WO2012002594A1 - Amorce destinée à diagnostiquer le virus de l'hépatite c, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite c utilisant le kit - Google Patents

Amorce destinée à diagnostiquer le virus de l'hépatite c, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite c utilisant le kit Download PDF

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WO2012002594A1
WO2012002594A1 PCT/KR2010/004275 KR2010004275W WO2012002594A1 WO 2012002594 A1 WO2012002594 A1 WO 2012002594A1 KR 2010004275 W KR2010004275 W KR 2010004275W WO 2012002594 A1 WO2012002594 A1 WO 2012002594A1
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hepatitis
virus
seq
probe
primer
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PCT/KR2010/004275
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English (en)
Korean (ko)
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이충현
김성열
박해준
박한오
변상진
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(주)바이오니아
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Priority to PCT/KR2010/004275 priority Critical patent/WO2012002594A1/fr
Priority to KR1020127028140A priority patent/KR101503039B1/ko
Publication of WO2012002594A1 publication Critical patent/WO2012002594A1/fr

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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • the present invention relates to a primer for the diagnosis of hepatitis C virus, a probe and a method for diagnosing hepatitis C virus using the same, and more particularly, a primer, a probe for detecting hepatitis C virus present in a biological sample and an environmental sample, and a method of using the same. It relates to a virus detection method that can be used for the presence and quantitative diagnosis of the hepatitis C virus infection based on the polymerase chain reaction.
  • Hepatitis C virus (also called HCV) is a small RNA virus of about 50 nm in the Flaviviridae family.
  • the entire genome consists of about 9,600 bases and has an untranslated region (UTR) at both ends of the genome, which plays an important role in transcription or translation.
  • the 5 'UTR has a site for initiating viral polyprotein translation and is also a transcription start site for starting RNA synthesis.
  • Hepatitis C virus is the only hepatitis C virus in the world, evenly distributed in one region. The incidence of hepatitis C is 1-3%, depending on the country, and about 1.5% in Korea. In some parts of Africa, the incidence rate is more than 10%, and in some countries, including Japan, hepatitis C virus accounts for 30-50% of all acute and chronic infections. In the United States, hepatitis C virus is a major pathogen of chronic infection, and more than 10,000 people are reported to die from hepatitis C virus-related liver disease every year, and the number of patients continues to increase.
  • Hepatitis C refers to a disease in which the liver is inflamed due to the body's immune response when hepatitis C virus is infected. Characterization of hepatitis C is chronic with 55 to 80% of infected people ( Less than 5%), and more than 20% of chronically infected people develop cirrhosis or liver cancer. Hepatitis C virus is spread by parenteral routes and is mainly transmitted by contaminated blood products, sexual intercourse, vertical infections from infected mothers, contaminated syringes, saliva, razors, toothbrushes, and the like. When infected with the hepatitis C virus, the incubation period averages 6-7 weeks.
  • Infections are latent, usually asymptomatic but can easily become fatigued, tasteless, and cause nausea and vomiting.
  • Myalgia and mild fever may occur, the color of urine may darken, jaundice with yellowing of the skin or eyes may occur in severe cases, and death may occur in fatal cases.
  • Hepatitis C virus diagnosis currently uses antibody and virus detection methods.
  • Antibody testing for hepatitis C virus is performed by enzyme-linked immunoassay.
  • the virus detection method is capable of detecting a small amount of virus in a small amount of serum by measuring a virus in a serum by real-time polymerase chain reaction, and thus can be used for early diagnosis and rapid treatment.
  • Hepatitis C virus has six genotypes and more than 50 subtypes. In Korea, genotypes 1b and 2a are predominant, and 70 to 80% of patients infected with hepatitis C virus are genotype 1 It is reported to be infected.
