WO2012002597A1 - Amorce destinée à diagnostiquer le virus de l'hépatite b, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite b utilisant le kit - Google Patents

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

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WO2012002597A1
WO2012002597A1 PCT/KR2010/004331 KR2010004331W WO2012002597A1 WO 2012002597 A1 WO2012002597 A1 WO 2012002597A1 KR 2010004331 W KR2010004331 W KR 2010004331W WO 2012002597 A1 WO2012002597 A1 WO 2012002597A1
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probe
seq
hepatitis
primer
virus
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PCT/KR2010/004331
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English (en)
Korean (ko)
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구완림
김성열
박해준
박한오
변상진
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(주)바이오니아
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Priority to PCT/KR2010/004331 priority Critical patent/WO2012002597A1/fr
Priority to KR1020127028141A priority patent/KR101498704B1/ko
Publication of WO2012002597A1 publication Critical patent/WO2012002597A1/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
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a hepatitis B virus primer, a probe and a method for diagnosing hepatitis B virus using the same, and more particularly, a primer, a probe for detecting hepatitis B 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 B virus infection based on the polymerase chain reaction.
  • Hepatitis B virus is an enveloped virus, which has a small genome of 3.2 kb, and is composed of partially duplex circular DNA. From this, four proteins are expressed, each of which is a surface antigen, a core protein, a DNA polymerase, and an X protein. After the virus enters the host cell, the genome is moved to the nucleus and the double stranded double stranded DNA (also called cccDNA, covalently closed circular DNA, or Relaxed circular DNA), which was partially part of the host cell's repair system, do. After the pregenomic RNA is transcribed and encapsulated into the core, a replication process occurs by hepatitis B virus DNA polymerase using the prezinomic RNA as a template. The mature viral particles then exit the host cell and a new infection cycle is repeated (Ganem D, Varmus HE., 1987, Ann. Rev. Biochem .. 56, 651-93).
  • Hepatitis B Virus (hereinafter also referred to as 'HBV') is a virus belonging to the Hepadnaviridae family, which is specifically infected only with human liver cells. It is estimated that there are about 350 million chronic carriers worldwide, and in Korea, about 7% of the population is estimated to be about 3 million people. The distribution of infected patients worldwide is large in Africa, Southeast Asia and China, and relatively small in Australia, Western Europe and North America. In the United States, an estimated 140,000 to 320,000 people a year are infected with the hepatitis B virus, with 1.2 million chronic hepatitis patients.
  • hepatitis Symptoms of hepatitis are mild, fatigue, jaundice in severe cases. In the late stage of chronic hepatitis, complications of cirrhosis occur, ascites, swelling, gastroesophageal variceal bleeding, hepatic encephalopathy, abnormal blood coagulation, spleen hyperplasia.
  • HBV infection can be determined by measuring HBsAg (HBV surface antigen) in serum.However, to distinguish between chronic and active infections, it can be determined by quantitatively measuring the levels of HBV DNA and liver enzymes circulating in the blood. HBV DNA testing is required. In addition, rapid and accurate quantitative testing of DNA is required for the rapid discovery of hepatitis B virus infections and for monitoring drug resistance or therapeutic effects.
  • the conventional methods not only require a lot of time and labor to purify the hepatitis B virus DNA, but also have a problem in that the reproducibility of the experiment is not high.
  • Korean Patent Registration No. 10-0194003 treats the cell culture with sodium hydroxide and beta-mercaptoethanol, and then neutralizes and heats the polymerase chain reaction (hereinafter, referred to as 'PCR').
  • 'PCR' polymerase chain reaction
  • the PCR reaction product was stained with electrophoresis and ethidium bromide on agarose gel. After analyzing the intensity with an image analyzer, it still takes a lot of time and labor when analyzing a large amount of compounds, and in this process, an experimental error occurs, which makes it difficult to obtain accurate results.
  • the present inventors have designed a novel primer and probe specific for hepatitis B 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 storage period is improved while the performance of the solution in the solution state is equal to that of the solution, and the error is simplified by the simplification of the mixing process.
  • the present invention has been completed by confirming that the results can be reduced as much as possible to obtain high reproducibility.
  • 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 probe for hepatitis B virus DNA diagnosis used in real time polymerase chain reaction.
