Oligonucleotide primers for amplifying HCV nucleic acid

The present invention provides improved primers for the polymerase chain reaction (PCR) amplification of a nucleic acid sequence from hepatitis C virus (HCV). The primers and amplification methods of the invention enable the detection of HCV with greatly increased sensitivity.

This application claims priority to U.S. provisional application No. 
60/007,739, filed Nov. 29, 1995. 
BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the field of molecular biology and nucleic 
acid chemistry. More specifically, it relates to methods and reagents for 
amplifying hepatitis C virus (HCV) nucleic acid. The invention therefore 
has applications in the detection of HCV, the field of medical diagnostics 
generally, and the field of molecular biology. 
2. Description of Related Art 
The Hepatitis C virus is a small RNA virus containing a single, positive 
sense, molecule of RNA about 10,000 nucleotides in length. The 
prototypical HCV was described in Choo et al., 1989, Science 244:359-362; 
Choo et al., 1991, Proc. Natl. Acad. Sci. USA 88:2451-2455; and European 
Patent Publication Nos. 318,216; 388,232; and 398,748. The genome is 
believed to contain a single, long, open reading frame that is translated 
into a single, large polyprotein and subsequently processed. The genome is 
known to contain a 5' untranslated region (UTR) upstream of the open 
reading frame. 
The genome of HCV exhibits a large degree of nucleic acid sequence 
heterogeneity among strains and isolates (see Simmonds, 1995, Hepatology 
21:570-583, and Bukh et al., 1994, Proc. Nati. Acad. Sci. USA 
91:8239-8243, both incorporated herein by reference). The 5' UTR sequence, 
however, is known to be relatively conserved. 
The invention of the polymerase chain reaction (PCR), a method for 
amplifying specific sequences of nucleic acids, makes possible the rapid 
detection of nucleic acids present in a sample in what was previously an 
undetectably low quantity (see U.S. Pat. Nos. 4,683,195; 4,683,202; and 
4,965,188, each of which is incorporated herein by reference). HCV genomic 
RNA can be detected by reverse transcribing HCV genomic RNA to form cDNA, 
amplifying the resulting cDNA by PCR, and detecting the presence of 
amplified product. 
HCV detection assays based on PCR amplification of HCV genomic sequences 
were described in copending U.S. patent application Ser. No. 08/240,547, 
now allowed; European Patent Publication No. 529,493; Young et al., 1993, 
J. Clin. Microbiol. 31(4):882-886; and Young et al., 1995, J. sin. 
Microbiol. 33(3):654-657, each incorporated herein by reference. As 
described therein, amplification of HCV RNA can be carried out using a 
combined reverse transcription-polymerase chain reaction (RT-PCR) 
amplification, in which a single enzyme catalyzes the primer extension 
both from the initial genomic RNA template (i.e., reverse transcription) 
and from the DNA templates synthesized in the amplification process. 
SUMMARY OF THE INVENTION 
The present invention provides improved oligonucleotide primers for the 
efficient reverse transcription-polymerase chain reaction (RT-PCR) 
amplification of a region of the 5' untranslated region of the hepatitis C 
virus (HCV) genome. 
An important advantage of the primers of the present invention over primers 
described in the prior art is that the present primers enable 
amplification of HCV nucleic acid with significantly higher efficiency. As 
shown in the examples, amplifications of HCV nucleic acid using the 
primers of the present invention are up to a 100-fold more efficient than 
amplifications using the primers described in the prior art. The 
significantly greater amplification efficiency obtained using the primers 
of the present invention is surprising and unexpected in view of the prior 
art. 
Another aspect of the invention relates to methods for amplifying a region 
of the HCV genome which comprise carrying out a polymerase chain reaction 
using the primers of the invention. Because of the significantly enhanced 
amplification efficiency obtained using the primers of the present 
invention, the amplification methods of the present invention provide 
significantly more amplified product while reducing the amount of 
primer-dimer formed. As a consequence, the methods of the present 
invention enable significantly more sensitive HCV detection assays. Thus, 
the present invention also provides methods for detecting the presence of 
HCV nucleic acid in a sample comprising: 
(a) treating said sample in a PCR reaction mixture containing the primers 
of the present invention under amplification conditions so that HCV 
nucleic acid, if present, is amplified; and 
(b) detecting if amplification has occurred, which indicates that HCV 
nucleic acid is present. 
Another aspect of the invention relates to kits which contain the 
amplification primers of the invention. These kits can include additional 
reagents, such as oligonucleotide probes for the detection of the 
amplified nucleic acid, and one or more amplification reagents, e.g., 
polymerase, buffers, and nucleoside triphosphates.

DETAILED DESCRIPTION OF THE INVENTION 
To aid in understanding the invention, several terms are defined below. 
The terms "nucleic acid" and "oligonucleotide" refer to primers, probes, 
and oligomer fragments to be amplified or detected, and shall be generic 
to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to 
polyribonucleotides (containing D-ribose), and to any other type of 
polynucleotide which is an N glycoside of a purine or pyrimidine base, or 
modified purine or pyrimidine base. There is no intended distinction in 
length between the terms "nucleic acid" and "oligonucleotide", and these 
terms will be used interchangeably. These terms refer only to the primary 
structure of the molecule. Thus, these terms include double- and 
single-stranded DNA, as well as double- and single-stranded RNA. 
