Classifications

• • 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
  • 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

Definitions

• the invention relates to a reagent for the amplification of nucleic acids of a virus associated with NonA / NonB / NonC / NonD / NonE (N-ABCDE) hepatitis and methods for detection using this reagent.
• hepatitis A virus HAV
• hepatitis B virus HB V
• HAV hepatitis A virus
• HB V hepatitis B virus
• HAV hepatitis C virus
• WO 94/18217 also describes a virus which cannot be assigned to the five groups mentioned.
• the nucleotide sequences used in the present invention are not similar to the sequences described in WO 94/18217.
• WO 95/21922 also describes a hepatitis reagent which does not have to be assigned to any of the five groups mentioned above.
• the group of hepatitis-associated viruses defined in the present invention is referred to as HGV.
• the information available so far from HGV suggests that HGV belongs to the Flaviviridiae family.
• the invention relates to a reagent for the amplification of HGV-specific nucleoside sequences, HGV being defined as a virus, the genome of which consists of RNA which contains a nucleoside sequence at the 5 'end which contains at least 80%, preferably at least 90 % is homologous to the nucleotides of SEQ ID NO 1, containing two HGV-specific primers, each with an extendable end, one of which contains a sequence of 15 to 30 bases, which is more than 80%, preferably at least 90% complementary to successive bases of the nucleotides of SEQ.ID NO 1, and of which the other contains a sequence of 15 to 30 bases which is more than 80%, preferably at least 90% homologous to successive bases of the nucleotides of SEQ. ID. NO. 1, the extendable ends of the primers being chosen so that in the case of the extension of each primer, each primer can hybridize with the extension product of the other primer and the respective extension products can serve as a template for the extension of the other primer.
• amplification is understood to mean a method for producing a large number of copies of a base sequence.
• Known methods are available for this, for example: B the polymerase chain reaction as described in US-A-4,683,202
• a primer is understood to mean a molecule which has a number of nucleobases on a backbone.
• a backbone is a basic polymer structure.
• the sugar-phosphate backbone is particularly well known, as occurs, for example, in nucleic acids such as DNA and RNA.
• Heterocyclic compounds which are capable of forming hydrogen bonds with complementary heterocycles are covalently bound to it.
• the best known are the naturally occurring bases adenine, guanine, cytosine, thymine and uracil. However, there are other bases in nature. The sequence of these bases is chosen so that they become successive bases of the nucleo- tide sequence are more than 90% complementary.
• This molecule has at least one extendable end.
• Extension is understood to mean in particular enzyme-catalyzed coupling of base units with the aid of mononucleoside triphosphates or oligonucleotides.
• a DNA polymerase is preferably used as the enzyme.
• the nucleic acid which contains nucleotide sequences which are to be amplified serves as a template or template for the specific incorporation of bases.
• the sequence of the template determines the sequence of the bases attached to the primer
• Molecules with between 15 and 30 bases are expediently used as primers, since these can be synthesized in a relatively simple manner by chemical synthesis
• the 3 'end is preferably used as the extendable end.
• reverse transcriptase RNA-dependent DNA- Polymerase
• reverse transcriptase also has DNA-dependent DNA polymerase activity, a large number of other DNAs are formed from the cDNA initially formed.
• another DNA-dependent DNA polymerase can also be used, eg. B from E coli or Thermus aquaticus
• RT-PCR reverse transcriptase
• a nucleic acid is formed, the length of which is determined by the ends of the two primers, which are preferably non-extendable from each other, and in principle the length of these nucleic acids, which are also referred to as ampiificates who- that contain the entire area of SEQJD.NO 1, ie be up to 348 bases long.
• the length of the amplificates is preferably> 100 bases.
• the amphficate length is particularly preferably between 150 and 200 nucleotides (nt).
• the amplificate contains an area which was newly formed by incorporating mononucleoside tri-phosphates or polynucleotides
• the primers according to the invention preferably do not hybridize with members from other hepatitis-associated virus groups, in particular not with hepatitis A, B, C, D or E viruses. Furthermore, they preferably do not hybridize with other nucleic acids occurring in human blood
• Primers that are completely complementary or homologous to a partial sequence of SEQ ID NO 1 are particularly preferred. However, it has been found that in some cases it is possible to select a few nucleotides so that they have a mismatch with respect to the sequence of SEQ ID NO 1. This mismatch is then preferably not at the 3 'end of the primer
