KR0152457B1 - Kit for the detection of hbv dna - Google Patents

Abstract

The present invention provides a simple way to detect HBV DNA in serum economically and with high sensitivity in a short time by simply obtaining a viral DNA from the serum, and using the same to confirm the PCR product by electrophoresis on agarose gel after two steps of PCR. A hepatitis B virus detection kit comprising a detection reaction primer and reagent suitable for use in a method that can be used.

Description

Non-B hepatitis virus DNA detection kit

FIG. 1 shows the results of detecting HBV DNA from the serum of a patient by PCR using a BCO1 / BCO2 primer set.

2 shows the detection limit of PCR technique using recombinant HBV DNA. A is the result of agarose gel electrophoresis and EtBr staining of the first PCR product using BSO1 / BSO2, and B is [γ- ³² P]. Southern blot hybridization using labeled probes, C shows agarose gel electrophoresis and EtBr staining of two-step PCR products using BSO1 / BSO2 and BSI1 / BSI2.

The present invention relates to a detection kit that can detect DNA of hepatitis B virus (hereinafter referred to as 'HBV') in serum economically, simply and quickly and with high sensitivity. More specifically, the present invention simply obtains the viral DNA from the serum, and uses it to perform PCR in one or two stages, and then confirms the PCR product by electrophoresis on agarose gel to shorten the HBV DNA in the serum without contamination. A hepatitis B virus detection kit comprising primers and reagents for detection reactions suitable for use in methods that can be detected economically and with high sensitivity in time.

The detection kit of the present invention is directed to a hepatitis B virus DNA detection kit comprising a primary PCR primer, a secondary PCR primer, a 10-fold concentration PCR buffer, dNTP (dATP, dCTP, dTTP and dGTP) and a heat resistant polymerase. In the primary PCR primers, BSO1 (5'-GACAAGAATCCTCACAATACC-3 ') and BSO2 (5'-ACCACATCATCCATATAACTGAAAG-3'), and the primers for the secondary PCR are BSI1 (5'-ACCTCCAATCACTCACCAACC-3 ') and BSI2 (5 '-AGGATGATGGGATGGGAATA-3'), and 10-fold concentration PCR buffer were 500 mM Tris / HCl, pH8.3, 200 mM KCl, 30 mM MgCl ₂ , 5% dimethyl sulfoxide and 2.5 mg / ml bovine serum albumin. Hepatitis B virus DNA detection kit, characterized in that using a buffer consisting of.

Another detection kit of the present invention comprises primer BCO1 (5'-TCTCTTGTTCATGTCCTACTGTTC-3 '), primer BCO2 (5'-AAGCTGGAGGAGTGCGAATC-3'), a 10-fold concentration PCR buffer of the composition, dNTP and heat resistant polymerase Hepatitis B virus DNA detection kit.

Hepatitis B virus (HBV) is a type of hepadna-viridae that is known to be one of the most important causes of various liver diseases, especially liver cancer. HBV has been actively studied in molecular biology, immunology and biochemical perspectives using genetic recombination techniques since it was first discovered in serum by Brauch, S. Blumberg in 1963 (Tiollas, P). , Science 213, 406-411 (1981).

In order to detect HBV DNA in the serum with high sensitivity, a DNA amplification method using PCR is generally used. For example, Kaneko et al. Detected HBV DNA by Southern blot hybridization after PCR (Kaneko et al., Proc. Natl. Acad. Sci. USA 86, 312-316 (1989)), or step 2 PCR the nested PCR (Kaneko et al., J. Clin. Microbiol. 27, 1930-33 (1989)) using the HBV DNA was detected in 10 ^-5 pg. In addition, HBV DNA in serum may be concentrated using IgM monoclonal antibodies against HBV coat proteins as a method of increasing sensitivity (Liang et al., J. Clin. Invest. 84, 1367-71). (1989)). In general, when PCR is used to detect HBV DNA, sensitivity varies by 10 to 100 times depending on the type of primer used, and it is known that analysis of PCR products by Southern blot hybridization can obtain the most sensitive results. Wang et al. J. Infect. Disease 163, 397-399 (1991). However, the above detection methods were used to incubate serum with protease K, sodium dodecyl sulfate (SDS), ethylenediamine tetraacetic acid (EDTA), and Tris buffer to obtain HBV DNA template for PCR. Extracting nucleic acid with phenol / chloroform and precipitating with ethanol requires much effort to obtain DNA template for PCR, and it is more difficult than general immunological measurement when searching a large number of patient samples. In addition, since the PCR device used here uses a plastic tube, the time required to carry out 30 cycles is too long (2 to 5 hours), and the yield is low and the method is inconvenient because the mineral oil is added to the reaction solution. 10μl is enough, but the reagents such as expensive enzymes and primers are big because more than 50μl of reaction products should be used as the conditions on the device.

