Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR SENSITIVE DETECTION
OF REVERSE TRANSCRIPTASE
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention provides a method for detecting the presence of a
retrovirus in a biological sample by detecting the presence of the enzyme,
reverse
transcriptase. The method utilizes a template and primers in conditions under
which a
DNA strand is synthesized and subsequently amplified by the polymerase chain
reaction,
if reverse transcriptase is present.
BACKGROUND ART
Retroviruses are widely distributed in vertebrates and are known to cause a
variety of diseases in man and animals including immunodeficiencies, leukemias
and
lymphomas (1). The entire retrovirus family is characterized by the presence
of a unique
enzyme, reverse transcriptase (RT), which transcribes the viral genomic RNA
into a
double-stranded DNA copy (1). This feature has led to studies of the unique
enzymatic
function of RT for two main applications. First, the presence of RT has been
the basis
for diagnosis of retroviral infection and for the generic detection of the
presence of
retroviruses in cell cultures and in the infected host. Second, the RT enzyme
constitutes
a primary target for antiviral drug intervention (1,2).
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The polymerase function of RT from several retroviruses has been well
characterized and shown to require an RNA template, a primer, triphosphate, a
divalent
cation and physiological salts (1). Therefore, assays for RT activity have
conventionally
used these reagents and conditions to measure the ability of a sample to
produce a DNA
copy of a known exogenous RNA template (e.g., poly rA) by synthesizing a
complementary DNA oligonucleotide primer with radiolabeled nucleotides (e.g.,
3H- or
32P-dTTP). RT synthesized DNA has been detected by measuring incorporation of
the
labeled nucleotide (3,4) and more recently, by assays which employ
nonradioactive
nucleotides in enzyme linked immunosorbent assay (ELISA) formats have been
developed (5-8) and by polymerase chain reaction (PCR) (17), as further
described
below.
Although RT assays continue to be an essential laboratory tool for the
identification of known and novel retroviruses (4-11), the successful use of
RT assays
has been limited to the detection of retroviral particles in culture
supernatant (4-8),
which has the disadvantage of requiring that a virus be cultured before
detection. By the
present. methods, lentiviruses, such as human immunodeficiency virus-i (HIV-1)
may be
readily detected, but oncoviruses, such as human T lymphocytic virus types I
and II
(HTLV-I and HTLV-II), are much more difficult to detect, presumably because of
poor
RT activity and because they are typically cell-associated, which means their
RT is less
accessible for detection in culture supernatants (6-8). This limitation has
rendered RT
testing of little value in the detection of HTLV infection.
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Despite multiple attempts to improve the sensitivity of RT assays, the direct
detection of retroviruses in clinical samples (e.g., serum) has been
unsuccessful (4-12).
For example, in studies of HIV-1 infected individuals, detection of virus in
plasma by
RT assays has been largely abandoned because of the low sensitivity of this
method (12).
The inability to detect RT activity in serum hinders the use of this
virological
marker in diagnosing disease, monitoring drug efficacy in patients, monitoring
virus
load and predicting disease progression. Present methods for qualitative and
quantitative detection of plasma HIV-1 include viral isolation and p24 antigen
capture
(13-15). However, these assays have disadvantages. Virus isolation from plasma
requires virus culture that is typically maintained for 14 to 28 days and is
labor intensive,
time consuming, fraught with biological variation and is not a universal
marker in the
infected population given the low levels of HIV-1 in patients with CD4+ T
lymphocyte
counts of >200/mm3= Similarly, p24 antigen, either free, virion-associated, or
immune
complexed, is not always present in the HIV-1 infected population.
RT-PCR permits the qualitative detection of the cell-free virus in plasma
using
an exogenous RT and a primer pair of known sequence to amplify viral RNA
sequences
in the plasma (15,16). Although RT-PCR has been reported to be highly
sensitive, this
assay requires RNA extraction and multiple sample manipulations that may
increase the
risks of PCR contamination. The RT-PCR assay may be complicated further by the
lack
of a standardized universal quantitative test and the variabilities that may
be incurred
during processing and storage with regard to the degradation of the genomic
RNA.
Although RT-PCR, like antigen capture, is highly specific, a knowledge of the
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nucleotide sequence of the target retrovirus fragment is necessary for primer
development. Given this linutation, RT-PCR is not suitable for detecting
variant, novel,
or unknown retroviruses.
Recently, two assays for RT, which use the RNA of bacteriophage MS2 or
brome mosaic virus (BMV) as a template and PCR as a detection system, have
been
reported to be highly sensitive for the detection of murine leukemia virus RT
and other
stocks of retroviruses in serum (18,19). However, the evaluation of the
specificity and
the sensitivity of these assays on adequate numbers of serum specimens was not
disclosed (18,19). In addition, in both of these assays, problems with
inhibitors of RT
activity in serum and nonspecific background RT activity have been described
(20),
raising serious questions about the diagnostic value of these assays.
