KR20020063123A - Kit for detecting hcv by rt-pcr and detection method thereof - Google Patents

Abstract

PURPOSE: Provided are a kit for detecting the nucleic acid of HCV with specificity and sensitivity and a detection method of HCV by using the kit. CONSTITUTION: The detection kit comprises: a RNA extract solution containing DEPC treated distilled water, Taq buffer, and DMSO; a primary PCR master mixed solution including DEPC treated distilled water, buffer, dNTP, a primer set having nucleotide sequences of SEQ ID NO:1 and 2, DTT, Taq/RNasin and reverse transcriptase; and a secondary PCR master mixed solution containing distilled water, buffer, dNTP, a primer set having nucleotide sequences of SEQ ID NO:3 and 4, Taq/UDG and cresol red. The detection method comprises the steps of: treating a sample with a RNA extract reagent and a primary PCR master mixed solution then performing PCR; treating the PCR products with a secondary PCR master mixed solution then performing PCR; and analyzing the presence of HCV on the gel.

Description

Kit for detecting hepatitis C virus by Altipial and its detection method

The present invention relates to a kit for detecting a nucleic acid in a sample using a nucleic acid amplification method, and more particularly, to a kit for detecting hepatitis C virus using the RT-PCR method and a detection method thereof.

In general, as a method of diagnosing various viral diseases, there is a method of diagnosing by using an antibody against a viral peptide antigen, but such an ELISA method is difficult to detect the viral protein present in the blood in small amounts, the early stage of the disease Not only is it difficult to diagnose viral infections at the stage, but the concentration of viral particles in the blood can change due to the course of the disease or the effects of treatment. In this case, false negative results obtained by ELISA can adversely affect the course of the disease.

The main focus of viral nucleic acid detection was focused primarily on qualitative analysis, such as the presence of specific gene mutations or pathogens in patient samples. This technique has been generalized in accordance with the development of amplification techniques such as PCR and NASBA, and an analysis system for infectious pathogens such as chlamydia and HIV has been developed.

Advances in PCR techniques have contributed significantly to lowering the detection limit necessary for the detection of nucleic acids, but are still a big problem when nucleic acid concentrations are particularly low, and the efficiency of PCR can vary from reactor to reactor, resulting in differences in these results. In some cases, the reproducibility of the quantitative data obtained by known methods is still insufficient.

To improve the reproducibility of PCR, Gililand uses a competitive PCR method that measures the amount of a sample by comparing the copy number of the sample with that of the standard dilution series, using a series of diluted internal standards that are amplified simultaneously with the sample. (PNAS 87: 2725 (1990)) and according to WO 93/23573 viral RNA concentrations can be determined in a range of 10 ² to 10 ⁹ copies by competitive PCR methods, and in WO 94/20460 in competitive RT-PCR. It is also described that 100 copies of HIV gene can be measured in plasma of HIV infected patients.

For improved detection of small amounts of PCR products, phosphor-labeled primers and automated laser fluorescence DNA analyzers have been developed by Forser et al. (BioTechniques 13 (1992), 106).

Hepatitis C virus is a sigle strand RNA virus with about 17,000 nucleic acids, which accounts for 70-80% of infections after transfusion, and 70% of chronic liver diseases with HBs Ag negative are detected.

There are two types of hepatitis C test methods: screening for antibodies using immunoassay (ELISA) and screening for hepatitis C virus itself using the PIAL method.

In the case of hepatitis B, antibody positive is usually an immune antibody that has been recovered from infection, whereas anti-HCV positive is a neutralizing antibody produced by the proliferation of HCV in a patient's body. No, anti-HCV positive means that you are more likely to be infected with HCV, but that does not mean hepatitis C.

For accurate diagnosis of hepatitis C, HCV PCR should be performed if the antibody is positive for EIA. Reportedly, 50% of NANBH patients with anti-HCV antibody negative showed HCV PCR positivity. It is estimated that many patients with hepatitis C are anti-HCV antibody negative. Therefore, if hepatitis is suspected in the high-risk HCV group, HCV PCR should be performed to confirm the viremia even if it is negative for EIA.