Oligonucleotides can be prepared by any suitable method, including, for 
example, cloning and restriction of appropriate sequences and direct 
chemical synthesis by a method such as the phosphotriester method of 
Narang et al., 1979, Meth. Enzyrol. 68:90-99; the phosphodiester method of 
Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethylphosphoramidite 
method of Beaucage et al., 1981, Tetrahedron Lett. 22:1859-1862; and the 
solid support method of U.S. Pat. No. 4,458,066, each incorporated herein 
by reference. Methods for synthesizing labeled oligonucleotides are 
described in Agrawal and Zamecnik, 1990, Nucl. Acids. Res. 
18(18):5419-5423; MacMillan and Verdine, 1990, J. Org. Chem. 55:5931-5933; 
Pieles et al, 1989, Nucl. Acids. Res. 17(22):8967-8978; Roget et al., 
1989,Nucl. Acids. Res. 17(19):7643-7651;and Tesler et al., 1989, J. Am. 
Chem. Soc. 111:6966-6976, each incorporated herein by reference. A review 
of synthesis methods is provided in Goodchild, 1990, Bioconiulate 
Chemistry 1(3):165-187, incorporated herein by reference. 
The term "hybridization" refers to the formation of a duplex structure by 
two single-stranded nucleic acids due to complementary base pairing. 
Hybridization can occur between fully (exactly) complementary nucleic acid 
strands or between "substantially complementary" nucleic acid strands that 
contain minor regions of mismatch. Conditions under which only fully 
complementary nucleic acid strands will hybridize are referred to as 
"stringent hybridization conditions" or "sequence-specific hybridization 
conditions". Stable duplexes of substantially complementary sequences can 
be achieved under less stringent hybridization conditions. Those skilled 
in the art of nucleic acid technology can determine duplex stability 
empirically following the guidance provided by the art (see, e.g., 
Sambrook et al., 1985, Molecular Cloning--A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, N.Y., incorporated herein by 
reference). 
Generally, stringent conditions are selected to be about 5.degree. C. lower 
than the thermal melting point (Tm) for the specific sequence at a defined 
ionic strength and pH. The Tm is the temperature (under defined ionic 
strength and pH) at which 50% of the base pairs have dissociated. Relaxing 
the stringency of the hybridization conditions will allow sequence 
mismatches to be tolerated; the degree of mismatch tolerated can be 
controlled by suitable adjustment of the hybridization conditions. 
The term "primer" refers to an oligonucleotide capable of acting as a point 
of initiation of DNA synthesis under conditions in which synthesis of a 
primer extension product complementary to a nucleic acid strand is 
induced, i.e., in the presence of four different nucleoside triphosphates 
and an agent for polymerization (i.e., DNA polymerase or reverse 
transcriptase) in an appropriate buffer and at a suitable temperature. A 
primer is preferably a single-stranded oligodeoxyribonucleotide. The 
appropriate length of a primer depends on the intended use of the primer 
but typically ranges from 15 to 35 nucleotides. Short primer molecules 
generally require cooler temperatures to form sufficiently stable hybrid 
complexes with the template. A primer need not reflect the exact sequence 
of the template nucleic acid, but must be sufficiently complementary to 
hybridize with the template. Primers can incorporate additional features 
which allow for the detection or immobilization of the primer but do not 
alter the basic property of the primer, that of acting as a point of 
initiation of DNA synthesis. For example, primers may contain an 
additional nucleic acid sequence at the 5' end which does not hybridize to 
the target nucleic acid, but which facilitates cloning of the amplified 
product. The region of the primer which is sufficiently complementary to 
the template to hybridize is referred to herein as the hybridizing region. 
As used herein, an "upstream" primer refers to a primer whose extension 
product is a subsequence of the coding strand; a "downstream" primer 
refers to a primer whose extension product is a subsequence of the 
complementary non-coding strand. A primer used for reverse transcription, 
referred to as an "RT primer", hybridizes to the coding strand and is thus 
a downstream primer. 
The term "oligonucleotide probe", as used herein, refers to a 
oligonucleotide which forms a duplex structure with a sequence of a target 
nucleic acid due to complementary base pairing. Probes are used for the 
detection or capture of the target nucleic acid. A probe is preferably a 
single-stranded oligodeoxyribonucleotide. The probe typically will consist 
of, or contain, a "hybridizing region" consisting preferably of from 10 to 
50 nucleotides, more preferably from 15 to 35 nucleotides, corresponding 
to a region of the target sequence. "Corresponding" means at least 
substantially complementary to either the designated nucleic acid or its 
complement. A probe need not reflect the exact sequence of the target 
nucleic acid, but must be sufficiently complementary to hybridize with the 
target under the hybridization conditions chosen. A probe oligonucleotide 
can contain, or be bound to, additional features which allow for the 
detection or immobilization of the probe but do not significantly alter 
the hybridization characteristics of the hybridizing region. For example, 
probes may be labeled by the incorporation of radiolabeled nucleotides or 
by being bound to a separate detectable moiety. 
