CA2124581A1 - One step pcr for detection of rare rna and dna - Google Patents

Abstract

A one step PCR technique for the detection of rare RNA and a combined one step PCR technique for the simultaneous detection of DNA
and RNA is provided which offers several important advantages over traditional PCR. The simplified procedure of the one step RNA
PCR and the combined one step PCR technique for the simultaneous detection of DNA and RNA and elimination of the potential for contamination with repeated opening of the reaction tubes reduces both labor intensity and the risk of false-positive results. In addition, the RT-PCR incubation program can be substantially shortened in the one step method and the combined one step PCR technique for the simultaneous detection of DNA and RNA. The increased intensity of reaction products using specimens containing both plus and minus stranded HCV RNA also favors the one step method. Thus, the one step RNA PCR is an attractive alternative to traditional RNA PCR for the detection of HCV RNA. And the combined one step PCR technique for the simultaneous detection of DNA and RNA is an attractive alternative to the separate use of traditional RNA
PCR and DNA PCR for the detection of HCV RNA and HBV DNA. The reduced risk of contamination and time required for each assay make these techniques particularly well suited for testing of multiple clinical specimens.

Description

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)).

.

`~ 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 ~) ~ 2 ~ 8 ~

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, ~12 l~rl~,l 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, 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~

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 .

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.

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.

'

Claims

Claims 1. A method for the detection of rare RNA
comprising a one step PCR assay wherein active reverse transcriptase and active heat stable DNA polymerase are present together in a reaction container.

2. The method of claim 1 wherein the reverse transcription reaction and the DNA amplification reaction are run sequentially.

3. The method of claim 2 wherein the reverse transcriptase is denatured before the DNA amplification reaction is initiated.

4. The method of claim 1 wherein the ratio of reverse transcriptase to heat stable DNA polymerase is between 2:1 and 10:1.

5. The method of claim 1 wherein the reaction container further contains MgCl2 at a concentration between 1 mM and 8 mM.

6. A one step PCR method for the detection of rare RNA in a sample wherein reverse transcriptase and heat stable DNA polymerase are added to the sample before incubation begins.

7. A one step PCR method for the detection of rare RNA in a sample wherein active reverse transcriptase and active heat stable DNA polymerase are present with the sample at the same time.

8. A one step PCR method for the detection of rare RNA in a sample wherein the same reaction solution is used for both reverse transcription and DNA amplification.

9. A method for the detection of HCV RNA comprising a one step PCR assay wherein active reverse transcriptase and active heat stable DNA polymerase are present together in the reaction container.

10. The method of claim 9 wherein the reverse transcription reaction and the PCR reaction are run sequentially.

11. The method of claim 10 wherein the reverse transcriptase is denatured before the DNA amplification reaction is initiated.

12. The method of claim 10 wherein the ratio of reverse transcriptase to heat stable DNA polymerase is between 2:1 and 10:1.

13. The method of claim 10 wherein the reaction container further contains MgCl2 at a concentration between 1.5 mM and 8 mM.

14. The method of claim 10 wherein the reverse transcription reaction and the PCR reaction are run in the same solution.

15. A kit for the detection of HCV RNA using one step PCR comprising a reaction vessel; separate containers containing reverse transcriptase and heat stable DNA
polymerase to be added to the reaction vessel at a ratio of between 2:1 and 10:1; dATP, dGTP, dTTP and dCTP;
antisense and sense primer specific for HCV; and standard buffer and salts for PCR.

16. A method for the simultaneous extraction of RNA
and DNA in a sample comprising an aqueous and an organic phase comprising the steps of:
a) deactivating the proteins in said sample;

b) performing an RNA extraction in said sample, by altering the pH of said sample so that said pH
is made acidic; and c) performing a DNA extraction in said sample by altering the pH of said sample so that said pH is made basic.
17. The method of claim 16 wherein said RNA is RNA
virus RNA, and said DNA is DNA virus DNA.
18. The method of claim 16 wherein said RNA is HCV
RNA, and said DNA is HBV DNA.
19. The method of claim 16 wherein said step of deactivating the proteins in said sample is performed using guanidinium isothiocyanate.
20. The method of claim 16 wherein said step of deactivating the proteins in said sample is performed using Proteinase K.
21. The method of claim 16 wherein said organic phase consists essentially of a phenol and chloroform mixture.
22. The method of claim 16 wherein said pH of said sample during said RNA extraction is in the range of 3.0 to 5.0 and said pH of said sample during said DNA
extraction is 7.1 to 9Ø
23. The method of claim 16 wherein said pH of said organic phase during said RNA extraction is 4 0 and said pH of said organic phase during said DNA extraction is 8Ø

24. The method of claim 16 wherein said pH of said sample during said RNA extraction is made acidic by addition of a buffer solution of acidic pH.

