KR100795704B1 - The primer and probe for detecting hbv-dna - Google Patents

Abstract

The present invention relates to primers and probes, which are used to quantify the gene of hepatitis B virus (HBV-DNA) via PCR hybridization. In particular, the hepatitis B virus is characterized in that the adr or adw subtype.

Hepatitis B virus gene (HBV-DNA), PCR, enzyme immunoassay, chemiluminescence

Description

Primer and probe for detecting hepatitis B virus gene {THE PRIMER AND PROBE FOR DETECTING HBV-DNA}

1 shows an outline of the color development method (using an enzyme immunoassay device).

2 shows an outline of a luminescence method (using a chemiluminescence measuring instrument).

Figure 3 is a photograph of the electrophoresis of the amplification product for the hybridization reaction shows an amplification product size of 173 bp.

4 shows a microplate for color reaction (absorbance measurement).

5 shows a microplate for a luminometer.

6 shows a standard calibration curve based on the results of reproducibility of the standard sample using color absorbance.

7 shows a standard calibration curve based on the results of reproducibility of standard samples using luminescence.

8 shows a comparison between the absorbance method according to the present invention and the product of Daijin Corporation.

Figure 9 shows a comparison of the results of the luminescence method according to the present invention and products made by Daijin Corporation.

Fig. 10 shows the correlation between the absorbance method and the invention method.

The present invention relates to primers and probes, which are used to quantify the gene of Hepatitis B virus (HBV) via PCR hybridization. In particular, the hepatitis B virus is characterized in that the adr or adw subtype.

HBV-DNA detection kit comprising the primers and probes of the present invention has high accuracy and precision for the detection of adr and adw, which are HBV subtypes (mainly found in Korea and East Asia), and immunoassay (enzyme) By providing a compatible system reagent that can be used with any of the instruments involved in immunoassay or chemiluminescence, the redundant investment in additional instrument purchases can be reduced and signal strength increased for increased sensitivity.

Hepatitis B virus (HBV) is a virus of the Hepadnaviridae strain that specifically infects the human body. It is known that about 500 million people worldwide are infected with HBV. The incubation period of HBV is 60-110 days, and in patients who do not recover from infection after 90-95% complete recovery through various clinical stages, HBV genomic DNA is inserted into and assimilated into the genomic DNA of human hepatocytes and chronically active hepatitis. , Cirrhosis, liver cancer, etc. will develop. Infection with HBV, like other diseases, causes chronic viral infections, lymphoma diseases and chronic kidney disorders. Thus, chronic infection is a disease with a very high mortality rate that develops into a more powerful form of disease and eventually leads to death of patients (Don Ganem et al., Ann. Rec. Biochem., 651 (1987): RPBeasley et al., Lancet, 1129 (1981): DAShafritz et al., New England J. of Medicine, 1067 (1981): SNZaman et al., Lancet 1, 1357 (1985)).

On the other hand, recent advances in molecular biology have enabled cloning sequences of the complete genome of HBV (Siddiqui, A. et. Al, Proc. Natl. Aca. Sci, USA Vol. 76, p4664, 1979; Sninsky, JJ et al, Nature.Vol. 279, p346, 1979; and Charnay, P. et al, Nucl.Acids Res., Vol 7, p335, 1979). Also coding regions of other viral proteins of HBV have been identified (European Patent Application Publication Nos. 13828, 20251 and 38765; Galibert et al. Nature, Vol. 281, p646-650, 1979; Pasek et al., Nature Vol. 282 , p575-579, 1979; and Valenzuela et al., Nature, Vol. 280, p815-819, 1979). HBV is a DNA virus, but has a reverse transcriptase activity of synthesizing DNA genes with RNA as a template similar to retroviruses (Ganem and Varmus, Annu. Rev. Biochem. 56: 651-693, 1987).

In Korea, hepatitis B virus (HBV) infection is on the decline these days, but it is still in the outbreak area where the number of carriers is estimated to be about 8%. In particular, chronic hepatitis B progresses to 50% in infancy and 20% in childhood, and is most acute in adults, but 3-5% progresses to chronic. Chronic hepatitis B is not easy to treat and is a serious disease with many developing cirrhosis or liver cancer.