  • the present inventors have designed a novel primer and probe specific for hepatitis C virus, by performing a real-time polymerase chain reaction using the primer, a probe and a kit comprising the same, compared to conventional methods
  • the present invention was completed by confirming that hepatitis virus RNA can be detected quickly and accurately, and that the polymerase chain reaction mixture required for the reaction can be dried, thereby improving the storage period while maintaining the same performance as the solution mixture. It was.
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a primer and a probe for diagnosing hepatitis C virus RNA for use in real time reverse transcription polymerase chain reaction.
  • Another object of the present invention is a kit for detecting hepatitis C virus RNA without cross-reacting with other hepatitis viruses, in which all reagents necessary for reverse transcription polymerase chain reaction are mixed, dispensed, and dried according to one test dose.
  • a hepatitis C virus RNA diagnostic kit that does not require the skill of the examiner for the purpose.
  • Another object of the present invention is to provide a rapid and accurate hepatitis C virus RNA diagnostic method.
  • the present invention provides primers and probes necessary for detecting hepatitis C virus RNA through real-time polymerase chain reaction or general polymerase chain reaction.
  • the real-time polymerase chain reaction of the present invention monitors the reaction results in real time by using oligonucleotide probes in which a primer and a fluorescent substance are chemically bound.
  • the probe binds to the complementary sequence in the nucleic acid of the sample, like two primers. The binding position is slightly away from the primer.
  • Probe of the present invention is a structure in which both the reporter (reporter) and the quencher (fluorescent material) is attached to both ends, if the reporter and the quencher is present in close proximity to each other to cancel the fluorescence of the reporter, but the amplification proceeds As the reporter falls from the quencher, the reporter's fluorescence is detected. Thus, the intensity of fluorescence increases gradually as the amplification cycle increases.
  • the inventors In order to detect hepatitis C virus of various subtypes compared to the conventional real-time polymerase chain reaction products, the inventors independently primers based on the specific sequence of the 5 'UTR gene (NCBI Accession No .; NC_004102) of hepatitis C virus. And probes were designed. Self-designed primers and probes are designed to not cross-react with other hepatitis viruses.
  • the primer sequence of the present invention comprises a part of hepatitis C virus or a part of its complementary nucleotide sequence, preferably 19 to 23 bases in the 50 to 360 bases of the 5 'UTR gene sequence of hepatitis C virus. Consists of nucleotide sequences. Especially preferably, it is a forward primer which is a base sequence described by SEQ ID NO: 1-SEQ ID NO: 3, and a reverse primer which is a base sequence shown by SEQ ID NO: 4-SEQ ID NO: 6.
  • the probe sequence of the present invention includes a part of the hepatitis C virus or a part of its complementary nucleotide sequence, preferably 20 to 50 in the 360 base of the 5 'UTR gene sequence of the hepatitis C virus. It consists of 23 base sequences.
  • the base sequence set forth in SEQ ID NO: 7 to SEQ ID NO: 9, preferably the base sequence set forth in SEQ ID NO: 7, is a forward probe.
  • the present invention provides a hepatitis C virus detection kit comprising the primer or probe.
  • the kit includes amplification buffers, dNTPs, controls, detection reagents, etc., in addition to the primers or probes of the present invention, and is preferably provided in a dry state, and includes additional components according to the purpose only when there is no effect on the reaction. can do.
  • the kit which is provided in a dry state, can be used for a long time due to improved storage stability, and a preparation process of the mixed solution can be omitted to obtain accurate and quantitative results with high reproducibility regardless of the skill of the experimenter.
  • the kit may further comprise primers and probes for internal control.
  • internal positive control hereinafter, also referred to as 'IPC'
  • the internal control primer comprises a part of the mouse dishevelled segment polarity protein (Dvl-1) (NCBI Accession No .; NC_005104) gene or a part of its complementary nucleotide sequence, preferably Is a forward primer which is composed of 5 to 40 base sequences in the 601th to 1400th bases of the nucleotide sequence, more preferably a forward primer which is the nucleotide sequence set forth in SEQ ID NO: 10, and a base sequence set forth in SEQ ID NO: 11 .
