Note: Descriptions are shown in the official language in which they were submitted.
W O 94/08032 P~r/US93/09233
- 3 ~. 2 ~
DESCRIPTION
One Step RNA and Combined One Ste~ RNA
and DNA Polymerase Chain Reaction for
Detection of Rare RNA or RNA and DNA
Related A~Plication
This application is a continuation-in-part of
Vierling and Hu, entitled "One Step RNA Polymerase Chain
Reaction for Detection of Rare RNA", filed September 29,
1992, U.S. Serial No. 07/954,35~, pending in the Patent
Office.
Background
PCR iB a well used technigue for amplification of
DNA. It is used in a variety of assays for the detection
of particular DNA sequences, ~uch as those associated with
bacterial or DNA virus caused diseases. Additionally, in
combination with reverse transcription of RN~ to DNA, PCR
can be used for the detection of RNA. Because of the
sensitivity of PCR, it is especially appropriate for the
detection of rare RNA or DNA.
The hepatitis C virus ~HCV), recognized as the
principal agent of non-A, non-B hepatitis (Choo, Q.-L. et
al.; Alter, H.J. et al.), is a positive-stranded RNA virus
related to human flaviviruses and animal pestiviruses
(Choo, Q.-L. et al.; Houghton, M. et al.). The HCV RNA
genome is approximately 10,000 nucleotides (nt) in lçngth
and contains a single open reading frame capable of
encoding a 3,100 amino acid polyprotein precursor of
individual structural and nonstructural proteins. A 5'
untranslated region (S'UTR) of approximately 324-341 nt is
highly conserved among different HCV strains and favored
for both dia~Fnostic HCV RNA PCR and HCV RNA hybridization
(Houghton, M. et al.; Hu, K.-Q. et al. ~1991); Bukh, J. et
al.; Hu, K.-Q. et al~ (1992)).
.
W094/08032 PCT/US93/09233
`~ 1 2 ~ 2
Detection of circulating anti-HCV antibodies or PCR
amplification of HCV RNA ("HCV RNA PCR~) are the two major
techniques currently used to diagnose HCV infection.
Since detection of HCV RNA by reverse transcription PCR is
more direct and sensitive than anti-HCV testing, it has
become the diagnostic standard for both acute and chronic
HCV infection ~Hu, K.-Q. et al. (l991); Bukh, J. et al.;
Houghton, M. et al.). Although HCV RNA PCR is a sensitive
and specific technique, extensive clinical application has
been thwarted by its labor-intensity, reaction time,
potential for contamination and disparate results among
laboratories due to variation in techniques and primers.
" Separate steps for reverse transcription (RT~ and the
subsequent addition of PCR reagents contribute to both the
labor intensity and potential for contamination. It would
be advantageous, therefore, to have a technique in which
both RT and PCR amplification could be accomplished in one
step.
One step PCR assays have successfully been performed
on abu~dant RNA such as bacterial ribosomal RNA. (Wang,
R.-F., et al.). However, the prior art describes a
serious obstacle to a one step RNA PCR assay for rare RNA.
Sellner et al. report that RTase severely inhibits Taq
polymerase. Because HCV is often present in very low copy
number, a large amount of RTase is necessary to ensure
that any HCV RNA is copied into DNA form before PCR.
Thus, the prior art teaches that a one step assay will not
be very accurate for detect-on of a rare RNA such as HCV
RNA.
Hepatitis B virus ("HBV") infection is a worldwide
human health problem that causes both acute and chronic
hepatitis and is associated with the development of
hepatocellu-lar carcinoma. Clinical diagnosis of HBV
infection has been based on detection of circulating HBV
antigens, antibodies against HBV viral peptides. There
has been recent enthusiasm for detection and ~uantitation
of HBV DNA by molecular hybridization. (Hoofnagle J.H. et
W094/08032 ` PCT/US93/09233
212'1 i8~
al.). HBV DNA PCR has been shown to be the most sensitive
technique for detection of even trace amounts of Hsv DN~.
(Monjardino J. et al and Kaneko S. et al.). However, as
with HCV RNA PCR, the labor-intensity, risk of contamina-
tion and time required for analysis have impeded itsclinical application.