This invention provides a RT assay that employs a PCR-based amplification
system to detect a known cDNA product of the RT reaction. The assay of the
present
invention, referred to hereafter as Amp-RT, is highly sensitive and specific,
requires no
knowledge of viral genomic sequence and allows the detection of RT activity in
samples of individuals infected with retroviruses or any other biological
entity that
produces RT.
SiJMMARY OF THE IlWENTION
The present invention provides a method for detecting the presence of a
retrovirus in a biological sample comprising the steps of a) contacting the
biological
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sample with an RNA template and a complementary DNA primer under conditions
whereby the RNA template and the DNA primer will anneal and a DNA strand will
be
synthesized as an extension from the DNA primer if reverse transcriptase is
present in
the sample; b) amplifying the synthesized DNA; and c) detecting the
amplification of the
5 synthesized DNA, the amplification of the synthesized DNA indicating the
presence of
reverse transcriptase in the biological sample, thus indicating the presence
of a retrovirus
in the biological sample.
The present invention also provides a method of detecting the presence of a
retrovirus in a biological sample comprising the steps of: a) contacting the
biological
sample with an RNA template, wherein the RNA template is a suitable region of
the
encephalomyocarditis virus which consists of the ribonucleotide of SEQ ID NO:4
and a
complementary DNA primer, wherein the primer is the oligonucleotide of SEQ ID
NO:2, under conditions whereby the RNA template and the DNA primer will anneal
and
a DNA strand will be synthesized as an extension from the DNA primer if
reverse
transcriptase is present in the sample; b) amplifying the synthesized DNA,
wherein the
amplification is by the polymerase chain reaction method whereby the
conditions for the
amplification comprise 30-40 cycles of heating the synthesized DNA and a
primer pair
to 93 to 96 C for 30 seconds to one minute, 53 to 56 C for 30 seconds to one
minute
and 70 to 74 C for 30 seconds to five minutes, wherein the primer pair
consists of the
oligonucleotide consisting essentially of SEQ ID NO:1 and the oligonucleotide
consisting essentially of SEQ ID NO:2; and c) detecting the amplification of
the
synthesized DNA, wherein the detection of the amplification of the synthesized
DNA is
by Southern blot hybridization assay, with a probe consisting essentially of
the
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oligonucleotide of SEQ ID NO:3, the amplification of the synthesized DNA
indicating
the presence of reverse transcriptase in the biological sample, thus
indicating the
presence of a retrovirus in the biological sample.
Additionally provided is a kit for detecting the presence of a retrovirus in a
biological sample, comprising a suitable region of the encephalomyocarditis
virus
genome as an RNA template and a complementary DNA primer for reverse
transcriptase
and a primer pair for polymerase chain reaction, whereby each component is
provided in
separate containers or any combination of the components is provided in a
single
container.
DETAILED DESCRIPTION OF THE INVENTION
As used in the specification and in the claims, "a" can mean one or more,
depending on the context in which it is used.
The present invention provides a method for detecting the presence of a
retrovirus in a biological sample comprising the steps of: a) contacting the
biological
sample with an RNA template and a complementary DNA primer under conditions
whereby the RNA template and the DNA primer will anneal and a DNA strand will
be
synthesized as an extension from the DNA primer if reverse transcriptase is
present in
the sample; b) amplifying the synthesized DNA; and c) detecting the
amplification of the
synthesized DNA, the amplification of the synthesized DNA indicating the
presence of
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reverse transcriptase in the biological sample, thus indicating the presence
of a retrovirus
in the biological sample.
The RNA template can comprise a suitable region of any ribonucleotide
sequence such as, for example, the encephalomyocarditis virus genome. As used
herein,
a "suitable region" means a region of the RNA sequence having no significant
secondary
structure, less than 50% G-C content and to which complementary DNA primers
can be
generated which have T. values within the range of reaction temperatures
appropriate
for the synthesis of a DNA strand, as further described herein. The RNA
template can
be of a length sufficient to produce a DNA product ranging in size from 100 to
500 base
pairs in length and most preferably about 300 base pairs in length. The RNA
template
can be the ribonucleotide of SEQ ID NO:4.
