Note: Descriptions are shown in the official language in which they were submitted.
CA 02528419 2005-11-30
COMPOSITIONS AND METHODS FOR REVERSE TRANSCRIPTASE-
POLYMERASE CHAIN REACTION (RT-PCR) OF HUMAN B-RETROVIRUS
The present invention relates to compositions, methods and kits for performing
reverse
transcriptase polymerase chain reaction (RT-PCR).
BACKGROUND OF THE INVENTION
PCR Amplification of RNA
Reverse transcriptases and methods for using them are known in the art for
reverse
transcribing RNA prior to PCR amplification. These methods, often collectively
referred
to as reverse transcriptase-PCR (RT-PCR), are widely used for detection and
quantification of RNA.
While many RT-PCR methods are known in the art, the efficient amplification of
a target
nucleic acid cannot be accomplished by a single standard protocol. In most
cases, a
variety of technical considerations and other problems are present that result
in less than
optimal amplification of target nucleic acids. In an attempt to address some
of the
technical problems often associated with RT-PCR, a nuinber of protocols have
been
developed taking into account the basic steps of the procedure: (a) the
isolation of RNA
and the hybridization of reverse primer to the target nucleic acid; (b) the
synthesis of
cDNA; and (c) PCR amplification.
In a two-step RT-PCR procedure, reverse transcription is performed as an
independent
step, preferably using the optimal buffer condition for reverse transcriptase
activity.
Typically after cDNA synthesis, the reaction mixture is diluted to decrease
the
concentration of salts, for example MgCl2 and other components, such as
deoxyribonucleoside triphosphate (dNTP) concentrations. The resulting
conditions are
better suited for Taq DNA Polymerase activity, and PCR is carried out
according to
standard conditions (see for example US 4,683,195 and US 4,683,202 which are
hereby
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incorporated by reference). In contrast to the two step procedure, one step RT-
PCR
methods usually employ a common buffer for both reverse transcriptase and Taq
DNA
Polymerase activities.
Attempts to enhance or streamline the process ofRT-PCR have been difficult,
and often
the end results depend on factors such as, but not limited to the choice of
primers and
target nucleic acid to be arnplified, the reaction and amplification
conditions, the
biological sample and relative abundance of the target nucleic acid in the
biological
sample. For exainple, the efficient detection, amplification and
quantification of viral
sequences from biological tissues and fluids can be difficult.
Primary Biliary Cirrhosis and Autoirnmunity
Primary biliary cirrhosis (PBC) is a progressive pluriglandular disease
affecting the liver,
pancreas, salivary and lachrymal glands (Neuberger, 1997, Lancet 850:875-79;
Epstein et
al., 1980, Lancet 1:1166-68). The hepatic disease is characterized by a florid
bile duct
lesion with lymphocytic infiltration and granulomatous destruction of 30 to 80
m sized
interlobular bile ducts (Rubin et al., 1965, Am. J. Pathol. 46:387-407). There
is no
curative therapy, apart from liver transplantation, and patients usually
develop cirrhosis
(Neuberger et al., 1997, Lancet 250:875-879). It is estimated to account for
approximately 2% of patients dying from cirrhosis in Europe and 10% of
patients that
requiring orthotopic liver transplantation in North America (Neuberger et al.,
1997,
Lancet 250:875-879).
The disease has been observed in all races and predominantly affects women
(Neuberger,
1997, Lancet 350: 875-879). To date, non-HLA genetic factors predisposing to
PBC have
not been identified but a positive family history provides the greatest risk
of developing
disease (Sherlock et al., 1993, Primary biliary cirrhosis: definition and
epidemiological
features. Kluwer Academic Publishers, Doredrecht/Boston/London, pp. 341-49).
There
are well documented cases of clustering in families and one report documented
a 2.4%
familial prevalence (Sherlock et al., 1993, Primary biliary cirrhosis:
definition and
epidemiological features. Kluwer Academic Publishers, DoredrechtBoston/London.
341-
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49 pp). No HLA class I alleles are associated with PBC but other immunogenetic
factors
appear to play an important role.
We have explored an infectious etiology of PBC. In the first instance, we used
representational difference analysis to identify retroviral sequences in the
liver of a
patient with PBC, we found that the majority of patients with PBC had antibody
reactivity to a retrovirus isolated from patients with Sjogren's syndrome and
then cloned
a retrovirus pol gene sequence from a PBC biliary epithelial cells cDNA
library (Mason,
A et al.,Lancet 1998;351:1620-24; Xu et al., Proc. Natl. Acad. Sci
2003;100:8454-8459.).
By BLASTN search, individual clones had a variable 91%-97% nucleotide homology
with the mouse mammary tumor virus (MMTV) and with retroviral sequences
derived
from human breast cancer samples (Xu L, et al., Hepatology 2004;39:151-156).