  • Dvl-1 mouse dishevelled segment polarity protein
  • the probe preferably has a nucleotide sequence set forth in SEQ ID NO: 12, all of which are forward probes.
  • the internal control primer comprises a part of the Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No .; FJ873800) or a part of its complementary base sequence, and preferably Is a forward primer which is composed of 5 to 40 base sequences within 37 to 1708 bases of the nucleotide sequence, more preferably a forward primer which is the nucleotide sequence shown in SEQ ID NO: 13, and a base sequence described in SEQ ID NO: 14. .
  • the probe preferably has a nucleotide sequence set forth in SEQ ID NO: 15, all of which are forward probes.
  • the internal control primers and probes are positive controls when tested, and when the real-time polymerase chain reaction was performed using the present invention, a negative judgment was obtained, that is, when the hepatitis C virus was not present in the sample, the result was experimental. It is necessary to verify whether the hepatitis C virus is absent or if the actual hepatitis C virus does not exist and should not interfere with the detection of hepatitis C virus when amplified with the hepatitis C virus primer set of the present invention. If the internal control is positive, the polymerase chain reaction itself indicates no problem.
  • the hepatitis C virus detection primers and probes can be any combination as long as the configuration of two primers (one forward, one reverse) and one probe, preferably a forward primer, SEQ ID NO: Reverse primers described as 5 and forward probes as shown in SEQ ID NO: 7 can be used (see Table 4).
  • the internal control primers and probes may also be any combination as long as it consists of two primers (one forward and one reverse).
  • a forward primer as set forth in SEQ ID NO: 10 when using a portion of the mouse dishevelled segment polarity protein (Dvl-1) (NCBI Accession No .; NC_005104) gene or a part of its complementary base sequence as the internal control primer, a forward primer as set forth in SEQ ID NO: 10, A reverse primer as set forth in SEQ ID NO: 11 and a forward probe as set forth in SEQ ID NO: 12 may be used, and the primer for the internal control may be a part of the Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No .; FJ873800) or its When using a part of the complementary nucleotide sequence, it is possible to use the forward primer of SEQ ID NO: 13, the reverse primer of SEQ ID NO: 14 and the forward probe of SEQ ID NO: 15.
  • MP Tobacco mosaic virus isolate Taigu movement protein
  • the primer of the present invention can be used not only for real-time polymerase chain reaction but also for general polymerase chain reaction.
  • a sample for use in the present invention may be obtained from a clinical sample or an environmental sample, but is not limited thereto.
  • the reporter of the hepatitis C virus probe is preferably FAM (6-carboxyfluorescein) and the quencher is BHQ1 (2,5-di-tert-butylhydroquinone-1), and the reporter of the internal control probe is TAMRA (Carboxy- tetramethyl-hod-amine), the matting agent is preferably used BHQ1, but is not limited thereto.
  • the detection method of the present invention even if a very small amount of nucleic acid of hepatitis C virus is present in the sample, hepatitis C virus can be detected, especially in the case of real-time polymerase chain reaction, during amplification. Amplification can be observed and the detection time can be reduced because no separate amplification product identification step is required.
  • the present polymerase chain reaction or real time polymerase chain reaction it is preferable to further use IPC, but is not limited thereto.
  • IPC in the polymerase chain reaction, it is easy to check whether the PCR was performed well by preparing the IPC template and the primers corresponding thereto.
  • the sample may be obtained from a clinical sample or an environmental sample, but is not limited thereto.
  • hepatitis C virus genes can be detected quickly and simply, and even at very low concentrations of hepatitis C virus present in the sample due to its high sensitivity.
  • the development of the hepatitis C virus RNA diagnostic kit of the present invention is expected to be able to accurately diagnose the initial stage of infection, and will contribute to the early confirmation and hepatitis C virus early expansion through widespread domestic and international kits. It is expected.