Accordingly, there is a need for reagents and kits comprising the same that can detect HBV DNA in serum with greater sensitivity and convenience.

Accordingly, the present inventors have steadily studied to solve the problems of the prior art and to develop an effective HBV DNA detection reagent, using a capillary thermal cycler after simply obtaining a DNA template without extracting DNA from serum. Primers and reagents suitable for use in the method of rapidly detecting HBV DNA in serum by performing PCR in one or two steps and then confirming the product separated by agarose gel electrophoresis by EtBr staining. Developed.

It is an object of the present invention to provide primers and reagents for detecting HBV DNA for the easy and rapid detection of HBV DNA in serum by one-step or two-step PCR.

Hereinafter, the present invention will be described in detail.

The present invention first provides a detection kit for use in detecting HBV DNA in serum using a one-step PCR method. In the detection kit of the present invention, primers BCO1 and BCO2 of the core region having the following sequence are used:

Primer BCO1 5'-TCTCTTGTTCATGTCCTACTGTTC-3 '

Primer BCO2 5'-AAGCTGGAGGAGTGCGAATC-3 '

In addition, the primers for the first PCR of the detection kit for use in the method of detecting the HBV DNA in the serum easily and quickly with high sensitivity using the first and second two-step PCR methods have the following sequence: The BSO1 and BSO2 parts of the surface are:

Primer BSO1 5'-GACAAGAATCCTCACAATACC-3 '

Primer BSO2 5'-ACCACATCATCCATATAACTGAAAG-3 '

In addition, primers for secondary PCR are BSI1 and BSI2 having the following sequences:

Primer BSI1 5'-ACCTCCAATCACTCACCAACC-3 '

Primer BSI2 5'-AGGATGATGGGATGGGAATA-3 '

The positions of the primers on the HBV DNA are expressed by the nucleotide number defined by Fujiyama et al. (A. Fujiyama et al., Nucleic Acids Res, 11, 4601-4610 (1983)).

The primers can be synthesized according to well-known DNA synthesis methods such as phosphoramidite method, in the present invention was synthesized by requesting Oligo company (Oligo Etc. Inc).

In addition, the detection kits of the present invention include a buffer for capillary PCR, which is provided in a buffer of 10-fold concentration and the composition is 200 to 500 mM Tris / HCl, pH8.3, 100 to 500 mM KCl, 15 to 40 mM MgCl, 3 To 6% dimethylsulfoxide and 1.0 to 5.0 mg / ml bovine serum albumin, preferably 500 mM Tris / HCl, pH8.3, 200 mM KCl, 30 mM MgCl, 5% dimethylsulfoxide and 2.5 mg / ml bovine serum Albumin.

The 10-fold concentration PCR buffer is added to 1/10 of the reaction volume when used, and the template DNA, the appropriate amount of heat-resistant DNA polymerase, and the concentration of dNTP (dGTP, dTTP and dCTP, respectively) of 10 μM to 500 μM, preferably 50 μM, respectively. 50 μM) is added.

HBV DNA detection method in the serum using the detection kit of the present invention is as follows.

Boil serum, dilute with distilled water and centrifuge to obtain supernatant; Performing a first polymerase chain reaction (PCR) using the supernatant; Amplifying the DNA by performing a secondary PCR using a portion of the PCR product; The amplified DNA can be confirmed by agarose gel electrophoresis. However, in the method for detecting HBV DNA by one step PCR, only the first polymerase chain reaction is performed.

This will be described in detail as follows.

First, the serum of the patient is boiled for several minutes and then quenched in ice, and the supernatant obtained by centrifugation is diluted 5 to 1000 times with distilled water and used as a DNA template.

In the above, the temperature of boiling the serum is preferably 90 ° C. to 98 ° C., particularly 94 ° C., and the boiling time is preferably 1 minute to 10 minutes, particularly 4 minutes. The method is much faster and more economical than conventional DNA extraction. The primary PCR is performed using the DNA template obtained above. At this time, primers BCO1 and BCO2 are used as primers for the first stage PCR, and primers BSO1 and BSO2 are used as the primers for the two-step PCR.