Since the hepatitis C virus itself is present in the blood in too small numbers, it cannot be detected by the commonly used enzyme immunoassay, and there may be hepatitis C patients among negative C100 antibodies. . The detection of HCV RNA by PSI is a direct indicator of hepatitis C. It is highly sensitive and specific and can be used to measure hepatitis C virus antigen in the early stage of infection. In addition, the hepatitis C antigen can be detected in patients with chronic hepatitis C, which is negative for C100 antibodies, and therefore the PI method is a useful indicator for the diagnosis and treatment of hepatitis C patients. However, the PCR method for testing the C virus itself is difficult to use because it is difficult to test.

Since hepatitis C virus is an RNA virus, the complementary DNA is synthesized using reverse transcriptase, and then HCV-RNA is measured using a fish response. Most clinical laboratories in research institutes and hospitals refer to thesis for HCV PSI and make experiments by modifying them according to their own methods to make temperature conditions, buffers, etc. Due to frequent contamination, it is difficult to make accurate measurements.

In the present invention, the primer is selected from the well-preserved nucleotide sequence in the hepatitis C virus, and the RNAV reagent, the buffer for performing PCR, the DNA polymerase, etc. are easily put into the HCV- It is kitted to detect RNA.

SUMMARY OF THE INVENTION The present invention solves the above problems and has been devised by the necessity of the above, and an object of the present invention is to provide a kit for detecting viral nucleic acids with simple, rapid and improved specificity and sensitivity.

Another object of the present invention to provide a method for detecting a viral nucleic acid using the kit.

1 is a photograph confirming the HCV secondary PCR product size 145 bp.

Figure 2 is a photograph showing the specificity of the kit of the present invention. In FIG. 2, lane 1 is a Phage 174 DNA / HaeIII molecular weight marker, lane 2 is a negative control, lane 3 is Hepatitis C virus ATCC 20827, lane 4 is Hepatitis B ATCC 31518, lane 5 is Hepatitis A ATCC VR-1357, lane 6 Chlamydia trachomatis ATCC VR-346, lane 7 is human papilloma virus type 18 ATCC 45152, lane 8 is Staphylococcus aureus ATCC 4012, and lane 9 is Staphylococcus epidermidis ATCC 9491.

Figure 3 is a photograph showing the sensitivity of the inventor kit. In Figure 3, lane 1 is Phage 174 DNA / HaeIII molecular weight marker, lane 2 is 1 ng, lane 3 is 100 pg, lane 4 is 10 pg, lane 5 is 1 pg, lane 6 is 100 fg, lane 7 is 10 fg, Lane 8 shows 1 fg, lane 9 shows 0.1 fg of RNA.

Figure 4 is a photograph of the results of the test by a third party HCV RT-PCR Kit. In addition to PCR bands specific to HCV, non-specific multibands appear in the picture. Lane 1 shows a Phage 174 DNA / HaeIII molecular weight marker, lanes 2 and 3 are HCV positive samples, lanes 4 and 5 are HCV negative samples, and lane 6 is a positive control.

In order to achieve the above object, the present invention provides a hepatitis C virus detection kit comprising an RNA extraction solution, a primary fish master mixture and a secondary fish master mixture. In the present invention, the RNA extract solution comprises DEPC treated distilled water, Taq buffer, DMSO, the primary fish master mixture contains DEPC treated distilled water, buffer, dNTP, primers, DTT, Taq / RNasin and reverse transcriptase, secondary fish Egg master mixtures include distilled water, buffers, dNTPs, primers and Taq / UDG, cresol red. In addition, the primer set of the primary mixed solution of the present invention has the nucleotide sequences shown in SEQ ID NOs: 1 and 2, the primer set of the secondary mixed solution has the nucleotide sequences shown in SEQ ID NOs: 3 and 4, the reverse transcriptase is AMV reverse transcriptase desirable.