As used herein, "complementary DNA primer" means an oligonucleotide which
anneals to the RNA template in a particular orientation to allow for the
synthesis of a
nascent DNA strand in the presence of RT in the biological sample under the
conditions
described herein. Also as used herein, the "conditions" under which a DNA
strand is
synthesized include the presence of nucleotides, cations and appropriate
buffering agents
in amounts and at temperatures such that the RNA template and the DNA primer
will
anneal and oligonucleotides will be incorporated into a synthesized DNA strand
if
reverse transcriptase is present. More specifically, an example of these
conditions is
provided in the Examples section. The described conditions have been optimized
from
other known RT/cDNA synthesis protocols. It is generally known that other
conditions
can be established for optimization of a particular RT reaction on the basis
of protocols
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well known to one of ordinary skill in the art. The DNA primer can be the
reverse
primer of a primer pair to be used in a subsequent amplification by PCR, such
as, for
example, the oligonucleotide of SEQ ID NO:2 (EMCR2).
The biological sample can comprise any biological tissue or body fluid (e.g.,
cells, serum, plasma, semen, urine, saliva, sputum, cerebrospinal fluid). The
synthesized
DNA strand can be amplified by any of the amplification protocols known in the
art now
or in the future, including but not limited to the polymerase chain reaction
(PCR) (17),
the ligation amplification reaction (LAR) (21), the ligase-based amplification
system
(LAS) (22), the self-sustained sequence replication (3SR) system (23), the
transcription-
based amplification system (TAS) (24) and the Qp replicase amplification
method (25).
For amplification of the synthesized DNA by PCR, the conditions for
amplification can include 30 to 40 (most preferably 35) cycles of heating the
synthesized
DNA and a primer pair to 93 to 96 C (most preferably 95 C) for 30 seconds to
one
niinute (most preferably one minute), 53 to 56 C (most preferably 55 C) for
30
seconds to one minute (most preferably one minute) and 70 to 74 C (most
preferably
72 C) for 30 seconds to five minutes (most preferably one minute).
As used herein, "a primer pair" refers to two primers, one having a forward
designation and the other having a reverse designation relative to their
respective
orientations on a double-stranded DNA molecule which consists of a sense and
antisense
sequence, such that under the amplification conditions described herein, the
forward
primer anneals to and primes amplification of the sense sequence and the
reverse primer
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anneals to and primes amplification of the antisense sequence. Primers can be
selected
for use in the amplification reaction on the basis of having less than 50% G-C
content,
having minimal complementarity with other primers in the reaction (to minimize
the
formation of primer dimers) and having T. values within the range of reaction
temperatures appropriate for PCR. In addition, primers can be selected to
anneal with
specific regions of the RNA template such that the resulting DNA amplification
product
ranges in size from 100 to 500 base pairs in length and most preferably around
300 base
pairs in length. For example, in the conditions described above, the primer
pair can
consist of the oligonucleotide of SEQ ID NO:1 (EMCF1) as the forward primer
and the
oligonucleotide of SEQ ID NO:2 (EMCR2) as the reverse primer.
As used herein, "detecting" or "detection" of the amplified DNA refers to
quantitatively or qualitatively determining the presence of the amplified DNA
strand
which is only synthesized if RT is present in the biological sample. The
amplification of
the synthesized DNA can be detected by any method for the detection of DNA
known in
the art. For example, detection of the amplified DNA can be by Southern blot
hybridization assay, by visualization of PCR products of specific molecular
weight on
ethidium bromide stained agarose gels, by measurement of the incorporation of
radiolabeled nucleotides into the synthesized DNA strand by autoradiography or
scintillation measurement, by ELISA modified for the capture of a detectable
moiety
bound to the amplified DNA, or any other detection method known to one of
ordinary
skill in the art.
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For detection by Southern blot hybridization assay, the synthesized DNA can be
detected by a probe specific for the DNA synthesized from the template. For
example,
such a specific probe can consist essentially of the oligonucleotide of SEQ ID
NO:3
(EMCP 1). A Southern blot hybridization protocol for use in the invention is
5 demonstrated in the Examples.
The present invention provides a method for detecting the presence of a
retrovirus in a biological sample comprising the steps of: a) contacting the
biological
sample with a suitable region of the encephalomyocarditis virus genome as an
RNA
10 template and a complementary DNA primer under conditions whereby the RNA
template and the DNA primer will anneal and a DNA strand will be synthesized
as an
extension from the DNA primer if reverse transcriptase is present in the
sample;
b) amplifying the synthesized DNA; and c) detecting the amplification of the
synthesized
DNA, the amplification of the synthesized DNA indicating the presence of
reverse
transcriptase in the biological sample, thus indicating the presence of a
retrovirus in the
biological sample.