The
human (3-retrovirus is flanked by 21ong terminal repeat regions and contains 5
potential
colinear open reading frames of 100 or more codons that encode Gag, protease
(Pro),
polymerase (Po1), envelope (Env) and superantigen (Sag) proteins. Like MMTV, a
-1
frame shift is required to generate the Gag-Pro polyprotein and a second -1
frame shift is
required to generate the Gag-Pro-Pol polyprotein, whereas the env and sag
genes are
translated as individual open reading frames (Xu L, et al., Hepatology
2004;39:151-156).
In reviewing the results from pilot studies of single and combination
antiretroviral
therapy in patients with primary biliary cirrhosis (Mason et al., Am. J.
Gastroenterol
2004; 99:2348-2355 which is hereby incorporated by reference), the authors
subject
serum samples from patients to RT-PCR before and after therapy. Using this
technique, it
was shown that 5 of 9 patients taking lamivudine had detectable virus in the
serum prior
to treatment and 4 had detectable virus in serum after treatment. While such a
method
may be used to determine if infection is eradicated or present in a subject,
it does not
suggest whether the present course of treatment may be working. Indeed, the
authors note
that the lack of sensitive and qualitative detection assays to link
diminishing viral load
with significant clinical improvements is a shortfall.
There is a need in the art for optimized assays for detecting, amplifying,
diagnosing and
quantifying human P-retrovirus nucleic acids in biological samples. Further,
there is a
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need in the art for optimized RT-PCR assays for detecting, amplifying,
diagnosing and
quantifying human retrovirus nucleic acids in human biological samples.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims,
the sub-
claims disclose further advantageous embodiments of the invention.
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SUMMARY OF THE INVENTION
The present invention relates to compositions, methods and kits for performing
reverse
transcriptase polymerase chain reaction (RT-PCR).
According to an embodiment of the present invention, there is provided a
method of
monitoring the therapy of a subject having a(3-retrovirus infection
comprising,
a) treating the subject with one or more therapeutics, and;
b) quantifying the level of human P-retrovirus in said subject.
In a preferred embodiment, the quantifying is performed by real time RT-PCR.
The present invention also contemplates a method as defined above, wherein the
one or
more therapeutics comprise lamivudine, zidovudine, combivir, ursodeoxycholic
acid or a
combination thereof.
The present invention also contemplates a method as defined above, wherein the
real time
RT-PCR employs a primer and probe as defined in Table 1. In a further
embodiment,the
RT-PCR is performed on nucleic acids obtained from a biological sample, such
as for
example a tissue, fluid or combination thereof isolated from a human subject.
In a
preferred embodiment, the biological sample is blood. In an alternate
embodiment,
which is not meant to be limiting, the biological sample is liver tissue froin
a biopsy.
The present invention also provides a method of monitoring the therapy of a
subject
having or suspected of having a human 0-retrovirus infection comprising:
a) quantifying one or more pretreatment levels of human 0-retrovirus in the
subject;
b) treating the subject with one or inore therapeutics, and;
c) quantifying one or more post-treatment levels of human (3-retrovirus in the
subject.
Further, it is contemplated that during said b) treating said subject with one
or more
therapeutics, one or more levels of human (3-retrovirus also may be
determined.
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The present invention also provides a method as defined above wherein the
level of
human (3-retrovirus is monitored in combination with one or more enzyme or
blood/tissue
component levels, for example, but not limited to bilirubin, serum amylase,
serum
bicarbonate, lactate, alkaline phosphatase, aspartate aminotransferase (AST),
alanine
aminotransferase (ALT) or a combination thereof.
The present invention also provides a method of screening a compound or
composition
for anti-human 0-retrovirus activity comprising,
a) administering the compound or composition to one or more subjects having
human (3-
retrovirus;
b) quantifying the level of human 0-retrovirus in said subject.
In the method as defined above, huinan (3-retrovirus infection may detected
and/or
quantified prior to said a) administering the compound or composition to one
or more
subjects.
The present invention also provides a kit for detecting and/or quantifying
human (3-
retrovirus in a biological sample comprising,
a) one or more primers for amplifying a human (3-retrovirus target sequence,
b) one or more reverse transcriptases,
c) one or more DNA polymerases,
d) one or more probes for performing real time RT-PCR,
e) one or more buffers for performing, terminating, inhibiting, diluting,
washing one or
more enzymatic reactions, or products produced or purified in a RT-PCR
reaction;
f) one or more restriction enzymes for cleaving DNA amplified from a PCR
reaction,
g) one or more hybridization probes for identifying DNA amplified from a PCR
reaction,
h) instructions for performing a RT-PCR reaction using the components of the
kit,
i) one or more therapeutics;
j) one or more dNTPs, or the like,
or any combination thereof.
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In a preferred embodiment, which is not meant to be limiting, the kit
comprises primers
that hybridize to the U5 long terminal repeat or pol regions of the human P-
retrovirus.
