WO2007078029A1

WO2007078029A1 - A quantitative method for hbv-dna, a primer and probe for detecting hbv-dna, and a detecting kit comprising the same

Info

Publication number: WO2007078029A1
Application number: PCT/KR2006/001424
Authority: WO
Inventors: Hyun Il Choi; Hyung Keun Park; Tae Woo Kim; Hye Young Lee; Hyo Kyung Cho
Original assignee: Catch By Gene Co., Ltd.
Priority date: 2006-01-05
Filing date: 2006-04-18
Publication date: 2007-07-12

Abstract

The present invention relates to a method for quantifying hepatitis B virus DNA (HBA-DNA) by means of the PCR hybridization process, and to primers and probes used for such method. The present invention also relates to a kit for detecting HBV-DNA comprising said primers and probes. Specifically, the present invention is characterized in that said HBV is either an adr sub-type or an adw sub-type.

Description

A QUANTITATIVE METHOD FOR HBV-DNA, A PRIMER AND PROBE FOR DETECTING HBV-DNA, AND A DETECTING KIT

COMPRISING THE SAME

Technical Field

[1] The present invention relates to a quantitative method for Hepatitis B Virus (HBV) by using a PCR hybridization process and also relates to primers and probes used for said quantitative method. The present invention also relates to a kit for detecting HBV- DNA comprising said primers and probes. Background Art

[2] Hepatitis B virus (HBV) is a virus from Hepadnaviridae family that specifically affects a human body. It is known that about five hundred millions of people over the world are infected with HBV. The latent period of HBV is for from 60 to 110 days. Passing through clinical periods with various levels, 90 to 95 % of patients infected with HBV fully recover, but in the case of patients remaining unrecovered from the infection, HBV genome DNA are inserted and incorporated into the patients hepatic cell-genome DNA, until it develops to a chronic active hepatitis, hepatocirrhosis, liver cancer, etc. HBV infection, like other diseases, causes chronic virus infectious diseases, lymphadenoma and chronic kidney failure. Accordingly, chronic infections of HBV have a very high fatality rate and could develop to more powerful forms of diseases and end up in death of patents (Don Ganem et al., Ann. Rec. Biochem., 651 (1987): R. P. Beasley et al., Lancet, 1129 (1981): D.A. Shafritz et al., new England J. of Medicine, 1067 (1981): S. N. Zaman et al., Lancet 1, 1357 (1985)).

[3] Meanwhile, due to recent developments in the molecular biology field, the complete sequence of the HBV genome can be cloned (Siddiqui, A. et al., Proc. Natl. Aca. Sci, U.S.A. Vol. 76, p4664, 1979; Sninsky, JJ. et al, Nature. Vol. 279, p346, 1979; and Charnay, P. et al, Nucl. Acids Res., VoI 7, p335, 1979). Moreover, coding regions of HBV s other virus proteins have been identified (European Patent 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 similarly to retroviruses, HBV has an activity of a reverse transcription activity that synthesizes DNA using RNA as a template (Ganem and Varmus, Annu. Rev. Biochem. 56:651-693, 1987).

[4] Although the infection rate of HBV has been lately decreasing in Korea, it is estimated that the HBV carrier rate reaches up to 8%, and thus Korea still belongs to one of the areas where HBV diseases frequently occur. Particularly, the development rate of HBV to chronic hepatitis B is 50% for babies and 20% for children. As for adults, HBV diseases mostly develop acutely, but with 3-5% of adults, such HBV diseases develop chronically. Chronic Hepatitis B is not easy to cure, and quite many cases show that HBV has developed to cirrhosis or liver cancer. Thus, HBV infection is a serious disease.

[5] The serotypes of HBV are classified into sub-types such as adw, adr, ayw, ayr, etc.

These sub-types have different genetic structures, and their distribution also shows differences by areas. Most sub-types found in the East Asia including Korea are adw- and adr sub-types.

[6] A quantitative test for HBV is a test of measuring the amount of DNAs existing in the serum of a patient infected with HBV. This test has been recently recognized as a good index for determining the replicating activity of HBV. The quantitative test for HBV is conducted by administering a therapeutic agent to an HBV patient and observing whether the amount of HBV DNA decreases or increases as a sign of cure. Thus the quantitative test for HBV is absolutely necessary for observing the development of the HBV infection with a patient and for verifying a therapeutic effect as an antiviral agent.