The antigenicity of HBV is represented by subtypes such as adw, adr, ayw, ayr, etc., each of which has a different genetic structure and a different distribution depending on the region. The adw and adr subtypes are mainly found in Korea and East Asia.

Genetic quantification of HBV is a test for measuring the amount of genes present in the serum of patients infected with HBV, and has recently been recognized as a good indicator for determining the replication activity of HBV. The quantitative test of HBV gene was followed by monitoring the progression of HBV-infected patients by checking whether the amount of HBV gene decreased after treatment with hepatitis B patients and the therapeutic effect was increased by increasing the amount of HBV gene. This test is necessary to verify the therapeutic effect of antiviral drugs.

Currently, most of the diagnostic reagents used in Korea for the diagnosis of HBV infection and observation of treatment effect are imported from foreign countries. In particular, the reagent for quantitative testing of the HBV gene (DNA), which observes whether the hepatitis virus has been cured or increased after administration of a therapeutic agent to a hepatitis B patient, has been treated with 100% foreign acid, 1-2 products are available exclusively.

Specifically, in the case of domestic companies, antigen-antibody reaction screening reagents confirming only HBV infection are produced by some companies (Dong-A Pharm, Green Cross, Budding Medics, etc.), and HBV-DNA of molecular biology methods for HBV-DNA quantitative test. There are no developers and producers of quantitative reagents.

On the other hand, for foreign companies, the HBV-DNA quantitative reagent is a branched DNA product produced by Chiron (supplied by BAYER) and a capture HBV-DNA product produced by Digen. And Roche's products are produced and supplied.

According to the Quantiplex HBV-DNA analysis of the chiron company, the amount of branched DNA is measured by chemiluminescence after PCR amplification of the labeled branch DNA, and the intensity of emitted light is proportional to the amount of HBV-DNA. In addition, according to Daizen Hybrid Capture II, RNA-DNA hybridization is performed by measuring the amount of DNA by chemiluminescence by combining RNA-DNA antibodies after hybridization with HBV-DNA extracted from RNA probes without amplification. The intensity of the reaction assay luminescence is proportional to the amount of HBV-DNA. In addition, according to Roche's Amplicor HBV monitoring test, HBV-DNA is hybridized with a probe after PCR amplification and the amount of DNA is measured by enzyme-immunoassay (chromophoresis).

However, the imported foreign products have a variety of detection range of HBV-DNA, but there is a problem that the specificity of the adr and adw subtype strains mainly found in Korea and East Asia are inferior in specificity and cause problems such as false positives. In addition, since the foreign products measure the amount of HBV-DNA only by chemiluminescence or enzymatic immunoassay, there is a problem in that the method of quantifying HBV-DNA and the device selection for it are narrow.

Accordingly, the present inventors have solved the problems of the prior art and completed the present invention by providing a novel hepatitis B virus (HBV) gene quantification method and detection kit using the same.

The present invention provides primers and probes, which are used to quantify the gene of hepatitis B virus (HBV-DNA) through PCR hybridization. The diagnostic reagent kit for detecting the HBV-DNA comprising the primer and the probe of the present invention has a high accuracy and precision specifically for the detection of the HBV subtypes adr and adw, which are mainly found in Korea and East Asia (technical competitiveness products) Development), and by providing a compatible system reagent that can be used with any of the devices involved in the immunoassay (enzyme immunoassay or chemiluminescence), it is possible to reduce redundant investments by purchasing additional equipment (enable reagents for compatibility). Two probes simultaneously participate in the hybridization reaction, increasing signal strength and improving sensitivity.

As described above, the diagnostic reagent for detecting the HBV gene of the present invention is a new diagnostic reagent localized with the above technical objective, and has the advantage of solving the technical and economic problems of the foreign products.

The present invention relates to a primer having a nucleotide sequence of SEQ ID NO: 1 or 2, which is used to quantify the gene of hepatitis B virus (HBV-DNA) through PCR hybridization.

The present invention also relates to a probe having a nucleotide sequence of SEQ ID NO: 3 or 4, which is used to quantify the gene of hepatitis B virus (HBV-DNA) by PCR hybridization method.

The present invention also relates to primers and probes, characterized in that the hepatitis B virus is adr or adw subtype.

