KR101503039B1 - Diagnostic primer for the heatitis c virus, probe, kit including same, and method for diagnosing the hepatitis c virus using the kit - Google Patents

Abstract

The present invention relates to a primer, a probe, and a detection method using the primer, the probe, and the internal control group for simultaneously diagnosing the gene of the hepatitis C virus by a polymerase chain reaction The present invention relates to a diagnostic kit comprising a primer and a probe for hepatitis C virus, and a diagnostic method for hepatitis C virus using the kit. According to the present invention, it is possible to rapidly and accurately detect in real time as compared with the conventional method for detecting hepatitis C virus, and the dried product of the polymerase chain reaction mixture containing the primer and the probe is improved in storage period Therefore, it can be useful for a kit for detecting hepatitis C virus.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a kit for diagnosing hepatitis C virus, a probe, a kit containing the kit, and a diagnostic method for hepatitis C virus using the kit. BACKGROUND ART }

The present invention relates to a primer, a probe for detecting hepatitis C virus, and a method for diagnosing hepatitis C virus using the same, and more particularly, to a primer, a probe and a probe for detecting hepatitis C virus present in a biological sample and environmental sample The present invention relates to a method for detecting a virus which can be used for the presence or absence of the hepatitis C virus infection and its quantitative diagnosis based on a polymerase chain reaction.

Hepatitis C virus (HCV) is a small RNA virus that belongs to the Flaviviridae family and is about 50 ㎚ in size. The entire genome consists of about 9,600 bases and has untranslated regions (UTRs) on both ends of the genome that play an important role in transcription or translation. The 5 'UTR has a site that initiates the viral polyprotein translation and is also the transcription initiation site to initiate RNA synthesis.

The hepatitis C virus is the only hepatitis virus that is spread evenly throughout the world, without prejudice to any one area. The incidence of hepatitis C is about 1-3% depending on the country and about 1.5% in Korea. In some parts of Africa, the incidence is more than 10%. In some countries, including Japan, hepatitis C virus accounts for 30% to 50% of all chronic and chronic infections. In the United States, hepatitis C virus is the main pathogen of chronic infection, and more than 10,000 people die every year from liver disease associated with hepatitis C virus, and the number of patients continues to increase.

Hepatitis C is a disease that causes inflammation in the liver due to the immune reaction of the body when it is infected with hepatitis C virus. The characteristic of hepatitis C is that 55 ~ 80% of the infected persons become chronic (hepatitis B Less than 5%), more than 20% of chronic infections progress to cirrhosis or liver cancer. Hepatitis C virus spreads on a parenteral route and is infected mainly by transfusions of contaminated blood products, sexual intercourse, vertical infections from infected mothers, contaminated syringes, saliva, razors, and toothbrushes. When hepatitis C virus is infected, the latency period is on average 6 to 7 weeks. Infection is sleeping. In the early stages of infection, there is usually no symptoms, but it can easily become tired, lack appetite, and cause nausea and vomiting. Myalgia and fever may occur, the color of the urine may become darker, severe jaundice may occur in the skin or eyes, and in the fatal case it can lead to death.

Currently, the diagnosis of hepatitis C virus uses antibody and virus detection methods. Antibody tests for hepatitis C virus are performed by enzyme immunoassay. However, the diagnosis by the antibody is difficult since the initial diagnosis can be performed after about 4 to 10 weeks after infection with the hepatitis C virus. The virus detection method can detect very small amount of virus in a small amount of serum by measuring the virus in the serum by real-time PCR, so that it can be used for early diagnosis and early diagnosis.

Hepatitis C virus has six genotypes and more than 50 subtypes. In Korea, chinotypes 1b and 2a are predominant, and 70-80% of patients infected with hepatitis C virus are genotype 1 It is reported to be infected.

In the case of a product that detects hepatitis C virus, it is necessary to mix and dispense various kinds of solutions in the initial stage of the reaction. Further, since the product is in the form of a solution, there is a disadvantage that it is difficult to store the product for a long period of time. For these reasons, it is difficult to effectively inspect a large number of people using the above kit. Therefore, development of primers and probes capable of detecting as many subtypes as possible is more effective.

Therefore, the present inventors have designed novel primers and probes specific for hepatitis C virus, and by performing real-time PCR using the above primers, probes and kits containing them, It is possible to rapidly and accurately detect the hepatitis virus RNA and to dry the polymerase chain reaction mixture necessary for the reaction, and it is confirmed that the storage period can be improved while maintaining the same performance as the mixed solution in the solution state. Respectively.

