EP0620859A1

EP0620859A1 - One step rna and combined one step rna and dna polymerase chain reaction for detection of rare rna or rna and dna

Info

Publication number: EP0620859A1
Application number: EP93922425A
Authority: EP
Inventors: John M. Vierling; Ke-Qin Hu
Original assignee: Cedars Sinai Medical Center
Current assignee: Cedars Sinai Medical Center
Priority date: 1992-09-29
Filing date: 1993-09-29
Publication date: 1994-10-26
Also published as: WO1994008032A1; JPH07501712A; AU5142393A; CA2124581A1

Abstract

Cette invention concerne une technique d'amplification en chaîne par polymérase (ACP) s'effectuant en une seule étape qui permet de détecter de l'ARN rare et une technique combinée d'ACP en une seule étape qui permet de détecter simultanément de l'ADN et de l'ARN et qui offre plusieurs avantages importants par rapport à l'ACP classique. La procédure simplifiée de l'ACP d'ARN en une seule étape et la technique combinée d'ACP en une seule étape permettent de détecter simultanément l'ADN et l'ARN et d'éliminer le risque de contamination induite par l'ouverture répétée des tubes de réaction, ce qui réduit l'intensité du travail et le risque de résultats positifs faux. En outre le programme d'incubation TI-ACP peut être sensiblement réduit dans le procédé en une seule étape et la technique combinée d'ACP en une seule étape permettant de détecter simultanément l'ADN et l'ARN. L'intensité accrue des produits de réaction utilisant des spécimens contenant de l'ARN du virus de l'hépatite C (VHC) à brins plus et moins favorise également le procédé en une seule étape. L'ACP d'ARN en une seule étape apparaît donc comme une technique intéressante pouvant remplacer l'ACP d'ARN classique utilisée pour détecter l'ARN de VHC, et la technique d'ACP combinée en une seule étape de détection simultanée d'ADN et d'ARN apparaît elle aussi comme une technique de rechange intéressante pour se substituer à l'utilisation séparée de l'ACP d'ARN et de l'ACP d'ADN classiques employées pour détecter l'ARN de VHC et l'ADN de VHC. Le risque de contamination réduit et le temps plus court requis pour effectuer chaque détermination permettent d'employer avantageusement ces techniques pour tester plusieurs spécimens cliniques.A one-step polymerase chain reaction (PCR) technique for detecting rare RNA and a combined one-step PCR technique for simultaneously detecting DNA and RNA and which offers several important advantages over conventional PCR. The simplified one-step RNA PCR procedure and the combined one-step PCR technique enable simultaneous detection of DNA and RNA and eliminate the risk of contamination induced by repeated opening reaction tubes, which reduces labor intensity and the risk of false positive results. Furthermore, the TI-PCR incubation schedule can be significantly reduced in the one-step method and the combined one-step PCR technique allowing DNA and RNA to be detected simultaneously. The increased intensity of reaction products using specimens containing plus- and minus-stranded hepatitis C virus (HCV) RNA also favors the one-step process. Single-step RNA PCR therefore appears as an interesting technique that can replace the classical RNA PCR used to detect HCV RNA, and the single-step combined PCR technique for simultaneous detection of HCV. DNA and RNA also appears to be an interesting alternative technique to replace the separate use of conventional RNA PCR and DNA PCR used to detect HCV RNA and DNA of HCV. The reduced risk of contamination and the shorter time required to perform each determination make it possible to advantageously employ these techniques to test multiple clinical specimens.

Description

DESCRIPTION

One Step RNA and Combined One Step RNA and DNA Polymerase Chain Reaction for

Detection of Rare RNA or RNA and DNA

Related Application

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. 07/954,359, pending in the Patent

Office.