Moon, I.G. et al. disclose a method for simultaneous
detection of HBV and HCV infection. However, the method
disclosed by Moon et al. differs in the method of
extraction of DNA and RNA from the sample. Also, Moon et
al. specifically teach that the extracted DNA and RNA
should be subjected to reverse transcription followed by
~, PCR ammplification and a second PCR amplification after
addition of a ~econd pair of "nested" primers specific for
HCV.
To overcome these drawbacks, it would be advantageous
to have a combined one step PCR for simultaneous detection
of HCV RNA and HBV DNA.
Summary of the Invention
The present invention provides a one step RNA PCR
method for the detection of rare RNA such as HCV RNA in
serum or tissues, and a combined one step HCV RNA PCR and
HBV DNA PCR method ("combined one step HBV-HCV PCR") for
the simultaneous detection of rare RNA such as HCV RNA and
HBV DNA in serum or tissues. These techniques are both
~en~itive and specific, substantially simplify the
traditional procedure, decrease the time necessary for
detection, and reduce the risk of contamination. These
techniques are an improvement over the traditional methods
for detectic of rare RNA such as HCV RNA PCR using
primers from _ne highly conser~ed 5'UTR of the HCV genome,
and over the se~arate steps previously required to detect
HBV DNA and HCV RNA. These techniques are suited to the
detection of RNA and DNA from any source, not only viral
RNA or DNA or HCV RNA or HBV DNA.
W094/08032 PCT/~S93/09233
~121~1
The one step method is a highly specific procedure.
The specificity of the one step method has been confirmed
by its 100~ concordance with traditional HCV RNA PCR in 50
serum samples, including positive and negative controls.
The one step method substantially reduces the time
reguired for analysis. The one step method is at least
three times as fast as traditional two step RTase plus PCR
procedures.
The sensitivity of the one step method for detection
of serially diluted hepatic RNA extracted from an HCV
infected liver is comparable to that of traditional ~CV
RNA PCR. Additionally, the one step method is more
~, sensitive than traditional PCR methods for detecting HCV.
In serum samples containing both plus and minus stranded
HCV RNA, the one step method consistently produced
stronger PCR product signals than traditional PCR. These
results indicated that both strands were reverse
transcribed in the one step technique.
The combined one step HBV-HCV PCR method is a highly
specific procedure. The specificity of the combined one
step HBV-HCV PCR method has been confirmed by its 100%
concordance with traditional HBV DNA PCR and HCV DNA PCR
in 28 serum samples. (See the Table set forth in Working
Example 15) Additionally, the expected 456 bp HBV DNA and
241 HCV CDNA bands were identified in the serum of a
patient with combined BV and HCV infection. Also, no
bands were identified in normal human serum. Finally,
Southern blots confirmed the specificity of the bands for
HBV or HCV.
The combined one step HBV-HCV PCR method also
substantially reduces the time required for analysis of
samples, and the sensitivity of the combined one step HBV-
HCV PCR method for detection of HBV DNA is greater than
the widely used HBV DNA slot hybridization diagnostic
technique. This was shown by the 100~ concordance between
the combined one step HBV-HCV PCR method and HBV DNA slot
hybridization among HBV-positlve sera. However~ among 12
W094/08032 ~J ~ ~ L~ PCT/US93/09233
patients with negati~e Hsv DNA slot hybridization assays,
3 patients were positi~e for HBV DNA in the combined one
step HBV-HCV PCR method.
Substantial reductions in risk of contamination make
these methods suitable for testing multiple clinical
samples. Because reagents are added only once, there is
less opportunity for impurities to enter the reactions.
This invention pro~ides more uniform results than
previously available methods because of the opportunity
for automation of many of the steps.
Brief Description of the Drawinqs
~, Figure 1 shows the effect of RTase concentration on
the one step HCV RNA PCR. A 241 bp HCV cDNA product
(lanes 1 and 1' to 5 and 5') was obtained from the
reactions of 2.5 U of Taq polymerase and 5, 10, 25, 50 and
100 U of RTase, respectively. MW: 123 bp ladder DNA
marker.