It is an object of the present invention to provide a primer and a probe for the detection of hepatitis C virus RNA used in real-time RT-PCR.

Another object of the present invention is a kit for detecting hepatitis C virus RNA without cross-reacting with other hepatitis viruses, wherein all the reagents necessary for the RT-PCR reaction are mixed, dispensed, A hepatitis C viral RNA diagnostic kit in which the skill level of an examiner is not required.

Another object of the present invention is to provide a rapid and accurate method for diagnosing hepatitis C virus RNA.

In order to achieve the above object, the present invention provides primers and probes necessary for detecting hepatitis C virus RNA through real-time PCR or general PCR.

The real-time PCR reaction of the present invention monitors the reaction result in real time by using an oligonucleotide probe in which a primer and a fluorescent substance are chemically bonded. In the polymerase chain reaction, the probe binds to the complementary sequence in the nucleic acid of the sample like the two primers, and the binding position is a part slightly separated from the primer. The probe of the present invention has a structure in which a fluorescent substance called a reporter and a quencher are attached to both ends of the probe. If a reporter and a quencher are present in close proximity, fluorescence of the reporter is not detected, , The reporter's fluorescence is detected when the reporter falls off the quencher. Therefore, the intensity of fluorescence gradually increases as the amplification cycle increases.

The inventors of the present invention have found that, in order to detect various subtypes of hepatitis C virus in comparison with conventional real-time PCR products, the inventors of the present invention independently prepared primers based on specific sequences of the 5 'UTR gene (NCBI Accession No. NC_004102) And probes. Self-designed primers and probes were designed to avoid cross-reactivity with other hepatitis viruses.

The primer sequence of the present invention includes a part of the hepatitis C virus or a part of the complementary base sequence thereof, preferably 19 to 23 nucleotides in the 50 to 360 base of the 5 'UTR gene sequence of hepatitis C virus Lt; / RTI > Particularly preferred are reverse primers, which are the nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3, and reverse primers, which are the nucleotide sequences shown in SEQ ID NO: 4 to SEQ ID NO: In addition, the probe sequence of the present invention includes a part of the hepatitis C virus or a part of the complementary base sequence thereof, and is preferably 20 to 50 nucleotides in the 50 to 360 bases of the 5 'UTR gene sequence of hepatitis C virus. It consists of 23 nucleotide sequences. The nucleotide sequence shown in SEQ ID NO: 7 to SEQ ID NO: 9, preferably the nucleotide sequence shown in SEQ ID NO: 7, is preferred and is a forward probe.

As a result of comparing the 100% match genes simultaneously with the primers (SEQ ID NO: 2 and SEQ ID NO: 5) and the probe (SEQ ID NO: 7) of the present invention, all of the hepatitis C virus subtypes reported to date (See Fig. 1).

In addition, the present invention provides a kit for detecting hepatitis C virus comprising the primer or the probe.

In addition to the primer or the probe of the present invention, the kit includes an amplification buffer solution, a dNTP, a control group, a detection reagent, and the like, and is preferably provided in a dry state. If the kit does not affect the reaction, can do. The kit provided in a dry state has improved storage stability and can be used for a long period of time. By omitting the manufacturing process of the mixed solution, it is possible to obtain an accurate quantitative result value with high reproducibility in a short period of time regardless of the proficiency of the experimenter.

The kit may further comprise an internal control primer and a probe. An internal positive control (hereinafter also referred to as 'IPC') template and primers suitable for the internal positive control (hereinafter also referred to as 'IPC') are prepared and put together when the PCR is performed, thereby easily confirming whether or not the PCR has been successfully performed. According to one embodiment of the present invention, the primer for internal control includes a part of the mouse disheveled segment polarity protein (Dvl-1) (NCBI Accession No. NC_005104) gene or a part of its complementary base sequence, Is a forward primer consisting of 5 to 40 nucleotides in the 601st to 1400th bases of the nucleotide sequence, more preferably a nucleotide sequence set forth in SEQ ID NO: 10, and a reverse primer set forth in SEQ ID NO: 11 . In addition, the probe preferably has the nucleotide sequence shown in SEQ ID NO: 12 and is a forward probe. According to another embodiment of the present invention, the primer for internal control comprises a part of the Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI deposit number: FJ873800) or a part of the complementary base sequence thereof, Is a forward primer consisting of 5 to 40 nucleotide sequences in the 37th to 1708th bases of the above base sequence, more preferably a nucleotide sequence set forth in SEQ ID NO: 13, and a reverse primer set forth in SEQ ID NO: 14 . Further, the probe preferably has the nucleotide sequence shown in SEQ ID NO: 15, and is a forward probe.