According to the present invention, after amplifying a specific region having the least variation of HBV using a specifically designed primer (SEQ ID NOs: 1 and 2), the amplified product is specifically designed in a microplate well. Hybridization with (SEQ ID NOS: 3 and 4), and the resultant reaction of the HBV-DNA by the amount of luminescence (absorbance) or luminescence (intensity of luminescence) exhibited by the enzymatic action of the enzyme by binding to the enzyme-labeled antibody. Measure the amount. Biotin is attached to the 5 'of the front primer, and the amplification product is attached to the microwell by binding to streptavidin. The test method may be selected whether the POD enzyme is attached to the DIG of the hybridized probe and measured by enzyme immunoassay or the chemiluminescence method by attaching the AP enzyme. In the multiple probe, two types of probes are hybridized simultaneously to the same DNA strand to increase signal intensity.

 The intensity of color development and the intensity of luminescence are proportional to the amount of HBV-DNA. Therefore, this reagent kit is an excellent reagent kit that can be converted into coloration method (using enzyme immunoassay device) or luminescence method (using chemiluminescence device) according to the user's intention. Quantification of the HBV-DNA is a test necessary for the observation of HBV-infected patients and the verification of the antiviral therapeutic effect.

An overview of the color development method (using an enzyme immunoassay device) is shown in FIG. 1. First, a DNA product of a specific sequence is placed in a microplate well, and double-stranded DNA is attached to a solid phase by binding of biotin and streptavidin. After removing the biotin-free DNA strand, the DIG-labeled probe is hybridized to the attached DNA strand. POD (peroxidase) binds to DIG and decomposes the substrate by the action of POD and develops the coloring agent TMB (tetra methyl benzidine). The intensity of color development is measured with an ELISA reader, and the intensity of absorbance is proportional to the amount of HBV-DNA.

An overview of the luminescence method (using chemiluminescence measuring instrument) is shown in FIG. First, a DNA product of a specific sequence is placed in a microplate well, and double-stranded DNA is attached to a solid phase by binding of biotin and streptavidin. After removing the biotin-free DNA strand, the DIG-labeled probe is hybridized to the attached DNA strand. The action of AP (alkaline phosphatase) on DIG causes the CDP luminescent substrate to shine. Light intensity is measured with a luminometer, and the light intensity is proportional to the amount of HBV-DNA.

The probes or primers for PCR hybridization or detection of HBV-DNA of the present invention were synthesized based on sequences that are specifically common to the adr and adw subtypes of HBV. The entire genome sequence of the adr subtype is J. Gen. Virol. 66 (PT1), 195-200 (1985), NCBI accession No. V00867 and the like, the entire genome sequence of the adw subtype is Dokl. Biochem. 271, 246-249 (1984), CBI accession No. V00866 and the like. The sense primer of the present invention (SEQ ID NO: 1) is located on the 1432-1453 position of the entire genome sequence of the HBV adr and adw subtype, the antisense primer (SEQ ID NO: 2) is located on the 1581-1604 position, probe 1 (SEQ ID NO: 3) ) Is based on the 1435-1460 position and probe 2 (SEQ ID NO: 4) is based on the nucleotide sequence on the 1550-1570. The probe or primer of the present invention can be synthesized using an automatic synthesis method or the like.

Probes or primers of the invention can be further modified to include detectable labels for diagnostics and probes. Such various labels are well known in the art, and suitable labels include, but are not limited to, biotin, DIG, and the like. Those skilled in the art can readily use these labels to obtain labeled variants of the probes or primers of the invention.

The primer used in the present invention is a mixture of the sense and antisense primers of SEQ ID NO: 1 and 2, the probe is a mixture of probe 1 and probe 2 of SEQ ID NO: 3 and 4, the base sequence thereof is shown in Table 1 below .

Item Sequence Sense primer 5'-ccg tcg gcg ctg aat ccc gcg g-3 '(SEQ ID NO: 1) Antisense primer Biotin-5'-acg tgc aga ggt gaa gcg aag tgc-3 '(SEQ ID NO: 2) Probe 1 DIG-5'-tcg gcg ctg aat ccc gcg gac gac cc-3 '(SEQ ID NO: 3) Probe 2 DIG-5'-cgt ctg tgc ctt ctc atc tgc-3 '(SEQ ID NO: 4)

The difference between the color development method and the light emission method is shown in Table 2 below, except that the inspection method is the same.

