WO1997019195A1

WO1997019195A1 - Amplification of nucleic acids and detection of a new non-a, non-b, non-c, non-d, non-e hepatitis virus

Info

Publication number: WO1997019195A1
Application number: PCT/EP1996/005108
Authority: WO
Inventors: Volker SCHLÜTER; Alfred Engel; Georg Hess; Beatus OFENLOCH-HÄHNLE
Original assignee: Boehringer Mannheim Gmbh
Priority date: 1995-11-21
Filing date: 1996-11-20
Publication date: 1997-05-29
Also published as: EP0809711A1; NZ322902A; MX9705464A; IL121249A0; KR100247215B1; AU730057B2; TW350876B; AU7693896A; ZA969715B; KR19980701553A; JPH10511003A; IL121249A

Abstract

The invention relates to a primer for carrying out a reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of the hepatitis G virus (HGV).

Description

Amplification of nucleic acids and detection of a new hepatitis NonA / NonB / NonC / NonD / NonE virus

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.

In addition to hepatitis A virus (HAV) and hepatitis B virus (HB V), which have been known and characterized for some time, some viruses have recently been characterized which are associated with hepatitis but which are independent groups of viruses. These groups are usually named so that the groups are assigned a consecutive capital letter. Each newly found hepatitis-associated virus is differentiated from the previous one by not belonging to the group of previously known viruses. For example, hepatitis C virus (HCV) was also referred to as NonA / NonB hepatitis virus. The present invention now relates to a virus which obviously cannot be classified into the group of the HAV, HBV, HCV, HDV and HEV viruses.

WO 94/18217 also describes a virus which cannot be assigned to the five groups mentioned. However, 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. In the following, 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.

For the purposes of the invention, 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. In the case of extension by mononucleoside triphosphate units, 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

In the case of a DNA polymerase, the 3 'end is preferably used as the extendable end. In the event that the reagent for the amplification of nucleotide sequences is to be used directly from the genomic RNA of the HGV, reverse transcriptase (RNA-dependent DNA- Polymerase) used. Since reverse transcriptase also has DNA-dependent DNA polymerase activity, a large number of other DNAs are formed from the cDNA initially formed. However, after the formation of DNA, another DNA-dependent DNA polymerase can also be used, eg. B from E coli or Thermus aquaticus

Methods for amplifying nucleotide sequences according to the principle of PCR using reverse transcriptase (RT-PCR) are described, for example, in US-A-5, 310,652 or WO 91/09944. For the purpose of defining the reaction conditions, the content of these patent applications is intended at this point be incorporated

With the help of the two primers, which are complementary to different strands of the nucleotide sequence, 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. However, the amphficate length is particularly preferably between 150 and 200 nucleotides (nt). In addition to the sequences of the primers, 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

An HGV-specific nucleotide sequence is understood to mean a sequence that only occurs in HGV, but not in other viruses and eukaryotes

An 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. In the case of subtype-specific detection, preference is therefore given to at least one of the primers or the probe is selected so that the sequence completely or partially spans one of these regions If one assumes that 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

In the case of subtype-specific detections, for example, a primer HGV subtype can be selected specifically and another HGV subtype-specifically

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

In addition to the HGV-specific nucleotide sequences, however, 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 However, the primers can also be modified further, for example by attaching a detectable or immobilizable group. Such groups can be, for example, directly or indirectly detectable groups, for example radioactive, colored or fluorescent groups. B immunologically or enzymatically active compounds, such as haptens or enzymes. These are detected in a subsequent reaction or reaction sequence. 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

If the 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. Such groups have already been particularly preferred among the primers 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. In the event that 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. In addition, 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.

If, in addition, an even more reliable diagnosis is desired, 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.

The following examples are intended to illustrate the invention:

Example 1 :

HGV-specific proof

Reagents

- QIAamp, HCV-Kit (QIAgen Cat. No. 29504)

- M-MuLV reverse transcriptase (Boehringer Mannheim, 1062603)

- PCR nucleotide mix (Boehringer Mannheim, 1581295)

- Hexamer random mix (Boehringer Mannheim, 1277081)

RNase inhibitor (Boehringer Mannheim, 799017)

- Sterilized double distilled water

- Primer 1 (SEQ.ID.NO. 2)

- Primer 2 (SEQ.ID.NO. 3)

- Biotinylated probe nucleic acid (SEQ.ID.NO. 4), biotinylated via amino linker (Applied Biosystems Inc.)

- PCR ELISA (DIG labeling kit, Boehringer Mannheim, 1636120) (or single reagents, PCR-DIG labeling mix (Boehringer Mannheim, 1585550) and Taq DNA polymerase (Boehringer Mannheim, 1146165))

- Enzymun® test DNA detection (Boehringer Mannheim, 1447777)

General recommendations

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. There should be separate sets at each work station of pipettes. Sealed, aerosol-tight pipette tips or special PCR pipettes should be used to avoid contamination. The reagents should be made fresh daily. The pipettes should be regularly contaminated. Fresh gloves should be used at every work station.