• HGV-specific nucleotide sequence is understood to mean a sequence that only occurs in HGV, but not in other viruses and eukaryotes
• HGV subtype-specific nucleotide sequence is understood to mean an HGV-specific sequence which does not occur in all HGV subtypes.
• a HGV subtype is understood to be a virus which has all the phenotypic properties of HGV, but whose nucleotide sequence differs from that of other HGVs. This deviation , preferably within nucleotides 64-348 of SEQ ID No. 1 However, at least 10%. It has been found that the subtypes of HGV differ from one another, particularly in the nucleotide regions 74-92, 186-223, 255-283 and 303-306, and particularly preferably from SEQ ID.NO 1.
• SEQ ID. No 1 represents a first subtype of HGV
• a subtype 2a and a subtype 2b can be defined on the basis of the alignment shown in FIG. 1. Only the nucleotides in which the sequences of the sequence in SEQ. Distinguish ID No 1
• a primer HGV subtype can be selected specifically and another HGV subtype-specifically
• FIG. 1 shows the nucleotides of HGV from different persons which correspond to nucleotides 64-348 of SEQ ID No. 1. It can be seen that, according to the above definition, the first-mentioned HGV (Sa 1134 and Sa 1172) belong to a first subtype (2a) , while the remaining HGV belong to their own subtype (2b). The occurrence of a deletion in pos 255 is remarkable compared to SEQ No 1. The conserved or variable regions can also be clearly seen from FIG. 1
• HGV subtype-unspecific sequences are understood to mean HGV-specific sequences which can hybridize with all known HGV subtypes. These are contained in particular in the conserved regions of SEQ ID NO 1, preferably between 92 and 186 and 223 and 255 and 315 and 348
• a detection of HGV is understood to mean a method in which it is determined whether HGV is contained in a sample.
• This sample is preferably a blood sample.
• a method for detecting an HGV subtype can be a method for determining the subtype on the one hand, but also act as such from HGV
• the primers according to the invention can also contain nucleotide sequences which are neither HGV-specific nor can hybridize with nucleic acids which are usually present in the sample. These are preferably at the non-extendable end of the primer of the amplificates are used
• the primers can also be modified further, for example by attaching a detectable or immobilizable group.
• groups can be, for example, directly or indirectly detectable groups, for example radioactive, colored or fluorescent groups.
• Haptens as described in EP-B-0 324 474 (digoxigenin), are particularly preferred.
• Biotin for example, has proven to be an immobilizable group, since it can be immobilized on surfaces which are coated with avidin or streptavidin This enables the amplificates to be captured on a solid phase and the removal of sample components or detection of disruptive components of the amplification reaction
• Particularly preferred numbers are those which contain a sequence of 6 successive bases which are contained in the sequences of SEQ.ID NO 2 or 3 or 5 to 20, or a sequence which is complementary thereto. Primers as described in SEQ ID NOS 2 or 3 or 5 to 20 are particularly preferred
• nucleotides Y and R appear in the SEQ ID, this should be understood to mean that the primers described are a mixture corresponding to the meaning of the letters Y and R.
• Another object of the invention is a reagent kit for the specific detection of HGV containing a reagent according to one of Claims 1 to 4 and a probe which contains a base sequence which lies within the extension product of the amplification reaction, ie between the extended ends of the primers
• a probe is understood to be a molecule which, like the primers, has a number of nucleobases on a backbone. However, the probe does not have to have an extendable end.
• the probe is characterized in that it contains a recognizable group. Such a recognizable group can be a detectable group or an immobilized or immobilizable group.
• the probe has an immobilizable residue, ie a residue which is in a subsequent or simultaneous reaction to a solid phase, a solid can be bound.
• the immobilizable group is a biotin residue
• the solid phase can be, for example, a surface of a microtiter plate or a tube coated with streptavidin or avidin.
• the reagent kit preferably contains the primers and the probe in separate containers.
• the reagent kit may contain other components required for amplification and detection or / and useful, e.g. B. a reverse transcriptase, mononucleoside triphosphates, preferably detectably labeled, a solid phase for trapping the hybrids from probe and amplificates and buffer substances.
• Another object of the invention is therefore a method for the detection of HGV in a sample, characterized by the formation of cDNA from part of the HGV RNA and amplification of the cDNA using a reagent according to one of claims 1 to 4, contacting the amplification products with a Probe containing a base sequence located between the extended ends of the primers and determining the formation of hybrids of the probe with the amplification products as a sign of the presence of HGV in the sample.