As used herein, an oligonucleotide primer or probe is "specific" for a 
target sequence if the number of mismatches present between the 
oligonucleotide and the target sequence is less than the number of 
mismatches present between the oligonucleotide and non-target sequences. 
Hybridization conditions can be chosen under which stable duplexes are 
formed only if the number of mismatches present is no more than the number 
of mismatches present between the oligonucleotide and the target sequence. 
Under such conditions, the target-specific oligonucleotide can form a 
stable duplex only with a target sequence. The use of target-specific 
primers under suitably stringent amplification conditions enables the 
specific amplification of those target sequences which contain the target 
primer binding sites. Similarly, the use of target-specific probes under 
suitably stringent hybridization conditions enables the detection of a 
specific target sequence. 
The terms "target region" and "target nucleic acid" refers to a region of a 
nucleic acid which is to be amplified, detected, or otherwise analyzed. 
The sequence to which a primer or probe hybridizes can be referred to as a 
"target". 
The term "thermostable DNA polymerase" refers to an enzyme that is 
relatively stable to heat and catalyzes the polymerization of nucleoside 
triphosphates to form primer extension products that are complementary to 
one of the nucleic acid strands of the target sequence. The enzyme 
initiates synthesis at the 3' end of the primer and proceeds in the 
direction toward the 5' end of the template until synthesis terminates. 
Purified thermostable DNA polymerases are commercially available from 
Perkin-Elmer, Norwalk, Conn. 
The terms "amplification reaction mixture" and "polymerase chain reaction 
mixture" refer to a combination of reagents that is suitable for carrying 
out a polymerase chain reaction. The reaction mixture typically consists 
of oligonucleotide primers, nucleotide triphosphates, and a DNA polymerase 
in a suitable buffer. Preferred amplification reaction mixtures are 
provided in the examples. 
The term "amplification conditions", as used herein, refers to reaction 
conditions suitable for the amplification of the target nucleic acid 
sequence. The amplification conditions refers both to the amplification 
reaction mixture and to the temperature cycling conditions used during the 
reaction. Under amplification conditions using the primers of the present 
invention, amplification of HCV nucleic acid, if present, will occur. 
Preferred amplification conditions are provided in the examples. 
The term "amplification efficiency", as used herein, refers to the amount 
of product produced from a given initial number of target sequences in a 
given number of amplification cycles. Thus, the amplification efficiencies 
of two reaction which differ only in the primers used are compared by 
quantitatively measuring the amount of product formed in each reaction. 
Conventional techniques of molecular biology and nucleic acid chemistry, 
which are within the skill of the art, are fully explained in the 
literature. See, for example, Sambrook et al., 1989, Molecular Cloning--A 
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid 
Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series, 
Methods in Enzynology (Academic Press, Inc.), all of which are 
incorporated herein by reference. All patents, patent applications, and 
publications mentioned herein, both supra and infra, are incorporated 
herein by reference. 
HCV Amplification Primers 
The nucleotide sequences of the primers are provided in Table 1, shown in 
the 5' to 3' orientation. Amplifications using the upstream primer with 
either of the downstream primers amplify a 240 base pair product from the 
5' untranslated region of the HCV genome. The primers hybridize to 
relatively conserved regions within the 5' untranslated region of the HCV 
genome and enable the amplification of nucleic acid from the known HCV 
isolates without the simultaneous amplification of non-target sequences 
from other viruses or from human genomic DNA. 
TABLE 1 
______________________________________ 
HCV Amplification Primers 
______________________________________ 
Upstream Seq. ID No. 
ST280A 1 5'-GCAGAAAGCGTCTAGCCATGGCGTTA 
Downstream (RT) 
ST778AA 2 5'-GCAAGCACCCTATCAGGCAGTACCACAA 
ST678A 3 5'-GCAAGCACCCTATCAGGCAGTACCACA 
______________________________________ 
Amplification 
The polymerase chain reaction (PCR) amplification process is well known in 
the art and described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 
4,965,188. Commercial vendors, such as Perkin Elmer (Norwalk, Conn.), 
market PCR reagents and publish PCR protocols. For ease of understanding 
the advantages provided by the present invention, a summary of PCR is 
provided. 
In each cycle of a PCR amplification, a double-stranded target sequence is 
denatured, primers are annealed to each strand of the denatured target, 
and the primers are extended by the action of a DNA polymerase. The 
process is repeated typically at least 25 times. The two primers anneal to 
opposite ends of the target nucleic acid sequence and in orientations such 
that the extension product of each primer is a complementary copy of the 
target sequence and, when separated from its complement, can hybridize to 
the other primer. Each cycle, if it were 100% efficient, would result in a 
doubling of the number of target sequences present. 
