
Kajal Arora
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Premas Biotech Pvt.Ltd
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Publication of WO2009087685A3 publication Critical patent/WO2009087685A3/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
    • C12Q1/6862—Ligase chain reaction [LCR]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method for detection, enumeration and/or identification of a disease related condition where the disease is caused by an infectious agent Hepatitis virus.
  • nucleic acid amplification technology has opened new avenues of detection and characterization of diseases as they provide a rapid and accurate way of assessing deviations in the physiology and pathophysiology in a given population or during developmental stages.
  • the molecular methods are becoming more popular due to their ease of performance, reproducibility, sensitivity and specificity of results obtained compared to traditional methods.
  • Various methods of amplifying nucleic acid sequences for disease detection are known in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • NASBA Self-Sustained Synthetic Reaction
  • Q.beta.-Replicase Q.beta.
  • NAATs With regard to infectious diseases, NAATs supersede the routine growth-based culture and microscopy methods in terms of their ease of use and a low turn-around time (Report on the "Evaluation of Diagnostic Tests for Detection of Genitourinary Chlamydia Infections by STD Control Branch, California Department of Health Services, Department of Epidemiology & Biostatistics, University of California, San Francisco; published in March 2001).
  • the NAATs provide substantial time and cost saving over traditional culture methods for determining the presence of a given pathogen in a clinical specimen. Certain NAATs also provide quantification of the pathogen thereby producing more efficient results. These technologies vary among themselves in their sensitivity and specificity to provide an accurate diagnosis. There is an increased demand for tests which maintain very high positive predictive value (PPV) and negative predictive value (NPV) for detection of all microorganisms and give reproducible results.
  • PPV positive predictive value
  • NPV negative predictive value
  • EP patent 0395292 (Barry et al, 1997) describes a method for generating DNA probes specific for an organism useful for distinguishing between genera and species.
  • the document provides the DNA probes obtained from a variable intergenic region intermediate the genes coding for 16S rRNA and 23S rRNA wherein the probes are specific for a variety of species including Aeromonas hydrophilla, A. salmonicida, Clostridium difficile, Mycobacterium bovis, M. tuberculosis and Salmonella typhimurium.
  • US 5,830,71 1 (Barany et al, 1998) describes a method for distinguishing a first nucleotide sequence which differs by at least a single base from a second nucleotide sequence by using ligase chain reaction (LCR) utilizing the thermophilic DNA ligase from Thermus aquaticus to detect a target DNA sequence.
  • LCR ligase chain reaction
  • the detection methods described in the prior art give variable results with respect to non- specificity, poor amplification signal and non reproducibility. It is therefore desirable to provide a method of detection of the target nucleic acid in a sample using ligase mediated amplification reaction that overcomes the above-mentioned disadvantages. It is an object of the present invention to provide a method, oligonucleotide sequences and a diagnostic kit for detection of a target nucleic acid in a sample with high sensitivity and efficacy.
  • the present invention relates to a method for detection, enumeration and/or identification of a disease related condition where the disease is caused by an infectious agent selected from a group consisting of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus in a sample.
  • the present invention particularly relates to a nucleic acid based detection method for diagnosis of infectious and genetically transmitted diseases, where the infectious agent is selected from a group consisting of Hepatitis B virus and Hepatitis C virus.
  • One aspect of the present invention relates to a process for detecting one or more than one target nucleic acids of Hepatitis virus in a sample, the process comprises of (a) providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acids of Hepatitis virus; one or more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein the nucleotide sequence of said one or more than one sets of oligonucleotides is selected from a group consisting of SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20, and SEQ ID NO: 22-25, and a thermostable ligase; (b) subjecting the reaction mixture under isothermal conditions to obtain a resultant reaction mixture thereby amplifying said one or more than one target nucleic acids; and (c) detecting the one or more amplified nucleic acids
  • Another aspect of the present invention relates to a set of oligonucleotides for detection of one or more than one target nucleic acids of Hepatitis virus in a sample, wherein the set of oligonucleotides is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Another aspect of the present invention relates to a set of oligonucleotides for detection of one or more than one target nucleic acids of Hepatitis B and C virus in a sample, wherein the set of oligonucleotides is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Another aspect of the present invention relates to a set of oligonucleotides for detection of one or more than one target nucleic acids of Hepatitis B virus in a sample, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10, wherein the set of oligonucleotides can be used in combination or separately.