In another aspect, the present invention is the step of performing a first fish after the RNA extraction reagent and the first fish master mixture to the sample, the second fish by treating the secondary fish master mixture to the first fish product and performing the second fish It provides a method for detecting hepatitis C virus comprising the step of confirming the presence of hepatitis C virus through a gel.

HCV fish kit of the present invention is a diagnostic kit that makes it easy to detect hepatitis C antigens in a general laboratory, the components include RNA extraction reagent for extracting hepatitis C virus from serum, and hepatitis C virus 5 '' In the non-coding region, the nucleotide sequence is well preserved, such as primers for primary and secondary fish reactions, and polymer buffers including various polymers necessary for conducting fish reactions. It is.

Commonly used HCV RT-PCR fish methods are as follows. First, reverse transcriptase is carried out using reverse transcriptase and cDNA primer to make RNA into DNA. Secondly, the first PCA reaction is performed using primers specific for hepatitis C using DNA synthesized by reverse transcription. Third, hepatitis C virus is detected by performing second fish again using a part of the product produced in the first fish reaction. On the other hand, the kit can reduce the time by reducing the reaction by one step compared to the method generally used by performing the RT reaction and the primary fish in the same tube at the same time with RNA fish. It has the advantage of saving labor.

The selective region of the primer used in the present invention is the 5'-noncoding region of hepatitis C virus, the HCV specific primer nucleotide sequence is shown in Table 1.

Table 1 HCV specific primer nucleotide sequence

primer Base Arrangement (HCV 5'-NCR) Primary Forward CTGTG AGGAA CTACT GTCTT Reverse AACAC TACTC GGCTA GCAGT Secondary Forward TTCAC GCAGA AAGCG TCTAG Reverse GTTGA TCCAA GAAAG GACCC

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

Example: Preparation of Kit Components

1) Preparation of Primary Fish Master Mixture

19 μl of RT-PCR solution and 0.5 μl of PCR Enzyme solution-1 and 2 were mixed to prepare as much primary PCR master mixture as necessary. The primary PCR master mixture uses 20 μl per sample. The method was prepared using 0.1% DEPC distilled water 11.5 ul, 10-fold buffer 3 μl (15 mM MgCl _2, 200 mM (NH ₄ ) ₂ SO _4, 725 mM Tris-HCl, pH 9.0, 0.1% Tween 20), 2 mM dNTP 1.5 μl 1.5 μl 10 pmol primer, 1.5 μl 0.1M DTT, Taq / RNasin (0.1 μl 5 U Taq polymerase, 0.2 μl 30U RNasin, 0.2 μl Taq polymerase dilution buffer), 0.5 μl AMV reverse transcriptase from Avian Myeloblastosis virus ( 0.3 μl 10 U AMV RT, 0.2 μl AMV dilution buffer). AMV RT dilution buffer was made by mixing 200 mM KH ₂ PO ₄ (pH 7.2), 0.2% Triton X-100, 2 mM DTT, 50% glycerol.

Taq polymerase dilution buffer was made by mixing 20 mM tris HCl (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP40, 50% glycerol.

2) Preparation of Secondary Fish Master Mixture

18 μl of the secondary PCR solution and 0.5 μl of the secondary PCR enzyme solution were mixed to prepare as many secondary PCR master mixtures as necessary (see Table 3). 18.5 μl per sample was used as the PCR master mixture. 13.8 μl of distilled water, 2.0 μl of 10-fold buffer, (15 mM MgCl _2, 200 mM (NH ₄ ) ₂ SO _4, 725 mM Tris-HCl, pH 9.0, 0.1% Tween 20), 2 μM dNTP 1.0 μl, 10 1.0 μl of pmol primer, Taq / UDG (0.1 μl of 5U Taq polymerase, 0.1 μl of 1U UDG, 0.3 μl of Taq polymerase dilution buffer), and 0.2 μl of 10 mg / ml cresol red were prepared.

3) Preparation of RNA Extraction Solution

875 μl of DEPC treated distilled water, 100 μl of Taq 10 × buffer and 25 μl of DMSO were mixed.