Either DNA or RNA target sequences can be amplified by PCR. In the case of 
an RNA target, such as in the amplification of HCV genomic nucleic acid as 
described herein, the first step consists of the synthesis of a DNA copy 
(cDNA) of the target sequence. The reverse transcription can be carried 
out as a separate step, or, preferably, in a combined reverse 
transcription-polymerase chain reaction (RT-PCR), a modification of the 
polymerase chain reaction for amplifying RNA. The RT-PCR amplification of 
RNA is well known in the art and described in U.S. Pat. Nos. 5,322,770 and 
5,310,652; Myers and Gelfand, 1991, Biochemistry 30(31):7661-7666; 
Copending U.S. patent application Ser. No. 08/240,547, now allowed; Young 
et al., 1993, J. Clin. Microbiol. 31(4):882-886; and Young et al., 1995, 
J. Clin. Microbiol. 33(3):654-657; each incorporated herein by reference. 
Various sample preparation methods suitable for RT-PCR have been described 
in the literature. For example, techniques for extracting ribonucleic 
acids from biological samples are described in Rotbart et al., 1989, in 
PCR Technology (Erlich ed., Stockton Press, N.Y.) and Han et al., 1987, 
Biochemistry 2:1617-1625, both incorporated herein by reference. The 
particular method used is not a critical part of the present invention. 
One of skill in the art can optimize reaction conditions for use with the 
known sample preparation methods. Preferred sample preparation methods for 
use in the detection of HCV RNA are described in Copending U.S. patent 
application Ser. No. 08/240,547, now allowed; Young et al., 1993, supra, 
and Young et al, 1995, supra. 
Due to the enormous amplification possible with the PCR process, low levels 
of DNA contamination from samples with high DNA levels, positive control 
templates, or from previous amplifications can result in PCR product, even 
in the absence of purposefully added template DNA. Laboratory equipment 
and techniques which will minimize cross contamination are discussed in 
Kwok and Higuchi, 1989, Nature, 339:237-238 and Kwok and Orrego, in: Innis 
et al. eds., 1990 PCR Protocols: A Guide to Methods and Applications, 
Academic Press, Inc., San Diego, Calif., which are incorporated herein by 
reference. Enzymatic methods to reduce the problem of contamination of a 
PCR by the amplified nucleic acid from previous reactions are described in 
PCT patent publication No. US 91/05210, U.S. Pat. No. 5,418,149, and U.S. 
Pat. No. 5,035,996, each incorporated herein by reference, and in Young et 
al., 1995, supra. 
Amplification reaction mixtures are typically assembled at room 
temperature, well below the temperature needed to insure primer 
hybridization specificity. Non-specific amplification may result because 
at room temperature the primers may bind non-specifically to other, only 
partially complementary nucleic acid sequences, and initiate the synthesis 
of undesired nucleic acid sequences. These newly synthesized; undesired 
sequences can compete with the desired target sequence during the 
amplification reaction and can significantly decrease the amplification 
efficiency of the desired sequence. Non-specific amplification can be 
reduced using a "hot-start" wherein primer extension is prevented until 
the temperature is raised sufficiently to provide the necessary 
hybridization specificity. 
In one hot-start method, one or more reagents are withheld from the 
reaction mixture until the temperature is raised sufficiently to provide 
the necessary hybridization specificity. Hot-start methods which use a 
heat labile material, such as wax, to separate or sequester reaction 
components are described in U.S. Pat. No. 5,411,876 and Chou et al, 1992, 
Nucl. Acids Res. 20(7):1717-1723, both incorporated herein by reference. 
In another hot-start method, a reversibly inactivated DNA polymerase is 
used which does not catalyze primer extension until activated by a high 
temperature incubation prior to, or as the first step of, the 
amplification (see copending U.S. patent application Ser. No. 60/002,673, 
filed Aug. 25, 1995, incorporated herein by reference). Non-specific 
amplification also can be reduced by enzymatically degrading extension 
products formed prior to the initial high-temperature step of the 
amplification, as described in U.S. Pat. No. 5,418,149, which is 
incorporated herein by reference. 
Analysis of Amplified Product 
In a preferred embodiment of the present invention, amplification of HCV 
genomic nucleic acid is carried out as part of an HCV detection assay. The 
amplification is carried out to increase the amount of HCV nucleic acid to 
a detectable level. Methods for detecting PCR amplified nucleic acids are 
well known in the art. For example, the presence and quantity of amplified 
product can be assayed directly using gel electrophoresis using protocols 
well known in the art (see, for example, Sambrook et al., 1989, supra). 
Detection of the amplified product can be carried using oligonucleotide 
probes which hybridize specifically to the amplified HCV nucleic acid. 
Suitable protocols for detecting hybrids formed between probes and target 
nucleic acid sequences are known in the art. HCV nucleic acid amplified 
using the primers of Table 1 can be detected using the probes and methods 
described in Copending U.S. patent application Ser. No. 08/240,547, now 
U.S. Pat. No. 5,527,669 Young et al., 1993, supra; and Young et al, 1995, 
supra. 
A preferred assay method, referred to as the 5'-nuclease assay, is 
described in U.S. Pat. No. 5,210,015 and Holland et al., 1991, Proc. Natl. 