[7] Most of the diagnostic reagents currently used in Korea for diagnosing a patient's

HBV infection and observing therapeutic effects thereon are imported from foreign countries. Particularly, all the reagents for the quantitative test for HBV-DNA, which are administered to a patient to observe the therapeutic effect as to whether the amount of HBV DNA has decreased as a sign of a successful treatment, or increased as a sign of failure of the treatment, are made in foreign countries. Among those, one or two products are exclusively provided in Korea.

[8] Specifically, Some of the Korean pharmaceutical companies (e.g., Dong-A Pharmaceutical, Green-Cross, Shin- Jin Medics, etc.) produce antigen-antibody reaction- screening reagents that can only confirm whether or not a patent has been affected by HBV, but no pharmaceutical companies have been developing and producing reagents for a quantitative test for HBV-DNA according to the molecular biological method for a quantitative test for HBV-DNA.

[9] Meanwhile, as for reagents for a quantitative test for HBV-DNA produced by foreign pharmaceutical companies, Chiron (provided by Bayer) produces branched DNA products; Digen produces capture HBV-DNA products; and Roche also produces such products.

[10] According to Chiron s Quantiplex HBV-DNA analysis, a labeled branch DNA is amplified according to the PCR- amplification process, and then the amount of said branch DNA is measured by means of a chemiluminescence method. Here, the luminosity is proportional to the amount of HBV-DNA. According to Hybrid Capture II of Digen, an RNA probe is hybridized with an HBV-DNA extracted without an amplification step, and an RNA-DNA antibody is conjugated thereto, and then the amount of the DNA is measured by means of the chemiluminescence method. The luminosity in this RNA-DNA hybridization reaction analysis is proportional to the amount of HBV-DNA. According to the monitoring test with Amplicor HBV produced by Roche, an HBV-DNA is amplified according to the PCR- amplification process, and the amplified HBV-DNA is hybridized with a probe, and then the amount of the DNA is measured by means of an enzyme immunoassay (chromogenic method).

[H]

Disclosure of Invention Technical Problem

[12] The above mentioned products may be able to detect various ranges of HBV-DNA, but those products have disadvantages such that they do not provide sufficient specificities to strains of the sub-types, adr and adw, which are mostly found in Korea and other areas of the East Asia; or that the products may cause a false positive reaction, etc. Further, since the above mentioned products use either the chemiluminescence method or the enzyme immunoassay for measuring the amount of HBV- DNA, choices in selecting quantitative methods for detecting HBV-DNA and equipments for conducting such detection are very narrow.

[13] In order to solve the above problems of the prior art, the present invention has been designed to provide a novel quantitative method for detecting HBV-DNA and a detecting kit by using the method. Technical Solution

[14] The present invention relates to a quantitative method for detecting HBV-DNA by using a PCR-hybridization process, and to primers and probes used for said method. The present invention is also designed to provide a kit for detecting HBV-DNA, comprising said primers and probes.

[15] The above diagnostic reagent kit for detecting HBV-DNA is characterized as follows:

[16] First, the diagnostic reagent kit has specificity with high accuracy and precision for the sub-types of HBV, adr and adw, mostly found in Korea and other areas in the East Asia (technologically competitive product development). Secondly, the kit provides a reagent that can be compatibly applied to any equipment related to immunity test (the enzyme immunoassay and the chemiluminescence method) so that a user does not have to pay extra costs for purchasing another equipment for each detecting method (compatible use of the reagent). Third, the diagnosing reagent kit provides a reagent in which two probes are involved in a hybridization reaction at the same time, which results in increasing signal intensity and thus enhancing the sensitivity of the reagent.

[17] As can be seen above, the diagnostic reagent of the present invention for detecting

HBV-DNA is a novel diagnostic reagent produced by domestic technology, having its technical purposes as shown above, and as such, the present invention has an advantage of solving technical and economical problems of foreign products.

[18]

[19] Constitutions of the Invention

[20] The present invention relates to a quantitative method for Hepatitis B Virus DNA

(HBV-DNA) by means of the PCR process, characterized in that said method uses primers having sequence numbers 1 and 2, and probes having sequence numbers 3 and 4.