The present invention also provides a method of detecting the presence of a
retrovirus in a biological sample comprising the steps of: a) contacting the
biological
sample with an RNA template and a complementary DNA primer under conditions
whereby the RNA template and the DNA primer will anneal and a DNA strand will
be
synthesized as an extension from the DNA primer if reverse transcriptase is
present in
the sample; b) amplifying the synthesized DNA by the polymerase chain reaction
method
whereby the conditions for the amplification comprise 30-40 cycles of heating
the
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synthesized DNA and a primer pair to 93 to 96 C for 30 seconds to one minute,
53 to
56 C for 30 seconds to one minute and 70 to 74 C for 30 seconds to five
minutes; and
c) detecting the amplification of the synthesized DNA, the amplification of
the
synthesized DNA indicating the presence of reverse transcriptase in the
biological
sample, thus indicating the presence of a retrovirus in the biological sample.
The present invention further provides a method for detecting the presence of
a
retrovirus in a biological sample comprising the steps of a) contacting the
biological
sample with a region of the encephalomyocarditis virus genome as an RNA
template and
a complementary DNA primer under conditions whereby the RNA template and the
DNA primer will anneal and a DNA strand will be synthesized as an extension
from the
DNA primer if reverse transcriptase is present in the sample; b) amplifying
the
synthesized DNA by the polymerase chain reaction method whereby the conditions
for
the amplification comprise 30-40 cycles of heating the synthesized DNA and a
primer
pair to 93 to 96 C for 30 seconds to one minute, 53 to 56 C for 30 seconds to
one
minute and 70 to 74 C for 30 seconds to five minutes; and c) detecting the
amplification
of the synthesized DNA, the amplification of the synthesized DNA indicating
the
presence of reverse transcriptase in the biological sample, thus indicating
the presence of
a retrovirus in the biological sample.
As an example, the present invention can provide a method for detecting a
retrovirus in a biological sample comprising the steps of: a) contacting the
biological
sample with a suitable region of the encephalomyocarditis virus genome as an
RNA
template, wherein the RNA template is the ribonucleotide of SEQ ID NO:4 and a
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complementary DNA primer, wherein the primer is the oligonucleotide of SEQ ID
NO:2, under conditions whereby the RNA template and the DNA primer will anneal
and
a DNA strand will be synthesized as an extension from the DNA primer if
reverse
transcriptase is present in the sample; b) amplifying the synthesized DNA,
wherein the
DNA is amplified by the polymerase chain reaction under conditions which
comprise
about 35 cycles of heating the synthesized DNA and a primer pair to 95 C for
one
minute, 55 C for one minute and 72 C for one minute, wherein the primer pair
consists
of the oligonucleotide consisting essentially of the SEQ ID NO:1 and the
oligonucleotide consisting essentially of the SEQ ID NO:2; and c) detecting
the
amplification of the synthesized DNA, wherein the amplification is detected by
Southern
blot hybridization assay with a probe consisting essentially of the
oligonucleotide of
SEQ ID NO:3.
Additionally provided is a kit for detecting the presence of a retrovirus in a
biological sample, comprising the enzymes, buffering agents, cations and
oligonucleotides well known in the art for carrying out RT and PCR reactions.
The kit
also comprises a suitable region of the encephalomyocarditis virus genome as
an RNA
template and a complementary DNA primer for reverse transcriptase and a primer
pair
for polymerase chain reaction. The RNA template, the complementary DNA primer
and
the primers of the primer pair can each be in separate containers or all or
any
combination of these components can be combined in a single container. The
complementary DNA primer can be the oligonucleotide of SEQ ID NO:2 (EMCR2),
the
RNA template can be the ribonucleotide of SEQ ID NO:4 and the primer pair can
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consist of the oligonucleotide consisting essentially of SEQ ID NO:1 (EMCF1)
and the
oligonucleotide consisting essentially of SEQ ID NO:2 (EMCR2).
The present invention has additional applications based on the basic inventive
principle that RT can be detected and quantitated in a biological sample. One
application of this principle is the differential identification of
retroviruses on the basis of
the specific reactivity of the detected RT with antibodies against different
retroviral RTs
(for example, antibodies which can distinguish RT produced by HIV-1 or HIV-2
in
infected individuals). Each retrovirus has a distinct RT, to which specific
antibodies can
be generated, thereby permitting identification of the specific retrovirus.
The method of
the present invention can be employed to first detect the presence of RT and
various
antibodies specific for RTs of different retroviruses can then be added to the
biological
sample to identify the type of retrovirus present. An antibody of known RT
specificity
will bind the RT present in the biological sample if the RT is produced by the
virus to
which the antibody is specific and will inhibit its activity, resulting in no
DNA synthesis
in the presence of antibody.