The primers may be as defined in Table 1.
This summary of the invention does not necessarily describe all necessary
features of the
invention but that the invention may also reside in a sub-combination of the
described
features.
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BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the
following
description in which reference is made to the appended drawings wherein:
FIGURE 1 shows a standard curve of real time RT-PCR assay for the U5 primers
demonstrating a linear range from about 4X 105 to about 4 DNA copies.
FIGURE 2 shows a standard curve of real time RT-PCR assay for the pol primers.
FIGURE 3 shows the detection of human (3-retrovirus RNA in samples derived
from
patients with PBC versus those from control non-PBC subjects. In panel (a), Ct
values
from 6 PBC patients' serum samples varied from 34.83 and 35.86 to Ct <= 37, or
negative. In panel (b) Ct values from liver sample of PBC patients (n=2,
Ct=28.49 and
30.06) and non-PBC subjects (n=6, Ct<=37, or negative) and not in control
samples.
FIGURE 4 shows a graph of human (3-retrovirus levels assessed by real-time RT-
PCR in
a patient with PBC unresponsive to UDCA treatment for 4 years (estimated viral
copy
number in Meq/ml shown by lower graph line). The subject experienced a partial
biochemical response in viral load, ALT and alkaline phosphatase levels after
3 months
of lamivudine therapy but then relapsed. Coinplete loss of virus with
normalization of
liver biochemistry was seen with Conlbivir treatment but biochemical and
virological
rebound was seen at month 9 of Combivir therapy.
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DESCRIPTION OF PREFERRED EMBODIMENT
The present invention relates to coinpositions and methods for performing
reverse
transcriptase polymerase chain reaction (RT-PCR).
The following description is of a preferred embodiment by way of example only
and
without limitation to the coinbination of features necessary for carrying the
invention into
effect.
The present invention is directed to coinpositions, methods and kits for
detecting human
(3-retrovirus including, but not limited to amplification of nucleic acid
molecules from
human (3-retrovirus by reverse transcriptase-polymerase chain reaction (RT-
PCR), and
identification of human (3-retrovirus proteins and antibodies thereto by
enzyme linked
immunosorbant assays (EL1SAs) or the like.
In an embodiment, the present invention is directed to compositions, methods
and kits for
use in reverse transcriptase-polymerase chain reaction (RT-PCR) for detection,
production, amplification, analysis and quantification of human (3-retrovirus
nucleic acids
from biological samples. ln particular, the invention provides compositions
comprising a
variety of components in various combinations. Such components may include,
but are
not limited to, one or more enzymes having reverse transcriptase activity, one
or more
DNA polymerases, one or more primers, one or more probes, one or more
nucleotides,
one or more buffers, or a combination thereof. These compositions may be used
in the
methods and kits of the invention to identify, diagnose, produce, amplify,
analyze,
quantitate and otherwise manipulate human (3-retrovirus nucleic acid molecules
using a
one- or two-step RT-PCR procedure. In addition, the composition, methods and
kits as
provided by the present invention may be employed for genetic testing and/or
the
prognosic evaluation of human 0-retrovirus infection prior to therapy, during
therapy,
post therapy, or any combination thereof.
As used herein, the term "human (3-retrovirus nucleic acids" refers to RNA
from the
human beta retrovirus viral genome, for example, as described in Xu et al.,
Hepatology
CA 02528419 2005-11-30
-10-
2004; 39:151-156, fragments thereof, transcripts thereof, and mutant sequences
derived
therefrom. Additional information concerning human (3-retrovirus nucleic acids
and
sequences may be found in Example 1.
As used herein, a"biological sample" refers to a sainple of tissue or fluid
isolated
from an individual. Thus, "biological sample" includes but is not limited to,
plasma,
serum, spinal fluid, lymph fluid, the external sections of the skin,
respiratory,
intestinal, and genitourinary tracts, tears, saliva, milk, blood cells,
tumors, organs, and
also samples of in vitro cell culture constituents (including, but not limited
to
conditioned medium resulting from the growth of cells in cell culture medium,
putatively virally infected cells, recombinant cells, and cell components).
Buffers
The one or more buffers in the compositions of the invention provide
appropriate pH and
ionic conditions for the enzymes as described herein. In an embodiment, which
is not
meant to be limiting, the enzymes may include one or more reverse
transcriptases, one or
more DNA polymerases, or a combination thereof. It is also contemplated that
nucleotides may used in the compositions, for example, but not limited to
deoxyribonucleoside triphosphates (dNTPs), and the primer nucleic acid
molecules
capable of hybridizing to the human 0-retrovirus nucleic acids molecules act
as substrates
for the synthesis or amplification of nucleic acid molecules in accordance
with an
embodiment of the invention. The compositions of the invention may also
include
additional components as described herein.
