Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-01-29
2003-07-01
Housel, James (Department: 1628)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024200, C536S024330, C435S005000, C435S006120, C435S069100, C435S091200, C435S091210, C435S450000
Reexamination Certificate
active
06586584
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to materials and methods for detection of Hepatitis C viral nucleic acids, in particular to probes and primers for detection of Hepatitis C in hybridization and amplification assays.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) is a member of the virus family Flaviviridae and infects at least 1% of the world's population. Infected individuals are at increased risk to develop cirrhosis of the liver and hepatocellular carcinoma. The viral genome is a single strand of RNA which contains a single gene. The polyprotein which is expressed is subsequently processed into at least ten functional proteins. The genome of HCV is highly heterogeneous and has an estimated mutation rate of about 10
−2
per base per generation. At least 100 strains have been identified and grouped into six major genotypes.
At the present time there is no reliable method for growth of HCV in vitro, which makes immunological methods of detection difficult to perform and the results unreliable. Quantitation of viral RNA in plasma is used extensively as a prognostic marker for patients undergoing treatment and as a means for monitoring their response to therapy. Due to the genomic heterogeneity, however, existing molecular assays for detection of HCV RNA are limited by their inability to detect all genotypes with equal efficiency. The probes and primers of the present invention may provide rapid and sensitive detection of HCV nucleic acids and offer an attractive alternative to immunological assays.
SUMMARY OF THE INVENTION
The present invention provides primers and probes derived from the 5′ untranslated region of the HCV genome which are predicted to facilitate detection and/or quantification of all presently known genotypes of HCV (1-6). That is, a single amplification primer pair according to the invention should efficiently amplify all known genotypes of HCV, which may then be detected in a single detection step using the detector probes and primers of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The primers, hybridization probes and detector primers of the present invention are based on portions of the 5′ untranslated region (UTR) of the HCV genome. Initially, design of the disclosed primers and probes was based on relatively conserved regions in an alignment of multiple HCV sequences. One goal was to develop probes and primers which, in spite of heterogeneity in the sequence, would be expected to provide amplification, detection and/or quantitation of all presently known HCV genotypes with approximately equal efficiency. In some cases this was accomplished by overlapping the hybridization site of the 5′ ends of certain of the detector probes with the hybridization site of the 3′ end an amplification primer. This approach took advantage of short stretches of relative sequence conservation in the primer hybridization region and avoided much of the sequence heterogeneity evident in the intervening region between the two amplification primers. This technique also allowed use of a smaller target sequence, thereby improving amplification efficiency.
As used herein, an amplification primer is an oligonucleotide for amplification of a target sequence by extension of the oligonucleotide after hybridization to the target sequence or by ligation of multiple oligonucleotides which are adjacent when hybridized to the target sequence. At least a portion of the amplification primer hybridizes to the target. This portion is referred to as the target binding sequence and it determines the target-specificity of the primer. In addition to the target binding sequence, certain amplification methods require specialized non-target binding sequences in the amplification primer. These specialized sequences are necessary for the amplification reaction to proceed and typically serve to append the specialized sequence to the target. For example, the amplification primers used in SDA (Strand Displacement Amplification) include a restriction endonuclease recognition site 5′ to the target binding sequence (U.S. Pat. Nos. 5,455,166 and 5,270,184). NASBA, (Nucleic Acid Sequence Based Amplification) 3SR (Self Sustaining Sequence Replication) and transcription based amplification primers require an RNA polymerase promoter linked to the target binding sequence of the primer. Linking such specialized sequences to a target binding sequence for use in a selected amplification reaction is routine in the art. In contrast, amplification methods such as PCR which do not require specialized sequences at the ends of the target, generally employ amplification primers consisting of only target binding sequence.
