WO2012002594A1 - Diagnostic primer for the hepatitis 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 hepatitis C virus gene-specific primer, to a probe, and to a detection method using same, and more particularly, to a primer for simultaneously diagnosing the hepatitis C virus gene by means of a polymerase chain reaction, to a probe, to a diagnostic kit including the primer and probe for an internal control group, and to a method for diagnosing the hepatitis C virus using the kit. According to the present invention, real-time detection that is faster and more accurate than existing hepatitis C virus detection methods is made possible, and as a dried polymerase chain reaction composition including the primer and probe has a longer storage time than one in solution form while having equal performance, it can be advantageously used in a kit for detecting the hepatitis C virus.

Description

Primer for hepatitis C virus, a probe, a kit comprising the same, and a method for diagnosing hepatitis C virus using the kit

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. However, since the diagnosis by the antibody can be measured from about 4 to 10 weeks after the hepatitis C virus infection, the initial diagnosis is difficult. 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.

Currently, products that detect hepatitis C virus have to be mixed and dispensed in advance at the initial stage of the reaction, so there is a high possibility of false result determination due to a tester's mistake. In addition, since the product composition is in the form of a solution, there is a disadvantage that it is difficult to use for a long time storage. For the above reasons, since it is difficult to effectively test a large number of people using the above kit, there is a demand for the development of primers and probes that are more effective and can detect as many subtypes as possible.

Accordingly, 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. To provide 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.

In order to achieve the above object, 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. During the polymerase chain reaction, 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.

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. In addition, 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.

When comparing genes that were 100% matched 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 the sequences of the hepatitis C virus subtype reported to date were searched. (See Figure 1).

In addition, 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. During the polymerase chain reaction, internal positive control (hereinafter, also referred to as 'IPC') template and primers prepared according to the same can be easily confirmed whether PCR was performed well. According to an embodiment of the present invention, 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 . In addition, the probe preferably has a nucleotide sequence set forth in SEQ ID NO: 12, all of which are forward probes. According to another embodiment of the present invention, 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. . In addition, 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). Preferably, 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. The primer of the present invention can be used not only for real-time polymerase chain reaction but also for general polymerase chain reaction. In addition, 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.

In addition, the present invention

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

2) provides a method for detecting hepatitis C virus comprising the step of examining the fluorescence value of the presence or absence of amplification or amplification product of step 1).

According to 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. In the present polymerase chain reaction or real time polymerase chain reaction, it is preferable to further use IPC, but is not limited thereto. 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. In addition, the sample may be obtained from a clinical sample or an environmental sample, but is not limited thereto.

Using the primers, probes, and detection methods of the present invention, 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. In addition, 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.

1 is a hepatitis C virus 5 'UTR gene (NCBI accession number; NC_004102) using BLAST of the National Center for Biotechnology Information (NCBI), a part of or a complementary base sequence of the 5' UTR gene sequence 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)

3: Amplification with primers of SEQ ID NOs: 2, 5 and probes of SEQ ID NO: 7 (set 2)

4: Amplification with primers of SEQ ID NOs: 3, 6 and probes of SEQ ID NO: 8 (set 3)

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%.

7 and 8 are graphs showing the results of real-time polymerase chain reaction using an Exicycler ^™ 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.

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. As a result of the experiment of FIG. 9, 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

Blue curve: Amplification curve by internally prepared RNA for internal control

NTC: blank as negative control

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. In FIG. 11, the blue line represents IPC, and the detected cycle value is the same within an error range of ± 1, and 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 ^™ 96 Real-Time Quantitative Thermal block. In FIG. 13, the blue line represents IPC, and the detected cycle value is the same within an error range of ± 1, and 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%.

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 ^™ 96 Real-Time Quantitative Thermal block. Obtained results. In FIG. 15, 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.

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 ^™ Quantitative Thermal Block. The blue line is the IPC signal, and the black line is the hepatitis C virus signal. As a negative sample, the amplification of the black graph, the hepatitis C virus signal, did not appear.

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.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Preparation of Template RNA and RNA for Internal Control

First, template DNA for real-time polymerase chain reaction was prepared. 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). Specifically, 5 μl of 2X Rapid Ligation Buffer (manufactured by Promega, USA), 1 μl T-Easy Vector (manufactured by Promega, USA), 1 μl T4 DNA ligase (manufactured by Promega, USA), 3 μl of gene synthesis material ( 8 ng) was mixed in the same tube, and then placed at a constant temperature at 37 ° C. for 1 hour. Thereafter, 5 µl of the reaction solution placed at the constant temperature was placed in 50 µl of E. coli pluripotent cells (competent cells), and placed on ice for 30 minutes, followed by incubation at 42 ° C. for 90 seconds, and then placed on ice for 2 minutes. The reaction solution was inoculated into LB plates containing ampicillin, IPTG, and X-Gal, followed by incubation at 37 ° C for 16 hours.