  • Still another aspect of the present invention relates to a set of oligonucleotides for detection of one or more than one target nucleic acids of Hepatitis C virus in a sample, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of one or more than one target nucleic acids of Hepatitis virus in a sample comprising a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of one or more than one target nucleic acids of Hepatitis B and C virus in a sample wherein the kit comprises a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of one or more than one target nucleic acids of Hepatitis B virus in a sample comprising a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of one or more than one target nucleic acids of Hepatitis C virus in a sample comprising a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Figure 1 shows amplification of target nucleic acid of Hepatitis virus using DINAR (Dual Isothermal Nucleic Acid Amplification Reaction)
  • the present invention relates to a method for detection, enumeration and/or identification of a disease related condition where the disease is caused by an infectious agent selected from a group consisting of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis
  • the present invention particularly relates to a nucleic acid based detection method for diagnosis of infectious and genetically transmitted diseases, where the infectious agent is selected from a group consisting of Hepatitis B virus and Hepatitis C virus.
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using isothermal nucleic acid amplification reaction (INAR) for detection of an infectious agent, wherein the infectious agent is selected from a group consisting of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus.
  • INAR isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using isothermal nucleic acid amplification reaction (INAR) for detection of an infectious agent, wherein the infectious agent is selected from a group consisting of Hepatitis B virus and/or Hepatitis C virus.
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using isothermal nucleic acid amplification reaction (INAR) for detection of an infectious agent such as Hepatitis B virus.
  • INAR isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using isothermal nucleic acid amplification reaction (INAR) for detection an infectious agent such as Hepatitis C virus.
  • INAR isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using dual isothermal nucleic acid amplification reaction (DINAR) for detection of an infectious agent, wherein the infectious agent is selected from a group consisting of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus.
  • DINAR dual isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using dual isothermal nucleic acid amplification reaction (DINAR) for detection of an infectious agent, wherein the infectious agent is selected from a group consisting of Hepatitis B virus and/or Hepatitis C virus.
  • DINAR dual isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using dual isothermal nucleic acid amplification reaction (DINAR) for detection of an infectious agent such as Hepatitis B virus.
  • DINAR dual isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting a target nucleic acid sequence in a sample using dual isothermal nucleic acid amplification reaction (DINAR) for detection an infectious agent such as Hepatitis C virus.
  • DINAR dual isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting one or more than one target nucleic acids in a sample using multiplex dual isothermal nucleic acid amplification reaction (MDINAR) for detection of an infectious agent, wherein the infectious agent is selected from a group consisting of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus.
  • One embodiment of the present invention is to provide a method for detecting one or more than one target nucleic acids in a sample using multiplex dual isothermal nucleic acid amplification reaction (MDINAR) for detection of an infectious agent, wherein the infectious agent is selected from a group consisting of Hepatitis B virus and/or Hepatitis C virus.
  • MDINAR multiplex dual isothermal nucleic acid amplification reaction
  • One embodiment of the present invention is to provide a method for detecting one or more than one target nucleic acids in a sample using multiplex dual isothermal nucleic acid amplification reaction (MDINAR) for detection of an infectious agent such as Hepatitis B virus.
  • One embodiment of the present invention is to provide a method for detecting one or more than one target nucleic acids in a sample using multiplex dual isothermal nucleic acid amplification reaction (MDINAR) for detection an infectious agent such as Hepatitis C virus.
  • MDINAR multiplex dual isothermal nucleic acid amplification reaction
  • Another embodiment of the present invention is to provide oligonucleotide sequences for nucleic acid detection of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus in a sample, wherein the set of oligonucleotides is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention is to provide oligonucleotide sequences for nucleic acid detection of Hepatitis B virus and/or Hepatitis C virus in a sample, wherein the set of oligonucleotides is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17- 20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention is to provide oligonucleotide sequences for nucleic acid detection of Hepatitis B virus in a sample, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2- 5 and SEQ ID NO: 7-10, wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention is to provide oligonucleotide sequences for nucleic acid detection of Hepatitis C virus in a sample, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25 wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention is to provide a diagnostic kit comprising oligonucleotide sequences and detection method for nucleic acid detection of Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus and Hepatitis E virus in a sample, wherein the kit comprises a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7- 10,
  • SEQ ID NO: 12-15 SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits comprising oligonucleotide sequences and detection method for nucleic acid detection of Hepatitis B virus and/or Hepatitis C virus in a sample
  • the kit comprises a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ , ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits comprising oligonucleotide sequences and detection method for nucleic acid detection of Hepatitis B virus in a sample
  • the kit comprises a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10, wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention is to provide a diagnostic kit comprising oligonucleotide sequences and detection method for nucleic acid detection of Hepatitis C virus in a sample, wherein the kit comprises a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • the diagnostic kit provided in the invention is highly sensitive, specific, easy to use, has a low turnaround time in comparison to conventional detection methods and capable of detecting low titers.