Acad. Sci. USA 88:7276-7280, both incorporated herein by reference. In the 
5'-nuclease assay, labeled detection probes are involved in the PCR 
amplification reaction mixture. The probes are modified so as to prevent 
the probes from acting as primers for DNA synthesis. Any probe which is 
hybridized to target DNA during a synthesis step, i.e., during primer 
extension, is degraded by the 5'-nuclease activity of the DNA polymerase, 
e.g., rTth DNA polymerase. The presence of degraded probe indicates both 
that hybridization between probe and target DNA occurred and that 
amplification occurred. Methods for detecting probe degradation are 
described in the '015 patent, and U.S. Pat. Nos. 5,491,063 and 5,571,673 
both incorporated herein by reference, and in the examples, below. 
The probe-based assay formats described above typically utilize labeled 
oligonucleotides to facilitate detection of the hybrid duplexes. 
Oligonucleotides can be labeled by incorporating a label detectable by 
spectroscopic, photochemical, biochemical, immunochemical, or chemical 
means. Useful labels include 32p, fluorescent dyes, electron-dense 
reagents, enzymes (as commonly used in ELISAS), biotin, or haptens and 
proteins for which antisera or monoclonal antibodies are available. 
Labeled oligonucleotides of the invention can be synthesized using the 
techniques described above. 
An alternative method for detecting the amplification of HCV nucleic acid, 
in which the increase in the total amount of double-stranded DNA in the 
reaction mixture is monitored, is described in Higuchi et al., 1992, 
Bio/Technology 10:413-417; Higuchi et al., 1993, Bio/Technology 
11:1026-1030; copending U.S. patent application Ser. No. 07/695,210, filed 
May 2, 1991; and European Patent Publication Nos. 487,218 and 512,334, 
each incorporated herein by reference. The detection of double-stranded 
target DNA relies on the increased fluorescence that ethidium bromide 
(EtBr) and other DNA binding labels exhibit when bound to double-stranded 
DNA. Amplification increases the amount of double-stranded DNA and results 
in a detectable increase in fluorescence. Because non-specific 
amplification and, in particular, primer-dimer, also results in the 
formation of double-stranded DNA, reduction of non-specific amplification 
is desirable. The primers of the present invention are particularly useful 
because they enable amplification with unexpectedly low levels of 
background non-specific amplification products. 
The amplification methods and primers of the present invention are not 
limited to use in detection assays. For example, as is well known in the 
art, amplified nucleic acid can be used in cloning or sequencing (see, for 
example, U.S. Pat. No. 4,683,195). The present primers are useful in 
general due to the higher yields of amplified nucleic acid and lower level 
of non-specific amplification products obtained. 
The present invention also relates to kits, multicontainer units comprising 
useful components for practicing the present method. A useful kit contains 
primers for the amplification of HCV nucleic acid. A kit can also contain 
means for detecting amplified HCV nucleic acid, such as oligonucleotide 
probes. Other optional components of the kit include, for example, an 
agent to catalyze the synthesis of primer extension products, substrate 
nucleoside triphosphates, appropriate buffers for amplification or 
hybridization reactions, and instructions for carrying out the present 
method. The examples of the present invention presented below are provided 
only for illustrative purposes and not to limit the scope of the 
invention. Numerous embodiments of the invention within the scope of the 
claims that follow the examples will be apparent to those of ordinary 
skill in the art from reading the foregoing text and following examples. 
EXAMPLE 1 
Amplification of HCV RNA 
Amplifications of HCV RNA were carried out using the following protocol. 
Sample Preparation 
Amplifications were carried out both using synthetic RNA templates and 
using RNA isolated from clinical samples. The use of a synthetic template 
allowed control over the number of target RNA molecules added to each 
reaction. Synthetic RNA templates were transcribed using an HCV RNA 
transcription vector as described in Young et al., 1993, supra. For 
amplifications of HCV RNA from clinical samples, RNA was isolated from 
serum as described in Young et al., 1995, supra. 
Ampliflcation 
Amplifications were carried out in 100 .mu.l reactions volumes. Each 
reaction contained the following reagents: 
HCV RNA template, 
400 nM each primer (except where noted), 
1 .mu.M labeled probe, 
50 mM Bicine (pH 8.3) 
100 mM KOAc, 
200 .mu.M each dATP, dCTP, dGTP, and dUTP, 
3.6 mM Mn(OAc).sub.2, 
8% glycerol, 
20 units of rTth DNA polymerase*, and 
2 units of UNG*. 
* manufactured and developed by Hoffmann-La Roche and marketed by Perkin 
Elmer, Norwalk, Conn. 
A detection probe was included in each reaction mixture to enable detection 
of the amplified product using the 5'-nuclease assay, as described below. 
The probe used was KY150, described in copending U.S. patent application 
Ser. No. 08/240,547, now U.S. Pat. No. 5,527,669, and Young et al., 1995, 
supra. The probe was synthesized with fluorescein (FAM) (Perkin Elmer, 
Applied Biosystems Division, Foster City, Calif.) bound at the 5' end and 
a 3'-PO.sub.4 instead of a 3'-OH to block any extension by the DNA 
polymerase. 