The primers and probes for the internal control were used as a positive control in the test. When a negative judgment was made when a real-time PCR was performed using the present invention, that is, when the sample was found to have no hepatitis C virus, Of the hepatitis C virus, or the absence of the actual hepatitis C virus, 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 is not a problem.

The primers and probes for detecting hepatitis C virus may be any combination of two primers (one forward direction and one reverse direction) and one probe, preferably a forward primer described in SEQ ID NO: 2, 5 and the forward probe described in SEQ ID NO: 7 can be used (see Table 4). Any combination of primers and probes for the internal control group can be used as long as it has two primers (one forward and one reverse) probes. Preferably, when a part of the mouse disheveled segment polarity protein (Dvl-1) (NCBI Accession No. NC_005104) gene or a part of its complementary base sequence is used as the internal control primer, the forward primer described in SEQ ID NO: 10, A reverse primer represented by SEQ. ID. NO. 11 and a forward probe represented by SEQ. ID. NO. 12 can be used. As a primer for internal control, a part of the Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No. FJ873800) When a part of the complementary base sequence is used, 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 can be used. The primers of the present invention can be used not only in real-time polymerase chain reaction but also in general polymerase chain reaction. In addition, the sample to be used in the present invention can be obtained from clinical samples or environmental samples, but is not limited thereto. Preferably, the reporter of the hepatitis C virus probe is FAM (6-carboxyfluorescein), the quencher is BHQ1 (2,5-di-tert-butylhydroquinone-1), the reporter of the internal control probe is TAMRA (Carboxy- tetramethyl-hod-amine), and BHQ1 is preferably used as the quencher, but the present invention is not limited thereto.

In addition,

1) performing a polymerase chain reaction or a real-time PCR using the sample as a template using the primer for detecting hepatitis C virus and the probe for detecting hepatitis C virus; And

2) detecting the presence or absence of amplification in step 1) or a fluorescence value for an amplification product.

According to the detection method of the present invention, it is possible to detect hepatitis C virus even if a very small amount of nucleic acid of hepatitis C virus is present in the sample because of its high sensitivity. In particular, in real- The detection time can be reduced since it is possible to observe whether amplification is performed and a separate amplification product confirmation step is not necessary. In the present polymerase chain reaction or real-time PCR, it is preferable to use IPC, but the present invention is not limited thereto. When the IPC template and the corresponding primer are prepared and put together, the PCR can be easily confirmed whether or not the PCR is performed well. In addition, the specimen can be obtained from clinical samples or environmental samples, but is not limited thereto.

By using the primer, probe and detection method of the present invention, the hepatitis C virus gene can be detected quickly and easily, and even a very low concentration hepatitis C virus present in the sample can be detected with high sensitivity. In addition, the development of the kit for the diagnosis of hepatitis C virus RNA of the present invention is expected to accurately diagnose the early stage of infection, and it will contribute greatly to the early diagnosis and further spread prevention of hepatitis C virus It is expected.