25. The method of claim 16 wherein said pH of said sample during said DNA extraction is made basic by addition of a buffer solution of basic pH.

26. The method of claim 16 wherein said RNA
extraction is performed before said DNA extraction.

27. The method of claim 16 wherein said DNA
extraction is performed before said RNA extraction.

28. A method for the simultaneous amplification and detection of a quantity of RNA and DNA in a sample comprising the steps of:
a) deactivating proteins in said sample;
b) performing an RNA extraction in said sample, by filtering the pH of said sample so that said pH
is made acidic; and c) performing a DNA extraction in said sample by altering the pH of said sample so that said pH is made basic;
d) pooling said resulting RNA and DNA
extracts;
e) adding active reverse transcriptase and active heat stable DNA polymerase to said sample containing said resulting RNA and DNA extracts;
f) first performing a reverse transcription reaction in said sample to yield a cDNA product, then inactivating said active reverse transcriptase, and activating said active heat stable DNA polymerase; and (g) detecting the presence of an amplified cDNA
product and detecting the presence of an amplified resulting DNA extract.

29. The method of claim 28 wherein the reverse transcriptase is denatured before the DNA amplification reaction is initiated.

30. The method of claim 28 wherein the ratio of reverse transcriptase to heat stable DNA polymerase is between 2:1 and 10:1.

31. The method of claim 28 wherein the reaction container further contains MgCl2 at a concentration between 1 mM and 8 mM.

32. The method of claim 28 wherein the same reaction solution is used for both reverse transcription and DNA
amplification.

33. The method of claim 28 wherein said RNA is DNA
virus RNA, and said DNA is DNA virus DNA.

34. The method of claim 28 wherein said RNA is HCV
RNA, and said DNA is HBV DNA.

35. The method of claim 28 wherein said step of deactivating the proteins in said sample is performed using guanidinium isothiocyanate.

36. The method of claim 28 wherein said step of deactivating the proteins in said sample is performed using Proteinase K.

37. The method of claim 28 wherein said organic phase consists essentially of a phenol and chloroform mixture.

38. The method of claim 28 wherein said pH of said sample during said RNA extraction is in the range of 3.0 to 5.0 and said pH of said sample during said DNA
extraction is 7.1 to 9Ø

39. The method of claim 28 wherein said pH of said sample during said RNA extraction is 4.0 and said pH of said sample during said DNA extraction is 8Ø

40. The method of claim 28 wherein said pH of said sample during said RNA extraction is made acidic by addition of a buffer solution of acidic pH.

42. The method of claim 28 wherein said pH of said sample during said DNA extraction is made basic by addition of a buffer solution of basic pH.

43. The method of claim 28 wherein said RNA
extraction is performed before said DNA extraction.

44. The method of claim 28 wherein said DNA
extraction is performed before said RNA extraction.

45. The method of claim 28 wherein following said step of pooling said resulting RNA and DNA extracts said extracts are co-precipitated.

46. The method of claim 28 wherein following said step of coprecipitating said resulting RNA and DNA
extracts said extracts are suspended in a solvent containing RNase inhibitors.

47. A kit for the simultaneous amplification of RNA
and DNA using one step PCR comprising; a container containing means for deactivating proteins, means for isolating DNA and RNA species from a sample, separate containers containing reverse transcriptase and heat stable DNA polymerase to be added to the reaction vessel at a ratio of between 2:1 and 10:1; dATP, dGTP, dTTP and dCTP; antisense and sense primer specific for HCV;
antisense and sense primer specific for HBV; and standard buffer and salts for PCR; and instructions indicating that protein denaturation must be performed first, followed by RNA extraction at acidic pH, and then DNA extraction at basic pH, followed by addition of the reverse transcriptase and the heat stable polymerase, followed by reverse transcription and PCR.

48. A kit for the simultaneous amplification of HCV
RNA and HBV DNA using one step PCR comprising; a container containing means for deactivating proteins, means for isolating DNA and RNA species from a sample, Separate containers containing reverse transcriptase and heat stable DNA polymerase to be added to the reaction vessel at a ratio of between 2:1 and 10:1; dATP, dGTP, dTTP and dCTP; antisense and sense primer specific for HCV;
antisense and sense primer specific for HBV; and standard buffer and salts for PCR; and instructions indicating that protein denaturation must be performed first, followed by RNA extraction at acidic pH, and then DNA extraction at basic pH, followed by addition of the reverse transcriptase and the heat stable polymerase, followed by reverse transcription and PCR.