• the present invention has the advantage of providing a specific method for the detection of HGV, which is nevertheless reliable in samples from different HGV-positive people and in which HCV is not also detected. Furthermore, it is possible to detect HGV with high sensitivity using the method according to the invention. The specificity in the Southern blot is high.
• the detection according to the method according to the invention can be combined with an RNA detection at another location in the HGV genome, in particular with an RT-PCR in the region which codes for non-structural proteins of the HGV .
• the sequence of the non-structure areas can be determined according to the method described in EP-B-0 318 216. For this, starting from sequence SEQJD.NO. 1 a primer extension in the 3 'direction of the genome carried out, and the sequence of the extension product determined. This sequence is in turn used to prepare a primer which is used in a renewed extension reaction in the 3 'direction of the genome, etc. The statement that at least one of the two amplification reactions was successful is then a sign of the presence of the HGV.
• RNA isolation Three separate work stations should be used for RNA isolation, preparation of the reverse transcriptions and amplification reactions as well as their implementation and detection of the amplification products.
• the reagents should be made fresh daily.
• the pipettes should be regularly contaminated.
• Fresh gloves should be used at every work station.
• RNA manufacturing processes such as Chomczynski acid phenol extraction or with guanidinium isothiocyanate, can also be used.
• a separate work station and a separate set of pipettes are used to produce the reverse transcription and amplification mixtures.
• the reagents used are shown in the table below.
• reaction vessels 40 ⁇ l of the amplification mixture are placed in reaction vessels. 10 ⁇ l of the solution from the cDNA synthesis are pipetted in. The mixture is briefly mixed and centrifuged in a vortex mixer. The reaction vessels are then transferred to a Perkin-Elmer thermal cycler (PE9600). 40 thermal cycles were carried out (15 seconds at 94 ° C, 30 seconds at 55 ° C. and 30 seconds at 72 ° C.) The reaction mixture was then stored at 4 ° C. until detection. The mixture should be kept at ⁇ 20 ° C. for longer storage Detection of the amplification products
• the digoxigenin-labeled amplification products were carried out using the enzyme detection DNA detection (on the ES devices, Boehringer Mannheim) or using the PCR-ELISA test (on microtiter plates, Boehringer Mannheim).
• the third work station is used for pipetting the amplification products and detection.
• the solution is briefly centrifuged before opening the reaction vessels.
• the solutions are diluted 1 • 10 with the denaturing solution from the above-mentioned kits.
• the concentration of the capture probe was preferably 75 ng / ml in the hybridization solution
• the primers of SEQ ID NO 5, 6, 7, 8 and 9 have also proven effective in the sense of the invention, in particular in the combinations 5/6, 5/7 and 2/9
• Certain pairs are used as primers (SEQ ID NO / SEQ ID NO), the results given in Table 1 having been obtained. For values (extinctions) of more than 100, detection was considered positive. It can be seen that the individual Certain pairs are used as primers (SEQ.ID.NO./SEQ.ID.NO.), The results given in Table 1 being obtained. In the case of values (extinctions) of more than 100, proof was assessed as positive. It can be seen that the individual subtypes can be specifically identified by selecting specific primer pairs (e.g. 2/14, 2/15 and 2/16).
• NAME OF THE INVENTION Reagent and method for the detection of NonA, NonB, NonC, NonD, NonE virus
• MOLECULE TYPE Genomic RNA

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Virology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Communicable Diseases (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=7777986

Family Applications (1)

Country Status (10)

Cited By (1)

Citations (5)

• 1996
  • 1996-11-20 NZ NZ322902A patent/NZ322902A/en unknown
  • 1996-11-20 ZA ZA9609715A patent/ZA969715B/xx unknown
  • 1996-11-20 EP EP96939857A patent/EP0809711A1/de not_active Withdrawn
  • 1996-11-20 WO PCT/EP1996/005108 patent/WO1997019195A1/de not_active Application Discontinuation
  • 1996-11-20 KR KR1019970704944A patent/KR100247215B1/ko not_active IP Right Cessation
  • 1996-11-20 TW TW085114275A patent/TW350876B/zh active
  • 1996-11-20 IL IL12124996A patent/IL121249A/xx not_active IP Right Cessation
  • 1996-11-20 AU AU76938/96A patent/AU730057B2/en not_active Ceased
  • 1996-11-20 JP JP9519389A patent/JPH10511003A/ja active Pending
• 1997
  • 1997-07-18 MX MX9705464A patent/MX9705464A/es unknown

Patent Citations (5)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events