Color development Luminescence Enzyme used Peroxidase (POD) Alkaline phosphatase (AP) Substrate Hydrogen peroxide CDP-star ^TM Device used Absorbance meter Luminous photometer Standard sample concentration (pg / ml) 0.5, 100, 1000, 2000 0.5, 100, 2000, 5000

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail based on an Example. The following examples are given for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Using Enzyme Immunoassay

Composition of Reagents

1. Extraction process of HBV-DNA

1) Dissolution solution (0.2N NaOH)

2) Neutralization solution (0.2N HCl)

2. Amplification and Hybridization

* Amplification of HBV-DNA

1) Lyophilized PCR reaction reagent test tube (polymerase 2.5U, 4 kinds of nucleic acid 250 uM, Tris-HCl 10 mM, KCl 40 mM, MgCl ₂ , stabilized pigments are stored in a dry state)

2) PCR buffer (purified water containing 1 pM primer and 100 uM d-UTP)

3) Low concentration positive control

4) Medium concentration positive control

5) Standard sample for preparing standard curve

Concentration of Standard Solution: 1.0.5 pg / ml

                2. 100 pg / ml

                3. 1000 pg / ml

                4. 2000 pg / ml

# Standard sample was purchased from ATCC 39630 HBV strain, US Pat. No. 4,942,125, Proc. Natl. Acad. Sci. It was produced by the method described in USA 78: 2606-2610, 1981 and the like.

* Hybridization reaction solution

1) Attachment Buffer (PBS, 0.05% Tween20)

2) DNA denaturation solution (0.2 N NaOH)

3) microwell plate coated with streptavidin

4) Hybridization Buffer (4X SSC, 20 mM HEPES, 2 mM EDTA, 0.15% tween 20, 0.5% BSA, pH 7.2)

5) DIG-labeled probe

6) Antibody-attached enzyme (Ab conjugated POD: peroxidase) and diluent (PBS)

Substrate: Hydrogen Peroxide

8) Coloring Agent (Tetra Methyl Benzidine; TMB)

9) Stopping solution: sulfuric acid

10) Wash solution (1X PBS, 0.05% tween20)

Inspection process

1. HBV-DNA Extraction

50 ul of the test serum and 50 ul of the dissolved solution were mixed and reacted at 37 ° C. for 30 minutes while stirring at 40 rpm. At this time, 2 to 4 standard samples were extracted in the same manner as the sample. 50 ul of neutralization buffer was added, mixed, and centrifuged for 10 minutes at 12000 G. 5 ul of supernatant was used for amplification.

2. PCR

5 ul of extracted DNA and 45 ul of PCR buffer were added to the dried preliminary mixture to prepare a master mixture (amplification reaction solution) with a total amount of 50 ul. Amplification reaction conditions were 1) 95 degreeC 3 minutes, 2) [94 degreeC 40 second, 60 degreeC 40 second, 72 degreeC 40 second] * 25 cycles, and 3) 72 degreeC 7 minutes. Amplification took about 1 hour.

In this regard, Figure 3 is a photograph of the electrophoresis of the amplification product for the hybridization reaction shows an amplification product size of 173 bp. Lane 1 is 100 bp ladder size marker DNA, lanes 2 and 3 are 0.5 pg / ml, lane 4 is 100 pg / ml, lane 5 is 1000 pg / ml, lane 6 is 2000 Standard sample at pg / ml concentration. Figure 3 shows a negative at less than 100 pg / ml and a distinct band at 1000 pg / ml showing the difference according to the amount of DNA.

After the amplification reaction, 150 ul of purified water was mixed into 50 ul of PCR amplification solution.