FIG. 1 shows that a 5 'UTR gene (NCBI Accession No. NC_004102) of the hepatitis C virus was found using BLAST of National Center for Biotechnology Information (NCBI), and a part of the 5' UTR gene sequence or its complementary base sequence Of the present invention and 100% of the nucleotide sequences of the hepatitis C virus registered in the NCBI.
FIGS. 2 to 4 show the results obtained by using an Exicycler ^™ Quantitative Thermal Block (manufactured by Bioneer, Korea) as a combination of all the combinations of the hepatitis C virus primer of the present invention and the partial probes of the present invention as shown in SEQ ID NO: 1 to SEQ ID NO: The results of the real-time PCR were shown in Fig.
2: amplification (set 1) with the primers of SEQ ID NOs: 1 and 4 and the probe of SEQ ID NO:
3: amplification (set 2) with the primers of SEQ ID Nos. 2 and 5 and the probe of SEQ ID NO:
4: amplification (set 3) with the primers of SEQ ID NOS: 3 and 6 and the probe of SEQ ID NO:
FIGS. 5 and 6 are graphs showing the results of testing the optimum concentration of the forward and reverse primers for the set of 2 that has the best PCR efficiency and the best results in the results of FIGS. 2 to 4. FIG. FIG. 6 shows the standard graph of FIG. 5, showing that the closer the R ² value is to 1, the closer the efficiency is to 100%, the higher the PCR efficiency. As a result of the experiment shown in FIG. 5, R ² Was 0.9998 and the efficiency was 91%.
FIGS. 7 and 8 are graphs showing the results of real-time PCR using Exicycler ^™ Quantitative Thermal Block (manufactured by Bioneer, Korea) as a combination of primers and probes of internal control RNA of the present invention. FIG. 7 shows a mouse disheveled segment polarity protein (Dvl-1) gene as an internal control RNA template, and FIG. 8 a graph showing a Tobacco mosaic virus isolate Taigu movement protein (MP) gene as an internal control RNA template.
FIG. 9 and FIG. 10 show the results of the real-time PCR of the HCV standard template using the Exicycler ^™ Quantitative Thermal Block using a primer of SEQ ID NOs: 2 and 5, a probe of SEQ ID NO: 7, an internal control primer and a probe, Lt; RTI ID = 0.0 > PCR < / RTI > FIG. 10 shows a standard graph of FIG. 5, showing that the closer the R ² value is to 1, the closer the efficiency is to 100%, the higher the PCR efficiency. As a result of the experiment shown in FIG. 9, R ² And the efficiency was 98%.
Black curves: Amplification curves of hepatitis C virus template RNA at 10 to 10 ⁷ copies per concentration
Blue curve: Amplification curves by internal self-made control RNA
NTC: Blank as a negative control group
FIGS. 11 and 12 are graphs showing experimental results of a standard template real-time RT-PCR using a dry-type polymerase chain reaction composition and the results obtained using an Exicycler ^™ Quantitative Thermal Block. In FIG. 11, the blue line indicates the IPC, the cycle value detected is the same within the range of ± 1 error, and the black line indicates the value of loading the hepatitis C viral RNA template by concentration. Fig. 12 shows the standard graph of Fig. 11, showing that the closer the R ² value is to 1, the closer the efficiency is to 100%, the higher the PCR efficiency. As a result of the experiment shown in FIG. 11, R ² And the efficiency was 97%.
Figure 13 is a concentration per the dynamic range of the standard HCV PCR template using a mixture of dry illustrates a graph of the real-time polymerase chain reaction Real-time PCR equipment Exicycler ^TM 96 Real-Time Quantitative Thermal block. In FIG. 13, the blue line indicates the IPC, the cycle value detected is the same within the range of ± 1 error, and the black line indicates the value of the hepatitis C viral RNA template loaded by concentration. FIG. 14 shows a standard graph of FIG. 13 showing that the closer the R ² value is to 1, the closer the efficiency is to 100%, the higher the PCR efficiency. As a result of the experiment shown in FIG. 13, R ² Was 0.9998 and the efficiency was 98%.
15 to 18 is a graph showing the results of real-time RT-PCR RNA extracts from 15 HCV positive specimens, and using the PCR mixture was dried, Exicycler ^TM 96 Real-Time Quantitative Thermal block. In Figure 15, the blue line shows the IPC signal and the black line shows the hepatitis C virus positive control. FIG. 16 is a graph showing the results obtained by combining the graphs of FIG. 17 and FIG. 18. In FIGS. 17 and 18, the black line is a signal appearing in the actual hepatitis C virus samples, and the blue line in FIGS. 16 and 18 represents the IPC signal.
FIG. 19 is a graph showing the results of real-time RT-PCR in the case of extracting RNA from 15 HCV negative specimens and using the dried PCR mixture, using the Exicycler ^™ Quantitative Thermal Block. The blue line is the IPC signal and the black line is the negative test for the hepatitis C virus signal. Therefore, it is confirmed that no amplification of the black graph of the hepatitis C virus signal is observed.
FIGS. 20 to 22 are graphs showing results obtained by confirming that cross-reacting with other hepatitis viruses does not occur as a result of experimenting with a standard template real-time RT-PCR using the polymerase chain reaction composition of the present invention of another hepatitis virus sample FIG. 20 shows the results of the hepatitis C virus sample, FIG. 21 shows the hepatitis A virus sample, and FIG. 22 shows the results of the hepatitis B virus sample.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Mold RNA  And internal For control RNA  Produce

First, template DNA was prepared for real-time PCR. The 5 'UTR portion of the hepatitis C virus registered in the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) is used for the gene synthesis method ( Biochem . Biophys . Res .. Commun 1998, and synthesized by a 248, 200-203), some of the pGEM-T-Easy Vector (Cat : was cloned into the A1360, Promega Co., USA). Specifically, 5 μl of 2 × rapid ligation buffer (Promega, USA), 1 μl of T-Easy Vector (Promega, USA), 1 μl of T4 DNA ligation (Promega, USA) 8 ng) were placed in the same tube and mixed, followed by incubation at 37 ° C for 1 hour. Thereafter, 5 μl of the reaction solution in which the above temperature was placed was added to 50 μl of E. coli competent cells, and the mixture was allowed to stand on ice for 30 minutes, followed by incubation at 42 ° C. for 90 seconds, and then placed on ice again for 2 minutes. The reaction solution was inoculated on an LB plate containing ampicillin, IPTG, and X-Gal and cultured at 37 DEG C for 16 hours.