4) Preparation of Enzyme Liquid-1

It was made by mixing 0.1 μl of 5U Taq, 0.2 μl of 30U RNasin, and 0.2 μl of Taq dilution buffer. Taq polymerase dilution buffer is prepared by mixing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP40, 50% glycerol.

5) Preparation of Enzyme Liquid-2

It was made by mixing 0.1 μl of 5U Taq, 0.1 μl of 1U UDG, and 0.3 μl of Taq dilution buffer.

6) As other positive control solution, nucleic acid positive for HCV PCR was used, and commercial oil (Sigma) was used.

Experimental Example 1 Detection of HCV Through the Kit

1) Extraction of HCV RNA

5 μl of HCV RNA extraction buffer is placed in a 0.5 ml tube, 10 μl of serum is mixed by pipetting and mixed well with HCV RNA extraction buffer, and the temperature of the PCR machine is adjusted to 92 ° C. Heated for a minute and then directly put on the prepared ice and centrifuged for 5-10 seconds.

2) Preparation and Conditions of HCV RT-Primary PCR Master Mixture

Table 2 HCV RT-Primary PCR Master Mix Solution (unit: μl) solution Sample number 1 Sample number 3 Sample Number 5 RT-Primary PCR Master Mixture 19.0 57 95 RT-Primary PCR Enzyme 1 0.5 1.5 2.5 RT-Primary PCR Enzyme 2 0.5 1.5 2.5 all 20 μl 60 μl 100 μl

After mixing 19.0 μl of HCV RT-primary PCR mixture and 0.5 μl of RT-primary PCR enzyme solution-1 and 2 to prepare as much RT-primary PCR master mixture as necessary (see Table 2), the RT-primary PCR master was prepared. 20 μl of the mixed solution is dispensed into each PCR tube, and the coagulated serum (with HCV RNA extracted) is crushed and mixed well, followed by dropping a drop of mineral oil on the upper layer, followed by reaction in a PCR machine (Table 3). Reference).

TABLE 3 HCV RT-Primary PCR Conditions Temperature / hour cycle 57 ° C / 3.0min 42 ° C / 30.0 min 95 ° C / 5.0 min 1 cycle 94 ℃ / 30 sec 51 ℃ / 30 sec 72 ℃ / 30 sec 35 cycles 72 ℃ / 5.0 minutes 1 cycle

3) Preparation and Conditions of Secondary PCR Master Mixture

After mixing 18 µl of the secondary PCR solution and 0.5 µl of the HCV secondary PCR enzyme solution to prepare as many secondary PCR master mixtures as necessary (see Table 4), dispense 18.5 µl into each PCR tube and pipetting 1.5 µl of the HCV primary PCR product. After mixing well, drop a drop of mineral oil on the upper layer, and reacted in a PCR machine (see Table 5).

Table 4 HCV secondary PCR master mixture (unit: μl) solution Sample number 1 Sample number 3 Sample Number 5 HCV Secondary PCR Mixture 18.0 54.0 90.0 HCV Secondary PCR Enzyme 0.5 1.5 2.5 all 18.5 μl 55.5 μl 92.5 μl

[Table 5] HCV secondary PCR conditions Temperature / hour cycle 95 ℃ / 5.0 min 1 cycle 94 ℃ / 30 seconds 53 ℃ / 30 seconds 72 ℃ / 30 seconds 30 cycles 72 ℃ / 5.0 minutes 1 cycle

4) Confirmation of PCR result

After preparing a 2% agarose gel, the PCR product was electrophoresed at 100-150 volt, and HCV secondary PCR product size 145 bp was confirmed by UV transilluminator (see Fig. 1).

Experimental Example 2: Data on the Effect of HCV PCR Kit of the Present Invention

(1) specificity

In order to see the specificity of the HCV PCR kit of the present invention, PCR was performed with HBV, HAV, and other kinds of viruses.