3. Hybridization reaction

150 ul of adhesion reaction buffer was added to the well, 10 ul of diluted PCR amplification solution was added and mixed, followed by reaction for 1 hour while stirring at 40 rpm in a water bath at 50 ° C. After adding 100 ul of the DNA denatured solution, the reaction was performed at room temperature for 20 minutes. At this time, the probe is prepared as needed in the reaction buffer at a ratio of 1 Ul / ml during the reaction time. After the reaction was washed five times with a wash solution. After rinsing off the washing solution cleanly, 100 ul of the probe preparation was added to the well, and reacted for 1 hour while stirring at 40 rpm in a water bath at 50 ° C. At this time, the enzyme is prepared as needed in the diluent at a ratio of 1: 1000 during the reaction time. After the reaction was washed five times with a wash solution. After diluting the washing solution cleanly, diluted enzyme solution (POD Conjugated anti-DIG ab) was added to each well by 100 ul and reacted for 30 minutes while stirring at 40 rpm in a 37 ° C water bath. At this time, the substrate solution and the colorant are prepared as necessary in a ratio of 1: 1 during the reaction time. After the reaction was washed five times with a wash solution. The washing solution was thoroughly shaken off, and the coloring solution prepared by diluting the same amount was dispensed into each well for 100 ul and reacted in the cow for 15 minutes. 100 ul of reaction stopper was added to each well to stop the reaction, and the absorbance (intensity of color development) was measured at 450 nm with reference to 620 nm in an enzyme immunoassay device.

In this regard, Figure 4 shows a microplate for color reaction (absorbance measurement), it can be seen that the light is made of a transparent material to transmit. The change of reaction can be seen with the naked eye. However, an ELISA reader is used for accurate measurements.

Result output

Sample 1: 0.5 pg / ml

Sample 2: 100 pg / ml

Sample 3: 1000 pg / ml

Sample 4: 2000 pg / ml

After preparing a standard calibration curve with the absorbance measurements of the standard samples 1 to 4, the absorbance measurements of each specimen were substituted into the standard calibration curve to determine the concentration of HBV-DNA (pg / ml).

Interpret the results

0.5 pg / ml or more is determined as positive, and an increase or decrease tendency is determined in comparison with the previous results and the like. If the measured concentration is significantly lower than the measurement result before administration or shows a negative result, it is determined that the drug (antiviral agent such as interferon) has a therapeutic effect against the virus. Conversely, if there is no reduction or increase in comparison with the result of pre-dose, it is determined that no therapeutic effect is observed. Clinicians have virtually no treatment effect at concentrations above 200 pg / ml, which translates to almost the same meaning as concentration values of 1000 pg / ml and 2000 pg / ml. The purpose of this test is to measure the degree of change in test values before and after dosing for follow-up rather than one-time test.

Example 2 Using a Chemiluminometer

Composition of Reagents

1. Extraction process of HBV-DNA

1) Dissolution solution (0.2N NaOH)

2) Neutralization solution (0.2N HCl)

2. Amplification and Hybridization

* Amplification of HBV-DNA

1) Lyophilized PCR reaction reagent test tube (polymerase 2.5U, 4 kinds of nucleic acid 250 uM, Tris-HCl 10 mM, KCl 40 mM, MgCl ₂ , stabilized pigments are stored in a dry state)

2) PCR buffer (purified water containing 1 pM primer and 100 uM d-UTP)

3) Low concentration positive control

4) Medium concentration positive control

5) Standard sample for preparing standard curve

Concentration of Standard Solution: 1.0.5 pg / ml

                2. 100 pg / ml

                3. 2000 pg / ml

                4. 5000 pg / ml

# Standard sample was purchased from ATCC 39630 HBV strain, US Pat. No. 4,942,125, Proc. Natl. Acad. Sci. It was produced by the method described in USA 78: 2606-2610, 1981 and the like.

* Hybridization reaction solution

1) Attachment Buffer (PBS, 0.05% Tween20)

2) DNA denaturation solution (0.2 N NaOH)

3) Streptavidin-Coated Microwell Plates

4) Hybridization Buffer (4X SSC, 20 mM HEPES, 2 mM EDTA, 0.15% tween 20, 0.5% BSA, pH 7.2)

5) DIG-labeled probe

6) Antibody-attached enzyme (Ab conjugated AP: alkaline phosphatase) and diluent (PBS)

7) Substrate: trade name CDP-star (Roche); Disodium 2-chloro-5- (4 [1,2-dioxytan-3,2 '-(5'-chloro) -tricyclo [3,3,1,1] decane] -4yl) phenyl Phosphate (disodium2-chloro-5- (4 [1,2-dioxetan-3,2 '-(5'-chliro) -tricyclo [3,3,1,1] decan] -4yl) phenyl phosphate)

8) Wash solution (1X PBS, 0.05% tween20)