  • Genomic DNA can be extracted from a sample by using methods well known in the art and used as a template DNA for isothermal nucleic acid amplification reactions, selected from a group consisting of INAR, DINAR and MDINAR.
  • Isothermal Nucleic Acid Amplification Reaction describes a reaction of 10-40 cycles in which both annealing and amplification are carried out at a temperature ranging from 55-74°C while denaturation is carried out at a temperature ranging from 90-97 0 C in the presence of one or more thermostable ligase enzymes.
  • DINAR Differenced Nucleic Acid Amplification Reaction
  • Multiplex Dual Isothermal Nucleic Acid Amplification Reaction describes two reactions of 10-40 cycles each comprising of one or more than .
  • one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids in which both annealing and amplification are carried out at a temperature ranging from 55-74 0 C while denaturation is carried out at a temperature ranging from 90-97°C in the presence of one or more thermostable ligase enzymes.
  • the oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 2-5 were synthesized using the methods well known in the art.
  • oligonucleotides were designed from 242 to 284 nucleotides (SEQ ID NO: 1) of the DNA sequence of Hepatitis B virus (Accession number NC 003977). This region is specific to detect Hepatitis B Virus. Region 1 CAG AGT Cl A GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA GGG G SEQ ID NO I
  • oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 7- 10 were synthesized using the methods well known in the art. These oligonucleotides were designed from 672 to 717 nucleotides (SEQ ID NO: 6) of the DNA sequence of Hepatitis B virus (Accession number NC 003977). This region is specific to detect Hepatitis B Virus.
  • oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 12- 15 were synthesized using the methods well known in the art. These oligonucleotides were designed from 48-106 nucleotides (SEQ ID NO: 11) of the DNA sequence of Hepatitis C virus (Accession number NC 004102). This region is specific to detect Hepatitis C Virus. Region
  • oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 17- 20 were synthesized using the methods well known in the art. These oligonucleotides were designed from 127-176 nucleotides (SEQ ID NO: 16) of the DNA sequence of Hepatitis C virus (Accession number NC 004102). This region is specific to detect Hepatitis C Virus.
  • oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 22- 25 were synthesized using the methods well known in the art. These oligonucleotides were designed from 204-261 nucleotides (SEQ ID NO: 21) of the DNA sequence of Hepatitis C virus (Accession number NC 004102). This region is specific to detect Hepatitis C Virus. Region
  • the present invention provides the oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 1-25, wherein the set of oligonucleotides can be used in combination or separately.
  • the present invention provides the oligonucleotide sets having nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention is to provide oligonucleotide set for nucleic acid detection of Hepatitis B and/or Hepatitis C virus, wherein the nucleotide sequence of the oligonucleotide set is as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Size of the oligonucleotide sequences provided in the present invention ranges from 15-50 bases.
  • Each oligonucleotide set provided in the present invention consists of at least 4 oligonucleotides (A, B, C and D) of the size ranging from 15-50 bases, wherein first oligonucleotide (A) is complementary to third oligonucleotide (C) and second oligonucleotide (B) is complementary to fourth oligonucleotide (D).
  • the second and third oligonucleotide (B and C) have a phosphate group attached to the 5' end and is optionally modified.
  • the 3' end of the second and third oligonucleotide (B and C) is optionally modified.
  • the second and third oligonucleotide (B and C) of each oligonucleotide set disclosed in the present invention are optionally modified by adding phosphate group at the 3' end.
  • the modification can also be carried out by adding, deoxy, alkyl or aryl groups at the 3' end.
  • One embodiment relates to a label or probe that can be attached to 5' end and/or 3' end of any of the four (A, B, C and D) oligonucleotides in each set.
  • Yet another embodiment of the present invention relates to the process for detecting a target nucleic acid of Hepatitis virus using the oligonucleotide set disclosed in the present invention, wherein at least one oligonucleotide of the set of oligonucleotides is optionally attached to a label.
  • Still yet another embodiment of the present invention relates to the process for detecting a target nucleic acid of Hepatitis virus using the oligonucleotide set disclosed in the present invention, wherein at least one oligonucleotide of the set of oligonucleotides is optionally attached to a label, wherein the label is selected from a group consisting of chromophores, fluorescent moieties, enzymes, antigens, and chemiluminescent moieties.