Background

PCR is a well used technique for amplification of DNA. It is used in a variety of assays for the detection of particular DNA sequences, such as those associated with bacterial or DNA virus caused diseases. Additionally, in combination with reverse transcription of RNA 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 length 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 (5'UTR) of approximately 324-341 nt is highly conserved among different HCV strains and favored for both diagnostic HCV RNA PCR and HCV RNA hybridization

(Houghton, . et al. ; Hu, K.-Q. et al. (1991); Bukh, J. et al.; Hu, K.-Q. et al. (1992)) . 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. (1991); 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 abundant 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 detection 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 hepatocellular 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 quantitation of HBV DNA by molecular hybridization. (Hoofnagle J.H. et al . ) . HBV DNA PCR has been shown to be the most sensitive technique for detection of even trace amounts of HBV DNA. (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 its clinical 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 a mplification and a second PCR amplification after addition of a second 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 FBV 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 sensitive 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 detection of rare RNA such as HCV RNA PCR using primers from the highly conserved 5'UTR of the HCV genome, and over the separate 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. 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 required 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 HCV 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 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. 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-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.

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 provides more uniform results than previously available methods because of the opportunity for automation of many of the steps.

Brief Description of the Drawings

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 traditional (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. 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 = 456 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. Detailed Description 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 temperature 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 37-42°C, 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. 94°C 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. 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 RNA, 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 sample. This extraction technique will work for any RNA or DNA species regardless of source. Methods which degrade or deactivate proteins, including DNases and RNases, 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 vary within the range of 3.0 to 5.0 and 7.1 to 9.0. 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 o^rdinary skil^": in the art. Both extracts are poo^' -d, 10 of yeast tl ,,A is added and the nucleic acids ar co-pre .pitated 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. 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 primers for

HBV DNA and HCV DNA are added in the reaction.

Enzymes Enzymes used in the reaction should be relatively pure. Any one of a variety of RTases can be used: Molony

Murine Leukemia 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 Instruments, Inc., Fullerton, CA. dNTPs 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 same concentration of dTNPs can be used for both the reverse transcription and the PCR 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.) .

Oligonucleotide 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. (1991), Hu, K.-Q. et al. (1992)), were used. They were derived from the HCV 5' UTR: 5' -ACTCCACCATAGATCATCCC-3' , 7-26 nt, sense; 5' -AACACTACTCGGCTAGCAGT-3' , 229-248 nt, antisense.

For HBV, a pair of oligonucleotide primers derived from HBV pre-S/S open reading frame were used. 5'- GTCTAGACTCGTGGTGGACT-3' , 119-139 nt, sense; 5'- AACCACTGTACAAATGGCAC-3' , 555-575 nt, antisense.

Following are working examples of the one step assay 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.

Example 1 RNA Extraction

The guanidinium isothiocyanate-acid-phenol technique

(Chomczynski, P., et al.) was used to extract RNA from either 0.1 ml aliquots of serum or from liver tissue.

Normal human serum and liver tissue from a patient with alpha-1-antitrypsin deficiency were used as negative controls, as previously reported (Hu, K.-Q., et al. (1992) ) . RNA extracted 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 step RNA PCR procedures.

Example 2 Traditional PCR

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 μl volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 500 μM dNTP, 20 U RNasin, 1 μM antisense primer and 25 U RTase. PCR was performed in a 50 μl volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 200 μM dNTP, 0.5 μM of each primer and 2.5 U Taq polymerase. For the traditional procedure, RT was performed at 42°C for 1 hr and PCR was done by denaturing single stranded cDNA and inactivating RTase at 94°C for 5 min followed by 30 cycles of PCR amplification (94°C, 1 min; 55°C, 1 min; 72°C, 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 μl volume containing lOmM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 0.5 μM of each primer, 200 μM each of dNTP, 20 U RNasin, 25 U RTase and 2.5 U Taq polymerase. To evaluate 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 concentrations for RT and PCR, variable concentrations of MgCl₂ were tested in the one step method. For the one step PCR, the incubation was programmed as foll_ s: RT reaction (42°C, lh) , RTase inactivity and DNA denaturation (94°C, 3 min) ; 30 cycles of PCR amplification (94°C, 1 min; 55°C, 1 min; 72°C, 2 min) . To determine the minimum time required for the one step PCR, different incubation periods were tested for both RT and PCR.