Inspection process

1. HBV-DNA extraction process and 2. PCR process are the same as in Example 1 (when using the enzyme immunoassay).

3. Hybridization reaction

150 ul of adhesion reaction buffer was added to the well, 10 ul of diluted PCR amplification solution was added and mixed, followed by reaction for 1 hour while stirring at 40 rpm in a water bath at 50 ° C. After adding 100 ul of the DNA denatured solution, the reaction was performed at room temperature for 20 minutes. At this time, the probe is prepared as needed in the reaction buffer at a ratio of 1 Ul / ml during the reaction time. After the reaction was washed five times with a wash solution. After rinsing off the washing solution cleanly, 100 ul of the probe preparation was added to the well, and reacted for 1 hour while stirring at 40 rpm in a water bath at 50 ° C. At this time, the enzyme is prepared as needed in the diluent at a ratio of 1: 1000 during the reaction time. After the reaction was washed five times with a wash solution. After washing off the wash solution cleanly, diluted enzyme solution (POD Conjugated anti-DIG ab) was added to each well by 100 ul and reacted for 30 minutes while stirring at 40 rpm in a 37 ° C water bath. At this time, the substrate solution and the colorant are prepared as necessary in a ratio of 1: 1 during the reaction time. After the reaction was washed five times with a wash solution. The washing solution was thoroughly shaken off, and the coloring solution prepared by diluting the same amount was dispensed into each well for 100 ul and reacted in the cow for 15 minutes. Luminance was measured at 466 nm wavelength of the luminometer within 15 minutes.

In this regard, Figure 5 shows a microplate for a luminometer, it can be seen that the light is made of an opaque material so that it does not affect other wells. It is not possible to notice the change in the reaction with the naked eye, and a luminometer must be used.

Result output

Sample 1: 0.5 pg / ml

Sample 2: 100 pg / ml

Sample 3: 2000 pg / ml

Sample 4: 5000 pg / ml

After preparing a standard calibration curve using the luminescence measurement values of the standard samples 1 to 4, the concentration of HBV-DNA (pg / ml) was obtained by substituting the luminescence measurement values of each sample into the standard calibration curve.

Interpret the results

It is the same as Example 1 (when using an enzyme immunoassay device).

Experimental Example 1. Quality Assurance

The detection range was 0.5-2000 pg / ml for colorimetric method and 0.5-5000 pg / ml for luminescence method (see FIGS. 6 and 7). Worse than the law. Therefore, a lower one than the case of the standard luminescence method is used.

Sensitivity

Sensitivity = (positive results-false negative results) / positive samples * 100%.

If the sensitivity of the positive sample is estimated based on the test results of 72 samples from 25 to 96 in Table 5, the sensitivity is (72-0) / 72 * 100 = 100%. As such, the sensitivity of HBV-DNA detection in accordance with the present invention showed very good results.

Specificity

Specificity = (number of negative results-number of false positive results) / number of negative samples * 100%.

The degree of negativeness of a negative sample is estimated based on the test results of 24 samples from 1 to 24 in Table 5 below: specificity = (24-1) / 24 * 100 = 95.8% to be. As such, the specificity of HBV-DNA detection also showed excellent results in accordance with the present invention.

Reproducibility

Reproducibility is repeated testing the same sample to determine how consistent the results of the sample are, and for quantitative reagents such as this reagent, the degree of reproducibility is very important.

Factors that represent reproducibility include: 1) mean value = total test result divided by the number of test iterations, 2) standard deviation = sum of the mean and the difference between each result divided by test iterations, and 3) variation coefficient. = Standard deviation divided by the mean.

The reproducibility is represented by the coefficient of variation. The larger the value, the lower the reproducibility, and the smaller the value of the coefficient of variation, the better the reproducibility.