  • the present invention relates to the process for detecting a target nucleic acid of Hepatitis virus using the oligonucleotide set disclosed in the present invention, wherein at least two oligonucleotides i. e. second and third oligonucleotide of the set of oligonucleotides are optionally modified at 3' end with phosphate, deoxy, alkyl or aryl group.
  • the present invention relates to the process for detecting a target nucleic acid of Hepatitis virus using the oligonucleotide set disclosed in the present invention, wherein at least two oligonucleotides second and third oligonucleotide of the set of oligonucleotides are modified at 5' end with phosphate group.
  • the amplified product obtained using the process disclosed in the present invention can be detected by various methods known in the art such as agarose gel electrophoresis, solution phase colorimetric detection using enzymes and photoactivity assays, solid phase colorimetric detection using enzymes and photoactivity assays, solution phase fluorimetric detection, solid phase fluorimetric detection, solution phase chemiluminiscent detection, solid phase chemiluminiscent detection, solution phase using nanoparticles, solid phase using nanoparticles, solution phase using radioactive detection and solid phase using radioactive detection method.
  • the kit disclosed in the present invention is based on INAR, DINAR and/or MDINAR for detection of the nucleic acid of infectious agent in a sample in one assay wherein the infectious agent may be present individually or in combination with each other.
  • Isothermal Nucleic acid Amplification Reaction Isothermal Nucleic acid Amplification Reaction using the oligonucleotides as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25 was carried out. Details are provided in Example 1.
  • the INAR disclosed in the present invention eliminates the problem of non-specificity, poor amplification signal and non reproducibility in detection of target nucleic acid in a sample for diagnosis of an infectious agent in a sample.
  • Dual Isothermal Nucleic acid Amplification Reaction using the oligonucleotides as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25 was carried out. Details are provided in Example 2.
  • the DINAR disclosed in the present invention eliminates the problem of non-specificity, poor amplification signal and non reproducibility in detection of target nucleic acid in a sample for diagnosis of an infectious agent in a sample.
  • MDINAR Dual Isothermal Nucleic acid Amplification Reaction
  • the MDINAR disclosed in the present invention eliminates the problem of non-specificity, poor amplification signal and non reproducibility in detection of target nucleic acid in a sample for diagnosis of an infectious agent in a sample.
  • the oligonucleotide sequences having nucleotide sequence as set forth in SEQ ID NO: 2- 5, SEQ ID NO: 7-10, SEQ ID NO: SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25 were synthesized using the methods well known in the art.
  • the present invention comprises the following embodiments:
  • one embodiment provides a process for detecting one or more than one target nucleic acids of Hepatitis virus in a sample, the process comprises (a) providing a reaction mixture comprising a sample containing or suspected of containing one or more than one target nucleic acids of Hepatitis virus; one or more than one set of oligonucleotides specific for one or more than one region of one or more than one target nucleic acids, wherein the nucleotide sequence of said one or more than one sets of oligonucleotides is selected from a group consisting of SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20, and SEQ ID NO: 22-25, and a thermostable ligase; (b) subjecting the reaction mixture under isothermal conditions to obtain a resultant reaction mixture thereby amplifying said one or more than one target nucleic acids; and;(c) detecting the one or more amplified nucleic
  • Another embodiment of the present invention provides a process for detecting one or more than one target nucleic acids of Hepatitis virus in a sample, wherein said process optionally comprises passing the said resultant reaction mixture through a purification column to obtain flow through and subjecting the whole- or part of the said flow through under isothermal conditions thereby amplifying said target nucleic acid.
  • Another embodiment of present invention provides a process of "Isothermal Nucleic Acid Amplification Reaction (INAR)" for detecting one or more than one target nucleic acids of Hepatitis virus in a sample as disclosed in the present invention, wherein the process comprises of one cycle of denaturation at a temperature ranging from about 90°C to 99°C for 1 to 10 minutes; and annealing and amplification at a temperature ranging from 55 0 C to 74°C for 1 to 3 minutes; and 10 to 40 cycles of denaturation at a temperature ranging from about 9O 0 C to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from about 55°C to 74°C for 30 seconds to 3 minutes.
  • INAR isothermal Nucleic Acid Amplification Reaction
  • the process of INAR for detecting one or more than one target nucleic acids of Hepatitis virus in a sample as disclosed in the present invention comprises one cycle of denaturation at 95°C for 10 minutes; and annealing and amplification at 65°C for 3 minutes; and 31 cycles of denaturation at 95°C for I minute and annealing and amplification at 65° for 2 minutes.
