+

WO1994008032A1 - Amplification en chaine par polymerase d'arn en une seule etape et d'arn et d'adn combines en une seule etape, employee pour detecter l'arn rare ou l'arn et l'adn - Google Patents

Amplification en chaine par polymerase d'arn en une seule etape et d'arn et d'adn combines en une seule etape, employee pour detecter l'arn rare ou l'arn et l'adn Download PDF

Info

Publication number
WO1994008032A1
WO1994008032A1 PCT/US1993/009233 US9309233W WO9408032A1 WO 1994008032 A1 WO1994008032 A1 WO 1994008032A1 US 9309233 W US9309233 W US 9309233W WO 9408032 A1 WO9408032 A1 WO 9408032A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
dna
pcr
sample
hcv
Prior art date
Application number
PCT/US1993/009233
Other languages
English (en)
Inventor
John M. Vierling
Ke-Qin Hu
Original Assignee
Cedars-Sinai Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cedars-Sinai Medical Center filed Critical Cedars-Sinai Medical Center
Priority to CA002124581A priority Critical patent/CA2124581A1/fr
Priority to AU51423/93A priority patent/AU5142393A/en
Priority to JP6509262A priority patent/JPH07501712A/ja
Priority to EP93922425A priority patent/EP0620859A1/fr
Publication of WO1994008032A1 publication Critical patent/WO1994008032A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • 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 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
  • HCV RNA PCR PCR amplification of HCV RNA
  • 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.
  • 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.
  • 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.) .
  • 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.
  • 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.
  • 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.
  • This invention provides more uniform results than previously available methods because of the opportunity for automation of many of the steps.
  • 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.
  • 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.
  • 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.
  • dNTPs deoxynucleotide triphosphates
  • RNase inhibitor(s) optionally RNase inhibitor(s) to protect the rare RNA from degradation
  • 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
  • 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.
  • 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.
  • RNA and DNA 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.
  • 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 r dinary skil " : in the art.
  • 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 r dinary 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.
  • DEPC diethyl pyrocarbonate
  • Reaction conditions for the one step PCR detection of rare RNAs are the same as those used in a traditional PCR reaction.
  • optimal salt and enzyme conditions can be readily determined.
  • Ribonuclease inhibitors such as RNasin TM (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.
  • HCV RNA the reaction conditions determined to be most favorable are found in Example 3.
  • 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
  • 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.) .
  • Standard primers for traditional PCR are used in the one step assay. They are added to the initial mix before incubation.
  • 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.
  • 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.
  • HCV RNA which often appears at low concentrations in patient samples, has been used as the test RNA.
  • test RNA 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.
  • RNA was extracted from either 0.1 ml aliquots of serum or from liver tissue.
  • RNA extracted from an HCV infected serum was used as a positive control.
  • Serum samples from 50 patients were tested using both the traditional and one step RNA PCR procedures.
  • 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 MgCl2, 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-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl 2 , 200 ⁇ M dNTP, 0.5 ⁇ M of each primer and 2.5 U Taq polymerase.
  • 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) .
  • 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 lOmM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl 2 , 0.5 ⁇ M of each primer, 200 ⁇ M each of dNTP, 20 U RNasin, 25 U RTase and 2.5 U Taq polymerase.
  • the one step HCV RNA PCR was performed using different concentration of both enzymes.
  • RT PCR uses different MgCl 2 concentrations for RT and PCR
  • variable concentrations of MgCl 2 were tested in the one step method.
  • 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) .
  • 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
  • M-MLV RTase GMBCO/BRL
  • AMV RTase Boehringer
  • M-MLV H " RTase yielded weaker PCR products. Comparable results were obtained using either of the two Taq polymerases (Perkin Elmer Cetus or Beckman) .
  • M-MLV RTase GMBCO/BRL
  • Taq Polymerase Perkin Elmer Cetus
  • the one step RNA PCR was performed using MgCl 2 concentrations of 1, 1.5, 2, 5 and 8 mM. Reaction products were comparable using 1.5, 2, 5 and 8 mM MgCl 2 in the one step HCV RNA PCR.
  • 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.
  • RNA PCR 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.
  • RTase is denatured by incubating RT reaction mixture at 95°C for 5 min in the absence of Taq polymerase.
  • 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.
  • RNA PCR can minimize the time required for HCV detection by both simplifying the procedure and shortening the programmed incubation times.
  • RNA PCR 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DEPC diethyl pyrocarbonate
  • 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 MgCl 2 ; 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.
  • 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) .
  • 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.
  • 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.
  • the HBV DNA PCR is more efficient because it does not involve the reverse transcription of RNA as is required for HCV.
  • 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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.
PCT/US1993/009233 1992-09-29 1993-09-29 Amplification en chaine par polymerase d'arn en une seule etape et d'arn et d'adn combines en une seule etape, employee pour detecter l'arn rare ou l'arn et l'adn WO1994008032A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002124581A CA2124581A1 (fr) 1992-09-29 1993-09-29 Pcr a une seule etape pour la detection de sequences d'arn et d'adn rares
AU51423/93A AU5142393A (en) 1992-09-29 1993-09-29 One step rna and combined one step rna and dna polymerase chain reaction for detection of rare rna or rna and dna
JP6509262A JPH07501712A (ja) 1992-09-29 1993-09-29 微量ｒｎａまはたｒｎａおよびｄｎａ検出用の一段ｒｎａおよび結合一段ｒｎａおよびｄｎａポリメラーゼ・チェイン・リアクション
EP93922425A EP0620859A1 (fr) 1992-09-29 1993-09-29 Amplification en chaine par polymerase d'arn en une seule etape et d'arn et d'adn combines en une seule etape, employee pour detecter l'arn rare ou l'arn et l'adn