Figure 2 shows RNA extracted from 1 ml of HCV-
positive serum that was serially diluted and tested by
both tradition,al (A) and one step (B) HCV RNA PCR. MW: 123
bp ladder DNA marker. Lanes 1 through 5 show agarose gels
containing the cDNA (241 bp) product of the HCV PCR for
specimens diluted 10-1, 10-2, 10-3, 10-4 and 10-5,
respectively.
2c Figure 3 shows the results of the combined one step
HBV-HCV PCR method, one step HCV RNA PCR and traditional
HBV DNA PCR. Lanes 1 and 2: One step HCV RNA PCR; lanes
3 and 4: Traditional HBV DNA PCR; lanes 5 and 6:
Combined one step HBV-HCV PCR; lane 7: Negative control.
MM: 123 bp ladder DNA marker. HBV DNA PCR products = 4S6
bp and HCV cDNA PCR products - 241 bp. Panel A is an
ethidium bromide stained agarose gel of electrophoresed
PCR products. Panel B is a Southern blot hybridization
using probe specific for HBV DNA. Panel C is a Southern
blot hybridization using probe specific for HCV cDNA.
W O 94/08032 PC~rtUS93/09233
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Detailed Descr ption of the Invention
This invention provides methods and means for rapid,
accurate, sensitive detection of rare RNAs in samples
through the use of a one step procedure wherein RTase and
PCR reactions are combined. The sample being subjected to
the assay for the rare RNA is combined with both RTase to
convert the RNA into DNA and a heat stable DNA polymerase
to perform the PCR, deoxynucleotide triphosphates (dNTPs),
optionally RNase inhibitor(s) to protect the rare RNA from
degradation, and the appropriate primer for the PCR
reaction, in a standard buffered salt solution. The
reactions are run sequentially. The one step nature of
~, the reaction removes the need to stop the first reaction,
extract the DNA, change buffer conditions, and add new
enzyme. Each one of these eliminated steps takes time and
introduces the opportunity for contamination of the
sample.
Additionally, the reaction can be automated once all
components have been added to the sample. A,controlled
tempe;-ature block such as a thermal cycler traditionally
used for PCR can be adapted to incubate the sample first
at a temperature appropriate for RTase, such as 37-42C,
followed by incubation for a period of time and at a
temperature sufficiently high to denature the RTase and
initially denature the DNA, e.g. 94C for 3 min.
Following this the temperature block cycles temperatures
as is standard for PCR. See, e.g., U.S.P.N. 4,683,195,
which is incorporated herein by reference.
The combined one step HBV-HCV PCR method encompasses
the aforesaid advantages of HCV RNA PCR with the
additional advantage that both HCV RNA and HBV DNA
detection can be carried out simultaneously resulting in
more efficient screening for HBV and HCV.
W O 94/08032 PC~r~US93/09233
Preparation of Sample
Nucleic Acid Extraction Methods
In the one step HCV RNA PCR method the sample to be
assayed is prepared by first extracting ~NA, by any of the
standard techniques such as guanidinium isothiocyanate
extraction (Sambrook, J., et al.; Chomczynski, P., et
al.). A prerequisite for the combined one step HBV-HCV
PCR method is the efficient extraction of both DNA and RNA
in amounts reflecting their relative quantities in a serum
~ample. This extraction technique will work for any RNA
or DNA species regardless of source~ Methods which
degrade or deactivate proteins, including DNases and
~, RNaRes, such as the guanidinium isothiocyanate or
Proteinase K method are suitable for use with the combined
one step HBV-HCV PCR method. For simultaneous extraction
of both RNA and DNA in the serum samples a repeated phenol
extraction method was developed. 150 ul of serum is
digested by 10-15 ul of proteinase K (10 mg/ml) at 50 C
for two hours. Phenol/chloroform extraction is carried
out first in an acid environment (pH 4.0) to isolate RNA,
and repeated after adjusting the pH of the phenol phase to
pH 8.0 for isolation of DNA. The acidity and basicity can
~ary within the range of 3.0 to 5.0 and 7.1 to 9Ø
Alternatively, the phenol/chloroform extraction could be
carried out at a basic pH to ext~act DNA followed by
adjustment of the pH to an acid environment to extract
RNA. Adjustment of the pH is carried out by the addition
of a buffer solution of the proper pH, appropriate for DNA
and RNA extractions; for example Tris-EDTA, and othe~s as
will be known to one of ordinary skill in the art. Both
extracts are pooled, 10 ug of yeast tRNA is added and the
nucleic acids are co-precipitated with isopropanol. The
pellets of extracted nucleic acids are resuspended in 10
ul of diethyl pyrocarbonate (DEPC) treated water and
stored at -70 C before use.