  • Another embodiment of the present invention provides a process of "Dual Isothermal Nucleic Acid Amplification Reaction (DINAR)” or “Multiplex Dual Isothermal Nucleic Acid Amplification Reaction (MDINAR)” for detecting one or more than one target nucleic acids of Hepatitis virus in a sample as disclosed in the present invention, wherein the process comprises of the following cycling conditions; Cycle 1 : denaturation at a temperature ranging from 90° to 99°C for 30 seconds to 10 minutes; and annealing and amplification at a temperature ranging from 55° to 74°C for 30 seconds to 3 minutes followed by Cycle 2 to 8 and upto 25: denaturation at a temperature ranging from 90 to 99°C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from 55° to 74°C for 30seconds to 3 minutes.
  • DINAR Dual Isothermal Nucleic Acid Amplification Reaction
  • MDINAR Multiplex Dual Isothermal Nucleic Acid Amplification Reaction
  • the resultant reaction mixture through a purification column to obtain flow through and subjecting the whole or part of the said flow through/reaction mixture to following isothermal conditions; 8-32 cycles of denaturation at a temperature ranging from 90 to 99 0 C for 30 seconds to 3 minutes and annealing and amplification at a temperature ranging from 55° to 74°C for 1 to 3 minutes and detecting the one or more amplified nucleic acids.
  • the process of DINAR and MDINAR describe in the present invention comprises of the following cycling conditions; Cycle 1 : denaturation at a temperature of 95°C for 10 minutes; and annealing and amplification at a temperature of 65° for 3 minutes followed by Cycles 2 to 15: denaturation at a temperature of 95 0 C for 1 minute and annealing and amplification at a temperature of 65° for 1 minute.
  • One embodiment of the present invention provides the sample of human or veterinary origin. Another embodiment of the present invention provides the samples, wherein the sample is selected from the group consisting of blood, sputum, tissue, saliva, cerebro-spinal fluid, semen, and other body fluids, milk and other body secretions, urine and other body excretions, and/or washings from a subject.
  • Still yet another embodiment of the present invention wherein at least two oligonucleotides are optionally modified at 3' end with phosphate, deoxy, alkyl or aryl groups.
  • Another embodiment of the present invention provides a set of oligonucleotides for detection of a target nucleic acid of Hepatitis virus in a sample, wherein the set of oligonucleotides is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Yet another embodiment of the present invention provides a set of oligonucleotides for detection of a target nucleic acid of Hepatitis B and C virus in a sample, wherein the set of oligonucleotides is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • Another embodiment of the present invention provides a set of oligonucleotides for detection of a target nucleic acid of Hepatitis B virus in a sample, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set
  • Yet another embodiment of the present invention provides a set of oligonucleotides for detection of a target nucleic acid of Hepatitis C virus in a sample, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of a target nucleic acid of Hepatitis virus in a sample comprising the set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2- 5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of a target nucleic acid of Hepatitis B and C virus in a sample comprising a set of oligonucleotides selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5, SEQ ID NO: 7-10, SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of a target nucleic acid of Hepatitis B virus in a sample comprising the set of oligonucleotides, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10, wherein the set of oligonucleotides can be used in combination or separately.
  • kits for detection of a target nucleic acid of Hepatitis virus C in a sample comprising the set of oligonucleotides, wherein the oligonucleotide set is selected from the group consisting of a nucleotide sequence as set forth in SEQ ID NO: 12-15, SEQ ID NO: 17-20 and SEQ ID NO: 22-25, wherein the set of oligonucleotides can be used in combination or separately.
  • An embodiment of the present invention is to provide a kit containing all the necessary reagents to perform the methods of detection disclosed herein.
  • the kit may contain specific oligonucleotide sequence sets optionally attached to a label, a suitable buffer and a thermostable ligase.
  • the kit may further contain a set of printed instructions indicating that the kit is useful for detection of the specific disease and/or non disease related conditions as disclosed in the present invention.
  • oligonucleotide sequences 1A-1D having nucleotide sequence as set forth in SEQ ID NO: 2-5 were synthesized using the methods well known in the art. These oligonucleotides were designed from 242 nucleotide position to 284 nucleotide position (Region 1 - SEQ ID NO: 1) of the DNA sequence of Hepatitis B virus (Accession number NC_003977).