Take white colonies, incubate for 16 hours in LB liquid medium, centrifuge, and discard supernatant, Accuprep  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.

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

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

Calculate the copy number of template RNA and use it in 10-degree dilution 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, manufactured by Bioneer, Korea) Stored at −70 ° C. until.

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). In the mouse dishevelled segment polarity protein (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. When the two internal control templates were reacted with the hepatitis C virus template, it was confirmed that both of the internal control amplifications were independently performed without affecting the amplification of the hepatitis C virus RNA template.

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.

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]

Calculate the copy number of template RNA and use it in 10-degree dilutions 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, manufactured by Bioneer, Korea). Stored at −70 ° C. until.

Example 2. Primer and Probe Design

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 ℃ to 60 ℃ optionally base sequence Was chosen to be a forward and reverse primer. In addition, between 50 and 360 base sequences, 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. As a result, the primers and probes shown in Table 1 were designed.

Select a base sequence between 19 and 21 bp in length and 55 to 62 ℃ in Tm between 601 and 1400 of the mouse dishevelled segment polarity protein (Dvl-1) gene for internal control. It was. In addition, between nucleotides 601 and 1400, the base length was 24 bp and the Tm value was selected between 67 and 72 ° C.

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).

Table 1 HCV sequence SEQ ID NO: Forward primer GCT CAA TGC CTG GAG ATT TG One GCG GAA CCG GTG AGT ACA C 2 TGG CGT TAG TAT GAG TGT CG 3 Reverse primer ACC CTA TCA GGC AGT ACC AC 4 TCA GGC AGT ACC ACA AGG C 5 GGC AAT TCC GGT GTA CTC AC 6 TaqMan Probe (Forward) CGT GCC CCC GCR AGA CTG CT 7 CCC CCT CCC GGG AGA GCC ATA GT 8 CGT GCC CCC GCG AGA TCA CT 9

TABLE 2 Base sequence for internal control SEQ ID NO: Forward primer ACG CAC CAG CTC TTC CTC ACC 10 Reverse primer ATG AGT GCC ATT GTC CGC G 11 TaqMan Probe (Forward) TAA CCA GCT CAG TGC CTG GCG CCC 12

TABLE 3 Base sequence for internal control SEQ ID NO: Forward primer AGA TTT CAG TTC AAG GTC GTT C 13 Reverse primer GAA ACC CGC TGA CAT CTT 14 TaqMan Probe (Forward) ACG CGA TGA AAA ACG TCT GGC AAG T 15

Example 3 Standard Template Real-Time Reverse Transcription Polymerase Chain Reaction

First, 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 ^™ 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 ^™ 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 μl of 10X RT Buffer, MMLV 600U, wTfi 10unit, 3 μl 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 μl 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

As a result of the reaction, 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. As a result of dilution of the hepatitis C virus prepared in Example 1 as a template and dilution by concentration, all three sets showed detection limits of equivalent concentrations (10 copies), and the number of detected cycles was 39-. The difference was between 41 Ct. In the polymerase chain reaction efficiency, 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.

Table 5 Set 1 Set 2 Set 3 Detection limit (LOD) 10copy (39.84Ct) 10copy (39.38Ct) 10copy (40.8Ct) PCR efficiency 90% 97% 92% Linearity 0.9994 0.9998 0.9996

Next, the primer and probe concentration combinations were optimized using 5 μl of 10X RT Buffer, MMLV 600U, wTfi 10unit, dNTP 20mM 3 μl, DTT 2.5mM, RNasin 9U, stabilizer, etc. A procedure was performed to demonstrate the condition. As a result, 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). When a standard graph of the standard template real-time reverse transcription polymerase chain reaction was prepared, the slope was -2.80 and the R ² value was 0.9998 (FIG. 6). Here, 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.

In addition, after combining the RNA for the internal control and the primer and the probe for the internal control synthesized in Example 2 in the set shown in Table 6 and proceeded the real-time polymerase chain reaction in the same manner as described above. 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).

Table 6 SEQ ID NO: Set 4 Set 5 Forward primer 10 13 Reverse primer 11 14 Forward probe 12 15

Example 4 Real-Time Reverse Transcription Polymerase Chain Reaction with Standard Template Including IPC

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.

As a result, as a result of calculating the number of copies according to the method of Example 1, 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). Here, 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. In addition, even though 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.