  • NCBI accession number NC_004102
  • NCBI National Center for Biotechnology Information
  • the primers and probes of the present invention which are composed of a part of the present invention, show a 100% match with the hepatitis C virus nucleotide sequence registered in the NCBI.
  • Figures 2 to 4 show the Exicycler TM Quantitative Thermal Block (Bioneer, Korea) instrument with all combinations of the hepatitis C virus primers and some probes of the present invention as set forth in SEQ ID NOs: 1-6 and SEQ ID NOs: 7-9 It is a graph showing the results of real-time polymerase chain reaction using.
  • Figure 2 Amplification with primers of SEQ ID NOs: 1, 4 and probes of SEQ ID NO: 7 (set 1)
  • FIGS. 5 and 6 are graphs showing the results of testing the optimal concentrations of the forward and reverse primers for the second set of PCR efficiency and the best result from the results performed in FIGS. 2 to 4.
  • FIG. 6 shows a standard graph of FIG. 5, wherein the closer the R 2 value is to 1, the higher the efficiency is to 100%, and the higher the PCR efficiency. As a result of the experiment of FIG. 5, R 2 was 0.9998 and Efficiency was 91%.
  • FIGS. 7 and 8 are graphs showing the results of real-time polymerase chain reaction using an Exicycler TM Quantitative Thermal Block (manufactured by Bioeer, Korea) instrument with a combination of primers and probes of the RNA for the internal control of the present invention.
  • Figure 7 is a mouse dishevelled segment polarity protein (Dvl-1) gene was used as the RNA template for the internal control
  • Figure 8 is a graph using Tobacco mosaic virus isolate Taigu movement protein (MP) gene as the RNA template for the internal control.
  • Dvl-1 mouse dishevelled segment polarity protein
  • MP Tobacco mosaic virus isolate Taigu movement protein
  • FIG. 9 and 10 are the real time of the HCV standard template using the real-time polymerase chain reaction device Exicycler TM Quantitative Thermal Block with a combination of the primers SEQ ID NO: 2, 5, the probe of SEQ ID NO: 7, the primer for the internal control and the probe
  • the graph of the polymerase chain reaction is shown.
  • FIG. 10 shows a standard graph of FIG. 5, wherein the closer the R 2 value is to 1, the higher the efficiency is to 100%, and the higher the PCR efficiency.
  • R 2 was 0.9994 and Efficiency was 98%.
  • Black curves Amplification curves of hepatitis C virus template RNA at 10 to 10 7 copy concentrations, respectively
  • FIG. 11 and 12 are graphs showing the experimental results of the standard template real-time reverse transcription polymerase reaction using the dry polymerase chain reaction composition, the results obtained using the Exicycler TM Quantitative Thermal Block.
  • the blue line represents IPC
  • the detected cycle value is the same within an error range of ⁇ 1
  • the black line represents the value of loading of hepatitis C virus RNA template by concentration.
  • FIG. 12 shows a standard graph of FIG. 11, wherein the closer the R 2 value is to 1, the higher the efficiency is to 100%, and the higher the PCR efficiency. As a result of the experiment of FIG. 11, R 2 was 0.9996 and Efficiency was 97%.
  • FIG. 13 shows a graph of real-time polymerase chain reaction of the dynamic range of each HCV standard template using a dry PCR mixture, using a real-time polymerase chain reaction apparatus Exicycler TM 96 Real-Time Quantitative Thermal block.
  • the blue line represents IPC
  • the detected cycle value is the same within an error range of ⁇ 1
  • the black line represents the value of loading of the hepatitis C virus RNA template by concentration.
  • FIG. 14 shows a standard graph of FIG. 13, wherein the closer the R 2 value is to 1, the higher the efficiency is to 100%, and the higher the PCR efficiency. 13 shows that R 2 is 0.9998 and Efficiency is 98%.
  • FIGS. 15 to 18 are graphs showing the results of real-time reverse transcription polymerase chain reaction using RNA extracted from 15 HCV positive samples and dried PCR mixture, using Exicycler TM 96 Real-Time Quantitative Thermal block. Obtained results.