  • Region 1 CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA GGG G SEQ ID NO: 1 IA 5' CAG AGT CTA GAC TCG TGG TGG S ' SEQ ID NO: 2
  • Cycle 1 o 95°C for 10 min-template denaturation o 65 0 C for 2 min-template annealing and ligation • Cycle 2 to 31 : o 95°C for 1 min- template denaturation o 65 0 C for 2 min- template annealing and ligation Reaction mixture (20 ⁇ l) comprising of:
  • Sample 1 Experimental Human Sample Bl (unknown blood Hepatitis sample)
  • Sample 2 Experimental Human Sample B2 (unknown blood Hepatitis sample)
  • Sample 3 Experimental Human Sample B3 (unknown blood Hepatitis sample)
  • Sample 4 Experimental Human Sample B4 (unknown blood Hepatitis sample)
  • Sample 5 Experimental Human Sample B5 (unknown serum Hepatitis sample)
  • Sample 7 Experimental Human sample B7 (unknown serum Hepatitis sample)
  • Sample 8 Negative control Human DNA (known Hepatitis negative sample)
  • Sample 9 Synthetic Positive control (Plasmid containing 513bp fragment from Hepatitis B virus; Accession number NC 003977)
  • Amplification of expected fragment of 43 bp was observed in positive control sample i.e. plasmid containing the 513 bp of hepatitis B nucleotide sequence, in B l, B2, B4, B5 and B7 samples. Fragment of 23 bp due to primer annealing only and no ligation in absence of target template was obtained in negative control sample. Fragment of 23 bp due to primer annealing and no fragment of 43 bp was obtained in B3 and B6 samples. This observation suggests that samples B l, B2, B4, B5, and B7 are positive for the presence of hepatitis B virus in the sample whereas samples B3 and B6 are not infected by hepatitis B virus. These results have been confirmed by the ELISA and PCR tests.
  • Dual isothermal nucleic acid amplification reaction for detecting Hepatitis B virus DNA using oligonucleotide set (Set 1)
  • the oligonucleotide set (Set 1) having nucleotide sequence as set forth in SEQ ID NO: 2-5 were synthesized using the methods well known in the art. These oligonucleotides were designed from nucleotide position 242 nucleotide position to 284 nucleotide position (Region 1 : SEQ ID NO: 1) of the DNA sequence of hepatitis B virus (Accession number NC_003977).
  • Region 1 CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA GGG G SEQ ID NO: 1
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ rD NO: 3 and SEQ ID NO: 4.
  • the oligonucleotide set as set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 were tested with blood and serum samples.
  • the DINAR for detecting Hepatitis B virus DNA was performed in two steps as given below.
  • the isothermal nucleic acid amplification reaction was performed using oligonucleotide having nucleotide sequences as set forth in SEQ ID NO: 2-5 for detection of Hepatitis B virus DNA in samples.
  • the total volume of the reaction mixture was 25 ⁇ l.
  • the final concentration of the components of the reaction mixture was 2.5 ⁇ l buffer (10X), 1 ⁇ l Thermostable ligase (5U/ ⁇ l), 1 ⁇ l of each oligonucleotide IA, I B, 1C and I D (SEQ ID NO: 2-5) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 25 ⁇ l.
  • isothermal nucleic acid amplification reaction was performed using the same primer set as used in the first step (SEQ ID NO: 2-5).
  • the reaction mixture used for performing the second isothermal nucleic acid amplification reaction was 10 ⁇ l of the final reaction mixture of reactions 1-9 of step 1, 2.5 ⁇ l buffer (10X), 1 ⁇ l Thermostable ligase (5U/ ⁇ l), l ⁇ l of oligonucleotides IA, IB, 1C and ID (SEQ ID NO: 2-5) at concentration 2.5ng each and ultrapure water to bring the reaction volume to 25 ⁇ l.
  • the cycling reaction was carried out as follows: 23 cycles at 95°C for 1 min and 65°C for 1 min.
  • the amplified DNA products obtained can also be detected by various other methods known in the art such as solution phase colorimetric detection using enzymes and photoactivity assays, solid phase colorimetric detection using enzymes and photoactivity assays, solution phase fiuorimetric detection, solid phase fluorimetric detection, solution phase chemiluminiscent detection, solid phase chemiluminiscent detection, solution phase using nanoparticles, solid phase using nanoparticles, solution phase using radioactive detection and solid phase using radioactive detection method.
  • solution phase colorimetric detection using enzymes and photoactivity assays solid phase colorimetric detection using enzymes and photoactivity assays
  • solution phase fiuorimetric detection solid phase fluorimetric detection
  • solution phase chemiluminiscent detection solid phase chemiluminiscent detection
  • solution phase using nanoparticles solid phase using nanoparticles
  • solid phase using nanoparticles solution phase using radioactive detection and solid phase using radioactive detection method.
  • the results are shown in Figure 1. Amplification of expected fragment of 43 bp was observed in positive control
  • reaction conditions employed for the above described process employed annealing and ligation temperatures varying from 55° to 74° C for duration of 1 min and using different primer sets. The results obtained were similar as for the above described reaction set up.