[21] The present invention further relates to a quantitative method for HBV-DNA, characterized by quantifying HBV-DNA by means of an enzyme immunoassay using peroxidase (POD) enzymes, or a chemiluminescence method using alkaline phosphatase (AP).

[22] Further, the present invention relates to primers having sequence number 1 or 2, and probes having sequence number 3 or 4, which are used for quantifying HBV-DNA by means of the PCR hybridization process.

[23] The present invention also relates to a kit for detecting HBV-DNA, characterized by comprising primers having sequence numbers 1 and 2, and probes having sequence numbers 3 and 4.

[24] The present invention further relates to a quantitative method for HBV-DNA, a kit for detecting HBV-DNA, and primers and probes, characterized in that said HBV is of the sub-type, adr or adw.

[25]

[26] According to the present invention, a base sequence in a specific area with the least

HBV variations is amplified by using specifically designed primers (sequence numbers 1 and 2), and the amplified product is hybridized with specifically designed probes (sequence numbers 3 and 4) in a microplate well, and then thus obtained product is conjugated to an antibody having an labeled enzyme in order to measure the amount of HBV-DNA by observing the amount of chromogenic color (absorbance) or the amount of luminescence (luminosity) brought out by the action of said conjugated enzyme. The 5' end of the front primer has a biotin attached, and the amplified product is conjugated to a streptavidin, and then attached to the microplate well. A probe having DIG attached to 5' end is complementary to the base sequence of the amplified strand of the front primer. A user can use as a detecting method either the enzyme immunoassay after attaching POD enzyme to DIG of the probe, or a chemiluminescence method after attaching AP enzyme to DIG of the probe. In said multi-probes, two kinds of probe are hybridized at a DNA strand at the same time, and thus the signal intensity is enhanced.

[27] The intensity of the chromogenic color and the luminosity are proportional to an

HBV-DNA amount. Thus, the reagent kit of the present invention proves its excellence in its compatible use for both the chromogenic method (by using an enzyme immunoassay) and the luminescence method (by using a chemilumometer) upon user's intention. Said quantitative method for HBV-DNA is absolutely necessary for observing the development of HBV-infected patients and for verifying the therapeutic effect of an anti- viral agent.

[28] An outline of the chromogenic method (using an enzyme immunoassay analyzer) is shown in Fig. 1. First, a DNA product of a specific base sequence is put in a microplate well, and then it is combined with biotin and streptavidin to have a DNA of double strand attached to a solid phase. After the DNA strand having no biotin is detached, the attached DNA stand is subjected to a hybridization reaction with the probe having a DIG labeled. DIG is conjugated to POD (Peroxidase), and the substrate is dissolved by the POD action, and then tetra methyl benzidine (TMB) which is a chromogenic agent is reacted to develop a color. The intensity of the chromogenic color is measured by a spectrophotometer (ELISA reader). The absorbance intensity is proportional to the amount of HBV-DNA.

[29] An outline of the luminescence method (using chemiluminometer) is shown in Fig.

2. First, a DNA product of a specific base sequence is put in a microplate well, and then it is combined with biotin and streptavidin to have DNA of double strand attached to a solid phase. After the DNA strand having no biotin is detached, the attached DNA strand is subjected to the hybridization reaction with the probe having a DIG labeled. The CDP luminescent substrate becomes luminant by the action of the DIG conjugated to AP (alkaline phosphatase). The luminosity is measured by a luminometer. The luminosity is proportional to the amount of HBV-DNA.

[30] Said probes or primers for a PCR hybridization reaction or for detecting HBV-DNA according to the present invention have been synthesized based on the common sequence specific to adr and adw which are the sub-types of HBV.

[31] The entire genome sequence of said sub-type adr is disclosed in J. Gen. Virol. 66

(PTl), 195-200 (1985), NCBI accession No. V00867, etc.; and the entire genome sequence of said sub-type adw is disclosed in Dokl. Biochem. 271, 246-249 (1984), CBI accession No. V00866, etc. In the present invention, the sense primer (sequence number 1) is based on the base sequence of position no. 1432-1453 of the entire genome sequence of adr and adw, which are the sub-types of said HBV; the antisense primer (sequence number 2) is based on the base sequence of position no. 1581-1604 thereof; probe 1 (sequence number 3) is based on the base sequence of position no. 1435-1460 thereof; and probe 2 (sequence number 4) is based on base sequence of position no. 1550-1570 thereof. The probes or primers of the present invention can be synthesized by using an automatic synthesis method, etc.