Reverse Transcriptase Enzymes
The compositions of the present invention may also comprise enzymes having
reverse
transcriptase activity. Any reverse transcriptase enzynle may be employed
provided that it
is capable of reverse transcribing human (3-retrovirus RNA following binding
of the
primer to the human (3-retrovirus primer target sequence. Enzymes having
reverse
transcriptase activity are commercially available. For example, but not
wishing to be
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limiting in any manner, SUPERSCRIPTTM, SUPERSCRIPT II"M, Moloney Murine
Leukemia Virus reverse transcriptases (M-MLV), and RSV reverse transcriptase
are
available from Life Technologies, Inc. (Rockville, Md). Any one or combination
of
reverse transcriptases enzymes may be contained or employed in the
compositions,
methods and kits of the present invention. In an embodiment of the present
invention,
which is not meant to be limiting, the enzymes exhibit reduced RNase H
activity. RNase
H is a processive 5' and 3' ribonuclease specific for the RNA strand for RNA-
DNA
hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley &
Sons
(1984)). Errors in transcription cannot be corrected by reverse transcriptase
because
known viral reverse transcriptases lack the 3'-5' exonuclease activity
necessary for
proofreading (Saunders and Saunders, Microbial Genetics Applied to
Bioteclinology,
London: Croom Helm (1987)).
By an enzyme "reduced in RNase H activity" it is meant that the enzyme has
less than
about 20%, more preferably less than about 15%, 10% or 5%, and most preferably
less
than about 2%, of the RNase H activity of a wildtype enzyme under specific
conditions.
The RNase H activity may be determined by a variety of assays, such as those
described,
for example, in US 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res.
16:265 (1988)
and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), the disclosures of all of
which are
fully incorporated herein by reference.
DNA Polymerases
The compositions of the invention may also comprise one or more DNA
polymerases,
which are preferably thermostable DNA polymerases. These DNA polymerases may
be
isolated from natural or recombinant sources, by techniques that are well-
known in the art
(See for example, but not limited to WO 92/06200, US 5,455,170, US 5,466,591,
WO
96/10640), from a variety of thermophilic bacteria that are available
commercially (for
example, but not limited to American Type Culture Collection (Rockville, Md.)
or may
be obtained by recombinant DNA techniques as described for example, but not
limited to
in WO 96/10640). Without wishing to be limiting, suitable sources of
thermostable
polymerases or the genes thereof for expression in recombinant systems are the
CA 02528419 2005-11-30
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thermophilic bacteria Thermus thermophilus, Thermococcus litoralis, Pyrococcus
furiosus, Pyrococcus woosii and other species of the Pyrococcus genus,
Bacillus
sterothermophilus, Sulfolobus acidocaldarius, Thermoplasma acidophilum,
Thermus
flavus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana, Thermotoga
maritima and other species of the Thermotoga genus, and Methanobacterium
thermoautotrophicum, and mutants, variants or derivatives thereof. It is to be
understood,
however, that thermostable DNA polymerases from other organisms may also be
used in
the present invention without departing from the scope or preferred
embodiments thereof.
As an alternative to isolation, thermostable DNA polymerases are available
commercially
from, for example, but not limited to Life Technologies, Inc. (Rockville,
Md.), New
England BioLabs (Beverly, Mass.), Finnzymes Oy (Espoo, Finland), Stratagene
(La Jolla,
Calif.), Boehringer Mannheim Biochemicals (Indianapolis, Ind.) and Perkin
Elmer Cetus
(Norwalk, Conn.).
In a preferred embodiment, the thermostable DNA polymerase or polymerases for
use in
the compositions, methods and kits of the present invention include, but are
not limited
to, Taq, Tne, Tma, Tli/VENTTM, DEEPVENTTM, Pfu, Pwo, Tfi or Tth DNA
polymerases,
or mutants or derivatives thereof. Taq DNA polymerase is commercially
available, for
example, from Life Technologies, Inc., or may be isolated from its natural
source, the
thermophilic bacterium Thermus aquaticus, for example, but not limited to, as
described
in US 4,889,818 and US 4,965,188. Tne DNA polymerase may be isolated from its
natural source, the thermophilic bacterium Thermotoga neapolitana, for
example, but not
Iimited to as described in WO 96/10640, and Tma DNA polymerase from its
natural
source, the thermophilic bacterium Thermotoga maritima, for example, but not
limited to
as described in US 5,374,553. Methods for producing mutants and derivatives of
thermophilic DNA polymerases are also contemplated by the present invention
and any
such teaching known in the art may be employed to produce such mutants. Tfi,
Tli/VENTTM and DEEPVENTTM are available commercially, for example, but not
limited
to from New England BioLabs, or may be produced as described (Bej and
Mahbubani, in:
PCR Technology: Current Innovations, Griffin, H. G., and Griffin, A. M., eds.,
CRC
Press, pp. 219-237 (1994) for Tli/VENTTM; Flaman, J.M., et al., Nucl. Acids
Res.