Example 5 Standard Template Real-Time Reverse Transcription Polymerase Chain Reaction Using Dry Type Polymerase Chain Reaction Composition

In order to examine the thermal stability of the 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 ^™ Quantitative Thermal Block (Bionia, Korea). It was. Specifically, 5 μl 10X RT Buffer, MMLV 600U, wTfi 10unit, 3 μl dNTP 20mM, DTT 2.5mM, RNasin 9U, a stabilizer and SEQ ID NO: 2 and SEQ ID NO: 5 selected in Examples 3 and 4 above Put the primer, the probe described in SEQ ID NO: 7 and the internal control primer (Bionia Co., Ltd.) and the probe into one tube, add distilled water to a total volume of 30 μL, and divide into a 96-well plate, and then dispense into a SuperCentra2 ( It was dried for 70 to 80 minutes using Bioneer Corporation, Korea). 5 μl of the hepatitis C virus RNA prepared in Example 1 was added to the dry polymerase chain reaction composition, 1 μl of the internal control RNA was added, and the total volume was divided into 50 μl by DW to dry the well. Mixed thoroughly. 45 cycles of real time reverse transcription polymerase chain reaction were carried out using the Exicycler ^™ Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions as in Example 4.

As a result, 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.

TABLE 7 RNA Copy No. Target 10 ⁷ 19.14 10 ⁶ 22.57 10 ⁵ 26.09 10 ⁴ 29.38 10 ³ 33.39 10 ² 35.89 10 38.51 NTC UD

Exicycler ^™ 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). When a standard graph of the standard template real-time reverse transcription polymerase chain reaction was prepared, the slope was -2.97 and the R ² value was 0.9998 (FIG. 14).

Example 6 Real-Time Reverse Transcription Polymerase Chain Reaction on Positive Specimens

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. Specifically, in 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 µl and thoroughly mixed to loosen the dry composition. In parallel, 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 ^™ Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions as in Example 3.

As a result, 15 positive samples obtained positive results that were 100% consistent with the antigen test results (FIGS. 15 to 18).

Example 7 Real-Time Reverse Transcription Polymerase Chain Reaction for Negative Specimen

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.

Specifically, in 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 µl and thoroughly mixed to loosen the dry composition. In parallel, 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 ^™ Quantitative Thermal Block (manufactured by Bioneer, Korea) under the same conditions as in Example 3.

As a result, a negative result was 100% consistent with the antigen test result in 15 negative samples (Fig. 19).

Example 8 Validation Test with Other Hepatitis Viruses Using Hepatitis C Virus Primer

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. For the hepatitis A sample and the hepatitis B sample which are representative samples of the hepatitis virus, 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.

 As a result, 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).

Claims (19)

A primer consisting of 19-23 nucleotide sequences in the 50-360th base of the hepatitis C virus 5 'UTR gene or complementary nucleotide sequences thereof. The method according to claim 1, A primer selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 6. A probe consisting of 20 to 23 base sequences within the 50 to 360 base of the hepatitis C virus 5 'UTR gene. The method according to claim 3, A probe selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 7 to SEQ ID NO: 9. The method according to claim 3 or 4, Probe with structure of reporter and quencher. The method according to claim 5, The reporter is a FAM probe. The method according to claim 5, The quencher is BHQ1. Hepatitis C virus detection kit comprising a primer for detecting hepatitis C virus of claim 1 and a probe for detecting hepatitis C virus of claim 3. The method according to claim 8, The primer has a nucleotide sequence described in SEQ ID NO: 2 and SEQ ID NO: 5, the probe is a hepatitis C virus detection kit having a nucleotide sequence described in SEQ ID NO: 7. The method according to claim 8, The hepatitis C virus detection kit is a dry hepatitis C virus detection kit. The method according to claim 8, The hepatitis C virus detection kit is a hepatitis C virus detection kit further comprises a gene for the internal control, primers and probes. The method according to claim 11, The internal control genes, primers and probes are hepatitis C virus detection kit is derived from Mouse dishevelled segment polarity protein (Dvl-1) (NCBI Accession No .; NC_005104) gene. The method according to claim 11, The internal control genes, primers and probes are hepatitis C virus detection kit is derived from Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No .; FJ873800). 1) performing a polymerase chain reaction or a real-time polymerase chain reaction using the hepatitis C virus detection primer of claim 1 and the hepatitis C virus detection probe of claim 3 as a template; And 2) Hepatitis C virus detection method comprising the step of examining the fluorescence value for the presence or absence of amplification or amplification product of step 1). The method according to claim 14, The primer is selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 6, The probe is hepatitis C virus detection method selected from the group consisting of nucleotide sequences set forth in SEQ ID NO: 7 to SEQ ID NO: 9. The method according to claim 15, The primer has a nucleotide sequence set forth in SEQ ID NO: 2 and SEQ ID NO: 5, the probe has a base sequence set forth in SEQ ID NO: 7 hepatitis C virus detection method. The method according to claim 14, The step of performing the chain reaction is a hepatitis C virus detection method performed by using a gene, a primer, a probe for the internal control. The method according to claim 14, The internal control gene, primers and probes are hepatitis C virus detection method is derived from Mouse dishevelled segment polarity protein (Dvl-1) gene (NCBI Accession No .; NC_005104). The method according to claim 14, The internal control genes, primers and probes are hepatitis C virus detection method is derived from Tobacco mosaic virus isolate Taigu movement protein (MP) gene (NCBI Accession No .; FJ873800).

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