[32] The probes or primers of the present invention can be additionally transformed so as to comprise a detectable label for diagnosis and probes. Such various labels are well known in the pertinent art, and appropriate labels comprise, but are not limited to, biotin, DIG, etc. A person skilled in the art can easily obtain such labeled variants of the probes or primers of the present invention by using said labels.

[33] The primers used for the present invention use a mixed solution of a sense primer having sequence number 1 with an antisense primer having sequence number 2 and a mixed solution of a probe 1 having sequence number 3 with a probe 2 having sequence number 4. The base sequence of the primers and the probes is shown in the following table 1.

[34] Table 1

[35] [36] Differences between the chromogenic method and the luminescence method are shown in table 2 below, and other than shown differences, the two methods are the same.

[37] Table 2

[38]

Brief Description of the Drawings [39] Figure 1 illustrates an outline of the chromogenic method (using an enzyme im- munoassay analyzer).

[40] Figure 2 illustrates an outline of a luminescent method (using a chemiluminometer).

[41] Figure 3 illustrates an electrophoresis picture of the amplified product for the hybridization reaction, showing the size of the amplified product being 173bp.

[42] Figure 4 illustrates a microplate for a color reaction (for measuring absorbance).

[43] Figure 5 illustrates a microplate for a luinometer.

[44] Figure 6 illustrates a standard calibration-curve drawn on the basis of the reproducibility result of a standard sample by means of the chromogenic method.

[45] Figure 7 illustrates a standard calibration-curve drawn on the basis of the reproducibility result of a standard sample by means of the luminescent method.

[46] Figure 8 illustrates a comparison of test results of the chromogenic method between the present invention and the product by Digen.

[47] Figure 9 illustrates a comparison of test results of the luminescent method between the present invention and the product of Digen.

[48] Figure 10 illustrates a correlation between the chromogenic method and the luminescent method.

[49]

Mode for the Invention

[50] Examples

[51] Hereinafter, the present invention is explained in more detail by way of working examples. The working examples shown below are merely for the purpose of giving examples, and as such the scope of the present invention is not limited to the shown examples.

[52]

[53] Example 1. Use of an enzyme immunoassay analyzer

[54] Composition of a reagent

[55] 1. Process for extracting HBV-DNA

[56] 1) Dissolution reagent (02.N NaOH)

[57] 2) Neutralization reagent (0.2N HCl)

[58]

[59] 2. Amplification and Hybridization Reaction

[60] * Amplification of HBV-DNA

[61] I) A test tube with freeze-dehydrated PCR reaction reagent (which comprises a polymerase of 2.5U, four kinds of nucleic acids of 25OuM, Tris-HCl of 1OmM, KCl of 4OmM, MgCl and stabilizing colorant, and is stored in a dehydrated state)

[62] 2) PCR buffer solution (a purified water containing primers of IpM and d-UTP of

10OuM) [63] 3) Low-concentrated positive control

[64] 4) Middle-concentrated positive control

[65] 5) A standard sample for drawing a standard curve

[66] Concentrations of standard solution: 1. 0.5 pg/ml

[67] 2. 100 pg/ml

[68] 3. 1000 pg/ml

[69] 4. 2000 pg/ml

[70] # The standard sample is made with a purchased ATCC 39630 HBV strain and by following the processes described in US Patent No. 4,942,125, Proc. Natl. Acad. Sci. USA 78:2606-2610, 1981, etc.