22(15):3259-3260 (1994) for DEEPVENTTM). Thermostable DNA polymerases are
CA 02528419 2005-11-30
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preferably added to the present compositions at a final concentration in
solution of about
0.1-500 units per milliliter, about 0.1 to 100 units per milliliter, about 0.1-
50 units per
milliliter, about 0.1-40 units per milliliter, about 0.1-36 units per
milliliter, about 0.1-34
units per milliliter, about 0.1-32 units per milliliter, about 0.1-30 units
per milliliter, or
about 0.1-20 units per milliliter, and most preferably at a concentration of
about 20 units
per milliliter. However, the present invention contemplates adding DNA
polymerase at a
final concentration of about 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20,
22.5, 25, 30, 35,
40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400 or 500
units per
milliliter. The amount of DNA polymerase inay also be defined by a range of
any two of
the values listed above.
In an alternate embodiment, which is not meant to be limiting in any manner,
the
concentration of DNA polymerases may be determined as a ratio of the
concentration of
the enzymes having reverse transcriptase activity.
dNTPs
The compositions of the invention may further comprise one or more
nucleotides, for
example, but not limited to deoxynucleoside triphosphates (dNTPs). The
nucleotide
components of the present compositions serve as building blocks for newly
synthesized
nucleic acids, being incorporated therein by the action of the reverse
transcriptases or
DNA polymerases. Examples of nucleotides suitable for use in the present
compositions
include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-
dGTP,
a-thio-dATP, a-thio-dTTP, a-thio-dGTP, a-thio-dCTP or derivatives thereof, all
ofwhich
are available commercially from sources including, but not limited to Life
Technologies,
Inc., New England BioLabs and Sigma Chemical Company (Saint Louis, Mo.). The
dNTPs may be unlabeled, or they may be detectably labeled by coupling them by
methods
known in the art with radioisotopes such as but not limited to 3H,14C, 32P or
35S, vitamins
such as but not limited to biotin, fluorescent moieties such as but not
limited to
fluorescein, rhodamine, Texas Red, or phycoerythrin, chemiluminescent labels,
dioxigenin and the like. Labeled dNTPs may also be obtained commercially, for
example,
but not limited to fi-om Life Technologies, Inc. or Sigma Chemical Company. In
the
CA 02528419 2005-11-30
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present compositions, the dNTPs are added to give a working concentration of
each
dNTP of about 5-5000 micromolar, about 10-500 micromolar, about 10-250
micromolar,
or about 10-100 nlicromolar, and most preferably a concentration of about 100
micromolar. However, the present invention contemplates a working
concentration of
dNTPs of about 5, 10, 25, 75, 100, 125,150, 175, 200, 250, 500, 600, 700, 800,
900,
1000, 2000, 3000, 4000, 5000 micromolar. Also, the concentration used may be
defined
by a range of any two of the values listed above.
Primers
In addition to nucleotides, the present compositions may also coinprise one or
more
primers which facilitate the synthesis of a first DNA molecule complementary
to all or a
portion of the human (3-retrovirus RNA template. Such primers may also be used
to
synthesize a DNA molecule complementary to al I or a portion of the first DNA
molecule,
thereby forming a double-stranded cDNA molecule. Additionally, these primers
may be
used in amplifying nucleic acid molecules in accordance with the invention.
Such primers
include, but are not limited to, human 0-retrovirus specific primers, meaning
that the
primers are capable of hybridizing to a target human (3-retrovirus RNA primer
sequence
and amplifying a target human 0-retrovirus sequence. The primer will be
completely or
substantially complementary to a region of the polynucleotide strand to be
copied. Thus,
under conditions conducive to hybridization, the primer will anneal to the
complementary
region of the analyte strand, and upon addition of suitable reactants (e.g., a
polymerase,
nucleotide triphosphates, and the like), the primer is extended by the
polymerizing agent
to form a copy of the analyte strand. Additional primers that may be used for
amplification of the DNA molecules according to the methods of the invention
will be
apparent to one of ordinary skill in the art.
Probes
The compositions of the present invention may further comprise one or more
probes to
monitor the PCR amplification reaction. For example, RT-PCR may employ a
probe, for
example, a fluorescently labelled probe to detect and quantify target
sequences of DNA.
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In an embodiinent of the present invention, the probe comprises a fluorescent
tag, for
example a dye or the like on one end and a suitable quencher on the other.