W094/08032 PCTiUSs3/0s233
i 8
Reaction Conditions
A. One Step HCV RNA PCR
Reaction conditions for the one step PCR detection of
rare RNAs are the same as those used in a traditional PCR
reaction.:: For the particular RNA being detected, optimal
salt and enzyme conditions can be readily determined.
Ribonuclease inhibitors such as RNasin ~ (obtained from
Promega Co., Madison, WI) increase the yield on the RTase
reaction. RNase inhibitor conditions for this reaction
are similar to those used in traditional RTase and PCR
reactions. For HCV RNA, the reaction conditions
determined to be most favorable are found in Example 3.
B. Combined One Step HBV-HCV PCR Method
The extracts of serum DNA and RNA (see Preparation of
Sample, B. above) are used as viral templates. Reaction
conditions are identical to those of the one step HCV RNA
PCR, except that two pairs of oligonucleotide primer for
HBV DNA and HCV DNA are added in the reaction.
Enzvmes
Enzymes used in the reaction should be relatively
pure. Any one of a variety of RTases can be used: Molony
Murine ~eukemia Virus RTase (MMLV), M-MLV RNase H- RTase
(M MLV H-) and Avian Myeloblastosis Virus (AMV) RTase were
successfully tested. RTase (for the PCR) reaction, can be
purchased from a number of commercial outlets (e.g.
GIBCO/BRL Lift Technologies, Inc., Gaithersburg~ MD;
Boehringer Mannheim Corporation, Indianapolis, IN; Perkin
Elmer Cetus, Emeryville, CA). A heat stable DNA
polymerase, such as Taq I, is used in the one step
reaction, just as it is used in the traditional PCR
reaction, for amplification. Such heat stable DNA
polymerases are available from many sources, including
Perkin Elmer Cetus, Emeryville, CA and Beckman
Instrumen~s, Inc., Fullerton, CA.
wo 94~08b32 '~ PC~rJUS93/09233
d~NTPs
dNTPs are used by both the RTase and heat stable DNA
polymerase. While concentrations of dNTPs vary between
traditional RTase reactions and PCR reactions, it has been
found that the ~ame concentration of dTNPs can be used for
both the reverse transcription and the PC~ portions of the
reaction. dNTPs can be mixed from individual sources, or
premixed solutions of the four dNTPs can be used (e.g.,
purchased from Pharmacia LKB Biotechnology Inc.,
Piscataway, NJ.).
Oliaonucleotide Primers
~, Standard primers for traditional PCR are used in the
one step assay. They are added to the initial mix before
incubation. For HCV, a pair of HCV oligonucleotide
primers, previously reported (Hu, K.-Q. et al. tl991), Hu,
K.-Q. et al. (1992)), were used. They were derived from
the HCV 5' UTR: 5'-ACTCC~CCATAGATCATCCC-3', 7-26 nt,
sense; 5'-AACACTACTCGGCTAGCAGT-3', 229-248 nt, antisense.
For B V, a pair of oligonucleotide primers derived
from HBV pre-S/S open reading frame were u~ed. 5'-
GTCTAGACTCGTGGTGGACT-3', 119-139 nt, sense; 5'-
AACCACTGTACAAATGGCAC-3', 555-575 nt, antisense.
Following are working examples of the one step as-~y
for rare RNA. HCV RNA, which often appears at low
concentrations in patient samples, has been used as the
test RNA. However, one of skill in the art will be able
to adapt the assay to whatever RNA is being assayed by
such steps as use of the appropriate PCR primer.