  • Another object of the present invention is a kit for detecting hepatitis B virus DNA without cross-reacting with other hepatitis viruses, in which all reagents required for the polymerase chain reaction are mixed, dispensed, and dried at one test dose. It is to provide a hepatitis B virus DNA diagnostic kit that does not require a tester's skill.
  • Still another object of the present invention is to provide a rapid and accurate hepatitis B virus infection and a quantitative diagnosis method of DNA.
  • the present invention provides primers and probes necessary for detecting hepatitis B virus DNA 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 designed primers and probes at specific sequences of the surface antigen S gene of hepatitis B virus to detect hepatitis B virus of various genotypes as compared to conventional real time polymerase chain reaction products. Self-designed primers and probes are designed to not cross-react with other hepatitis viruses.
  • Primers and probes of the present invention include a portion of the hepatitis B virus surface antigen S gene (GeneBank Accession No .; X04615) or a portion of its complementary sequence, preferably within the 155th to 835th bases of the base sequence. It is a forward primer which comprises 5-40 base sequences, More preferably, it is a forward primer which is a base sequence shown by SEQ ID NO: 1-4, and a base sequence shown by SEQ ID NOs: 5-8.
  • the probe is preferably a nucleotide sequence set forth in SEQ ID NOs: 9 to 12, all of which are forward probes.
  • the present invention provides a hepatitis B 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'
  • primers include, for example, a part of Mus musculus dishevelled, dsh homolog 1 (Drosophila) (Dvl1) gene (GenBank. Accession No. NM010091) or a part of its complementary sequence, preferably from 942 of the base sequence.
  • Dvl1 dsh homolog 1 gene
  • It is a forward primer which contains 5 to 40 base sequences in the 1708th base, more preferably a base sequence described in SEQ ID NO: 13 to 15 and a base sequence described in SEQ ID NO: 16 to 18.
  • the nucleotide sequence shown in SEQ ID NO: 19-21 is preferable, and a probe is all a forward probe.
  • the internal control primers and probes are positive controls in the test, and when the (real-time) polymerase chain reaction is performed using the present invention, a negative judgment is obtained, that is, when the hepatitis B virus is not present in the sample. Is necessary to verify whether it is an experimental mistake or that no actual hepatitis B virus is present, and should not interfere with the detection of hepatitis B virus gene when amplified with the hepatitis B virus gene primer set of the present invention. If the internal control is positive, the polymerase chain reaction itself indicates no problem.
  • the primers and probes may be any combination as long as two primers (one forward and one reverse) and one probe, but are preferably a forward primer as shown in SEQ ID NO: 1 and a reverse primer as shown in SEQ ID NO: 5 And forward probes set forth in SEQ ID NO: 9.
  • 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 B virus probe is preferably FAM (6-carboxyfluorescein), the quencher is BHQ1, the reporter of the internal control probe is TAMRA (Carboxy-tetramethyl-hod-amine), and the quencher is BHQ1.
  • FAM 6-carboxyfluorescein
  • TAMRA Carboxy-tetramethyl-hod-amine
  • BHQ1 BHQ1
  • TAMRA Carboxy-tetramethyl-hod-amine
  • the detection method of the present invention even if a very small amount of hepatitis B virus is present in the sample due to its high sensitivity, especially in real time polymerase chain reaction, the amplification can be observed immediately during the amplification process. There is no need for a separate amplification product identification step, which reduces detection time.
  • 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 B virus genes can be detected quickly and simply, and even at very low concentrations of hepatitis B virus present in the sample with high sensitivity can be detected accurately.
  • the development of the hepatitis B virus DNA diagnostic kit of the present invention is expected to be able to accurately diagnose the initial stage of infection, it is expected to greatly contribute to the confirmation of drug resistance and treatment effect of hepatitis B virus through monitoring the drug treatment. do.
  • Figures 1 and 2 align the hepatitis B virus surface antigen S sequence using BLAST of the National Center for Biotechnology Information (NCBI) to identify the high homology parts, 100% matched to black Indicated.
  • NCBI National Center for Biotechnology Information
  • the primers and probes of the present invention were prepared based on the homologous sequences.
  • FIG. 1 confirms homology based on the genotype C sequences found in Korea
  • FIG. 2 compares the sequences of primers and probes prepared based on genotype C with various genotype sequences.