  • the blue line represents the IPC signal and the black line represents the hepatitis C virus positive control.
  • FIG. 16 shows the graphs of FIGS. 17 and 18, in which black lines in FIGS. 17 and 18 represent signals of actual hepatitis C virus samples, and blue lines in FIGS. 16 and 18 represent IPC signals.
  • FIG. 19 is a graph showing the results of real-time reverse transcription polymerase chain reaction using RNA extracted from 15 HCV-negative samples and dried PCR mixture, which is obtained using Exicycler TM Quantitative Thermal Block.
  • the blue line is the IPC signal
  • the black line is the hepatitis C virus signal.
  • the hepatitis C virus signal did not appear.
  • FIG. 20 to 22 are different hepatitis virus samples tested using a standard template real-time reverse transcription polymerase reaction using the polymerase chain reaction composition of the present invention, a graph showing the results confirming that no cross-reaction with other hepatitis viruses occurs As shown in FIG. 20, the hepatitis C virus sample, FIG. 21 is the hepatitis A virus sample, and FIG. 22 is the result of the hepatitis B virus sample.
  • Hepatitis C virus can be synthesized from the 5 'UTR portion of the hepatitis C virus registered in the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov ( Biochem. Biophys. Res). Commun. 1998, 248, 200-203), and some of them were cloned into pGEM-T-Easy Vector (Cat: A1360, manufactured by Promega, USA).
  • NCBI National Center for Biotechnology Information
  • Plasmid DNA was extracted from the pellets using a plasmid DNA prep kit (manufactured by Bioneer, Korea). Plasmid DNA was measured using a UV spectrometer (manufactured by Shimazu, Japan) to measure the concentration and purity, and confirmed that the purity was between 1.8 and 2.0. Plasmid DNA was transcribed into RNA using an in vitro transcription kit (Ambion, USA). After the transcription, the concentration and purity were measured by UV spectrometer, and when the purity was between 1.8 and 2.0, it was used as template RNA in the subsequent real-time polymerase chain reaction. RNA copy number was calculated by the following formula.
  • the internal control RNA was prepared in the same manner as the template RNA preparation.
  • the internal control RNA is needed to confirm that when a negative result is obtained, the negative result is not due to an amplification error.
  • the internal control RNA was used as two types of mouse dishevelled segment polarity protein (Dvl-1) gene (NCBI Accession No .; NC_005104) and Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No .; FJ873800).
  • Dvl-1 gene 767 bp region, which is the 601th to 1400th sequence including the primer and probe sequences, was synthesized and used to prepare RNA for internal control.
  • Tobacco mosaic virus isolate Taigu movement protein (MP ) Gene was synthesized from 37th to 1708 including the primer and probe sequence in the gene was used to prepare the RNA for the internal control.
  • Plasmid DNA was measured using a UV spectrometer (manufactured by Shimazu, Japan) to measure the concentration and purity, and confirmed that the purity was between 1.8 and 2.0. Plasmid DNA was transcribed into RNA using an in vitro transcription kit (Ambion, USA). After the transcription, the concentration and purity were measured by UV spectrometer, and when purity was between 1.8 and 2.0, it was used as template RNA for internal control in the real-time polymerase chain reaction. RNA copy number was calculated by the following formula.
  • Hepatitis C Virus Hepatitis C Virus 5 'UTR gene (NCBI Accession No .; NC_004102) between 50 and 360, the length of 19-23 bp, Tm value of 55 °C to 60 °C optionally base sequence was chosen to be a forward and reverse primer.
  • the length was between 20 and 23 bp, and the Tm value was arbitrarily selected as a probe to select a base sequence, and the Tm value was checked using a Primer3Plus program.
  • the primers and probes shown in Table 1 were designed.