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US95435992A 1992-09-29 1992-09-29
US07/954,359 1992-09-29
US5871693A 1993-05-06 1993-05-06
US08/058,716 1993-05-06

Publications (1)

Publication Number Publication Date
WO1994008032A1 true WO1994008032A1 (fr) 1994-04-14

Family

ID=26737960

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/009233 WO1994008032A1 (fr) 1992-09-29 1993-09-29 Amplification en chaine par polymerase d'arn en une seule etape et d'arn et d'adn combines en une seule etape, employee pour detecter l'arn rare ou l'arn et l'adn

Country Status (5)

Country Link
EP (1) EP0620859A1 (fr)
JP (1) JPH07501712A (fr)
AU (1) AU5142393A (fr)
CA (1) CA2124581A1 (fr)
WO (1) WO1994008032A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003528A2 (fr) * 1994-07-27 1996-02-08 Cambridge University Technical Services Limited Oligonucleotides et leur utilisation
GB2351559A (en) * 1999-06-29 2001-01-03 Advanced Biotech Ltd Reverse transcriptase formulations
EP1513869A2 (fr) * 2002-05-02 2005-03-16 Abbott Laboratories Polynucleotides pour la detection et la quantification des acides nucleiques du virus de l'hepatite b
EP1978109A1 (fr) 2007-04-05 2008-10-08 Roche Diagnostics GmbH RT-PCR rapide à étape unique
US7785844B2 (en) 2002-06-14 2010-08-31 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
CN102286622A (zh) * 2011-08-02 2011-12-21 王有福 番茄溃疡病菌分子标准样品及其制备方法
WO2016118085A1 (fr) * 2015-01-21 2016-07-28 Agency For Science, Technology And Research Pcr sur arn de cellule unique et analyse mutationnelle (scrm-pcr) : un procédé d'analyse simultanée de l'adn et de l'arn au niveau d'une cellule unique
US11338296B2 (en) 2018-07-26 2022-05-24 Lex diagnostics Ltd. Variable temperature reactor, heater and control circuit for the same
WO2022138887A1 (fr) 2020-12-25 2022-06-30 株式会社ニッポンジーン Adn polymérase modifiée

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0388232A1 (fr) * 1989-03-17 1990-09-19 Chiron Corporation Diagnostics et vaccins de NANBV
US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5077192A (en) * 1988-10-25 1991-12-31 The General Hospital Corporation Method of detecting antigenic, nucleic acid-containing macromolecular entities
EP0388232A1 (fr) * 1989-03-17 1990-09-19 Chiron Corporation Diagnostics et vaccins de NANBV