[71]

[72] * Hybridization Reaction test solution

[73] 1) Buffer solution for attaching reaction (PBS, Tween20 of 0.05%)

[74] 2) DNA denaturing test solution (0.2 N NaOH)

[75] 3) A microplate well coated with strep tavidin

[76] 4) Buffer solution for hybridization reaction (SSC of 4X, HEPES of 2OmM, EDTA of 2mM, tween20 of 0.15%, BSA of 0.5% and pH 7.2)

[77] 5) DIG-labeled probe

[78] 6) Antibody conjugated POD (peroxidase) and deluted solution (PBS)

[79] 7) Substrate solution: hydrogen peroxide

[80] 8) A chromogenic agent (Tetra Methyl Benzidine: TMB)

[81] 9) Stopping solution: sulphuric acid

[82] 10) Washing solution (PBS of IX, 0.05% tween20)

[83]

[84] Test Process

[85] 1. Extracting HBV-DNA

[86] A test serum of 50ul is mixed with a dissolution test solution of 50ul. Stirred at 40 rpm, the mixture is reacted at a temperature of 37⁰C for 30 minutes. Here, two of each of standard samples 1 to 4 are extracted in the same way as with a test body. A neutralization buffer solution is added as much as 50ul and mixed, and then the mixture is precipitated by centrifugation at 12000G for 10 minutes. The supernatant of 5 ul is used in the amplification reaction.

[87]

[88] 2. PCR

[89] The extracted DNA of 5ul and the PCR buffer solution of 45ul are mixed with a dry preparatory mixture to obtain a master mixture solution (amplification reaction solution) of 50ul in total. The amplification conditions are set to be (1) at a temperature of 95⁰C for 3 minutes, (2) [at 94⁰C for 40 seconds, at 6O⁰C for 40 seconds and 72⁰C for 40 seconds] *25 cycles and (3) at a temperature of 72⁰C for 7 minutes. The amplification takes about one hour.

[90] In this regard, Fig. 3 illustrates an electrophoresis picture of the amplified product for the hybridization reaction, showing the size of the amplified product being 173bp. Lane 1 is a marker DNA having the size of lOObp ladder, lanes 2 and 3 are standard samples having concentration of 0.5 pg/ml each, and lanes 4, 5 and 6 also standard test solutions having concentration of 100pg/ml, 1000pg/ml and 2000pg/ml, respectively. Fig. 3 illustrates a test result being negative when the concentration of the standard sample is not greater than 100pg/ml, and it also shows that at the concentration of 1000pg/ml, a clearly visible band appears to demonstrate differences depending on the amount of DNAs.

[91] After the amplification reaction, purified water of 150ul is mixed with the PCR amplified solution of 50ul.

[92]

[93] 3. Hybridization Reaction

[94] The buffer solution of 150ul for the attachment reaction is put into the well, and the deluted PCR amplification solution of lOul is added and mixed. Stirred at 40rpm, the mixture is reacted in a tank having a temperature of 5O⁰C for one hour. A DNA de- naturation solution of lOOul is added to the mixture, which then is reacted at room temperature for 20 minutes. Here, probes are prepared as much as needed in the ratio of lUl/ml to the hybridization reaction buffer solution during the reaction. After the reaction is completed, the product is washed five times with washing solution, and the washing solution is cleanly shaken off. Then, the prepared probe solution of lOOul is put into the well. Stirred at 40rpm in the water tank having a temperature of 5O⁰C, it is reacted for one hour. Here, an enzyme is prepared as much as needed by being mixed in the deluted solution in the ratio of 1:1000 during the reaction. After the reaction is completed, the reaction product is washed five times with washing solution. After the washing solution is cleanly shaken off, said deluted enzyme solution (POD Conjugated anti-DIG antibody) is added into each well by lOOul. Stirred at 40rpm in a water tank having a temperature of 37⁰C, it is reacted for 30 minutes. Here, a substrate solution and a chromogenic agent are prepared as much as needed by being mixed at a ratio of 1 : 1 during the reaction. After the reaction is completed, the reaction product is washed five times with washing solution. After the washing solution is cleanly shaken off, the same amount of a chromogenic test solution prepared by being deluted is injected by lOOul into each well, and then the product is reacted in a darkroom for 15 minutes. The reaction stop solution is added into each well by lOOul to stop the reaction, and the absorbance (intensity of the chromogenic color) is measured at 450nm wavelength with the enzyme immunoassay analyzer, by referring to 620nm. [95] In this connection, Fig. 4 illustrates a microplate for coloring reaction (for measuring absorbance), and in Fig. 4, the microplate is made of a transparent material so as to let the light penetrate. The change in the reaction is also visible to naked eyes. However, in order to conduct an accurate measurement, a spectrophotomer (ELISA reader) is used.