Another application of the present invention is screening patients for
resistance
to drug therapy. The susceptibility of RT to anti-RT drugs can be monitored
over time
in a patient receiving anti-RT drug therapy. The present invention can be
employed to
detect the emergence of anti-RT drug resistance in a patient by direct testing
of the
patient's serum for RT activity as an indicator of the susceptibility of the
RT in the
patient's serum to the anti-RT drug(s) used. If drug resistance is detected,
alternative
treatment strategies may be implemented. This invention obviates the need for
the
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lengthy and labor-intensive culture methods currently used to study drug
resistance in
patients.
A third application of the present invention is the monitoring of virus load
in
patients infected with biological entities which produce RT. Quantitative
measurement
of RT over time can be correlated with survival and/or recovery rates in
patients with
illnesses caused by theses entities, for the purpose of following disease
progression and
prognosing the patient's illness.
The present invention is more particularly described in the following examples
which are intended as illustrative only since numerous modifications and
variations
therein will be apparent to those skilled in the art.
EXAMPLES
The following examples are intended to illustrate, but not limit, the
invention.
While the protocols described are typical of those that might be used, otlier
procedures
known to those skilled in the art may be alternatively employed.
Viruses
The retroviruses used in this study were prepared as follows. IHIV-1 Lai and
simian immunodeficiency virus (SIVB670) were propagated in peripheral blood
lymphocytes (PBLs). Caprine arthritis encephalitis virus (CAEV-63) was
propagated in
fetal goat synovial membrane cells. HTLV-I and HTLV-II were obtained from
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supernatants of MT-2 and Mo-T cell lines; simian retroviruses types 1 and 2
(SRV-1
and SRV-2) were grown in Raji cells; gibbon ape leukemia virus (GALV) was
grown in
Jurkat cells and simian foamy virus type 3 (SFV-3) was grown in the Cf2Th cell
line.
5 In vitro transcription of the RNA template
An RNA sequence from the encephalomyocarditis virus (EMCV) was used as
template and was generated from a plasmid vector obtained from Novagen
(Madison,
WI, USA). A small EMCV sequence (350 bp) (SEQ ID NO:4) was amplified from the
plasmid, using standard PCR conditions and the primer pair T7-EMCF1 (SEQ ID
NO:6)
10 and EMCR2 (SEQ ID NO:2). The sequence of T7-EMCF 1(SEQ ID NO:6) was:
5'GGTACCTAATACGACTCACTATAGGGAGACATTAGCCATTTCAACCCAT3'
and that of EMCR2 (SEQ ID NO:2) was:
5'GTTCATGACAGGCCGATACAGAGG3'.
15 To allow the in vitro transcription of this PCR product, the sense primer,
EMCF1: 5'CATTAGCCATTTCAACCCAT3' (SEQ ID NO:1), was modified at the 5'
end by adding a T7 promoter sequence: 5'GGTACCTA.ATACGACTCACTAT-3' (SEQ
ID NO:5).
The EMCV-amplified product was then transcribed with the large scale T7
transcription kit from Novagen according to the manufacturer's instructions.
The DNA
in the RNA preparation was subsequently digested twice with 20 units of RNase-
free
DNase (Promega) at 37 C for 60 minutes and the DNase was inactivated by
heating at
95 C for ten minutes. The purity of the RNA preparation was checked for
residual DNA
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contanvnation by PCR amplification with EMCF 1(SEQ ID NO: 1) and EMCR2 (SEQ
ID NO:2) and subsequent Southern blot hybridization to the 32P-end-labeled
internal
probe EMCP1 (SEQ ID NO:3): 5'TGCTCTCACCTTATCAAAATCCAAT3'.
Amp-RT
Twenty ul or less of the biological sample to be tested was added to 40 ul of
RT
buffer containing 10 ng of RNA template, 10 units of RNasi[,~.06% NP-46,M200
ng
primer (EMCR2, SEQ ID NO:2), 0.8mM EGTA, 2mM DTT, 50mM Tris-HCI, 50mM
KCI, and 10mM MgC12. The reaction was incubated at 37 C for two hours and
then
heated at 95 C for ten minutes to inactivate any RT activity. A volume of 50
ul of
standard PCR buffer (17) containing 0.5 units of Taq polymerase and 200 ng of
the
sense primer without the T7 sequence (EMCF1, SEQ ID NO:1) was added to this
mixture. The reaction was cycled 35 times at 95 C for one minute, 55 C for one
minute
and 72 C for one minute. Twenty ul of the reaction product was electrophoresed
in
1.8% agarose gels and Southern blot hybridized to a 32P-end-labeled internal
probe
(EMCP1, SEQ IDNO:3) at 42 C overnight. The blots were washed in 2X SSC (NaCI
and sodium citrate), 0.5% sodium dodecyl sulfate (SDS) for one to two hours.