Sample preparation

The QIAamp HCV kit according to the manufacturer's instructions was used for the production of total RNA from human sera. Other RNA manufacturing processes, such as Chomczynski acid phenol extraction or with guanidinium isothiocyanate, can also be used.

cDNA synthesis (reverse transcription)

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.

Table 1

10 μl of the RT mix are pipetted into PCR reaction tubes. 10 μl of the RNA solution are then added. It is briefly treated in a vortex mixer and centrifuged. It is incubated for 10 minutes at room temperature, 30 minutes at 42 ° C and 5 minutes at 95 ° C. The reaction mixture is then stored at 4 ° C. until further processing

Labeling reaction

A further work station and a separate set of pipettes are used to produce the amplification mixture. It is not necessary to pipette the amplification mixture on ice. The reagents used are given in the table below Table 2

40 μl of the amplification mixture are placed in reaction vessels. 10 μl 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 detailed protocols for the detection of amplification products are given in the above-mentioned kits (enzyme test DNA detection) for the ES system (ES 300, ES 600, ES 700) and PCR-Elisa for the microtiter plate format. With the primer pairs 2 / 3 and 2/8, a lower detection limit of less than 10 to 30 copies / batch was reached

Alternative primers

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

With the primer combination 2/8, 3 different genotypes could be detected, so it was an HGV subtype-unspecific detection

Example 2

HGV subtyping

With the exception of the primers, the conditions described in Example 1 were used

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).

Table 3

Values deviating from the expected values were found in the underlying values, but these are correctly obtained when the experiments are repeated under better adapted stringency conditions (2/12 negative and 2/20 positive). The primer sequences and their individual HGV specificity are given in Table 4.

Table 4

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Boehringer Mannheim GmbH

(B) STREET: Sandhoferstr. 116

(C) LOCATION: Mannheim

(E) COUNTRY: DE

(F) POSTAL NUMBER: 68305

(G) TELEPHONE: 0621 759 4348 (H) TELEFAX: 0621 759 4457

(ii) NAME OF THE INVENTION: Reagent and method for the detection of NonA, NonB, NonC, NonD, NonE virus

(iii) NUMBER OF SEQUENCES: 20

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 348 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic RNA

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: YES

(viii) POSITION IN THE GENOME:

(A) CHROMOSOME / SEGMENT: untranslated region

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ACGTGGGGGA GTTGATCCCC CCCCCCCGGC ACTGGGTGCA AGCCCCAGAA ACCGACGCCT 60

ATCTAAGTAG ACGCAATGAC TCGGCGCCGA CTCGGCGACC GGCCAAAAGG TGGTGGATGG 120

GTGATGACAG GGTTGGTAGG TCGTAAATCC CGGTCACCTT GGTAGCCACT ATAGGTGGGT 180

CTTAAGAGAA GGTTAAGATT CCTCTTGTGC CTGCGGCGAG ACCGCGCACG GTCCACAGGT 240 GTTGGCCCTA CCGGTGGGAA TAAGGGCCCG ACGTCAGGCT CGTCGTTAAA CCGAGCCCGT 300

TACCCACCTG GGCAAACGAC GCCCACGTAC GGTCCACGTC GCCCTTCA 348

(2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

CGGCCAAAAG GTGGTGGATG 20

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide (C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CGACGAGCCT GACGTCGGG 19

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(üi) HYPOTHETICAL: NO

(ix) FEATURE:

(A) NAME / KEY: miscjeature (B) LOCATION:!. 19

(D) OTHER INFORMATION: / note = "G at the 5 'end is covalently linked to biotin via aminolink"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GGTAGCCACT ATAGGTGGG 19

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CCAGAAACCG ACGCCTATC 19

(2) INFORMATION ON SEQ ID NO: 6: (i) SEQUENCE LABEL:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CTTATTCCCA CCGGTAGGGC 20

(2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE LABEL:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM. Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CAAGAGAGAC ATTGAAGGGC 20

(2) INFORMATION ABOUT SEQ LD NO: 8:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

AACACCTGTG GACCGTGCG 19

(2) INFORMATION ON SEQ ID NO: 9:

(i) SEQUENCE LABEL:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQIDNO: 9:

CCACTGGTCCTTGTCAACTC 20

(2) INFORMATION QEQIDNO: 10:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQIDNO: 10:

CAATGACTCGGCGCCGAC 18 (2) SPECIFICATIONS: QIDNO: 11:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

TGATGGCCCY GCGCCRAA 18th

(2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE LABEL:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide" (iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

AGAGGAATCT TAACCTTCTC 20

(2) INFORMATION ON SEQ ID NO: 13:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

AGAGGGACCG TAGCCTCCC 19

(2) INFORMATION ON SEQ ID NO: 14:

(i) SEQUENCE LABEL: (A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION, / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQIDNO: 14:

GGTCTCGCCGCAGGCACA 18

(2) INFORMATION ZSQIDNO: 15:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CGTTCTCGCC ACGGGCATT 19

(2) INFORMATION ON SEQ ID NO: 16:

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other nucleic acid

(A) DESCRIPTION: / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQIDNO: 16:

CTTTCYCTCCRTAAGCGCG 19

(2) INFORMATIONS QIDNO: 17

(i) SEQUENCE LABEL:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear 2 B

(ii) MOLECULE TYPE Other nucleic acid

(A) DESCRIPTION / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICALLY NO

(xi) SEQUENCE DESCRIPTION SEQ ID NO 17

TCGGGCCCTT ATTCACACC 19

(2) INFORMATION ON SEQ ID NO 18

(i) SEQUENCE LABEL

(A) LONG. 18 base pairs

(B) ART. Nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(ii) MOLECULE TYPE Other nucleic acid

(A) DESCRIPTION / desc = "oligodeoxynbonucleotide"

(iii) HYPOTHETICALLY NO

(xi) SEQUENCE DESCRIPTION SEQ ID NO 18

CCGGGYCCTT ATTACACC 18

(2) INFORMATION ON SEQ ID NO 19 (i) SEQUENCE LABEL

(A) LONG 19 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(ii) TYPE OF MOLECULE Other nuclear acid

(A) DESCRIPTION / desc = "Ohgodeoxynbonucleotide"

(iii) HYPOTHETICALLY NO

(xi) SEQUENCE DESCRIPTION SEQ ID NO 19

TAACGACGAG CCTGACGTC 19

(2) INFORMATION ON SEQ ID NO 20

(l) SEQUENCE LABEL

(A) LONG 19 base pairs

(B) ART nucleotide

(C) STRANDFORM single strand

(D) TOPOLOGY linear

(ii) MOLECULE TYPE Other nucleic acid

(A) DESCRIPTION / desc = "oligodeoxyribonucleotide"

(iii) HYPOTHETICALLY NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

TAACGGCGTG CCTAGCGCC 19

Claims

PATENT CLAIMS

Reagent for the amplification of HGV-specific nucleotide sequences, wherein HGV is defined as a virus whose genome consists of RNA which contains at the 5 'end a nucleoside sequence which is at least 80% homologous to 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% complementary to successive bases of SEQ ID NO 1, and the other of which a sequence of 15 to 30 Contains bases that are more than 80% homologous to successive bases 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.

Reagent according to Claim 1, characterized in that at least one of the primers has a sequence of 6 successive bases which are in the sequences of the

SEQ.ID NO 1 are contained, or contains a sequence complementary thereto

Reagent according to one of claims 1 or 2, characterized in that at least one of the primers contains a sequence of SEQ ID NO 2 or 3 or 5 to 20

Reagent according to one of the preceding claims, characterized in that at least one of the primers is detectably labeled

Reagent according to one of the preceding claims, characterized in that one or more of the primers are HGV-subtype-unspecific

Reagent according to one of claims 1 to 4, characterized in that one or more of the primers are HGV-specific 7. Reagent according to one of claims 5 or 6, characterized in that one primer is HGV subtype-specific and another HGV subtype-specific.

8. reagent kit for the specific detection of HGV containing a reagent according to one of the preceding claims and a probe which contains a base sequence which lies in the extension product formed between the extended ends of the primers.

9. Reagent kit according to claim 8, characterized in that the base sequence has at least 6 successive bases which are in the sequences of SEQ.ID.NO. 2 to 20 are contained, or contains a sequence complementary to this.

10 reagent kit for the specific detection of HGV containing a reagent according to one of claims 1 to 7 and a further reagent containing further primers for the amplification of HGV nucleotide sequences.

11. Reagent kit according to claim 10, containing a probe for the detection of the extension products for each pair of primers.

12. A method for the detection of HGV in a sample, characterized by the formation of cDNA from part of the HGV-RNS and amplification of the cDNA with extension of the primers of a reagent according to one of claims 1 to 7 with formation of amplification products, bringing the amplification products into contact with one Probe which contains a base sequence which lies between the extended ends of the primers and determination of the formation of hybrids of the probe with amplification products as a sign of the presence of HGV in the sample.

13. Method for the detection of HGV in a sample characterized by

Formation of cDNA from two or more parts of the HGV-RNA and amplification of the cDNA using a reagent according to one of claims 1 to 7 and using another pair of primers which is HGV-specific, Contacting the amplification products with one probe per amplified part,

- Determination of the formation of hybrids of the probes with the amplification products and

- Evaluation of the hybrid formation between the probes and amplification products, the determination of the hybrid formation of an amplification product being evaluated as a sign of the presence of HGV in the sample.