  • the nucleotide sequence of the Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No .; FJ873800) for internal control was randomly selected to have a length of 18 to 22 bp and a Tm value of 55 to 62 ° C. Selection was made into forward and reverse primers. The base sequence was randomly selected between the base sequences 942 and 1708 at 25 bp in length and the Tm value was 67 to 72 DEG C. (Table 3).
  • hepatitis C virus primers and all the probes were combined in a set, and then real-time reverse transcription polymerase chain reaction was performed using Exicycler TM Quantitative Thermal Block (Bionia, Korea).
  • RNA of the hepatitis C virus prepared in Example 1 was used as a template, and the hepatitis C virus primers and probes designed in Example 2 were combined into several sets, respectively, and then Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) Real time polymerase chain reaction was carried out using () (Table 4). Specifically, hepatitis C virus template RNA synthesized in Example 1, 5 ⁇ l of 10X RT Buffer, MMLV 600U, wTfi 10unit, 3 ⁇ l of dNTP 20mM, DTT 2.5mM, RNasin 9U, stabilizer, etc.
  • Primer for hepatitis virus detection, probe and distilled water were added and mixed to a total volume of 50 ⁇ l and then aliquoted into 96-well plates. At this time, the concentration of the forward primer and the reverse primer included in the total dose was used 20 pmole, respectively, the concentration of the probe was used 20 pmole.
  • the reaction was carried out at 45 ° C. for 15 minutes to synthesize cDNA, and after 5 minutes of modification at 95 ° C., 45 cycles of 5 seconds at 95 ° C. and 5 seconds at 55 ° C. were performed. The amplified fluorescence value was continuously measured once after 55 ° C. 30 seconds reaction as each PCR cycle progressed.
  • Table 4 SEQ ID NO: Set 1 Set 2 Set 3 Forward primer One 2 3 Reverse primer 4 5 6 Forward probe 7 7 8
  • PCR primers having the highest PCR amplification efficiency were found to be set 2, that is, the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 5, and the forward probe of SEQ ID NO: 7 (FIGS. 2 to 4).
  • Two sets of experimental graphs are shown in FIGS.
  • set 2 was 97% and PCR efficiency was the best. If a standard graph is drawn using the cycle value detected by diluting the virus template by concentration, the closer the value is to 1, the straight line of the graph is better. Comparing the three sets, all of them had a value greater than 0.999 and showed equivalence.
  • the primer and probe concentration combinations were optimized using 5 ⁇ l of 10X RT Buffer, MMLV 600U, wTfi 10unit, dNTP 20mM 3 ⁇ l, DTT 2.5mM, RNasin 9U, stabilizer, etc.
  • a procedure was performed to demonstrate the condition.
  • 30 pmole of the primer of SEQ ID NO: 2 when the 30 pmole of the primer of SEQ ID NO: 2, 30 pmole of the primer of SEQ ID NO: 5 and 20 pmole of the probe of SEQ ID NO: 7 had a good copy number limit of 10 copies, the real time reverse transcription polymerase chain reaction. The efficiency of was 98% (Fig. 5).
  • R 2 is a correlation coefficient representing the linearity of the graph when the standard graph of the real-time polymerase chain reaction is drawn. The closer to 1 (the closer to the straight line), the polymerase chain reaction proceeded properly.
  • CDNA was synthesized by reacting at 45 ° C. for 15 minutes, denatured at 95 ° C. for 5 minutes, and reacted with 45 cycles of 5 seconds at 95 ° C. and 5 seconds at 55 ° C.
  • the amplified fluorescence value was continuously measured once after 55 ° C. and 30 seconds as each PCR cycle proceeded (FIGS. 7 and 8).
  • Hepatitis C virus RNA and RNA for the internal control prepared in Example 1 as a template Hepatitis C primers of SEQ ID NO: 2, 5 selected in Example 2, probe of SEQ ID NO: 7, primer for the internal control and Apply Probes to Exicycler TM Real-time polymerase chain reaction was performed using Quantitative Thermal Block (Bionia, Korea). 45 cycles of real-time polymerase chain reaction were carried out under the same conditions and components as in Example 3.