[96]

[97] Output Result

[98] Standard sample 1: 0.5pg/l

[99] Standard sample 2: lOOpg/ml

[100] Standard sample 3: lOOOpg/ml

[101] Standard sample 4: 2000pg/ml

[102] Based on the above measured absorbance values of the standard samples 1 to 4, a standard calibration-curve is drawn, and then the absorbance value of each test body is applied to the calibration-curve to calculate the concentration (pg/ml) of HBV-DNA.

[103]

[104] Interpretation of Results

[105] The concentration of 0.5pg/ml and above is considered as positive. In order to determine whether the concentration tends to increase or decrease, the results are compared with each other. If the measured concentration has remarkably decreased or shows negative reaction compared with the value measured before the administration, the pharmaceutical (antiviral agent such as interferon, etc.) is decided as having therapeutic effects on the virus. To the contrary, if the concentration does not reduce at all or rather increases compared with the value measured before the administration, it is determined that the pharmaceutical does not have therapeutic effect at all. Clinical doctors practically interpret that when the concentration is greater than or equal to 200pg/ml, the pharmaceutical has no therapeutic effect, which is as good as concentrations of 1000pg/ml, 2000pg/ml, etc. The purpose of this test is to measure the degree of change in the measured values before and after the administration to observe the development of the virus, and as such, it is not for a one-time test.

[106]

[107] Example 2. Use of the Chemiluminometer

[108] Composition of the reagent

[ 109] 1. Process for Extracting HBV-DNA

[110] 1) Dissolution test solution (0.2N NaOH)

[111] 2) Neutralization test solution (0.2N HCl)

[112] 2. Amplification and Hybridization reaction

[113] * Amplification of HBV-DNA

[114] I) A test tube with freeze-dehydrated PCR reaction reagent (which comprises a polymerase of 2.5U, four kinds of nucleic acids of 25OuM, Tris-HCl of 1OmM, KCl of

4OmM, MgCl and stabilizing colorant, and is stored in a freeze-dehydrated state) [115] 2) PCR buffer solution (a purified water containing primers of IpM and d-UTP of

10OuM)

[116] 3) Low-concentrated positive control

[117] 4) Middle-concentrated positive control

[118] 5) A standard sample for drawing a standard curve

[119] Concentrations of standard solution: 1. 0.5 pg/ml

[120] 2. 100 pg/ml

[121] 3. 2000 pg/ml

[122] 4. 5000 pg/ml

[123] # The standard sample is made of a purchased ATCC 39630 HBV strain and by following the processes described in US Patent No. 4,942,125, Proc. Natl. Acad. Sci.

USA 78:2606-2610, 1981, etc. [124]

[125] * Hybridization Reaction test solution

[126] 1) Buffer solution for attachment reaction (PBS, 0.05% Tween20)

[127] 2) DNA denaturing test solution (0.2 N NaOH)

[128] 3) A microplate well coated with streptavidin

[129] 4) Buffer solution for hybridization reaction (SSC of 4X, HEPES of 2OmM, EDTA of 2mM, tween20 of 0.15%, BSA of 0.5% and pH 7.2) [130] 5) DIG-labeled probe

[131] 6) Antibody conjugated AP (Alkaline phosphatase) and deluted solution (PBS)

[132] 7) Substrate solution: Product name CDR-star (by Roche); disodium

2-chloro-5-(4[l,2-dioxetan-3,2 -(5 -chliro)-tricyclo[3,3,l,l]decan]-4yl)phenyl phosphate

[133] 8) Washing solution (PBS of IX, 0.05% tween20)

[134]

[135] Test Process

[136] The process for extracting HBV-DNA and the PCR process (1. and 2.) are the same as shown in Example 1 above. [137]