Reproducibility of Standard Samples Using Color Absorbance

Repeat count Standard sample 1 (0.5 pg / ml) Standard Sample 2 (100 pg / ml) Sample 3 (1000 pg / ml) Standard Sample 4 (2000 pg / ml) One 0.273 0.682 1.359 2.151 2 0.290 0.677 1.389 2.235 3 0.298 0.654 1.454 2.274 4 0.291 0.674 1.278 2.138 5 0.252 0.671 1.411 2.214 6 0.318 0.706 1.419 2.128 7 0.306 0.691 1.338 2.126 8 0.281 0.675 1.285 2.185 9 0.286 0.665 1.433 2.179 10 0.368 0.711 1.289 2.246 11 0.308 0.707 1.321 2.244 12 0.327 0.874 1.364 2.155 13 0.264 0.883 1.401 2.201 14 0.344 0.692 1.406 2.173 15 0.277 0.687 1.287 2.113 16 0.295 0.663 1.293 2.219 17 0.273 0.682 1.359 2.151 18 0.290 0.677 1.389 2.235 19 0.298 0.654 1.454 2.274 20 0.291 0.674 1.278 2.138 21 0.252 0.671 1.411 2.214 22 0.318 0.706 1.419 2.128 23 0.306 0.691 1.338 2.126 24 0.281 0.675 1.285 2.185 25 0.286 0.665 1.433 2.179 26 0.368 0.711 1.289 2.246 27 0.308 0.707 1.321 2.244 28 0.327 0.874 1.364 2.155 29 0.264 0.883 1.401 2.201 30 0.344 0.692 1.406 2.173 31 0.277 0.687 1.287 2.113 32 0.295 0.663 1.293 2.219 Average 0.29 0.71 1.36 2.19 Standard Deviation 0.02884 0.066711 0.05812 0.047571 Coefficient of variation 9.66% 9.43% 4.28% 2.17%

* The above figures are absorbance, no unit

Table 3 shows good variation coefficients of 9.66% and 9.43% in standard samples 1 and 2, and extremely good coefficients of variation of 4.28% and 2.17% in standard samples 3 and 4, respectively. This result can be judged by very good reproducibility.

The standard calibration curve based on the results of the standard sample was also good (Figure 6).

Reproducibility of standard samples using chemiluminescence (luminescence photometer).

Repeat count Standard sample 1 (0.5 pg / ml) Standard Sample 2 (100 pg / ml) Sample 3 (2000 pg / ml) Standard Sample 4 (5000 pg / ml) One 85 10482 119311 201971 2 92 8194 122077 199432 3 88 10194 120303 206453 4 102 10199 109997 199932 5 77 9399 121212 198063 6 96 10301 119880 200112 7 92 10188 117678 219832 8 72 11298 117566 198875 9 70 11298 120399 197887 10 99 7898 123765 204056 11 77 10192 120626 206789 12 78 12770 117678 199467 13 87 10832 119878 198997 14 89 9669 109978 201005 15 77 9878 103020 210042 16 69 10189 124011 198997 17 76 9697 119098 199807 18 87 9898 108788 201426 19 93 9980 120303 200443 20 88 10990 130119 199427 21 80 10814 112989 203008 22 81 9299 123511 199887 23 98 10277 118998 202145 24 79 9477 130208 199942 25 78 10585 119402 198889 26 86 10929 120302 200543 27 84 10987 123118 201024 28 79 10982 121233 198989 29 92 11298 118789 197889 30 69 10979 111920 206654 31 89 11988 120119 199876 32 88 10189 123224 200987 Average 84.28125 10354.6875 119046.875 201651.4375 Standard Deviation 8.970681038 978.3711799 5716.028626 4461.690156 Coefficient of variation 10.64% 9.45% 4.80% 2.21%

* The above values indicate the intensity of luminescence and are commonly referred to as RLU values.

Table 4 shows good variation coefficients of 10.64% and 9.45% in standard samples 1 and 2, and the extremely good coefficients of variation of 4.80% and 2.21% in standard samples 3 and 4, respectively. It can be judged by the excellent reproducibility.

A standard calibration curve based on the results of the standard sample was also well prepared (FIG. 7).

Correlation with Existing Test Reagents

As a result of comparing with the product of the US Daijin (luminescence method), the correlation coefficient between the two methods was 0.9554 in the case of the reagent absorbance method (Fig. 8). Even in this case, the correlation coefficient between the two methods was high as 0.9798 (FIG. 9).