  • hepatitis C virus template RNA was detected up to 10 copies (Fig. 9), and when a standard graph of the standard template real-time polymerase chain reaction was prepared. , The slope was -2.96, the R 2 value was 0.9994 (Fig. 10).
  • R 2 is a correlation coefficient indicating the linearity of the graph when the standard graph of the real-time polymerase chain reaction is drawn, which means that the closer to 1 (the closer to the straight line), the PCR proceeded properly.
  • 10 5 copies of the internal control RNA was reacted together, it was confirmed that internal control amplification was independently performed without affecting the amplification of the hepatitis C virus RNA template.
  • PCR mixture premix dried product
  • a PCR mixture of the same composition as in Example 3 was prepared, dried, and then subjected to a real-time polymerase chain reaction using an Exicycler TM Quantitative Thermal Block (Bionia, Korea). It was.
  • Example 4 5 ⁇ l of the hepatitis C virus RNA prepared in Example 1 was added to the dry polymerase chain reaction composition, 1 ⁇ l of the internal control RNA was added, and the total volume was divided into 50 ⁇ l by DW to dry the well. Mixed thoroughly. 45 cycles of real time reverse transcription polymerase chain reaction were carried out using the Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions as in Example 4.
  • hepatitis C virus template RNA was stably detectable up to 10 copies (Fig. 11 and Table 7), and when the standard graph of the standard template real-time reverse transcription polymerase chain reaction was prepared, the slope was -2.94, R The value of 2 was 0.9996 (FIG. 12).
  • Table 7 shows the numerical value of the graph of FIG. 11, and when the amount of HCV RNA as a positive control is 10 7 , it indicates that a signal can be seen even if only 19.14 cycles of Realtime PCR is turned, and NTC is a negative sample as a negative control. Indicates. From the above results, it can be seen that the equivalent performance was maintained in the two methods of real-time reverse transcription polymerase chain reaction using the polymerase chain reaction mixture in the solution state and the dry mixture.
  • Exicycler TM Real-Time Quantitative Thermal Block (manufactured by Bioneer) for final dynamic range (concentration range of hepatitis C virus RNA that can be detected in one reaction) using a dry PCR mixture in the same manner as above. , Korea) was subjected to 45 cycles of real-time polymerase chain reaction under the same conditions as in Example 3.
  • Hepatitis C virus template RNA with the calculated copy number according to the method of Example 1 was used by diluting 10 times in the range of up to 10 10 copies at the lowest 10 copy concentration, and hepatitis C virus RNA detection was normally performed in the 10 log range. It was confirmed (FIG. 13).
  • the slope was -2.97 and the R 2 value was 0.9998 (FIG. 14).
  • Detection test was performed on hepatitis C virus samples using the dry type PCR mixture prepared in Example 3. The sample was positively determined by the antibody diagnostics using the serum of the patient, real time by the method of Example 5 for 15 hepatitis C positive samples using the hepatitis C virus detection primer / probe set of the present invention Reverse transcription polymerase chain reaction was performed.
  • Example 1 to a dry type PCR composition comprising a hepatitis C virus primer and a probe described in SEQ ID NO: 7 and the internal control primer and a probe described in SEQ ID NO: 2, 5 prepared in Example 2
  • the prepared hepatitis C virus RNA and the internal control RNA were added as a template, and the mixture was dispensed with distilled water to have a total volume of 50 ⁇ l and thoroughly mixed to loosen the dry composition.
  • RNA extracted from each sample was added to the same components as above except for template RNA for the specimen test. 45 cycles of real-time polymerase chain reaction were carried out using Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions as in Example 3.
  • Detection test was performed on hepatitis C virus samples using the dry type PCR mixture prepared in Example 3. The sample was negatively determined by antibody diagnostics using patient serum, and the hepatitis C virus detection primer / probe set of the present invention was used for the 15 hepatitis C negative samples by the method of Example 5 in real time. Reverse transcription polymerase chain reaction was performed.