Colonies of white color were taken and cultured in LB liquid medium for about 16 hours. The supernatant was discarded by centrifugation and the plasmid DNA was extracted from the pellet using Accuprep ^Ð plasmid DNA prep kit (Bioneer, Korea). Using the plasmid DNA is UV spectrophotometer (manufactured by Shimazu, Japan) at a concentration and then measuring the purity confirmed to be between purity of 1.8 to 2.0, and MAXIscipt ^Ð In vitro transcription Kit (Ambion Co., USA) and plasmid DNA into RNA Was transferred. The concentration and purity were measured with a UV spectrophotometer after the transcription and when the purity was between 1.8 and 2.0, they were used as template RNA in the subsequent real-time PCR. The RNA copy number was calculated by the following formula.

6.02 × 10 ²³ × concentration (concentration g / ml measured by UV spectrometer) / (3,015 + 400) × 340

[3,015 bp: size of T-easy vector, 400 bp: size of hepatitis C virus template RNA]

The copy number of the template RNA was calculated and then 10-diluted with 1X TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.6% BSA) containing 0.6% BSA (Bovine Serum Albumin, Lt; RTI ID = 0.0 > -70 C. < / RTI >

An internal control RNA was prepared in the same manner as the template RNA preparation. Internal control RNA is necessary to ensure that when negative results are obtained, the negative results are not due to amplification errors.

Two types of internal control RNAs were used: mouse disheveled 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). In the mouse disheveled segment polarity protein (Dvl-1) gene, the 767 bp region from the 601th to 1400th sequence including the primer and the probe sequence was used for the internal control RNA preparation and the Tobacco mosaic virus isolate Taigu movement protein (MP ) Genes were synthesized from 37th to 1708th, including primers and probe sequences, and used for internal control RNA preparation. When two internal control templates were reacted with the hepatitis C virus template, both of them showed independent amplification of the internal control without affecting the amplification of the hepatitis C virus RNA template.

Using the plasmid DNA is UV spectrophotometer (manufactured by Shimazu, Japan) at a concentration and then measuring the purity confirmed to be between purity of 1.8 to 2.0, and MAXIscipt ^Ð In vitro transcription Kit (Ambion Co., USA) and plasmid DNA into RNA Was transferred. Concentration and purity were measured with a UV spectrometer after transcription and when the purity was between 1.8 and 2.0, they were used as template RNAs for internal control in later real-time PCR. The RNA copy number was calculated by the following formula.

6.02 × 10 ²³ × concentration (concentration g / ml measured by UV spectrometer) / (3,015 + 767) × 340

[3,015 bp: size of T-easy vector, 767 bp: size of template RNA for internal control]

The copy number of the template RNA was calculated and then 10-diluted with 1X TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.6% BSA) containing 0.6% BSA (Bovine Serum Albumin, Lt; RTI ID = 0.0 > -70 C. < / RTI >

Example  2. primer  And Probe  design

The nucleotide sequence of the hepatitis C virus 5 'UTR gene (NCBI Accession No. NC_004102) is selected from the nucleotide sequence 50 to 360, the length is 19 to 23 bp, the Tm value is 55 to 60 ° C, Were selected as forward and reverse primers. In addition, a base sequence was arbitrarily selected between 50 and 360 nucleotides, between 20 and 23 bp in length, and between 67 and 72 ° C, and the Tm value was checked using the Primer3 Plus program. As a result, the primers and probes shown in Table 1 were designed.

The nucleotide sequence of the mouse disheveled segment polarity protein (Dvl-1) gene for internal control was arbitrarily selected from 601 to 1400 in such a manner that the length was 19 to 21 bp and the Tm value was 55 ° C to 62 ° C. A forward and reverse primer Respectively. Between 601 and 1400 nucleotides, the length was 24 bp and the Tm value was between 67 and 72 ° C.