[138] 3. Hybridization Reaction

[139] The buffer solution of 150ul for the attachment reaction is put into a well, and the deluted PCR amplification solution of lOul is added and mixed. Stirred at 40rpm, the mixture is reacted in a tank having a temperature of 5O⁰C for one hour. A DNA denaturing solution of lOOul is added to the mixture, which is then reacted at room temperature for 20 minutes. Here, probes are prepared as much as needed in the ratio of lUl/ml to the hybridization reaction buffer solution during the reaction. After the reaction is completed, the product is washed five times with washing solution, and the washing solution is cleanly shaken off. Then, the prepared probe solution of lOOul is put into the well. Stirred at 40rpm in the water tank having a temperature of 5O⁰C, it is reacted for one hour. Here, an enzyme is prepared as much as needed by being mixed in the deluted solution at a ratio of 1 : 1000 during the reaction. After the reaction is completed, the reaction product is washed five times with washing solution. After the washing solution is cleanly shaken off, said deluted enzyme solution (POD Conjugated anti-DIG antibody) is added into each well by lOOul. Stirred at 40rpm in a water tank having a temperature of 37⁰C, it is reacted for 30 minutes. Here, a substrate solution and a chromogenic agent are prepared as much as needed by being mixed at a ratio of 1 : 1 during the reaction. After the reaction is completed, the reaction product is washed five times with washing solution. After the washing solution is cleanly shaken off, the same amount of the chromogenic test solution prepared by being deluted is injected by lOOul into each well, and then the product is reacted in a darkroom for 15 minutes. Within said 15 minutes, the luminosity is measured at 466nm wavelength of the chemi- luminometer.

[140] In this connection, Fig. 5 illustrates the microplate for measuring luminescence, and it can be understood that the microplate is made of an opaque material so that the light does not penetrate, to prevent it from affecting other wells. The change in reaction is invisible to naked eyes, and thus in order to observe such changes, a user must use a luminometer.

[141]

[142] Output Result

[143] Standard sample 1: 0.5pg/l

[144] Standard sample 2: lOOpg/ml

[145] Standard sample 3: 2000pg/ml

[146] Standard sample 4: 5000pg/ml

[147] Based on the above measured luminosities of standard samples 1 to 4, a standard calibration-curve is drawn, and then the luminosity of each test body is applied to the calibration-curve to calculate the concentration of HBV-DNA (pg/ml).

[148]

[149] Result Interpretation

[150] It is same as in Example 1 above.

[151]

[152] Experimental example 1. Quality assurance

[153] The detected range of the concentration is 0.5-2000pg/ml in the chromogenic method, and 0.5-5000 pg/ml in the luminescent method (see Figs. 6 and 7). However, the linearity of the chromogenic method is lower at a concentration of 2000pg/ml and above than that of the luminescent method. Thus, the concentration of the standard sample for the chromogenic method should be lower than that of the luminescent method. [154]

[155] Degree of Sensitivity

[156] The degree of sensitivity is defined as follows:

[157] Degree of sensitivity = (the number of positive results - the number of negative results) / the number of positive test bodies * 100% [158] Sensitivity indicating how positive the positive sample results are can be calculated based on the results of the test conducted with 72 samples from number 25 to number

96 in table 5 as below:

[159] Sensitivity = (72-0)/72* 100=100%

[160] As can be seen above, the degree of sensitivity in detecting HBV-DNA according to the present invention shows a very excellent result. [161]

[162] Degree of Specificity

[163] The degree of specificity is defined as follows:

[164] Degree of specificity = (the number of negative results the number of positive results) / the number of negative test bodies * 100%. [165] Specificity indicating how negative the negative sample results are can be calculated based on the results of the tests conducted with 24 samples from number 1 to number 24 in table 5 as below:

[166] Degree of specificity = (24-1) / 24 * 100 = 95.8%.

[167] As can be seen above, the degree of specificity in detecting HBV-DNA according to the present invention shows a very excellent result. [168]

[169] Reproducibility

[170] Reproducibility determines how consistent the test results are when the same test bodies are repeatedly tested. Reproducibility is very important to a quantitative test reagent such as the present invention. [171] Factors influencing reproducibility are :

[172] Mean value of the results = the value obtained by dividing the total sum of each test result by the number of tests repeated; [173] Standard deviation = the value obtained by adding each difference between the average value and each of the test result values and then dividing the added sum by the number of tests repeated; and [174] Coefficient of variation = the ratio obtained by dividing the standard deviation by the mean value. [175] Reproducibility is represented by the coefficient of variation, and as the coefficient of variation becomes greater, the reproducibility becomes lower, and as the coefficient of variation becomes smaller, the reproducibility becomes greater. [176] Reproducibility of a standard sample by using a chromogenic absorbance (enzyme immunoassay analyzer) [177] Table 3

[178] * Above numeric values indicate absorbance, and there is no measuring unit applied.