  • FIG. 3 to 8 is a real time polymerase chain reaction using a 7500 Fast Real-Time PCR System (manufactured by Applied Biosystems, USA) with all combinations of the HBV primers and probes of the present invention as set forth in SEQ ID NO: 1 to SEQ ID NO: 8
  • Figure 9 is a graph showing the results
  • Figure 9 is a combination of primers and probes of the DNA for the internal control of the present invention described in SEQ ID NO: 13 to SEQ ID NO: 21 7500 Fast Real-Time PCR System (manufactured by Applied Biosystems, USA) instrument Graph showing the results of real-time polymerase chain reaction using.
  • 10 to 12 is a combination of the primers and probes of the present invention described in SEQ ID NO: 4, 8, 12 in which 1 base of SEQ ID NO: 1, 5, 9 is replaced with a mixbase Real-time polymerase chain reaction apparatus Exicycler TM
  • This graph shows the results of real-time polymerase chain reaction of HBV standard template using 96 Real-Time Quantitative Thermal Block.
  • FIG. 10 to 12 is a combination of the primers and probes of the present invention described in SEQ ID NO: 4, 8, 12 in which 1 base of SEQ ID NO: 1, 5, 9 is replaced with a mixbase Real-time polymerase chain reaction apparatus Exicycler TM
  • This graph shows the results of real-time polymerase chain reaction of HBV standard template using 96 Real-Time Quantitative Thermal Block.
  • FIG. 18 shows the results of real-time polymerase chain reaction of HBV standard templates with a dry PCR composition comprising primers and probes of the invention set forth in SEQ ID NOs: 1, 5, 9 and SEQ ID NOs: 13, 16, 19
  • a real-time polymerase chain reaction device Exicycler TM 96 Real-Time Quantitative Thermal block was used.
  • FIG. 19 shows a standard curve of a real-time polymerase chain reaction graph applying HBV standard template by concentration to a dry PCR mixture using Exicycler TM 96 Real-Time Quantitative Thermal Block (Slope: ⁇ 0.3024, R 2 : 0.9994) .
  • FIG. 20 is a graph showing the results of real-time polymerase chain reaction of HBV standard template using a dry PCR mixture, using a real-time polymerase chain reaction device Exicycler TM 96 Real-Time Quantitative Thermal block.
  • 21 to 25 are real-time polymerase chains using PCR mixtures immediately after drying and at 40 ° C. constant temperature storage days (2 days, 4 days, 6 days, 8 days, respectively) for storage stability testing of PCR mixtures in a dry state.
  • This graph shows the result of the reaction.
  • the formula at the bottom of the graph shows the standard curve of the real-time polymerase chain reaction graph applying the HBV standard template for each concentration.
  • 26 and 27 are graphs showing the results of DNA extraction from HBV genotype panel and real-time polymerase chain reaction using a dried PCR mixture. Results obtained using Exicycler TM 96 Real-Time Quantitative Thermal block to be.
  • template DNA was prepared first.
  • the surface antigen S sequence of the hepatitis B virus was aligned to identify a high homology portion (FIG. 1).
  • the hepatitis B virus surface antigen S gene (GeneBank Accession No .; X04615), one of the highly homologous sequences, 680 bp, the 155 th to 835 th sequences including the primer and probe sequences, were synthesized ( NBiochem. Biophys). Res. Commun. 1998, 248, 200-203) and cloned into pGEM-T-Easy Vector (Cat: A1360, manufactured by Promega, USA).
  • Plasmid DNA was measured by UV spectrometer (manufactured by Shimazu Co., Japan) and the purity was confirmed to be between 1.8 and 2.0. Based on the concentration measurement results, the DNA copy number was calculated by the following formula. It was.
  • the copy number of the template DNA was calculated and then diluted 10 ⁇ with 1 ⁇ TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) and stored at ⁇ 70 ° C. until use.
  • Internal control DNA was prepared in the same manner as the template DNA preparation. Internal control DNA is needed to confirm that when a negative result is obtained, the negative result is not due to an amplification error.
  • Dvl1 dsh homolog 1 (Drosophila) (Dvl1) gene (GenBank Accession No. NM010091) using the 767 bp region, which is the 928th to 1647th sequence including the primer and probe sequences, for the internal control DNA preparation. Based on the concentration measurement results of the extracted plasmid DNA, DNA copy number was calculated by the following formula.
  • the copy number of the DNA for the internal control was calculated and then diluted 10 ⁇ with 1 ⁇ TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) and stored at ⁇ 70 ° C. until use.