Experimental method was tested according to Experimental Example 1, the experimental results are shown in FIG. In FIG. 2, lane 1 is a Phage 174 DNA / HaeIII molecular weight marker, lane 2 is a negative control, lane 3 is Hepatitis C virus ATCC 20827, lane 4 is Hepatitis B ATCC 31518, lane 5 is Hepatitis A ATCC VR-1357, lane 6 Chlamydia trachomatis ATCC VR-346, lane 7 is human papilloma virus type 18 ATCC 45152, lane 8 is Staphylococcus aureus ATCC 4012, and lane 9 is Staphylococcus epidermidis ATCC 9491. As can be seen in Figure 2, it showed PCR positive only in HCV and not positive in other viruses (see Figure 2).

(2) sensitivity

HCV RNA was purified purely from HCV ATTCC Strain 20827 to make a 1 ng PCR mixture, which was then diluted 10 times to make a concentration of 0.1 fg, followed by PCR using this kit to measure sensitivity. Experimental method was tested according to Experimental Example 1, the experimental results are shown in the photograph in FIG. In Figure 3, lane 1 is Phage 174 DNA / HaeIII molecular weight marker, lane 2 is 1 ng, lane 3 is 100 pg, lane 4 is 10 pg, lane 5 is 1 pg, lane 6 is 100 fg, lane 7 is 10 fg, Lane 8 shows 1 fg, lane 9 shows 0.1 fg of RNA. As can be seen in FIG. 3, PCR positivity was shown up to 10 fg of HCV RNA tubes (see FIG. 3).

(3) reproducibility

5 HCV positive control and 5 negative control HCV of the present invention The PCR kit was used for in-day and bet-day experiments. The method was performed by two researchers divided by morning and afternoon by PCR for 5 days. Experimental method was tested according to Experimental Example 1, the experimental results are shown in Table 6. As can be seen in Table 6, 100% reproducibility was shown in both the PCR positive control and the negative control.

Table 6 HCV PCR reproducibility results

1 day 2 days 3 days 4 days 5 days Positive control 1 One experiment (A) positivity positivity positivity positivity positivity 2 experiments (B) positivity positivity positivity positivity positivity Positive control 2 One experiment (A) positivity positivity positivity positivity positivity 2 experiments (B) positivity positivity positivity positivity positivity Positive control 3 One experiment (A) positivity positivity positivity positivity positivity 2 experiments (B) positivity positivity positivity positivity positivity Positive Control 4 One experiment (A) positivity positivity positivity positivity positivity 2 experiments (B) positivity positivity positivity positivity positivity Positive Control 5 One experiment (A) positivity positivity positivity positivity positivity 2 experiments (B) positivity positivity positivity positivity positivity Negative control 1 One experiment (A) voice voice voice voice voice 2 experiments (B) voice voice voice voice voice Negative control 2 One experiment (A) voice voice voice voice voice 2 experiments (B) voice voice voice voice voice Negative control 3 One experiment (A) voice voice voice voice voice 2 experiments (B) voice voice voice voice voice Negative control 4 One experiment (A) voice voice voice voice voice 2 experiments (B) voice voice voice voice voice Negative control 5 One experiment (A) voice voice voice voice voice 2 experiments (B) voice voice voice voice voice

(4) Clinical utility test

In order to see the clinical usefulness of the HCV RT-PCR kit of the present invention it was compared to the standard diagnostic method HCV EIA kit (Dong A Co., Korea). Serum was used as sample, 34 positive specimens from EIA test and 28 negative specimens were used. Experimental method was tested according to the HCV RT-PCR test of the present invention according to Experimental Example 1, HCV EIA test according to the kit instructions. In the test method of EIA test, serum of a patient is added to a plate containing hepatitis C virus and incubated for 1 hour. Next, after washing, hepatitis C antigen-HRP conjugate was added to bind, and then the color absorbent was added to measure absorbance at 450 nm. The color develops darker in proportion to the amount of hepatitis C antibody.