  • Other primer set i.e. set 2 consisting of oligonucleotide as set forth in SEQ ID NO: 7-10 were tested with the same set of samples with similar results.
  • the enzyme concentration for thermostable ligase was varied from 1-10 units with consistent and reproducible results.
  • Example 3 Multiplex Dual Isothermal Nucleic acid Amplification Reaction (MDINAR) for detecting Hepatitis B virus DNA using oligonucleotide set (Set 1 and 2)
  • the oligonucleotide sets (Set 1 and Set 2) having nucleotide sequence as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10 were synthesized using the methods well known in the art.
  • the oligonucleotides set forth in SEQ ID NO: 2-5 were designed from nucleotide position 242 to nucleotide position 284 (Region 1 : SEQ ID NO: 1) of the DNA sequence of hepatitis B virus (Accession number NC_003977). This region is specific to detect Hepatitis B Virus.
  • oligonucleotides set forth in SEQ ID NO: 7-10 were designed from nucleotide position 672 to nucleotide position 717 (SEQ ID NO: 6) of the DNA sequence of hepatitis B virus (Accession number NC_003977). This region is specific to detect Hepatitis B Virus.
  • Region 1 CAG AGT CTA GAC TCG TGG TGG ACT TCT CTC AAT TTT CTA GGG G SEQ ID NO: 1
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 3 and SEQ ID NO: 4.
  • a phosphate group was attached to the 5' end of the oligonucleotides as set forth in SEQ ID NO: 8 and SEQ ID NO: 9.
  • the MDINAR for detecting Hepatitis B virus DNA was performed in two steps as given below.
  • STEP l The isothermal nucleic acid amplification reaction was performed using oligonucleotide having nucleotide sequences as set forth in SEQ ID NO: 2-5 and SEQ ID NO: 7-10 for detection of Hepatitis B virus DNA in samples.
  • the total volume of the reaction mixture was 25 ⁇ l.
  • the final concentration of the components of the reaction mixture was 2.5 ⁇ l buffer (10X), 1 ⁇ l Thermostable ligase (5U/ ⁇ l), 1 ⁇ l of each oligonucleotide I A, I B, 1 C and ID (SEQ ID NO: 2-5) and 2A, 2B, 2C, 2D (SEQ ID NO: 7-10) at concentration 2.5ng each, lOOng of template DNA and ultrapure water to bring the reaction volume to 25 ⁇ l.
  • the cycling reaction employed is as follows: first cycle at 95°C for 10 min and 65°C for 3 min followed by 14 cycles at 95°C for 1 min and 65°C for 1 min.
  • Various samples and controls were used for detection of Hepatitis B viral DNA.
  • the description of the different reactions (reactions 1-9) used as template DNA is given below:
  • Sample 1 Experimental Human Sample Bl (unknown blood Hepatitis sample)
  • Sample 2 Experimental Human Sample B2 (unknown blood Hepatitis sample)
  • Sample 3 Experimental Human Sample B3 (unknown blood Hepatitis sample)
  • Sample 4 Experimental Human Sample B4 (unknown blood Hepatitis sample)
  • Sample 5 Experimental Human Sample B5 (unknown serum Hepatitis sample)
  • Sample 6 Experimental Human sample B6 (unknown serum Hepatitis sample)
  • Sample 7 Experimental Human sample B7 (unknown serum Hepatitis sample)
  • Sample 8 Negative control Human DNA (known Hepatitis negative sample)
  • Sample 9 Synthetic Positive control (plasmid containing 513bp fragment from Hepatitis B virus; Accession number NC 003977)
  • isothermal nucleic acid amplification reaction was performed using the same primer set as used in the first step (SEQ ID NO: 2-5 and SEQ ID NO: 7-10).
  • the reaction mixture used for performing the second isothermal nucleic acid amplification reaction was 10 ⁇ l of the final reaction mixture of reactions 1-9 of step 1, 2.5 ⁇ l buffer (10X), 1 ⁇ l Thermostable ligase (5U/ ⁇ l), l ⁇ l each of oligonucleotides IA, IB, 1C and I D (SEQ ID NO: 2-5) and 2A, 2B, 2C, 2D (SEQ ID NO: 7-10) at concentration 2.5ng each and ultrapure water to bring the reaction volume to 25 ⁇ l.
  • the cycling reaction was carried out as follows: 23 cycles 95 0 C for 1 min and 65°C for 1 min. After completion of the 23 cycles of reaction in the thermocycler, the entire reaction mixture of reactions 1 -9 was electrophoresed on a 2.5% agarose gel at 100V for 45 min and the DNA products were identified using methods known in the art. The gel picture was captured on a gel documentation system.