Tobacco mosaic virus isolate for internal control The nucleotide sequence of the Taigu movement protein (MP) gene (NCBI Accession No. FJ873800) was arbitrarily set to be between 18 and 22 bp in the nucleotide sequence of 37 to 799 and a Tm value of 55 to 62 ° C. Were selected as forward and reverse primers. Between 942 and 1708, the length was 25 bp, and the Tm value was arbitrarily selected between 67 and 72 ° C to select probes (Table 3).

Example  3. Standard mold  real time Reverse transcription Polymerase chain reaction

First, the hepatitis C virus primer and all the probes were combined in a set, and real-time RT-PCR was performed using Exicycler ^™ Quantitative Thermal Block (Bionics Co., Ltd., Korea).

Using the RNA of the hepatitis C virus prepared in Example 1 as a template, the Hepatitis C virus primers and probes designed in Example 2 were combined in various sets, and then quantified using Exicycler ^™ Quantitative Thermal Block ) Were used to perform real-time PCR (Table 4). Specifically, 5 μl of 10 × RT buffer, 3 μl of MMLV 600 U, 10 μl of wTfi, 20 μl of dNTP, 2.5 mM of DTT, 9 U of RNasin, and a stabilizer were placed in a tube and the hepatitis C virus template RNA, C type A primer, a probe for detection of hepatitis virus, and distilled water were added, and the mixture was mixed to a total volume of 50 μl, and the mixture was dispensed into a 96-well plate. At this time, the concentration of the forward primer and the reverse primer contained in the total volume was 20 pmole, respectively, and the concentration of the probe was 20 pmole. The cDNA was synthesized by reacting at 45 ° C for 15 minutes, denatured at 95 ° C for 5 minutes, and reacted at 95 ° C for 5 seconds and at 55 ° C for 5 seconds for 45 cycles. The amplified fluorescence values were continuously measured once at 55 ° C for 30 seconds after each PCR cycle.

As a result, it was found that the primer and the probe having the highest PCR amplification efficiency were the set 2, 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 show the experiment graphs of the three sets. As a result of detecting the detection limit by diluting the hepatitis C virus as prepared in Example 1 as a template, the detection limit of the equivalent concentration (10 copies) was observed in all three sets, and the number of cycles detected was 39- Respectively. The efficiency of PCR was 97% for Set 2 and the PCR efficiency was the best. When the virus template is diluted by concentration and the standard graph is drawn using the detected cycle value, the closer the numerical value representing the straight line of the graph is to 1, the better. When all three sets were compared, all values were equal to or greater than 0.999.

Next, the primer and probe concentration combinations optimized by using 5 μl of 10X RT Buffer, 10 μl of MMLV 600 U, 10 μl of wTfi, 3 μl of dNTP, 2.5 mM of DTT, 9 U of RNasin, The process to verify the condition was performed. As a result, the copy number limit of the detectable virus was good at 10 copies when 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, and real-time reverse transcription polymerase chain reaction Was 98% (FIG. 5). When a standard graph of the standard template real-time RT-PCR was drawn, the slope was -2.80, the R ² The value was 0.9998 (Fig. 6). Here, R ² is a correlation coefficient indicating the linearity of the graph when the standard graph of the real-time PCR is drawn. The closer to 1 (closer to the straight line) the correlation coefficient of the graph indicates that the polymerase chain reaction has proceeded properly.

The internal control RNA synthesized in Example 2, internal primers for internal control, and probes were combined with the sets shown in Table 6, and real-time PCR was performed in the same manner as described above. The cDNA was synthesized by reacting at 45 ° C for 15 minutes, denatured at 95 ° C for 5 minutes, and reacted at 95 ° C for 5 seconds and at 55 ° C for 5 seconds for 45 cycles. Amplified fluorescence values were continuously measured once at 55 < 0 > C for 30 seconds after each PCR cycle (Figs. 7 and 8).

Example  4. IPC Including Standard mold  real time Reverse transcription Polymerase chain reaction

Using the hepatitis C viral RNA prepared in Example 1 and the internal control RNA as templates, the hepatitis C primer of SEQ ID NOs: 2 and 5 selected in Example 2, the probe of SEQ ID NO: 7, the primer for internal control, The probe was applied and real-time PCR was performed using Exicycler ^™ Quantitative Thermal Block (Bioneer, Korea). 45 cycles of real-time PCR were carried out under the same conditions and components as in Example 3. [

As a result, the number of copies was calculated according to the method of Example 1. As a result, it was possible to detect up to 10 copies of hepatitis C virus template RNA (FIG. 9), and when a standard graph of a standard template real- , The slope was -2.96, and the R ² The value was 0.9994 (Fig. 10). Here, R ² is a correlation coefficient indicating the linearity of the graph when the standard graph of the real-time PCR is drawn. It means that the closer to 1 (closer to the straight line) the PCR proceeds. In addition, it was confirmed that internal control amplification was independently performed without affecting the amplification of the hepatitis C virus template even when 10 ⁵ copies of the internal control RNA were reacted together.