Exam~le 1
3G RNA Extraction
The guanidinium isothiocyanate-ac,id-phenol technique
(Chomczynski, P., et al.~ was used to extract RNA fro~
either 0.1 ml aliquots of serum or from liver tissue.
Normal human serum and liver tissue from a patient with
Wos4/08032 PCT/US93/09233
L ~
alpha-l-antitrypsin deficiency were used as negative
controls, as previously reported (Hu, K.-Q., et al.
(1992)). RNA extract~d from an HCV infected serum was
used as a positive control. Serum samples from 50
patients (33 patients with proven chronic HCV infection
and 17 patients with acute or chronic liver diseases of
other etiologies) were tested using both the traditional
and one ~tep RNA PCR procedures.
Example 2
Traditional P~R
Traditional HCV RNA PCR was performed as previously
reported ~Hu, K.-Q., et al. (1991); Hu, K.-Q., et al.
(1992)). Briefly, RNA extracted from 0.1 ml of serum was
reversely transcribed in a 20 ~l volume containing 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgC12, 500 ~M dNTP, 20
U RNasin, 1 ~M antisense primer and 25 U RTase. PCR was
performed in a 50 ~l volume containing 10 mM Tris-HC1 (pH
8.3), 50 mM KCl, 2 mM MgCl2, 200 ~M dNTP, 0.5 ~M of each
primer and 2.5 U Taq polymerase. For the traditional
procedure, RT was performed at 42C for 1 hr and PCR was
done by denaturing single stranded cDNA and inactivating
RTase at 94C for 5 min followed by 30 cycles of PCR
amplification (94C, 1 min; 55C, 1 min; 72C, 2 min).
Example 3
One Step Assay
The one step HCV RNA PCR procedure sequentially
accomplishes both RT and PCR in a single step. The
reaction was carried out in a 50 ~l volume containing 10mM
Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.5 ~M of each
primer, 200 ~M each of dNTP, 20 U RNasin, 25 U RTase and
2.5 U Taq polymerase. To evaIuate the possible inhibition
of Taq polymerase by RTase and to optimize results, the
one step HCV RNA PCR was performed using different
concentration of both enzymes. Since traditional RT PCR
uses different MgCl2 concentratlonæ for RT and PCR,
W094/08032 PCT/US93/09233
~12 l~rl~,l
11
variable concentrations of MgCl2 were tested in the one
step method. For the one step PCR, the incubation was
programmed as follows: RT reaction (42C, lh3, RTase
inactivity and DNA denaturation (s4oc~ 3 min); 30 cycles
of PCR amplification (94C, 1 min; 55C, 1 min; 72C, 2
min). To determine the minimum time required for the one
step PCR, different incubation periods were tested for
both RT and PCR.
Exam~le 4
Evaluation of HCV RNA PCR Results
Ten ~l of each PCR product was electrophoresed
~, through a 1.5~ agarose gel, stained with ethidium bromide
and photographed under W light. Molecular weights were
determined by a 123-bp ladder DNA marker from GIBCO/BRL,
Gaithersburg, MD. The specificity of HCV RNA PCR products
was demonstrated by Southern blot hybridization (Hu, K.-
Q., et al. (1991); Hu, K~-Q., et al. (1992)).
Example 5
Variation in Enzyme~E~ee and Source
When three different RTases were compared in the one
step RN~ PCR, M-MLV RTase (GIBCO/BRL) and AMV RTase
(Boehringer) produced comparable results. M-MLV H- RTa e
(GIBCO/BRL) yielded weaker PCR products. Comparable
results were obtained using either of the two Taq
polymerases (Perkin Elmer Cetus or Beckman). For
convenience, we used 25 U of M-MLV RTase (GIBCO/BRL) and
2.5 U of Taq Polymerase (Perkin Elmer Cetus) for
subsequent studies.
Example 6
Variation in MgCl2 Concentration
Since traditional HCV ~NA PCR uses different concen-
trations of MgCl2 for RT and PCR steps, the one step ~NA
PCR was performed using MgCl2 concentrations of 1, 1.5, 2,
W094/08032 PCT/US93/09233
5 and 8 mM. Reaction products were ~omparable using 1.5,
2, 5 and 8 mM MgCl2 in the one step HCV RNA PCR.