  • the nucleotide sequence 155 to 835 of the hepatitis B virus surface antigen S gene (GeneBank Accession No .; X04615) has a length of 19 to 27 bp and a Tm value of 55 to 65 ° C. It was set as. In addition, between nucleotide sequences 155 to 835, the length was between 20 and 30 bp, and the Tm value was arbitrarily selected as a probe, and the Tm value was checked using the Primer3Plus program (Table 1). .
  • Dvl1 Internal length of the musculus dishevelled, dsh homolog 1 (Drosophila) (Dvl1) gene (GenBank. Accession No. NM010091), between 942 and 1708, length 17-23 bp, Tm value 55-62 °C
  • the base sequence was arbitrarily selected to be a forward and reverse primer.
  • the length was between 19 and 30 bp, and the Tm value was selected between 67 and 72 ° C. at random, and the Tm value was checked using the Primer3Plus program (Table 2). .
  • the concentration of the forward primer, the reverse primer and the probe contained in the total dose of 50 ⁇ l was used 15pmole, respectively.
  • the amplified fluorescence value was continuously measured once after 55 ° C. 30 second reaction as each PCR cycle proceeded.
  • the PCR amplification efficiency of the primers and probes was the highest of the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 5, the forward probe of SEQ ID NO: 9 (Table 3, Figures 3 to 13) .
  • the 20th base of the forward primer of SEQ ID NO: 1 having the highest PCR amplification efficiency, the 18th base of the reverse primer of SEQ ID NO: 5, and the 7 of the forward probe of SEQ ID NO: 9 Real-time polymerase chain reaction was carried out in the same manner as described above using the forward primer of SEQ ID NO: 4, the reverse primer of SEQ ID NO: 8, and the forward probe of SEQ ID NO: 12 in which each base of the first base was replaced with mixbase (Table 5 ).
  • the substituted sequences as described above there is an advantage in that various subtypes can be detected by additionally matching sequences that are not 100% identical by 1base difference (FIG. 2).
  • the reaction conditions were denatured at 95 ° C for 10 minutes, and then reacted with 45 cycles of 20 seconds at 95 ° C and 30 seconds at 55 ° C.
  • primers and probes for the internal control were selected for efficient PCR amplification (Table 4), and the highest PCR amplification efficiency among the primers and probes was the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 16 It was found that the forward probe of SEQ ID NO: 19 (FIG. 9).
  • the forward probe of SEQ ID NO: 9 has a structure in which FAM is bound as a reporter at the 5 'end and BHQ1 is bound as a quencher at the 3' end.
  • an inner dT probe having a quencher coupled to the eighth base sequence T of the probe sequence was prepared, and real-time polymerase chain reaction was performed by the same method.
  • the primer was set with the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 5 having the highest amplification efficiency, and performed a real-time polymerase chain reaction using Exicycler TM Quantitative Thermal Block (Bionia, Korea). The reaction conditions were denatured at 95 ° C.
  • HBV DNA and internal control DNA prepared in Example 1 as a template, HBV primers and probes described in SEQ ID NO: 1, 5, and 9 selected in Example 2, and SEQ ID NO: 13, 16 and 19 Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) real-time polymerase chain reaction was performed by applying primers and probes for internal control described. This is because the amplification of the internal control DNA is independent without affecting the amplification efficiency of the HBV DNA even if the polymerase chain reaction is performed by mixing the internal control DNA, the internal control primer and the probe with the HBV DNA, the HBV primer and the probe. This is to confirm the occurrence. Reaction conditions were carried out 45 cycles of real-time polymerase chain reaction in the same composition and method as in Example 2.
  • the number of copies was calculated according to the method of Example 1, HBV template DNA was able to detect up to 10 copies (Fig. 14), the standard template
  • the slope was -0.3121 and the R 2 value was 0.9992 (FIG. 15).
  • 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.
  • PCR mixtures of the same composition as in Example 2 were prepared, dried, and subjected to real-time reverse transcription polymerase chain reaction using an Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea). Was executed.
  • HBV DNA and internal control DNA prepared in Example 1 were added to the dry PCR mixture 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 matter. 45 cycles of real-time polymerase chain reaction were performed using Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions and components as in Example 2.
  • HBV template DNA was detected as low as 10 copies by counting the copy number according to the method of Example 1 (FIG. 18).
  • the slope was ⁇ 0.3024
  • the R 2 value was 0.9994 ( Figure 19).
  • 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.