The
blots were exposed from six to 24 hours.
To check the integrity and the purity of the RNA template preparation, RT
reactions were carried out using the EMCV RNA template (SEQ ID NO:4) and an
HIV-1 virus stock as the RT source. While the HIV-1 reactions were positive,
control
reactions which had no HIV-1, or which were pretreated with RNase before the
addition
of HIV-1, were all negative. These negative results indicated that the
underlying
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reaction of Arnp-RT was RNA-dependent and that the RNA template and other
assay
components were free of any contaminating target DNA. To further confirm that
the
reaction was mediated by RT, Amp-RT reactions were prepared by using HIV-1
culture
supernatant in the presence of 2 ug of tetrahydroimidazo-benzodiazepin (TIBO)
compounds, which are non-nucleoside RT inhibitors (2). The results
demonstrated
inhibition of the Amp-RT reaction only in the presence of the TIBO compounds,
while
the DNA polymerase activity of Taq in the control amplification reactions was
not
affected.
Comparative sensitivity analysis of Amp-RT
Many specific and generic assays are currently used to detect the presence of
retroviruses. To compare the relative sensitivities of these assays to Amp-RT,
serial
10-fold dilutions of an HIV-1 culture supernatant were made in culture medium
and
then divided into equal aliquots. Separate aliquots were subjected to testing
by standard
RT assay, branched DNA detection, p24 antigen capture, 50% tissue culture
infective
dose (TCID50) determination, RT-PCR and Amp-RT.
Standard RT assay
RT activity was measured in culture supernatant with the template primer of
poly(rA).oligo dT according to the methods of Willey et al. (3). The enzymatic
activity
was assessed by measuring the incorporated tritiated thymidine monophosphate.
Branched DNA (bDNA) detection of HIV-1
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Branched DNA (bDNA) detection of HIV- I was performed according to the
manufacturer's instructions (Chiron, Emryville, California, USA). A cutoff of
5,000
RNA equivalents/ml was used, as recommended by the manufacturer.
p24 antigen capture
Levels of base dissociated-p24 antigens of HIV-1 were determined by a
commercially available ELISA kit from Organon Technika (North Carolina, USA).
TCID50 determination
The TCIDso of the HIV-1 viral stock was determined on PBLs as previously
described by McDougal et al. (26).
RT-PCR
Particle-associated HIV-1 genomic RNA in culture supernatant was extracted
with phenol/chloroform, precipitated with ethanol and reconstituted in 40 ul
of RT
buffer [50mM Tris-Cl (pH 8.3), 20mM KCI, 10mM MgCIj (15). RNA was further
digested with 5 units of DNase-I (Promega) in the presence of 10mM sodium
acetate at
37 C for 30 minutes. DNase was inactivated by heating at 95 C for ten minutes.
For
reverse transcription, 10 ul of RNA was added to 20 ul of the RT mixture,
containing
10mM DTT, 20 units RNasin, 0.875mM each of GTP, ATP, CTP and TTP, 200 ng of
reverse primer (SK39, SEQ ID NO:8) and 0.02 units of AMV-reverse
transcriptase. The
mixture was reverse transcribed at 42 C for 45 minutes. Controls included 10
ul
aliquots that were not reverse transcribed. All samples were then amplified by
PCR in
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100 ul reaction volumes for 35 cycles, usino, SK38/SK39 (SEQ ID NOS:7 and 8)
as
primers and the amplified products were Southern blot hybridized to the 32P-
labeled
SK19 (SEQ ID NO:9) probe (27).
The results, shown in Table 1, demonstrated that Amp-RT was the most
sensitive assay and was 100,000 times more sensitive than the standard RT
assay,
10,000 times more sensitive than the bDNA detection system and the p24 antigen
capture assay and 100 times more sensitive than TCID50 and RT-PCR. The p24
concentration in the undiluted HIV-1 sample was found to be 1.6 ng.
Testing of clinical samples with Amp-RT
Forty-two serum samples from HIV-1 seropositive individuals enrolled in a
study
of the natural history of HIV in homosexual men in Atlanta, Georgia, USA (28)
were
tested by Amp-RT. The clinical stage of the HIV-1 infection in these men was
determined on the basis of the Centers for Disease Control and Prevention
(CDC)
revised classification system (29). Laboratory markers, including CD4+
lymphocyte
counts and percentage of CD4+ lymphocytes in the blood, were determined by
standard
flow cytometric methods. Serum samples from 20 healthy individuals who were
HTLV-I/II and HIV-1 seronegative were included as controls.