In previous studies of one step RNA PCR methods for
amplification of Ross River virus ~Sellner, L.N., et al.)
and bacterial ribosomes (Wang, R.-F., et al.), the optimal
concentration of MgCl2 was the subject of controversy. In
contrast to the report of Wang, et al. which indicated
that only a relatively narrow range of MgCl2 concentrations
was feasible, the one step HCV RNA PCR produced reaction
products over a range of MgCl2 concentrations from 1 to 8
mM. However, 5 or 8 mM concentrations were sometimes
associated with nonspecific signals on the agarose gel,
" and using 1 mM MgCl2, the PCR productq were uniformly less
intense. Thus, 2 mM MgCl2 was the preferred concentration.
Example 7
Inhibition-of Taq Polymerase bv RTase
Since RTase can inhibit Taq polymerase activity
(Sellner, L.N., et al.; GeneAmp RNA PCR Kit instructions,
Perkin Elmer Cetus (1990)), this possible deleterious
interaction was extensively studied in the one step RNA
PCR. Varying concentrations of either RTase or Taq
polymerase were employed to achieve different ratios of
these two enzymes. As shown in Figure 1, reactions using
2.5 U of Taq polymerase and a wide range of RTase
concentrations (from 5 U to 100 U) produced detectable PCR
products. Results were optimal using 25 U RTase. Using
2S U of RTase, concentrations of 2.5 to 10 U of Taq
polymerase produced PCR products. Optimal results were
achieved using 2.5 U of Taq polymerase. Thus, a ratio of
RTase to Taq polymerase as high as 10:1 was feasible, and
deleterious effects of the inhibition of Taq polymerase by
RTase were not observed.
W094/08032 2 ~ PCT/US93/09233
Example 8
RTase Reaction Conditions
To study further RT in the one step HCV RNA PCR the
temperature and duration of incubation were varied.
Incubation at 42C for 1 hr appeared to be optimal, but
periods as short as 15 min yielded PCR products comparable
to thoQe observed with longer incubations. In traditional
PCR, RTase is denatured by incubating RT reaction mixture
at 95C for 5 min in the absence of Taq polymerase. In
the one step RNA PCR, however, RTase denaturation occurs
in the prePence of Taq polymerase, which could decrease
the acti~ity of Taq polymerase and the sensitivity of PCR
amplification. Denaturation for 2-4 min in the one step
RNA PCR produced results comparable to those of
traditional PCR.
Example 9
Reaction Time
To determine the minimum time required for accurate
HCV RNA PCR, the time periods for denaturing, annealing
and elongating were varied. When the RT reaction was
fixed at 15 min, the PCR program could be shortened to
94C, 30 sec; 55C, 30 sec; and 72C, 45 sec for 30
cycles. Thus, one step RNA PCR can minimize the time
required for HCV detection by both simplifying the
procedure and shortening the programmed incubation times.
Exam~le 10
Concordance and S~ecificity
When one step RNA PCR was used to detect HCV RNA, the
expected 241 bp HCV cDNA was identified in ~NA extracted
from the serum of a patient with HCV infection (positive
control). The HCV specificity of the cDNA generated in
the one step RNA PCR was confirmed by Southern blot assay
using cloned HCV cDNA as the probe. In contrast, the one
step RNA PCR was negatiYe using RNA extracted from normal
human serum or the Iiver of a patient with alpha-1-
. ' .
W094/08032 PCT/~S93/09~33
~2~
14
antitrypsin deficiency. To assess concordance andspecificity further, traditional PCR and one step RNA PCR
were performed in parallel using RNA extracted from 50
serum samples: 33 previously confirmed as positive and 17
as negative for H~V RNA. One hundred percent concordance
between the one step RNA PCR and traditional PCR was
observed, and the specificity of the cDNA was confirmed by
Southern blotting.