Amplifications were carried out in a GeneAmp TC9600 DNA thermal cycler 
using thin-walled MicroAmp reaction tubes (both from Perkin Elmer, 
Norwalk, Conn.), using the following temperature profile: 
Pre-reaction incubation 50.degree. C. for 2 minutes; 
Reverse transcription 60.degree. C. for 30 minutes 
2 cycles: denature 95.degree. C. for 15 seconds, anneal/extend 60.degree. 
C. for 20 seconds 
46 cycles: denature 90.degree. C. for 15 seconds, anneal/extend 60.degree. 
C. for 20 seconds 
Hold 72.degree. C. for no more than 15 minutes 
Following the temperature cycling, the reactions were held at -20.degree. 
C. before analysis. 
Detection of Amplified Product 
Amplified HCV nucleic acid was analyzed both by gel electrophoresis and 
using the 5'-nuclease assay. Gel electrophoresis provided an easily 
visualized confirmation of the presence of amplified product and a rough 
estimate of the relative amount of amplification product produced. The 
5'-nuclease assay was used to provide an accurate quantitative estimate of 
the amount of amplification product produced. 
A. Gel Electrophoresis 
The presence of amplified product was detected by gel electrophoresis as 
follows. Reaction products were fractionated using an agarose gel (3% 
NuSieve and 1% SeaChem) and 1X TBE (0.089 M Tris, 0.089 M boric acid, 
0.0025 M disodium EDTA) running buffer. Electrophoresis was carried out at 
100 volts for approximately 1 hour. Ethidium bromide (0.5 .mu.g/ml) was 
added following electrophoresis to stain any DNA present. The gel was 
destained briefly in water and the ethidium bromide-stained bands of DNA 
were visualized using UV irradiation. 
B. 5'-Nuclease Assay 
As described above, amplifications were carried out in the presence of a 
fluorescein-labeled, HCV-specific detection probe modified so as to 
preclude extension by the DNA polymerase. The probe, which was 
complementary to a region of the HCV target sequence located between the 
two primer binding sites, was cleaved by the 5'-nuclease activity of the 
rTth DNA polymerase during primer extension. Following amplification, 
residual uncleaved probe was separated from reaction mixture and the 
fluorescence of the remaining cleaved probe fragments was then measured as 
an indication of the amount of amplification product synthesized. 
Uncleaved probes were extracted from the reaction mixture following 
amplification using beads coated with polyethyleneimine (PEI), which bind 
to the full-length, uncleaved probe, but which do not bind appreciably to 
cleaved probe fragments. The PEI beads are added to the reaction mixture, 
allowed to bind to the uncleaved probes, and the resulting PEI-probe 
complexes are removed by centrifugation. The amount of probe cleavage 
fragments remaining is determined by measuring the fluorescence. Details 
of the PEI bead extraction are described below. 
Prior to use, PEI beads (Baker Bond wide-pore PEI beads from J. T. Baker, 
Phillipsburg, N.J.) were soaked in distilled, deionized (dd) water at 
least for several hours (or overnight) at 4.degree. C. The PEI beads were 
washed sequentially using the following: (1) dd water, (2) ethanol, (3) dd 
water, (4) 1M Tris (pH 8.3), (5) 50 mM Tris (pH 8.3), 1M NaCl, and (6) 
binding buffer (10 mM Tris, 50 mM KCl, 1 mM EDTA, 500 mM NaCl and 8 M 
Urea). After the last wash, the beads were resuspended in binding buffer 
at 60 mg (wet weight) of PEI beads per 300 pi of binding buffer. 
To capture the unbound probe, 75 .mu.l of the PCR reaction mixture were 
added to 300 .mu.l of the PEI bead suspension (about 60 mg of PEI beads). 
The mixture was vortexed for 10 minutes to mix and allow binding of the 
PEI beads to the undegraded probes. Following centrifugation in a 
Microfuge at maximum speed for 2 minutes to remove the PEI-probe 
complexes, 200 pl1 of supernatent were transferred by pipette to the well 
of a microwell plate. The fluorescence was measured in a 
CytoFluor.TM.microtiter plate reader (Perceptive Biosystems, Bedford, 
Mass.) at room temperature using a 485 nm excitation filter (20 nm band 
pass width) and 530 nm emission filter (25 rim band pass width). 
EXAMPLE 2 
Comparison with Prior Art Primers--Analysis by Gel Electrophoresis 
This example describes a comparison of the primers of the present invention 
to the primers described in the prior art which are most similar to the 
present primers. The property of the primers compared was the 
amplification efficiency, defined as the amount of product produced in a 
given number of amplification cycles. 
The primers described in the prior art which are most similar to the 
primers of the present invention are KY80 (SEQ ID NO: 4) and KY78 (SEQ ID 
NO: 5) described in copending U.S. patent application Ser. No. 08/240,547, 
now U.S. Pat. No. 5,521,669; European Patent Publication No. 529,493; and 
Young et al., 1993, supra. A sequence comparison of these prior art 
primers and the primers of the present invention is provided below. 
Comparison of Primer Sequences 
______________________________________ 
Upstream Seq ID No. 