During each
amplification cycle, the probe attaches to a target nucleotide sequence of
human (3-
retrovirus RNA (or DNA made therefrom), followed by attachment ofthe primers
to the
target human (3-retrovirus nucleotide sequences. As the targeted nucleotide
strand is
copied, the reporter dye is released from the probe and emits a fluorescent
signal. The
amount of fluorescence signal increases with each cycle of PCR in proportion
to the
amount oftarget DNA. This permits direct detection and quantification ofthe
target DNA
with a high degree of specificity, accuracy and sensitivity. In a preferred
embodiment the
RT-PCR composition, methods and kits of the present invention comprise or
employ
TaqMan technology to detect and quantify human J3-retrovirus RNA. However,
other
types of probe technologies may also be employed, for example, but not limited
to
Molecular Beacons, Scorpions , or SYBROx Green.
Additional Reagents
In accordance with the compositions, methods and kits of the invention, one or
more
additional reagents may be include or employed therein. Such reagents include,
but are
not limited peptides, polypeptides and proteins such as, human serum albumin,
bovine
serum albumin, ovalbumin, Albumax, casein, gelatin, collagen, globulin,
lysozyme,
transferrin, myoglobin, hemoglobin, lactalbumin, fumarase, glyceraldehyde-3-
phosphate
dehydrogenase (GAPDH), amyloglucosidase, carbonic anhydrase, lactoglobulin,
aprotinin, soybean trypsin inhibitor, trypsinogen, phosphorylase b, myosin,
actin,
galactosidase, catalase, tryptic soy digests, tryptose, lectins and the like,
or fragments or
derivatives thereof. When added to the RT-PCR reactions, the reagents are
preferably
used in low ainount, for example, in the range of about 0.001 to about 100
g/ml. In
specific embodiments, not meant to be limiting in any manner,, the reagents
may be
present in an amount of 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 15, 20,25,
50, 75 or 100
g/ml. Alternatively, the additional reagents may be present in a range defined
by any two
of the values listed above.
Methods of RT-PCR
CA 02528419 2005-11-30
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In the RT-PCR reaction, the reaction mixtures are incubated at a temperature
sufficient to
synthesize a DNA molecule complementary to all or portion of the human 0-
retrovirus
RNA template. Such conditions typically range from about 20 C to 75 C, more
preferably
from about 35 C to 60 C and most preferably from about 45 C to about 55 C.
However,
the method of the present invention contemplates incubating the reaction
mixture at about
20 C, 25 C, 30 C, 35 C, 40 C, 45 C, 50 C, 55 C, 60 C, 65 C or 70 C. The
present
invention also contemplates that the reaction mixture may be incubated at any
temperature within a range defined by any two of the values listed above.
After the reverse transcription reaction, the reaction is incubated at a
teinperature
sufficient to amplify the synthesized DNA molecule. Preferably, the
amplification is
accomplished via one or more polymerase chain reactions (PCRs). Preferred
conditions
for amplification comprise thermocycling, which may coinprise alternating
heating and
cooling of the mixture sufficient to amplify the DNA molecule and which most
preferably comprises alternating from a first temperature range of from about
90 C to
about 100 C, to a second temperature range of from about 45 C to about 75 C,
more
preferably from about 50 C to about 75 C or from about 55 C to about 75 Cand
most
preferably from about 65 Cto about 75 C. According to the invention, the
thermocycling
may be performed any number of times, preferably from about 5 to about 80
times, more
preferably greater than about 10 times and most preferably greater than about
20 times.
However, the method of the present invention specifically contemplates
thermocycling
10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41,
42, 43, 44, 45,
50, 55, 75 or 100 times. Further, the ainount of thermocycling may be defined
by a range
of any two of the values listed above.
In an embodiment of the present invention, there is provided a method for the
production,
detection, amplification, or quantification (or any combination thereof) of
human (3-
retrovirus comprising the steps of:
a) obtaining a biological sample from a subject;
b) performing RT-PCR on RNA isolated from said sample and;
CA 02528419 2005-11-30
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c) determining if said RT-PCR has amplified a nucleic acid, or fragment
thereof from
said human (3-retrovirus.
The method as provided above may also include one or more steps relating to
quantification of the human (3-retrovirus nucleic acid, or fragment thereof.
In such an
instance, it is possible for a clinician or the like to estimate the relative
amount of human
0-retrovirus infection in a subject.
As provided above, the compositions and methods of the present invention may
be used
for diagnostic PCR analysis for the identification of retroviral nucleotides
which correlate
with the presence of a human 0-retrovirus. In alternate embodiments, which are
not meant
to be limiting in any manner, the compositions and methods of the present
invention may
be employed for the identification of retroviral nucleotides which correlate
with
associated disorders, such as, but not limited to primary biliary cirrhosis
(PBC), Sjogren's
syndrome, scleroderma, SLE, autoimmune thyroiditis, various other connective
tissue
disorders and lymphoma. In yet another embodiment, the compositions and
methods of
the present invention can be used in therapeutic PCR analysis to monitor the
presence of
a human 0-retrovirus and determine the effectiveness of a therapeutic
protocol. For
example, but not wishing to be limiting in any manner, the methods and
compositions of
the present invention may be employed to determine the effectiveness of
antiviral or other
therapy. Representative examples of antiviral drugs that may be employed in
therapy
include, but are not limited to lamivudine, zidovudine, Combivir or any other
antiviral or
combination of antiviral drugs.