  • Example 1 to a dry type PCR composition comprising a hepatitis C virus primer and a probe described in SEQ ID NO: 7 and the internal control primer and a probe described in SEQ ID NO: 2, 5 prepared in Example 2
  • the prepared hepatitis C virus RNA and the internal control RNA were added as a template, and the mixture was dispensed with distilled water to have a total volume of 50 ⁇ l and thoroughly mixed to loosen the dry composition.
  • RNA extracted from each sample was added to the same components as above except for template RNA for the specimen test. 45 cycles of real-time polymerase chain reaction were carried out using Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions as in Example 3.
  • the Green Cross Medical Foundation received RNA extracted from samples of patients identified through clinical symptoms and gene sequencing and was used in the experiment.
  • a verification experiment was carried out for the cross-reaction with the hepatitis virus by the method of Example 5 using the hepatitis C virus detection primer of the present invention.
  • the hepatitis C virus detection primer / probe set of the present invention was confirmed that the real-time reverse transcriptase polymerase chain reaction to the hepatitis C virus, the reaction does not occur in other hepatitis virus samples (Fig. 20 to 22).

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Abstract

La présente invention concerne une amorce spécifique d'un gène du virus de l'hépatite C, une sonde, et un procédé de détection utilisant celles-ci, et plus particulièrement, une amorce destinée à diagnostiquer simultanément le gène du virus de l'hépatite C par le biais d'une réaction en chaîne par polymérase, une sonde, un kit de diagnostic comprenant l'amorce et la sonde pour un groupe de contrôle interne, et un procédé de diagnostic du virus de l'hépatite C utilisant le kit. Selon la présente invention, il est possible d'utiliser une détection en temps réel qui est plus rapide et plus précise que les procédés de détection du virus de l'hépatite C existants, et, comme une composition de réaction en chaîne par polymérase sèche comprenant l'amorce et la sonde présente un temps de stockage plus long que celle sous une forme en solution tout en ayant une performance égale, elle peut être utilisée de manière avantageuse dans un kit de détection du virus de l'hépatite C.
PCT/KR2010/004275 2010-07-01 2010-07-01 Amorce destinée à diagnostiquer le virus de l'hépatite c, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite c utilisant le kit WO2012002594A1 (fr)

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PCT/KR2010/004275 WO2012002594A1 (fr) 2010-07-01 2010-07-01 Amorce destinée à diagnostiquer le virus de l'hépatite c, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite c utilisant le kit
KR1020127028140A KR101503039B1 (ko) 2010-07-01 2010-07-01 Ｃ형 간염 바이러스 진단용 프라이머, 프로브, 이를 포함하는 키트 및 상기 키트를 이용한 ｃ형 간염 바이러스 진단 방법

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WO2020160502A1 (fr) * 2019-01-31 2020-08-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés et compositions destinés à détecter des pathogènes transmis par tranfusion
CN111560475A (zh) * 2020-05-20 2020-08-21 曲靖市第一人民医院 一种Redondovirus病毒核酸检测试剂盒

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643799A (zh) * 2012-04-23 2012-08-22 中国水产科学研究院淡水渔业研究中心 建鲤GAPDH基因含内含子的部分序列的克隆方法及其realtimePCR方法
CN102643799B (zh) * 2012-04-23 2013-06-05 中国水产科学研究院淡水渔业研究中心 建鲤GAPDH基因含内含子的部分序列的克隆方法及其realtimePCR方法
WO2020160502A1 (fr) * 2019-01-31 2020-08-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédés et compositions destinés à détecter des pathogènes transmis par tranfusion
CN111560475A (zh) * 2020-05-20 2020-08-21 曲靖市第一人民医院 一种Redondovirus病毒核酸检测试剂盒

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