Example 11
10 SensitivitY
Since PCR is a very sensitive technique, specificity
~, of the assay is a constant concern (Kwok, S., et al.). To
assess the relative sensitivity of one step RNA PCR, RNA
extracted from HCV infected liver was serially diluted and
tested by both the traditional and one step RNA PCR
techniques. As shown in Figure 2, both traditional PCR
and one step RNA PCR detected comparable dilutions of HCV
RNA. Using RNA extracted from serum specimens containing
the HCV r-plicating intermediate (minus strand), the one
step RNA PCR uniformly produced stronger signals on
agarose gel than traditional PCR. This suggests that the
initial RT occurs in both orientations in the one step
method and increa9es the quantity of cDNA available for
PCR amplification. Since minus stranded HCV RNA is
present in the sera of approximately 50% of chronically
infected patients, the one step RNA PCR may be more
sensitive for the detection of this subgroup than
traditional PCR.
When HCV primers from NS3 and NS4 (Hu, K.-Q. et al.
(1991)) were used, the intensity of reaction products was
inferior to that obtained with HCV 5'UTR primers in either
the one step or traditional HCV RNA PCR. These results
are consistent with published reæults comparing NS3/NS4
and 5' UTR primers in traditional HCV RNA PCR (Hu, K.-Q.
et al. (1991); Bukh, J., et al.). Primers from the HCV
5'UTR are favored since this region of the genome is
W094/08032 2 ~ PCT/US93/09233
highly conserved among different HCV isolates (Houghton,
M., et al.), and PCR sensitivity is greatest (Hu, K~-Q. et
al. (1991); Bukh, J., et al.).
Working examples for the combined one step HBV-HCV
PCR method are set forth below.
Example 12
combined one ste~ HBV-HCV PCR Method
The combined one step HBV-HCV PCR method sequentially
accomplishes both RT and PCR of HCV ~NA and PCR of HBV DNA
all in one reaction vessel. Nucleic acid extraction is
performed a~ follows. 150 ul of serum i8 digested by 10-
~, 15 ul of Proteinase K (10 mg/ml) at 50 C for two hours.
Phenol/chloroform extraction is carried out first in an
acid environment (pH 4.0) to isolate RNA, and repeated
after adjusting the pH of the phenol phase to pH 8.0 for
isolation of DNA. The acidity and basicity can vary
within the range of 3.0 to 5.0 and 7.1 to 9Ø
Alternatively, the phenol/chloroform extraction could be
carried out at a basic pH to extract DNA followed by
adjustment of~the pH to an acid environment to extract
RNA. Adjustment of the pH is carried out by the addition
of a buffer solution of the proper pH, appropriate for DNA
and RNA extractions, for example Tris-EDTA, and others as
will be known to one of ordinary skill in the art. Both
extracts are pooled, lQ ug of yeast tRNA is added and the
nucleic acids are co-precipitated with isopropanol. The
pellets of extracted nucleic acids are resuspended in 10
ul of diethyl pyrocarbonate (DEPC) treated water and can
be stored at -70 C before use.
The nucleic acid extracts are then denatured and the
RT-PCR reaction is carried out in a volume of 25 ul
containing 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2 mM MgCl2;
0.5 uM of oligonucleotide primers specific for HBV or HCV;
200 uM of each dNTP; 6.25 U RTase; 20 U RNasin and 1.25 U
Taq polymerase.
W094/08032 pcT~us~3tos233
J ~ 1 .
16
For the combined one step HBV-HCV PCR, the incubation
was programmed as follows: RT reaction (42C, lh), RTase
inactivity and DNA denaturation (94C, 3 min); 30 cycles
of PCR amplification (94C, 1 min; 55C, 1 min; 72C, 2
min).
Example 13
Evaluation of Combined One Step HBV-HCV PCR Results
Ten ~l of each PCR product was electrophoresed
through a 1.5~ agarose gel, stained with ethidium bromide
and photographed under W light. Molecular weights were
determined by a 123-bp ladder DNA marker from GIBCO/BRL,
~, Gaithersburg, MD. The specificity of HBV DNA or HCV RNA
PCR products was demonstrated by Southern blot
hybridization (Hu, K.-Q., et al. (1993 in press); Hu, K.-
Q., et al. (1992)). Two plasmids, pGHCVlA containing HCV5' UTR fragment (Hu, K-Q., et al. (1992) and pNER
containing HBV genome (Hu, K-Q., et al. (1~90) were used
as the probe sources for the Southern blot hybridization.