It is also contemplated that subjects may be treated with ursodeoxycholic
acid, alone or
in combination with one or more antiviral drugs as known in the art and/or as
provided
above.
In an embodiment wherein lamividine is employed as an antiviral drug,
generally it is
used in an amount of about 50 to 300 mg per day, more preferably about 100 mg
to 200
mg per day, still more preferably about 150 mg per day. In an embodiment
wherein
combivir combination therapy is employed as an antiviral composition,
generally, it
CA 02528419 2005-11-30
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comprises about 150mg of lamivudine and 300mg of zidovudine per day, more
preferably
twice a day. In an embodiment wherein ursodeoxycholic acid (UDCA) is employed
in
therapy, generally it is used in an ainount of about 5 to 30 mg/kg/day, more
preferably
about 10-20 mg/kg/day, still more preferably about 13-15 mg/kg/day.
The present invention contemplates a method of monitoring the therapy of a
subject
having a 0-retrovirus infection comprising:
a) treating the subject with one or more therapeutics, and;
b) quantifying the level of human 0-retrovirus in said subject.
The method may additionally employ the step of comparing the level of human (3-
retrovirus in said subject after treatment with the results of one or more
prior tests
wherein the level of human 0-retrovirus in the subject is determined. In this
regard, a
particular therapy may be effective if the levels of human (3-retrovirus are
maintained,
more preferably reduced over the course of the subject's treatment regimen. In
such a
manner, it is possible to monitor the effectiveness of a particular treatment.
In an alternate embodiment, there is provided a method monitoring the therapy
of a
subject having a(3-retrovirus infection comprising:
a) identifying and/or quantifying a pretreatment level of human 0-retrovirus
in the
subject;
b) treating the subject with one or more therapeutics, and;
c) identifying and/or quantifying the post treatment level of human 0-
retrovirus in the
subject.
In still a further embodiment, which is not meant to be limiting, the subject
may be
monitored by, or subjected to one or more additional tests, for example, but
not limited to
liver tests, biopsies, blood tests or the like. In a preferred embodiment, the
level of human
0-retrovirus is monitored in combination with one or more enzyme or
blood/tissue
cornponent levels, for example, but not limited to bilirubin, serum amylase,
serwn
bicarbonate, lactate, alkaline phosphatase, aspartate aminotransferase (AST),
alanine
aininotransferase (ALT) or the like. It is also contemplated that the level of
human 0-
CA 02528419 2005-11-30
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retrovirus may be monitored in combination with one or more histological
examinations,
biopsies or the like, for example, but limited to qualitative and/or
quantitative
examination of ductopenia, qualitative and/or quantitative examination of
inflammation,
for example, but not limited to necroinflammatory scores. Biopsies may be
assessed for
ductopenia, staged for fibrosis, graded for necroinflammatory activity, and/or
evaluated
for bile duct injury using the Ishak scoring system (Ishak et al., J. Hepatol
1995, 22:696-
9, which is herein incorporated by reference).
In a preferred embodiment, the primers for amplifying a human 0-retrovirus
target
sequence comprise nucleotide sequence that hybridize to the U5 long terminal
repeat and
pol regions of the human 0-retrovirus.
The above description is not intended to limit the claimed invention in any
manner,
furthermore, the discussed combination of features might not be absolutely
necessary for
the inventive solution.
The present invention will be further illustrated in the following examples.
However it is
to be understood that these examples are for illustrative purposes only, and
should not be
used to limit the scope of the present invention in any manner.
Examples
Example 1: Human (3-retrovirus sequence and additional information
The nucleotide sequence of human 0-retrovirus has been previously described.
9690 base
pairs of the proviral genome is provided in GenBank accession nos AF513913-
AF513923. Additional information concerning the isolation and characterization
of the
human (i-retrovirus is provided in Xu et al., PNAS July, 2003 Vol 100 No. 14
8454-8459;
Mason et al., Hepatology 2004; 39:151-156; and US 6,468,737 the disclosures of
which
are herein incorporated by reference.
CA 02528419 2005-11-30
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Example 2: Representative Example of RT-PCR for Human (3-retrovirus
Serum samples were assessed by RT-PCR for evidence of human (3-retrovirus
infection.