Preliminary experiments using normal human serum spiked with HIV-1 were all
Amp-RT negative and indicated that preparation of serum samples for Amp-RT
requires
special conditions different from those described for culture supernatant. For
instance,
even small volumes (5-20 ul) of serum or plasma could not be directly tested
by
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Amp-RT because of protein precipitation that develops during the high
temperature
stage of the PCR cycle. Therefore, to avoid this problem, sera were
ultracentrifuged
and the Amp-RT assay was performed using the viral pellet.
5 For testing of culture supernatant, 20 ul or less was used for the RT
reaction.
For testing serum. or plasma samples, 0.5 ml was diluted 10-fold in DEPC-
treated
phosphate buffered saline, clarified by centrifugation at 1,000 g for 10
minutes and then
ultracentrifuged at 44,000 g for one hour. The pellet was suspended in 50 ul
of RT
buffer containing 0.6% NP-40 for 15 minutes and aliquots of 5-45 ul were used
in the
10 Amp-RT assay.
The results of testing serum samples indicated that Amp-RT was able to detect
RT activity in 36 (85.7% ) serum samples (Table 2), 31 of which (86.1%) had
detectable
p24 antigen. This high sensitivity was also coupled with a high specificity
since none of
15 the HIV-1/HTLV seronegative samples were positive. Of the six samples from
patients
classified as Al, three were Amp-RT positive (50%), while five of seven
(71.4%) and
of 26 (96.1%) of the samples from patients classified as B2 and C3,
respectively,
were Amp-RT positive.
20 Testing of the hepatitis B virus (HBV) by Amp-RT
Because the polymerase gene of HBV has been reported to possess RT-like
activity (30), Amp-RT was employed to detect the HBV-associated RT activity.
Serum
samples from 21 HBV-infected individuals were selected, including 11 samples
with
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21
detectable HBe antigen (Abbott HBe EIA, Chicago, USA) and ten other samples
with
no detectable HBe antigen. .
Despite the documented presence of I-iBV virions in I 1 serum specimens, none
of the samples yielded positive results by Amp-RT analysis. These negative
results
exclude the possibility of the HBV-associated RT interfering in the Amp-RT
assay, at
least under these conditions, and therefore confirm the specificity range of
the assay for
the detection of only retroviral RTs.
Detection of different retroviruses by Amp-RT.
The ability of Amp-RT to detect a wide variety of retroviruses representing
members of all three retroviral subfamilies was investigated. The lentiviruses
tested
included HIV-1, SIV and CAEV. HTLV-I, HTLV-II, GALV, SRV-1 and SRV-2 were
representative of the oncoviruses and SFV-3 was representative of the
spumaviruses.
Amp-RT was capable of detecting all retrovinises tested. Most importantly,
viruses such
as IFTI,V-I, HTLV-II and GALV, which are typically difficult to detect with
the
standard RT methodology, were easily identified with this assay.
These viruses could be detected by Amp-RT in dilutions containing 0.04 to
0.00004 ul of the original unconcentrated culture supernatant. The serial
dilutions of the
tested retroviruses were made in supernatant from uninfected cell lines (Hut-
78, A301
or U937). All three supernatants consistently tested negative, as can be seen
from the
end point dilutions of the tested retroviruses.
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22
Side by side comparison of previously published RT assays
To demonstrate that the assay of the present invention provides increased
specificity and sensitivity in comparison to other published RT assays [22,
23], a side by
side comparison of these assays can be performed. These assays differ in the
particular
RNA template sequence, primer sequences, RT buffer compositions, amplification
conditions, detection conditions and sample preparation used. Therefore, for a
comparison
of the two assays, a panel of serum samples from HIV-1 seropositive
individuals and from
HIV-1 and HTLV-I/II negative individuals can be tested according to the
teachings of the
present assay and each other assay. Positive controls can include normal human
serum
spiked with HIV-1 virus particles. The specificity and sensitivity of these
assays can then
be computed and compared.
Throughout this application various publications are referenced by numbers
within parentheses. Full citations for these publications are as follows. The
disclosures of
these publications in their entireties may be referred to for further details
of the state of
the art to which this invention pertains.
Although the present process has been described with reference to specific
details of certain embodiments thereof, it is not intended that such details
should be
regarded as limitations upon the scope of the invention except as and to the
extent that
they are included in the accompanying claims.
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Table 1. Comparison of the sensitivity of Arnp-RT in the detection of HIV-1
with other
generic and virus-specific methods.
HIV-1 Std.
dilution RT bDNA p24 ag TCID50 RT-PCR Amp-RT
+ + + + + +
10-' + + + + + +
10-Z + + + + + +
10-3 - + + + + +
10-4 - - - + + +
10's - - - + + +
10-6 - - - - - +
io-7 - - - - - +
10'g - - - - - -
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Table 2. Detection of RT activity by Amp-RT in serum samples from HIV-1
infected
individuals characterized by clinical stage, CD4+ lymphocyte counts in the
peripheral
blood and amount of p24 antigen in the serum.