ST280A 1 5'-GCAGAAAGCGTCTAGCCATGGCGTTA 
KY80 4 5'-GCAGAAAGCGTCTAGCCATGGCGT 
Downstream 
ST778AA 2 5'-GCAAGCACCCTATCAGGCAGTACCACAA 
ST678A 3 5'-GCAAGCACCCTATCAGGCAGTACCACA 
KY78 5 5'-CTCGCAAGCACCCTATCAGGCAGT 
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Amplifications were carried out as described in Example 1 using samples 
containing 0, 10, 25, and 100 copies of synthetic HCV RNA template. 
Reactions were carried out using the primer combinations shown below. 
Reactions (a), (b), and (c) contained 400 nM of each primer. Reaction (d) 
differed from (c) in that the primer concentrations were increased to 600 
nM. 
(a) KY80 (SEQ ID NO: 4) and KY78 (SEQ ID NO: 5) (b) KY80 (SEQ ID NO: 4) and 
ST778AA (SEQ ID NO: 2) (c) ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 
2) (d) ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 2) 
Amplifications with each primer pair and input target number were carried 
out in triplicate. The amplified products were analyzed by gel 
electrophoresis, as described above. The results are presented in FIG. 1. 
The bands corresponding to the amplified HCV target sequence are 
indicated; the lower bands corresponds to non-specific amplification 
products (primer dimer). 
For each HCV target concentration, the most intense bands, corresponding to 
the greatest amount of amplification product, were produced using the 
primer pair ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 2). Only this 
primer pair yielded a detectable amount of amplification product from 10 
copies of target. Also apparent, particularly in the amplifications using 
100 copies of target, is a significant decrease in amount of non-specific 
amplification product using ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 
2). A comparison of amplifications (c) and (d) indicates that a primer 
concentration of 400 nM provided better results than a primer 
concentration of 600 nM. 
The amplifications carried out using KY80 (SEQ ID NO: 4) and ST778AA (SEQ 
ID NO: 2) were included to assess the improvement attributable to the RT 
primer of the present invention. The results obtained from amplifications 
(a) and (b) with 25 and 100 copies of the target sequence indicate that 
use of the RT primer of the present invention in combination with the 
prior art upstream primer resulted in a significant improvement in product 
yield, although not as great as obtained from amplifications (c) and (d) 
using a combination of both the RT and upstream primers of the present 
invention. 
Although gel analysis provides easily visualized evidence of the 
superiority of the primers ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 
2), the intensity of the bands do not provide an accurate quantitative 
comparison of the amplification products. For a quantitative comparison, 
the 5'-nuclease assay was used. 
EXAMPLE 3 
Comparison with Prior Art Primers--5.dbd.-nuclease Analysis 
This example describes amplifications using the primer combinations 
described in Example 2 wherein the analysis of the amplification products 
was carried out using the 5'-nuclease assay. Amplifications were carried 
out as described in Example 1 using samples containing 0, 10, 25, 
10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, and 10.sup.6 copies of synthetic 
HCV RNA template and primer concentrations of 400 nM for each primer. 
Amplifications with each primer pair and input target number were carried 
out in triplicate. As in example 2, above, the following primer 
combinations were used. 
(a) KY80 (SEQ ID NO: 4) and KY78 (SEQ ID NO: 5) (b) KY80 (SEQ ID NO: 4) and 
ST778AA (SEQ ID NO: 2) (c) ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 
2) 
The amplified product was analyzed using the 5'-nuclease assay, as 
described in example 1. The data are presented in FIG. 2, plotted as the 
fluorescence signal of the cleaved probe fragments versus the logarithm of 
the initial HCV target sequence copy number. Each fluorescence value is 
the average of the replicate measurements. The standard error for each 
value is indicated in FIG. 2. 
The data presented in FIG. 2 provide confirmation of the significant 
improvement in amplification efficiency obtained using the primers of the 
present invention, as observed in the gel electrophoretic analysis 
presented in FIG. 1, described above. The fluorescence signals generated 
from amplifications of 10--10.sup.5 copies of the target sequence using 
primer pair ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 2) were 
significantly greater than the fluorescence signals generated from the 
corresponding amplifications using primer pair KY80 (SEQ ID NO: 4) and 
KY78 (SEQ ID NO: 5). Furthermore, as also seen in FIG. 1, amplifications 
using the RT primer of the present invention, ST778AA (SEQ ID NO: 2), in 
combination with the prior art upstream primer, KY80 (SEQ ID NO: 4), also 
resulted in a significant improvement in product yield, although not as 
great as obtained using a combination of both the RT and upstream primers 
of the present invention. 
One measure of the relative amplification efficiency is provided by 
comparing the HCV input copy number required to obtain a given 
fluorescence signal. As seen in FIG. 2, the average signal obtained from 
the amplifications of 10 copies of HCV target using primer pair ST280A 
(SEQ ID NO: 1) and ST778AA (SEQ ID NO: 2) was approximately equal to the 
average fluorescence signal obtained from amplifications of 10.sup.3 
copies of HCV using primer pair KY80 (SEQ ID NO: 4) and KY78 (SEQ ID NO: 
5). Thus, a similar amount of amplification product was obtained from a 
100-fold lesser input copy number, or, in other words, the amplifications 
using primer pair ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 2) were 
about 100-fold more efficient than the amplifications using the prior art 
primers. 