RT-PCR was performed with nested oligonucleotide primers complementary to the
of
human (3-retrovirus long terminal repeat (Xu et al.., Proc. Natl. Acad Sci
2003; 100:8454-
51). Total RNA was extracted from serum with Trizol (Gibco BRL, Grand Island,
NY),
and random primers were used to make cDNA template for PCR. Nested PCR was
performed using 5'-AGAAATGGTTGAACTCCCGAGAG-3' and 5'-GGC
TTGTAAGAGGAAGTTGGCTG-3' for external primers, and 5'-
TTGTTTCCCACCAAGGACGAC-3' and 5'-GAAGGTCGAGTTCTCCGAATCG-3' as
internal primers to provide a 216 bp product. Both PCR and reactions were
performed in
with 2.5 mM Mg+2 using cycling parameters of 94 C, 52 C, and 72 C for 20s at
each
temperature in first PCR and for 10 s at each temperature in the nexted PCR.
Example 3: Representative Examples of Primers and Probes for Real Time RT-
RCR of human (3-retrovirus nucleic acids.
Representative examples of primers and probes that may be used in real timeRT-
PCR are
provided in Table 1. The table indicates the location of the human (3-
retrovirus primer or
probe target sequence (i.e U5 or Pol), forward or reverse primers, the 5' to
3' sequence of
the primers/probe, amplicon in base pairs, location thereof and gene including
GenBank
accession number. A person of skill in the art will recognize that a variety
of other
primers and probes may be used in real time RT-PCR.
Table 1. Sequences of the primers and probes for detection and quantification
of human
betaretrovirus
Name of primes Sequence Amplicon Location Gene (clone)
and probes (5'-3') (bp)
US-F GCCCATCAGACAAAGACATACTCA 72 44-67 U5 (AF513913)
U5-R CCAATAGCCCCAGGCAAA 115-98 U5 (AF513913)
U5-probe TCTCTGCTGCAAACTTGGCATAGCTCTG 69-96 U5 (AF513913)
Pol-Fl TCACATGACCTCTACCACACCACTA 74 398-422 Pol (AF513922)
CA 02528419 2005-11-30
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Pol-Rl CCGAACGGCCATTTGC 471-456 Pol (AF513922)
Pol-probel AGAAAGGAATTGTGATTTTTACGGACGGGTC 424-454 Pol (AF513922)
Pol-F2 GCTGTTACAAGACCTACGTGCAGTT 71 1161-1185 Pol(AF248269)
Pol-R2 ACGGCAAGCCTGGTTGTAAT 1231-1212 Pol(AF248269)
Pol-probe2 TGCCACAATGCACGATATGGGA 1188-1209 Pol(AF248269)
Example 4: Human (3-retrovirus specific real time RT-PCR assay
Two quantitative Real Time RT-PCR assays have been developed using the TaqMan
technology to detect and quantify human 0-retrovirus RNA. Primers and pi-obe
complementary to the U5 long terminal repeat and pol regions of the human (3-
retrovirus
as discussed above were employed. To assess the sensitivity of each assay, 10-
fold serial
dilutions of a plasmid containing the human (3-retrovirus long terminal repeat
and pot
regions were prepared using an approximate range of 4x 105 to 4 DNA copy
number
(Figures 1 and 2). Each assay has the capability to detect less than about 10
plasmid
copies.
Preliininary studies revealed that both assays are functional and
complementary with
regard to detecting viral levels in tissue and serum of patients with PBC. It
has been
observed that human 0-retrovirus RNA in the liver is detected in a 103 higher
viral load
compared to serum from subjects with PBC. Also the appropriate control
subjects either
have low or undetectable viral load (Figure 3).
Preliminary studies have been conducted to determine whether the real-time RT-
PCR
assays will be functional to measure viral load in response to antiviral
treatment. To date,
we have observed that most patients have biochemical responses with
improvement in the
liver function tests, alkaline phosphatase and alanine aminotransferase (ALT)
corresponding with the initiation of antiviral therapy. By testing cDNA from
serial serum
samples derived from patients undergoing lamivudine, or Combivir therapy, we
have
been able to verify the utility of both assays by demonstrating that the
reduction of viral
CA 02528419 2005-11-30
-22-
levels corresponding with antiviral treatment and improvement in liver
biochemistry
studies (Figure 4).
All citations are herein incorporated by reference.
The present invention has been described with regard to preferred embodiments.
However, it will be obvious to persons skilled in the art that a number of
variations and
modifications can be made without departing froin the scope of the invention
as described
herein.
CA 02528419 2007-01-04
SEQUENCE LISTING
APPLICANT NAME: Rev Tree
TITLE: Compositions And Methods For Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR) Of Human beta-retrovirus
FILE REFERENCE: 08904086CA
CURRENT APPLICATION DATA:
APPLICATION NUMBER: 2,528,419
FILING DATE: 2005-11-30
NUMBER OF SEQ ID NO.: 13
SOFTWARE: PatentIn version 3.3
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CA 02528419 2007-01-04
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CA 02528419 2007-01-04
ORGANISM: Artificial
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CA 02528419 2007-01-04
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22/4