Example 14
Optimization of the HBV Si~nal Relative to the HCV Siqnal
If the ratio of HBV DNA to HCV cDNA is not
appropriately adjusted, the intensity of the HBV signal is
so much greater than that of the HCV cDNA signal that the
region of 456 bp (B V DNA band) can be smeared and it may
not be possible to determine the exact molecular size of
the PCR product. Two major factors probably contribute to
the disparate intensity of the HBV DNA and HCV cDNA
signals. First, a greater titer of HBV than HCV viruses
in the serum results in an increased number of templates
of HBV DNA compared to HCV RNA. Second, the HBV DNA PCR
is more efficient because it does not involve the reverse
transcription of RNA as is required for HCV.
Optimization of the HBV DNA to HCV cDNA ratio was
carried out as follows. RNA was first extracted from 0.15
ml of serum from a patient with combined HBV and HCV
W094/08032 PCT~US93~09233
;~ l 2 ~
infection. DNA was then extracted from the phenol phase
by neutralization of the pH. The DNA was collected into
a separate tube. The RNA extract was combined with a
different amount of extracted DNA. The RNA and DNA were
co-precipitated in the same tube with 10 ug tRNA. The
combined HBV-HCV PCR was performed as descl.bed above.
The reduced amount of DNA templates in the PCR reaction
prQduced a sharp band of HBV DNA without affecting the
intensity of the HCV cDNA signal. Comparison of the
different ratios of HBV DNA and HCV RNA templates showed
that a 1:7.5 to 1:15 ratio of DNA to RNA extraction (i.e.
entire HCV RNA extract pooled with 1/7.5 to 1/15 of the
DNA extract from 0.15 ml serum) produced optimal results.
The dilution of the DNA extract did not reduce the
sensitivity of HBV DNA detection.
Example 15
Concordance and Specificity of Combined One Step HBV-HCV
PCR
The combined one step HBV-HCV PCR method is a highly
~0 specific procedure. The expected 456 bp HBV DNA and 241
HCV cDNA band8 were identified in the serum of a patient
with combined HBV and HCV infection. Also, no bands were
identified in normal human serum. Finally, Southern blots
confirmed the specificity of the bands for HBV or HCV. To
assesQ sensitivity and specificity, traditional HBV DNA
PCR, one step HCV RNA PCR and combined one step HBV-HCV
PCR were performed in parallel using 28 serum samples.
The specificity of the combined one step HBV-HCV PCR
method was conflrmed by its 100~ concordance with
tradi:ional HBV DNA PCR and HCV DNA PCR in the 28 serum
samples.
W094/08032 PCT/VS93/09233
81 18
Concordance of the Traditional and One Step HBV-HCV PCR
.
Chronic Liver Combined HBV/HCV PCR
Disease Groups~ (n=) HBV(+) HCV(+)
HBV (+) and HCV (~) 6 6 6
HBV (+) and HCV (-) 6 6 0
HBV (-) and HCV (+) 9 0 9
HBV (-) and HCV (-) 7 0 0
~ HBV DNA was detected by B V DNA PCR; HCV RNA, by a one
~, step RNA PCR.
ExamDle 16
Concordance of HBV Slot HYbridization with
Combined One Ste~ HBV-HCV PCR
HBV DNA slot hybridization is widely used for the
diagnosis of HBV infection. Therefore, the concordance of
HBV DNA slot hybridization with the combined one step HBV-
HCV PCR method was examined using 34 serum samples. 100%
concordance was observed between the combined one step
HBV-HCV PCR me~hod and HBV DNA slot hybridization.
Example 17
Se~si~ivitv of Combined One Step HBV-HCV PCR
The sensitivity of the combined one step HBV-HCV PCR
method for detection of B V DNA is greater than the widely
used HBV DNA slot h~bridization diagnostic technique.
This was shown by the 100~ concordance between the
combined one step HBV-HCV PCR method and HBV DNA slot
hybridization among HBV-positive sera. However, among 12
patients with negative HBV DNA slot hybridization assays,
3 patients were positive for HBV DNA in the combined one
step HBV-HCV PCR method.
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