  • the amplified DNA products obtained can also be detected by various other methods known in the art such as solution phase colorimetric detection using enzymes and photoactivity assays, solid phase colorimetric detection using enzymes and photoactivity assays, solution phase fluorimetric detection, solid phase fluorimetric detection, solution phase chemiluminiscent detection, solid phase chemiluminiscent detection, solution phase using nanoparticles, solid phase using nanoparticles, solution phase using radioactive detection and solid phase using radioactive detection method.
  • Amplification of expected fragment of 43bp for region 1 as set forth in SEQ ID NO: 1 and 45 bp for region 2 as set forth in SEQ ID NO: 6 was observed in positive control sample i.e. plasmid containing the 513 bp of hepatitis B nucleotide sequence, in B l , B2, B4, B5 and B7. Fragment of 23 bp due to primer annealing only and no ligation in absence of target template was obtained in negative control (NC) sample. Fragment of 23 bp due to primer annealing and no fragment of 43 bp was obtained in B3 and B6 samples.
  • NC negative control
  • samples Bl, B2, B4, B5, and B7 are positive for the presence of hepatitis B virus in the isolated sample whereas samples B3 and B6 are not infected by hepatitis B virus.
  • Other reaction conditions employed for the above described process employed annealing and ligation temperatures varying from 55° to 74° C for duration of 1 min and using different primer sets. The results obtained were similar as for the above described reaction set up. Other primer sets were tested with the same set of samples with similar results. The enzyme concentration for thermostable ligase was varied from 1 -10 units with consistent and reproducible results.

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Abstract

L'invention concerne un procédé de détection, de dénombrement et/ou d'identification d'un état pathologique associé à une maladie causée par un agent infectieux sélectionné dans un groupe comprenant: le virus de l'hépatite A, le virus de l'hépatite B, le virus de l'hépatite C, le virus de l'hépatite D et le virus de l'hépatite E; ce procédé étant mis en oeuvre dans un échantillon. En particulier, l'invention concerne un procédé de détection fondé sur l'acide nucléique servant à diagnostiquer des maladies infectieuses et des maladies transmises de manière génétique, l'agent infectieux étant sélectionné dans un groupe comprenant le virus de l'hépatite B et le virus de l'hépatite C.
PCT/IN2009/000013 2008-01-04 2009-01-05 Procédé de détection d'acide nucléique de l'hépatite et ses utilisations WO2009087685A2 (fr)

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US20140134611A1 (en) * 2012-10-18 2014-05-15 Roche Molecular System, Inc. Dual probe assay for the detection of heterogeneous amplicon populations
WO2014144578A1 (fr) * 2013-03-15 2014-09-18 The Usa, As Represented By The Secretary, Department Of Health And Human Services Détection sélective des virus des hépatites a, b, c, d ou e ou de leurs combinaisons
WO2014136124A3 (fr) * 2013-03-05 2014-12-24 Indian Council Of Medical Research Trousse d'essai de pcr en temps réel multiplexe pour la détection simultanée du virus de l'hépatite
WO2020160502A1 (fr) * 2019-01-31 2020-08-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés et compositions destinés à détecter des pathogènes transmis par tranfusion
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
CN102146486A (zh) * 2011-04-01 2011-08-10 上海邃志生物科技有限公司 一组用于检测hbv基因突变的核苷酸序列及其应用
US20140134611A1 (en) * 2012-10-18 2014-05-15 Roche Molecular System, Inc. Dual probe assay for the detection of heterogeneous amplicon populations
US9963737B2 (en) * 2012-10-18 2018-05-08 Roche Molecular Systems, Inc. Dual probe assay for the detection of heterogeneous amplicon populations
US11274998B2 (en) 2012-12-26 2022-03-15 Ventana Medical Systems, Inc. Specimen processing systems and methods for holding slides
WO2014136124A3 (fr) * 2013-03-05 2014-12-24 Indian Council Of Medical Research Trousse d'essai de pcr en temps réel multiplexe pour la détection simultanée du virus de l'hépatite
WO2014144578A1 (fr) * 2013-03-15 2014-09-18 The Usa, As Represented By The Secretary, Department Of Health And Human Services Détection sélective des virus des hépatites a, b, c, d ou e ou de leurs combinaisons
WO2020160502A1 (fr) * 2019-01-31 2020-08-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés et compositions destinés à détecter des pathogènes transmis par tranfusion

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