[179] [180] Table 3 above shows the result of the reproducibility of the standard samples by using the chromogenic absorbance. From the table, standard samples 1 and 2 indicate 9.66% and 9.43%, respectively, which are good as a coefficient of variation, and standard samples 3 and 4 indicate 4.28% and 2.17%, respectively, which are extremely good as a coefficient of variation. Such results can be recognized as an excellent reproducibility.

[181] The standard calibration-curve made based on the test results of the standard samples shows also an excellent result (Fig. 6). [182] Reproducibility of standard samples by using chemiluminometer (luminescent luminosity measuring equipment) [183] Table 4

[184] * Above numeric values indicate luminosity, and RLU value is used as its measuring unit.

[185] [186] The above table 4 shows the results of the reproducibility of the standard samples by using a luminometer. From the table, standard samples 1 and 2 indicate 10.64% and 9.45%, respectively, which are good as a coefficient of variation, and standard samples 3 and 4 indicate 4.80% and 2.21%, respectively, which are extremely good as a coefficient of variation. As such, the results can be recognized as an extremely excellent reproducibility.

[187] The standard calibration-curve made based on the result of the standard samples shows also an excellent result (Fig. 7).

[188] [189] Correlation with conventional test reagents [190] In comparison with products by the American company, Digen, which are based on the luminescent method, the correlation coefficient between the chromogenic method of the test reagent according to the present invention and the luminescent method of Digen is 0.9554, showing a high correlation (Fig. 8), and the luminescent method of the present invention also shows a high correlation with that of the Digen as 0.9798 (Fig. 9).

[191] [192] Comparison of test results of the detecting methods using the test reagent of the present invention

[193] Table 5

[194]

Industrial Applicability

[195] The present invention is designed to provide probes and primers within the HBV- DNA sequence having no variants, found through comparative searches for the base sequences of as many strains of adr and adw sub-types as possible. As such, the present invention has high sensitivity and high specificity so as to detect possibly all strains of adr and adw sub-types. Thus, the kit for detecting HBV according to the present invention has excellent specificity compared to products which are able to detect various ranges of virus but have low specificity to strains of adr and adw subtypes mostly found in Korea and other areas in the East Asia, and which may cause a false positive reaction.

[196] Moreover, a user of the present invention can choose as a detecting method, the enzyme immunoassay method wherein a POD enzyme is conjugated to DIG of a hybridized probe, or the chemiluminescent method wherein AP enzyme is attached. Thus, the present invention provides a user with a financial advantage since the user does not have to additionally purchase another detecting equipment.

[197] In addition, the multi-probe used in the present invention has an effect of enhancing the signal intensity because two types of probes are hybridized at the same DNA strand at the same time. Thus, the present invention can enhance the detection rate not only at a high concentration but also at a low concentration.

Claims

[1] A quantitative method for Hepatitis B Virus (HBV-DNA) by means of

Polymerase Chain Reaction (PCR) hybridization process, characterized by using primers having sequence numbers 1 and 2 and probes having sequence numbers of 3 and 4. [2] The method according to claim 1, characterized in that HBV-DNA is quantified by means of an enzyme immunoassay using peroxidase (POD) enzymes. [3] The method according to claim 1, characterized in that HBV-DNA is quantified by means of a chemiluminescent method using alkaline phosphatase (AP) enzymes. [4] The method according to any one of claims 1 to 3, characterized in that HBV is either an adr subtype or an adw subtype. [5] Primers having base sequence number 1 or 2 and being used for quantifying

HBV-DNA by the PCR hybridization process. [6] Primers according to claim 5, characterized in that HBV is either an adr subtype or an adw subtype. [7] Probes having base sequence number 3 or 4 and being used for quantifying

HBV-DNA by the PCR hybridization process. [8] Probes according to claim 7, characterized in that HBV is either an adr subtype or an adw subtype. [9] A kit for detecting HBV-DNA, characterized by comprising the primers of claim

5 and the probes of claim 7. [10] The kit for detecting HBV-DNA according to claim 9, characterized in that HBV is either an adr subtype or an adw subtype.