No Patient ID p24 (pgJml) %CD4 CD4 count Clinical Stage* Amp-RT
1 69877 NEG 27 830 Al NEG
2 82850 NEG 23 699 Al P
3 69878 141 33 566 Al p
4 77913 NEG 25 599 Al P
111380 49 27 778 Al NEG
6 111318 49 28 539 Al NEG
7 85987 141 23 348 A2 p
8 66583 55 13 286 A2 p
9 62303 42 12 341 B2 P
71172 56 22 281 B2 NEG
11 71245 100 11 208 B2 P
12 77149 44 16 282 B2 P
13 86563 141 14 286 B2 P
14 66994 NEG 25 260 B2 NEG
57589 141 18 370 B2 P
16 21149 NEG 10 97 B3 P
17 70145 8 5 26 C3 NEG
18 84300 141 1 4 C3 P
19 111207 N 10 103 C3 P
67365 141 5 24 C3 P
21 96215 40 6 80 C3 P
22 120979 141 3 19 0 P
23 20525 73 3 22 C3 P
24 736 103 8 50 C3 P
1837 158 3 6 C3 P
26 3125 150 5 12 0 P
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27 3369 33 1 1 C3 P
28 20577 85 6 123 C3 P
29 20525 73 3 22 C3 P
20577 85 6 123 C3 P
31 20709 158 10 42 C3 P
32 20876 80 7 57 C3 P
33 21156 124 14 106 C3 P
34 21196 24 8 48 C3 P
21684 45 4 25 C3 P
36 23505 64 4 19 C3 P
37 23507 15 3 52 C3 P
38 24462 149 6 41 C3 P
39 24705 149 5 128 C3 P
25238 98 5 13 C3 P
41 23209 71 5 10 C3 P
42 53416 NEG 14 81 0 P
*Al and A2 refer to asymptomatic, acute (primary) or persistent generalized
lymphadenopathy with CD4+ lymphocyte counts of >500/ul or 200-499/ul,
respectively.
B2 and B3 refer to symptomatic conditions different from those in stage A or
AIDS,
with CD4+ lymphocyte counts of 200-499/ul and <200u1, respectively. C3 refers
to
AIDS, with CD4+ lymphocyte counts of <200/ul (29).
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immunodeficiency virus type 1 RNA in plasma: Application to acute retroviral
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: HENEINE, WALID
FOLKS, THOMAS M.
SWITZER, WILLIAM M.
YAMAMOTO, SHINJI
(ii) TITLE OF INVENTION: METHODS FOR SENSITIVE DETECTION OF
REVERSE TRANSCRIPTASE
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Spratt, Gwendolyn D.
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(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CATTAGCCAT TTCAACCCAT 20
(2) INFORMATION FOR SEQ ID NO:2:
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(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:2:
GTTCATGACA GGCCGATACA GAGG 24
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGCTCTCACC TTATCAAAAT CCAAT 25
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
CAUUAGCCAU UUCAACCCAU GCGUUUGAGG AGAAGCGCUU UCUGAUAACC GGUGGUCUCC 60
CAUCAGGUUG UGCAGCGACC UCAAUGCUAA ACACUAUAAU GAAUAAUAUA AUAAUUAGGG 120
CGGGUUUGUA UCUCACGUAU AAAAAUUUUG AAUUUGAUGA UGUGAAGGUG UUGUCGUACG 180
GAGAUGAUCU CCUUGUGGCC ACAAAUUACC AAUUGGAUUU UGAUAAGGUG AGAGCAAGCC 240
UCGCAAAGAC AGGAUAUAAG AUAACUCCCG CUAACACAAC UUCUACCUUU CCUCUUAAUU 300
CGACGCUUGA AGACGUUGUC UUCUUAAAAP. GAAAGUUUAA GAAAGAGGGC CCUCUGUAUC 360
GGCCUGUCAU GAAC 374
(2) INFORMATION FOR SEQ ID NO:5:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GGTACCTAAT ACGACTCACT AT 22
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGTACCTAAT ACGACTCACT ATAGGGAGAC ATTAGCCATT TCAACCCAT 49
(2) INFORMATION FOR SEQ ID NO:7:
(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:7:
ATAATCCACC TATCCCAGTA GGAGAAAT 28
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDED2v`ESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
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TTTGGTCCTT GTCTTATGTC CAGAATGC 28
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATCCTGGGAT TAAATAFIAAT AGTAAGAATG TATAGCCCTA C 41