Another measure of the relative amplification efficiency is provided by 
comparing the lowest HCV target number detectable. Using primer pair 
ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID NO: 2), amplifications of 10 
copies of HCV RNA provided a clearly detectable signal. In contrast, using 
primer pair KY80 (SEQ ID NO: 4) and KY78 (SEQ ID NO: 5), amplifications of 
10 copies of HCV target RNA did not result in a detectable signal. No 
detectable signal was generated using primer pair KY80 (SEQ ID NO: 4) and 
KY78 (SEQ ID NO: 5) below 100 copies of HCV. 
Given the sequence similarity of the upstream primers ST280A (SEQ ID NO: 1) 
and KY80 (SEQ ID NO: 4), and the sequence similarity between the 
downstream primers ST778AA (SEQ ID NO: 2) and KY78 (SEQ ID NO: 5), there 
would be no reason to expect such a dramatic improvement in amplification 
efficiency. The observed improvement obtained using the primers of the 
present invention was surprising and unexpected in view of the prior art. 
EXAMPLE 4 
Comparison with Prior Art Primers: 5'-nuclease Assay 
This example describes comparisons of amplifications using the following 
primer combinations. 
(a) KY80 (SEQ ID NO: 4) and KY78 (SEQ ID NO: 5) (b) ST280A (SEQ ID NO: 1) 
and ST678A (SEQ ID NO: 3) (c) ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID 
NO: 2) 
Amplifications were carried out as described above using samples containing 
0, 10, 25, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.6 and 10.sup.7 copies of 
synthetic HCV RNA template and primer concentrations of 400 nM for each 
primer. Amplifications with each primer set and input target number were 
carried out in triplicate. Amplified product was analyzed using the 
5'-nuclease assay, as described above. The data are presented in FIG. 3, 
plotted as the fluorescence signal of the cleaved probe fragments versus 
the logarithm of the initial HCV target sequence copy number. Each 
fluorescence value is the average of the replicate measurements. The 
standard error of each value is indicated in FIG. 3. 
The fluorescence signals generated from amplifications of 10-10.sup.4 
copies of target using either primer pair ST280A (SEQ ID NO: 1) and ST678A 
(SEQ ID NO: 3) or primer pair ST280A (SEQ ID NO: 1) and ST778AA (SEQ ID 
NO: 2) exceeded the fluorescence signals generated from the corresponding 
amplifications using primer pair KY80 (SEQ ID NO: 4) and KY78 (SEQ ID NO: 
5). The results indicate that both primer pairs of the present invention 
amplified the HCV target RNA with greater efficiency than the prior art 
primers. 
A comparison of the average signal obtained from amplifications of 10 
copies of HCV target using primer pair ST280A (SEQ ID NO: 1) and ST778AA 
(SEQ ID NO: 2) to the average signal obtained from amplifications of 102 
copies of HCV target using primer pair KY80 (SEQ ID NO: 4) and KY78 (SEQ 
ID NO: 5) shows that amplifications using primer pair ST280A (SEQ ID NO: 
1) and ST778AA (SEQ ID NO: 2) were over 10-fold more efficient. Similarly, 
amplifications using primer pair ST280A (SEQ ID NO: 1) and ST678A (SEQ ID 
NO: 3) were nearly 10-fold more efficient within the same range of initial 
HCV target copy number. 
EXAMPLE 5 
Amplification in a Tricine Buffer 
The 5'-nuclease assays described above used probes labeled with fluorescein 
(FAM) at the 5' end and a 3'-PO4 instead of a 3'-OH to block any extension 
by the DNA polymerase. Amplifications were also carried out using probes 
labeled with hexachlorofluorescein (HEX), also obtained from Perkin Elmer, 
Applied Biosystems Division (Foster City, Calif.). Unlike FAM-labeled 
probes, HEX-labeled probes were unstable in the Bicine amplification 
buffer described in example 1. However, HEX-labeled probes were found to 
be stable in Tricine amplification buffers. 
Amplifications using Bicine and Tricine are essentially equivalent, 
although a routine reoptimization of the reagent concentrations is 
recommended. The reaction mixture found to be optimal for amplifications 
using a Tricine buffer is described below. No change of the temperature 
cycling was necessary. 
HCV RNA template, 
400 nM each primer, 
1 .mu.M HEX-labeled probe, 
200 .mu.M each dATP, dCTP, dGT?, and dUTP, 
55 mM Tricine (pH 8.3), 
90 mM KOAc, 
3.0 mM Mn(OAc).sub.2, 
8% glycerol, 
20 units of rTth DNA polymerase*, and 
2 units of UNG*. 
* manufactured and developed by Hoffinann-La Roche and marketed by Perkin 
Elmer, Norwalk, Conn. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCAGAAAGCGTCTAGCCATGGCGTTA26 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GCAAGCACCCTATCAGGCAGTACCACAA28 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 27 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GCAAGCACCCTATCAGGCAGTACCACA27 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GCAGAAAGCGTCTAGCCATGGCGT24 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CTCGCAAGCACCCTATCAGGCAGT24 
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