  • the Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea) was used to finally determine the dynamic range (concentration range of HBV DNA that can be detected in one reaction). 45 cycles of real time reverse transcription polymerase chain reaction were carried out under the same conditions as in Example 2. HBV template DNA was calculated by copying the number of copies according to the method of Example 1 from 10 to 10 copies at the lowest 10 copy concentration range was used, it was confirmed that the HBV DNA detection is normally performed in the 10 log range (Fig. 20) .
  • PCR in dry state comprising HBV primers and probes as set forth in SEQ ID NOs: 1, 5 and 9 and primers and probes for internal control as set forth in SEQ ID NOs: 13, 16 and 19, using the same composition and method as in Example 4 above
  • the mixture was placed at a constant temperature for 8 days at 40 ° C. for 2 days, and the dry type PCR composition for each storage period was subjected to the same conditions as in Example 2 using Exicycler TM Quantitative Thermal Block (manufactured by Bioneer, Korea). Real time polymerase chain reaction was performed.
  • the dry-type PCR composition was prepared in 8 batches at once in the same batch, and then a part of the mixture immediately after drying was used for 45 cycles of real-time polymerase chain reaction under the same conditions as in Example 2 to obtain a control result. Got it. All other dry type PCR compositions were placed in a 40 ° C. incubator at the same time, and were taken out at intervals of 2 days as needed for the reaction, and each polymerase chain reaction was performed in real time. At this time, the HBV template DNA was counted in the number of copies according to the method of Example 1, and the reaction was carried out using 7 concentrations from the highest 10 7 to the lowest 10 1 copy.
  • N.Temp Normal temperature means normal temperature.
  • HBV gene detection was possible in all of PHD201-02 to PHD201-09 which were positive samples of the A to F genotype (Figs. 26 and 27). This means that detection of various HBV genotypes is possible by using dry PCR mixtures comprising HBV primers and probes set forth in SEQ ID NOs: 1, 5 and 9.

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Abstract

La présente invention concerne une amorce destinée à diagnostiquer le virus de l'hépatite B, une sonde, et un procédé de détection utilisant celles-ci, et plus particulièrement, une amorce spécifique d'un gène S d'un anticorps de surface du virus de l'hépatite B, une sonde, et un procédé de diagnostic du virus de l'hépatite B utilisant l'amorce et la sonde. Lors de l'utilisation de l'amorce et de la sonde de la présente invention, une petite quantité de l'ADN du virus de l'hépatite B présente dans un échantillon biologique peut être détectée de manière rapide et précise, la stabilité au stockage peut être améliorée par le biais de l'utilisation d'un kit sec, et des résultats très reproductibles peuvent être obtenus.
PCT/KR2010/004331 2010-07-02 2010-07-02 Amorce destinée à diagnostiquer le virus de l'hépatite b, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite b utilisant le kit WO2012002597A1 (fr)

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PCT/KR2010/004331 WO2012002597A1 (fr) 2010-07-02 2010-07-02 Amorce destinée à diagnostiquer le virus de l'hépatite b, sonde, kit comprenant celles-ci, et procédé de diagnostic du virus de l'hépatite b utilisant le kit
KR1020127028141A KR101498704B1 (ko) 2010-07-02 2010-07-02 Ｂ형 간염 바이러스 진단용 프라이머, 프로브, 이를 포함하는 키트 및 상기 키트를 이용한 ｂ형 간염 바이러스 진단 방법

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Publication number Priority date Publication date Assignee Title
CN103616510A (zh) * 2013-12-06 2014-03-05 苏州长光华医生物医学工程有限公司 乙型肝炎表面抗体测定试剂盒以及检测方法
CN108330212A (zh) * 2018-03-09 2018-07-27 佛山市优特医疗科技有限公司 一种用于检测乙肝病毒的引物组、组合物及试剂盒
CN113249526A (zh) * 2021-06-30 2021-08-13 瑞博奥(广州)生物科技股份有限公司 用于检测乙型肝炎病毒的pcr反应序列组合及其试剂盒
CN115873993A (zh) * 2021-12-31 2023-03-31 广西壮族自治区疾病预防控制中心 一种检测乙型肝炎病毒9个基因型的试剂盒及其应用
CN115873993B (zh) * 2021-12-31 2023-07-21 广西壮族自治区疾病预防控制中心 一种检测乙型肝炎病毒9个基因型的试剂盒及其应用

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