The experimental results are shown in Table 7. As can be seen from Table 7, there were 4 cases of inconsistencies when the two methods were used. One case was positive in EIA, negative in PCR, and negative in EIA and positive in PCR. It was three examples that came out. This sample was tested with a third-party PCR kit that amplified by PCR in the HCV gene region of the HCV RT-PCR kit and other sites of the present invention showed the same results as the results of this kit. If positive in EIA and negative in PCR, HCV antigen is expected to be seroconversion when HCV antigen is positive, and HCV antibody is expected. It is expected that the sensitivity of the EIA method is not measured because the number is small. Therefore, the gene diagnosis method using PCR is more useful than the EIA method for accurate HCV test.

Table 7 Results for Clinical Usefulness

HCV EIA Experiment + - Of the present invention HCV RT-PCR + 33 A B 3 - 1 C D 25 +/- all 34 28

1) Agreement is = 93.6%,

2) Sensitivity = 97.1%,

3) Specificity = 89.3%,

4) positive predictive value = 91.7%,

5) The negative predictive value is = 96.2%.

(5) Comparison with other products

Table 8 shows the results of comparing the PCR kit of the present invention with products of other companies that are commercially available in Korea.

[Table 8] Comparison with other companies' products

Contents HCV PCR Kits of the Invention Third party HCV PCR kit Nucleic Acid Extraction Reagent Inexpensive and convenient because a special nucleic acid extracting reagent is included in the kit It is a cost increaser and not convenient because it is not included in the kit and must be purchased separately. responsiveness Measures up to 10 fg RNA No clear mention of sensitivity Kit composition The method is simple because the primer and PCR mixtures are contained in one kit and used without the need to dilute the primer concentration separately. It is inconvenient because you have to purchase primer and PCR mixture separately, and every time you use it, you have to dilute the concentration of primer as much as you need. Experiment time RNA extraction time: 10 minutes Primary PCR reaction time: 1 hour 10 minutes Secondary PCR reaction time: 60 minutes Electrophoresis time: 20 minutes Total time required 2 hours 40 minutes RNA extraction time: 1 hour Primary PCR reaction: 2 hours 30 minutes Secondary PCR reaction: 1 hour 20 minutes Electrophoresis time: 20 minutes Total time required: 5 hours 10 minutes Electrophoretic Dyeing Reagent No need to add loading dye during electrophoresis by adding cresol red to the second PCR mixture Hassle to add dyeing reagent every time electrophoresis PCR method Simultaneous experiment of transcriptional reaction and primary PCR, simple and fast Long time due to separate transcription reaction

Kit of the present invention having the configuration as described above has the advantage that do not put a separate dye indicator when the electrophoresis of the final fish product, put the cresol red in the second fish master mixture, HCV RNA extraction reagent is included in the kit It is very simple. In addition, it is more economical than other detection kits, and has a 50% improvement in rapidity by simultaneously performing transcription and primary fish reactions, and has excellent sensitivity and accuracy of 10 fg. This can be useful for

Claims (5)

a) RNA extract solution containing DEPC treated distilled water, Taq buffer, DMSO; b) a primary fish master mixture comprising DEPC treated distilled water, buffer, dNTP, primers, DTT, Taq / RNasin and reverse transcriptase; And c) A kit for detecting hepatitis C virus comprising a distilled water, buffer, dNTP, primers and a secondary fish master mixture comprising Taq / UDG, cresol red. The method of claim 1, Hepatitis C virus detection kit, characterized in that the primer set of the primary mixed solution has a nucleotide sequence described in SEQ ID NO: 1 and 2. The method of claim 1, Hepatitis C virus detection kit, characterized in that the primer set of the secondary mixed solution has a nucleotide sequence described in SEQ ID NO: 3 and 4. The method of claim 1, The reverse transcriptase is a hepatitis C virus detection kit, characterized in that AMV reverse transcriptase. a) subjecting the sample to an RNA extraction reagent of claim 1 and a primary fish master mixture followed by a primary fish; b) treating the primary fish product with the secondary fish master mixture of claim 1 to perform the secondary fish; And c) A method for detecting hepatitis C virus comprising the step of confirming the presence of hepatitis C virus through a gel.