As used herein, the terms “primer” and “probe” refer to the function of the oligonucleotide. A primer is typically extended by polymerase or ligation following hybridization to the target but a probe typically is not. A hybridized oligonucleotide may function as a probe if it is used to capture or detect a target sequence, and the same oligonucleotide may function as a primer when it is employed as a target binding sequence in an amplification primer. It will therefore be appreciated that any of the target binding sequences disclosed herein for amplification, detection or quantitation of HCV may be used either as hybridization probes or as target binding sequences in primers for detection or amplification, optionally linked to a specialized sequence required by the selected amplification reaction or to facilitate detection.
Based on the alignment of the 5′ untranslated regions of multiple HCV genotypes, the following amplification primers were designed for testing in SDA reactions. Target binding sequences are underlined. The remaining 5′ portion of the sequence comprises the restriction endonuclease recognition site (RERS) that is required for the SDA reaction to proceed plus a generic non-target-specific tail sequence. It will be readily apparent that the target binding sequences may be used alone to amplify the target in reactions which do not require specialized sequences or structures (e.g., PCR) and that other specialized sequences required by amplification reactions other than SDA (e.g., an RNA polymerase promoter) may be substituted for the RERS-containing sequence shown below. “S1” and “S2” in the primer name indicates “right” and “left” primers, respectively, when the oligonucleotides are used in amplification reactions:
AMPLIFICATION PRIMERS FOR HCV REGION 1
1S1.1
CGATTCGCCTCCAGACTTCTCGGG
TGGTCTGCGGAAC
SEQ ID NO:1
1S1.2
CGATTCGCCTCCAGACTTCTCGGGA
TGGTCTGCGGAAC
SEQ ID NO:2
1S2.1
ACCGCATCGAATGACTGTCTCGGG
GAAAGGACCCGGT
SEQ ID NO:3
1S2.1a
ACTCGCATCGAATGACTGTCTCGGG
GAAAGGACCCGGT
SEQ ID NO:4
1S2.2
ACCGCATCGAATGACTGTCTCGGG
GAAAGGACCCAGT
SEQ ID NO:5
1S2.2a
ACTCGCATCGAATGACTGTCTCGGG
GAAAGGACCCAGT
SEQ ID NO:6
1S2.3
ACCGCATCGAATGACTGTCTCGGG
GAAAGGACCC(T)GTC
SEQ ID NO:7
1S2.3a
ACTCGCATCGAATGACTGTCTCGGG
GAAAGGACCC(T)GTC
SEQ ID NO:8
AMPLIFICATION PRIMERS FOR HCV REGION 3
3S1a.1
CGATTCCGCTCCAGACTTCTCGGG
TGGGT(A)GCGAAAGGC
SEQ ID NO:9
3S1b.1
CGATTCCGCTCCAGACTTCTCGGG
TGGGT(A)GCGAAAGG
SEQ ID NO:10
3S1c.1
CGATTCCGCTCCAGACTTCTCGGG
GGGT(A)GCGAAAGGC
SEQ ID NO:11
3S2a.1
ACCGCATCGAATGCATGTCTCGGG
CTCC(T)GGGGCACT
SEQ ID NO:12
3S2b.1
ACCGCATCGAATGCATGTCTCGGG
CCTCC(T)GGGGCACT
SEQ ID NO:13
3S2c.1
ACCGCATCGAATGCATGTCTCGGG
CCTCC(T)GGGGCAC
SEQ ID NO:14
3S2d.1
ACCGCATCGAATGCATGTCTCGGG
GGCACTCGCAAGC
SEQ ID NO:15
(X) = Deliberately mismatched bases
The following detector primers were also designed for detection of amplification products produced using the amplification primers. They hybridize to the target sequence downstream of amplification primers so that they are displaced during the amplification reaction. An advantage of this detection method is that the target sequence can be detected and/or quantified as the amplification reaction is occurring, i.e., in “real-time” rather than at an endpoint, as is known in the art. The target binding sequences of the primers are underlined. The remaining portion of the sequence forms a hair
Hellyer Tobin J.
McMillian Ray A.
Becton Dickinson and Company
Housel James
Kiang Allan M.
Li B Q
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