Comparison of results between test methods of this reagent

 No. The present invention  Dijin  No. The present invention  Dijin EIA reader Luminometer EIA reader Luminometer One <0.5 <0.5 <0.5 49 664 705 789 2 <0.5 <0.5 <0.5 50 712 676 554 3 <0.5 <0.5 <0.5 51 177 203 251 4 <0.5 <0.5 <0.5 52 312 344 372 5 <0.5 <0.5 <0.5 53 947 1097 1032 6 <0.5 <0.5 <0.5 54 87.3 76.5 109 7 <0.5 <0.5 <0.5 55 1399 1374 1276 8 0.5 0.6 <0.5 56 1093 1209 1324 9 <0.5 <0.5 <0.5 57 1293 1377 1566 10 <0.5 <0.5 <0.5 58 1976 1889 1728 11 <0.5 <0.5 <0.5 59 312 338 243 12 <0.5 <0.5 <0.5 60 225 214 228 13 <0.5 <0.5 <0.5 61 696 776 897 14 <0.5 <0.5 <0.5 62 768 887 908 15 <0.5 <0.5 <0.5 63 1101 1068 998 16 <0.5 <0.5 <0.5 64 878 991 1099 17 <0.5 0.5 <0.5 65 479 524 398 18 <0.5 <0.5 <0.5 66 412 398 447 19 <0.5 <0.5 <0.5 67 911 899 778 20 <0.5 <0.5 <0.5 68 765 778 754 21 0.7 <0.5 <0.5 69 887 1021 1002 22 <0.5 <0.5 <0.5 70 434 446 428 23 <0.5 <0.5 <0.5 71 552 643 724 24 <0.5 <0.5 <0.5 72 756 677 576 25 22.4 18.9 12.2 73 More than 2000 3210 3789 26 11.9 36.4 27.4 74 More than 2000 4787 5544 27 23.7 28.1 16.5 75 More than 2000 3240 3155 28 33.2 27.5 36.7 76 More than 2000 4294 3988 29 37.4 38.7 42.3 77 More than 2000 More than 5000 4192 30 66.3 52.1 44.1 78 More than 2000 3322 2998 31 8.9 10.4 7 79 1899 2078 2342 32 23.6 21.5 12.9 80 More than 2000 3687 2988 33 98.4 117.2 78.3 81 More than 2000 3421 4576 34 90.5 97.2 87.3 82 More than 2000 1994 2564 35 26.5 27.4 23.2 83 More than 2000 2898 3129 36 45.7 42.8 35.7 84 1653 1879 2657 37 19.4 22.3 14.2 85 More than 2000 More than 5000 More than 6000 38 13.1 11.5 9 86 More than 2000 2943 2234 39 52.9 56.4 67.5 87 More than 2000 3287 3434 40 36.7 41.1 54.4 88 1789 2212 3098 41 18.4 19.2 22.1 89 More than 2000 4856 More than 6000 42 14.1 11.9 15.8 90 More than 2000 4321 3465 43 39.4 38.2 42.7 91 More than 2000 3425 3996 44 69.1 76.4 56.9 92 More than 2000 2778 2443 45 24.1 28.9 21.1 93 More than 2000 2162 2998 46 46.3 52.5 66.4 94 More than 2000 2375 2879 47 28.9 34.1 44.1 95 More than 2000 3476 2878 48 12.1 17.4 18.9 96 More than 2000 1992 1654

The present invention has designed probes and primers within the HBV-DNA sequence without mutations by comparing and searching the sequences of as many strains as possible of the adr and adw subtypes, so that the sensitivity and specificity can be detected to detect all possible strains of the adr and adw subtypes. The degree is high. Therefore, the specificity of the HBV detection kit of the present invention compared to the imported foreign products, which vary in the range of detection, but the specificity of the adr and adw subtypes mainly found in Korea and East Asia is lowered and causes problems such as false positives. Is very excellent.

In addition, the test method may be selected whether the POD enzyme is attached to the DIG of the hybridized reaction probe and measured by enzyme immunoassay or by the chemoluminescence method by attaching the AP enzyme. Therefore, the economic burden due to the additional introduction of the device can be reduced.

In addition, the multiple probe used in the present invention has the effect of increasing the signal strength by hybridizing two types of probes to the same DNA strand at the same time. Therefore, the detection rate can be increased at high concentrations as well as at low concentrations.

Attach sequence list electronic file

Claims (4)

delete delete A reagent for quantification of hepatitis B virus adr or adw subtype gene (HBV-DNA) through a PCR hybridization method, comprising a probe having a nucleotide sequence of SEQ ID NO: 3 and a probe having a nucleotide sequence of SEQ ID NO: 4. delete

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