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Analytical Biochemistry, Vol. 162, issued 1987, P. CHOMCZYNSKI et al., "Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction", pages 156-159, especially pages 156-157. *
Analytical Biochemistry, Volume 168, issued 1988, V.T.-W. CHAN et al., "Simultaneous Extraction from Clinical Biopsies of High-Molecular-Weight DNA and RNA: Comparitive Characterization by Biotinylated and 32P-Labeled Probes on Southern and Northern Blots", pages 16-24, especially pages 17-18. *
H.A. ERLICH, "PCR Technology, Principles and Applications for DNA Amplifications", published 1989 by M Stockton Press (N.Y.), pages 7-16, see entire document. *
Nucleic Acids Research, Volume 17, No. 5, issued 1989, C. GOBLET et al., "One-step amplification of transcripts in total RNA using the polymerase chain reaction", page 2144, see entire document. *
Nucleic Acids Research, Volume 18, No. 5, issued 1990, J. SINGER-SAM et al., "Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase and Zfy gene transcripts during mouse spermatogenesis", pages 1255-1259, see especially page 1256. *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996003528A2 (fr) * 1994-07-27 1996-02-08 Cambridge University Technical Services Limited Oligonucleotides et leur utilisation
WO1996003528A3 (fr) * 1994-07-27 1996-04-18 Lynxvale Ltd Oligonucleotides et leur utilisation
GB2351559A (en) * 1999-06-29 2001-01-03 Advanced Biotech Ltd Reverse transcriptase formulations
GB2351559B (en) * 1999-06-29 2001-09-05 Advanced Biotech Ltd Improved reverse transcriptase formulations
EP1513869A2 (fr) * 2002-05-02 2005-03-16 Abbott Laboratories Polynucleotides pour la detection et la quantification des acides nucleiques du virus de l'hepatite b
EP1513869A4 (fr) * 2002-05-02 2006-03-15 Abbott Lab Polynucleotides pour la detection et la quantification des acides nucleiques du virus de l'hepatite b
EP2292801A2 (fr) 2002-06-14 2011-03-09 Gen-Probe Incorporated Compositions et procédé pour la détection du virus de l'hépatite B
US7785844B2 (en) 2002-06-14 2010-08-31 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
US9109264B2 (en) 2002-06-14 2015-08-18 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
US9914982B2 (en) 2002-06-14 2018-03-13 Gen-Probe Incorporated Compositions and methods for detecting hepatitis B virus
EP1978109A1 (fr) 2007-04-05 2008-10-08 Roche Diagnostics GmbH RT-PCR rapide à étape unique
US8119353B2 (en) 2007-04-05 2012-02-21 Roche Diagnostics Operations, Inc. Rapid one-step reverse transcriptase PCR
CN102286622A (zh) * 2011-08-02 2011-12-21 王有福 番茄溃疡病菌分子标准样品及其制备方法
WO2016118085A1 (fr) * 2015-01-21 2016-07-28 Agency For Science, Technology And Research Pcr sur arn de cellule unique et analyse mutationnelle (scrm-pcr) : un procédé d'analyse simultanée de l'adn et de l'arn au niveau d'une cellule unique
CN107208154A (zh) * 2015-01-21 2017-09-26 新加坡科技研究局 单细胞rna和突变分析pcr(scrm‑pcr)：用于在单细胞水平上同时分析dna和rna的方法
US11186865B2 (en) 2015-01-21 2021-11-30 Agency For Science, Technology And Research Single cell RNA and mutational analysis PCR (SCRM-PCR): a method for simultaneous analysis of DNA and RNA at the single-cell level
US11338296B2 (en) 2018-07-26 2022-05-24 Lex diagnostics Ltd. Variable temperature reactor, heater and control circuit for the same
WO2022138887A1 (fr) 2020-12-25 2022-06-30 株式会社ニッポンジーン Adn polymérase modifiée

Also Published As

Publication number Publication date
CA2124581A1 (fr) 1994-04-14
JPH07501712A (ja) 1995-02-23
EP0620859A1 (fr) 1994-10-26
AU5142393A (en) 1994-04-26

Similar Documents

Publication Publication Date Title
Young et al. Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay
Jin et al. Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase
JP2656996B2 (ja) Ｎａｎｂｖの診断用薬
Han et al. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5'untranslated region and poly (A) tails at the 3'end.
Koizumi et al. Diversity of quasispecies in various disease stages of chronic hepatitis C virus infection and its significance in interferon treatment
JP4031511B2 (ja) Ｃ型肝炎ウイルスゲノムの新規な３’末端配列並びにその診断及び治療使用
Wongteerasupaya et al. Detection of yellow-head virus (YHV) of Penaeus monodon by RT-PCR amplification
Krug et al. Hepatitis C virus genotypes in Southern Brazil
CN117683910B (zh) 血卵涡鞭虫的检测引物组和探针及其应用
WO1994008032A1 (fr) Amplification en chaine par polymerase d'arn en une seule etape et d'arn et d'adn combines en une seule etape, employee pour detecter l'arn rare ou l'arn et l'adn
Saito et al. Sequence analysis of PePHD within HCV E2 region and correlation with resistance of interferon therapy in Japanese patients infected with HCV genotypes 2a and 2b
Hu et al. One-step RNA polymerase chain reaction for detection of hepatitis C virus RNA
RU2145635C1 (ru) Олигомер (варианты), способ обнаружения последовательности hcv (варианты), набор для обнаружения, способ получения крови
Vašková et al. Real-time NASBA for detection of strawberry vein banding virus
Doglio et al. Rapid genotyping of hepatitis C virus by direct cycle sequencing of PCR-amplified cDNAs and capillary electrophoresis analysis
Mihm et al. A reevaluation of the association of hepatitis C virus replicative intermediates with peripheral blood cells including granulocytes by a tagged reverse transcription/polymerase chain reaction technique
Hosoda et al. Detection and partial sequencing of hepatitis C virus RNA in the liver
CN106702024B (zh) 检测寨卡病毒的试剂盒及其应用
Soguero et al. Assessment of genotype and molecular evolution of hepatitis C virus in formalin-fixed paraffin-embedded liver tissue from patients with chronic hepatitis C virus infection
Tajbakhsh et al. Determination of hepatitis C virus genotypes among HCV positive patients in Shahrekord, Iran
KR0135621B1 (ko) 씨(c)형 간염 바이러스 알엔에이(rna) 검출키트
US6946245B2 (en) Oligonucleotides and methods for detecting hepatitis C viral nucleic acids
Olson et al. Detection of dengue viral RNA in mosquito vectors by mixed phase and solution hybridization
Hu et al. Simultaneous detection of both hepatitis B virus DNA and hepatitis C virus RNA using a combined one-step polymerase chain reaction technique
Kawano et al. Self-priming of hepatitis C virus RNA

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

WWE Wipo information: entry into national phase

Ref document number: 1993922425

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2124581

Country of ref document: CA

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWP Wipo information: published in national office

Ref document number: 1993922425

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1993922425

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载