Example  5. Dry type Polymerase chain reaction  Using the composition Standard mold  real time Reverse transcription Polymerase chain reaction

PCR Mixture (Premix) For the purpose of examining the thermal stability of the dried product, a PCR mixture having the same composition as in Example 3 was prepared and dried, and then a real-time PCR was performed using Exicycler ^™ Quantitative Thermal Block Respectively. Specifically, 5 占 퐇 of 10 占 RT Buffer, 10 占 퐇 of MMLV 600U, 10 占 퐇 of wTfi, 20 占 퐇 of dNTP, 2.5 占 퐉 of DTT, 9U of RNasin, a stabilizer, and SEQ ID NO: 2 and SEQ ID NO: 5 selected in Examples 3 and 4 Primer, a probe described in SEQ ID NO: 7, and an internal control primer (Bioneer Co., Ltd.) and a probe were placed in a tube and mixed with distilled water so as to give a total volume of 30 μl. The mixture was dispensed into a 96- Bioneer, Korea) for 70 to 80 minutes. 5 μl of the hepatitis C virus RNA prepared in Example 1 was added to the dried polymerase chain reaction composition as a template, 1 μl of internal control RNA was added, and the mixture was divided into 50 μl of total volume by DW. . 45 cycles of real-time RT-PCR were performed under the same conditions as in Example 4 using Exicycler ^™ Quantitative Thermal Block (Bionics Co., Ltd., Korea).

As a result, when a standard graph of a standard template real-time RT-PCR was prepared, the slope was -2.94, and the R (SEQ ID NO: ² The value was 0.9996 (FIG. 12). Table 7 shows the values of the graph of FIG. 11 as numerical values. When the amount of HCV RNA as a positive control group is 10 ⁷ , signals can be seen even when only 19.14 cycles of Realtime PCR is performed. NTC is a negative control group . From the above results, it was found that the same performance was maintained in the two methods of real-time RT-PCR using the mixture of the solution-type polymerase chain reaction and the dry mixture.

To confirm the dynamic range (range of concentration of hepatitis C virus RNA that can be detected in one reaction) using the dried PCR mixture in the same manner as described above, an Exicycler ^TM Real-Time Quantitative Thermal Block , Korea) was used to carry out 45 cycles of real-time PCR under the same conditions as in Example 3. [ Using the method of Example 1, the hepatitis C virus template RNA was diluted 10 times at a concentration of 10 ¹⁰ copies at a maximum of 10 copies, and the hepatitis C virus RNA was detected normally in 10 log range (Fig. 13). When a standard graph of the standard template real-time RT-PCR was drawn, the slope was -2.97, the R ² The value was 0.9998 (Fig. 14).

Example  6. Real time for positive samples Reverse transcription Polymerase chain reaction

A detection test was performed on the hepatitis C virus sample using the dry type PCR mixture prepared in Example 3 above. The test sample was determined to be positive by the antibody test method using the patient serum. Using the primer / probe set for detecting hepatitis C virus of the present invention, 15 samples of the hepatitis C positive sample were analyzed in real time A reverse transcription polymerase chain reaction was performed. Specifically, a dry type PCR composition comprising the hepatitis C virus hepatitis C primer described in SEQ ID NOs: 2 and 5 and the probe described in SEQ ID NO: 7 and the primer and probe for the internal control prepared in Example 2, The hepatitis C viral RNA and the internal control RNA were added as a template and mixed with distilled water so that the total volume was 50 쨉 l to thoroughly mix the dried composition. In parallel with this, except for the template RNA, the RNA extracted from each sample was substituted for the same components as above for the sample test. 45 cycles of real-time PCR were performed under the same conditions as in Example 3 using Exicycler ^™ Quantitative Thermal Block (manufactured by BIONIA Co., Ltd., Korea).

As a result, 100 positive results were obtained in 15 positive samples (Fig. 15 to Fig. 18).

Example  7. On the negative specimen  Real time Reverse transcription Polymerase chain reaction

A detection test was performed on the hepatitis C virus sample using the dry type PCR mixture prepared in Example 3 above. The sample was a sample which was judged to be negative by the antibody diagnostic method using the patient's serum. Using the primer / probe set for detecting hepatitis C virus of the present invention, 15 samples of the hepatitis C negative sample were analyzed in real time A reverse transcription polymerase chain reaction was performed.