Example 4 Evaluation of HCV RNA PCR Results

Ten μl of each PCR product was electrophoresed through a 1.5% agarose gel, stained with ethidium bromide and photographed under UV 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 Type and Source When three different RTases were compared in the one step RNA PCR, M-MLV RTase (GIBCO/BRL) and AMV RTase (Boehringer) produced comparable results. M-MLV H^" RTase (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 MgCl-, Concentration

Since traditional HCV RNA PCR uses different concen¬ trations of MgCl₂ for RT and PCR steps, the one step RNA PCR was performed using MgCl₂ concentrations of 1, 1.5, 2, 5 and 8 mM. Reaction products were comparable 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 products were uniformly less intense. Thus, 2 mM MgCl₂ was the preferred concentration.

Example 7

Inhibition of Tag Polymerase by 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 25 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. 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 42°C for 1 hr appeared to be optimal, but periods as short as 15 min yielded PCR products comparable to those observed with longer incubations. In traditional PCR, RTase is denatured by incubating RT reaction mixture at 95°C for 5 min in the absence of Taq polymerase. In the one step RNA PCR, however, RTase denaturation occurs in the presence of Taq polymerase, which could decrease the activity 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 94°C, 30 sec; 55°C, 30 sec; and 72°C, 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.

Example 10

Concordance and Specificity

When one step RNA PCR was used to detect HCV RNA, the expected 241 bp HCV cDNA was identified in RNA 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 negative using RNA extracted from normal human serum or the liver of a patient with alpha-1- antitrypsin deficiency. To assess concordance and specificity 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 HCV 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 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 replicating 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 increases 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 results 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 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 step HBV-HCV PCR Method

The combined one step HBV-HCV PCR method sequentially accomplishes both RT and PCR of HCV RNA and PCR of HBV DNA all in one reaction vessel. Nucleic acid extraction is performed as follows. 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 vary within the range of 3.0 to 5.0 and 7.1 to 9.0. 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 p per 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, 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 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. For the combined one step HBV-HCV PCR, the incubation was programmed as follows: RT reaction (42°C, lh) , RTase inactivity and DNA denaturation (94°C, 3 min) ; 30 cycles of PCR amplification (94°C, 1 min; 55°C, 1 min; 72°C, 2 min) .

Example 13 -r

Evaluation of Combined One Step HBV-HCV PCR Results

Ten μl of each PCR product was electrophoresed through a 1.5% agarose gel, stained with ethidium bromide and photographed under UV 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 HCV 5' UTR fragment (Hu, K-Q. , et al. (1992) and pNER containing HBV genome (Hu, K-Q., et al. (1990) were used as the probe sources for the Southern blot hybridization.

Example 14 Optimization of the HBV Signal Relative to the HCV Signal

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 (HBV 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 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 described above. The reduced amount of DNA templates in the PCR reaction produced 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 specific procedure. The expected 456 bp HBV DNA and 241 HCV cDNA bands were ic tified 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 assess 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 confirmed by its 100% concordance with traditional HBV DNA PCR and HCV DNA PCR in the 28 serum samples. Concordance of the Traditional and One Step HBV-HCV PCR

^*HBV DNA was detected by HBV DNA PCR; HCV RNA, by a one step RNA PCR.

Example 16

Concordance of HBV Slot Hybridization with Combined One Step 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 method and HBV DNA slot hybridization.

Example 17

Sensitivity of Combined One Step HBV-HCV PCR

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-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

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 simultaneours 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 9.0.

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 8.0. 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 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; 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 RNA 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 9.0.

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 8.0.

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.

V

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.