Specifically, a dry type PCR composition comprising the hepatitis C virus hepatitis C primer described in SEQ ID NOs: 2 and 5 and the probe described in SEQ ID NO: 7 and the primer and probe for the internal control prepared in Example 2, The hepatitis C viral RNA and the internal control RNA were added as a template and mixed with distilled water so that the total volume was 50 쨉 l to thoroughly mix the dried composition. In parallel with this, except for the template RNA, the RNA extracted from each sample was substituted for the same components as above for the sample test. 45 cycles of real-time PCR were performed under the same conditions as in Example 3 using Exicycler ^™ Quantitative Thermal Block (manufactured by BIONIA Co., Ltd., Korea).

As a result, a result of 100% identical voice to the result of antigen test was obtained in 15 negative samples (FIG. 19).

Example  8. Hepatitis C virus Primer  Verification tests with other hepatitis viruses used

The Green Cross Medical Foundation received RNA extracted from a patient's sample identified through clinical symptoms and gene sequence analysis and used in the experiment. Verification experiments were conducted on the cross-reactivity with the hepatitis virus by the method of Example 5 using the primer for detecting hepatitis C virus of the present invention for hepatitis A virus samples and hepatitis B samples which are representative samples of hepatitis viruses.

As a result, in the primer / probe set for detecting hepatitis C virus of the present invention, it was confirmed that a real-time RT-PCR reaction occurred for hepatitis C virus, but no reaction occurred for other hepatitis virus samples 22).

Claims (19)

A composition for detecting hepatitis C virus comprising a forward primer of SEQ ID NO: 2, a reverse primer of SEQ ID NO: 5, and a probe of SEQ ID NO: 7. The method according to claim 1,
A composition for detecting hepatitis C virus, which further comprises at least one set selected from the group consisting of the following set 1 and set 3:
Set 1: the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 4 and the probe of SEQ ID NO: 7; And
Set 3: the forward primer of SEQ ID NO: 3, the reverse primer of SEQ ID NO: 6, and the probe of SEQ ID NO: 8. delete delete The method according to claim 1,
Wherein the probe is a structure in which a reporter and a quencher are attached. The method of claim 5,
Wherein said reporter is FAM. The method of claim 5,
Wherein the quencher is BHQ1. A kit for detecting hepatitis C virus comprising a composition for detecting hepatitis C virus comprising a forward primer of SEQ ID NO: 2, a reverse primer of SEQ ID NO: 5, and a probe of SEQ ID NO: 7. The method of claim 8,
Kit for detecting hepatitis C virus further comprising at least one set selected from the group consisting of the following set 1 and set 3:
Set 1: the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 4 and the probe of SEQ ID NO: 7; And
Set 3: the forward primer of SEQ ID NO: 3, the reverse primer of SEQ ID NO: 6, and the probe of SEQ ID NO: 8. The method of claim 8,
The kit for detecting hepatitis C virus is a dry type kit for detecting hepatitis C virus. The method of claim 8,
The kit for detecting hepatitis C virus further comprises a gene for internal control, a primer and a probe. The method of claim 11,
Wherein the internal control gene, primer and probe are derived from a mouse disheveled segment polarity protein (Dvl-1) (NCBI Accession No. NC_005104) gene. The method of claim 11,
Wherein the internal control gene, primer and probe are derived from Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No. FJ873800). 1) A polymerase chain reaction or a real-time PCR was carried out using a composition for detecting hepatitis C virus comprising the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 5 and the probe of SEQ ID NO: ; And
2) detecting the presence or absence of amplification in step 1) or a fluorescence value for an amplification product. 15. The method of claim 14,
Wherein the composition for detecting hepatitis C virus further comprises at least one set selected from the group consisting of the following set 1 and set 3:
Set 1: the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 4 and the probe of SEQ ID NO: 7; And
Set 3: the forward primer of SEQ ID NO: 3, the reverse primer of SEQ ID NO: 6, and the probe of SEQ ID NO: 8. delete 15. The method of claim 14,
Wherein the step of performing the chain reaction is performed by further using an internal control gene, a primer, and a probe. 18. The method of claim 17,
Wherein the internal control gene, primer and probe are derived from mouse disheveled segment polarity protein (Dvl-1) gene (NCBI Accession No. NC_005104). 18. The method of claim 17,
Wherein the internal control gene, primer and probe are derived from Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No. FJ873800).

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