In the first PCR, primers BSO1 and BSO2 are used in the range of 0.5 μM to 50 μM, preferably 1 μm, based on 10 μl of the reaction volume. The PCR buffer solution 1 μl and 1 μl of 10-fold PCR buffer solution, 0.4 μl, respectively. To 0.8 units of heat resistant DNA polymerase, 10-500 μM of dNTP and residual volume of distilled water. The PCR reaction is carried out in a known reaction cycle using a capillary temperature cycler. For the detection method by one-step PCR, the first PCR product was electrophoresed on agarose gel and stained with EtBr to confirm the presence of HBV DNA and continued in the two-step PCR method for more sensitive detection. Thus, a part of the primary PCR product is used as a template to perform secondary PCR. At this time, primers BSI1 and BSI2 are used as primers, and the reaction conditions are the same as those of the primary PCR.

The PCR reactions use a capillary temperature cycler (FTC 2000, manufactured by Daehan Medical Systems), so the amount of reactants required is small, the reaction is completed in a short time, and the probability of contamination is greatly reduced. After completion of the second PCR, the product was electrophoresed on agarose gel and stained with EtBr to confirm the presence of hepatitis B virus DNA in serum.

HBV DNA detection method by PCR using the detection kit of the present invention can detect HBV DNA without contamination problem, in particular, by the two-step PCR detection method by the two-step PCR detection kit 10 It is very economical and simple to detect sensitively up to pg.

Hereinafter, the method for detecting HBV DNA in the serum with high sensitivity using the diagnostic kit of the present invention will be described in more detail as an example of use. The following uses and comparisons are given merely to illustrate the invention and are intended to limit the scope of the invention in any aspect.

[Example]

(Step 1) Preparation of template DNA

5 μl of serum of hepatitis B patient was boiled at 94 ° C. for 4 minutes, quenched in ice, and centrifuged at 12,000 rpm for 5 minutes using an Eppendorf microcentrifuge. 10 µl of distilled water was added and diluted to 5 µl of the supernatant obtained above.

(Step 2) Primary PCR

1μM of primer BSO1, 1μM of primer BSO2 was added to the capillary, and 1μl of the DNA solution obtained above was added as a template. 1 μl of dimethyl sulfoxide (DMSO) and 2.5 mg / ml bovine serum albumin (BSA) were added and 50 μM dNTP (50 μM dGTP, 50 μM dATP, 50 μM dTTP, 50 μM dCTP), 0.4 unit of Taq DNA polymerization, respectively. Enzyme was included and distilled water was added to make the total volume to 10 mu l. Place the capillary containing the reaction solution into a capillary thermal cycler (FTC 2000, manufacturer: Korea Medical Systems) 95 ℃, 5 seconds; 55 ° C., 10 seconds; 30 cycles of PCR were conducted at 72 ° C for 15 seconds.

(Step 3) Secondary PCR

PCR was carried out in the same manner as in Step 2 using 1 µl of the primary PCR product as a template and BSI1 and BSI2 as primers.

(Step 4) Agarose gel electrophoresis and EtBr staining

The PCR product of step 3 was electrophoresed on 2% agarose gel and stained with EtBr to confirm the presence of HBV DNA.

In order to determine the detection limit of the method, the result of using the recombinant HBV DNA (type: adr, source: Genetic Engineering Center) in 10-fold dilutions in order as a template for PCR is shown in FIG. In Figure 2, A was able to detect up to 0.01 pg as a result of staining with EtBr after agarose gel electrophoresis of the product of primary PCR using primers BSO1 / BSO2, and B was primary PCR using primers BSO1 / BSO2. Product of [γ- P] As a result of Southern blot hybridization using a labeled probe 10 Up to pg, C was detected, and C was electrophoresed on agarose gel, and the product produced through two-step PCR was stained with EtBr. Up to pg could be detected. In FIG. 2, column 1 is ØX 174 DNA digested with HaeIII, column 2 is a reagent control that does not contain DNA, column 3 is a negative control, columns 4 to 12 are 1 pg, 0.1 pg, 10 pg, 10 pg, 10 pg, 10 pg, 10 pg, 10 pg, and 10 This is the result of amplifying the DNA of pg.

Meanwhile, a template DNA solution was prepared as in Step 1, PCR was performed as in Step 2 using primers BCO1 and BCO2, and then agarose gel electrophoresis and EtBr staining were performed as in Step 4 to prepare a template DNA solution. The same result as 1 degree was obtained.

In FIG. 1, columns 1, 2, and 3 are the same samples as in FIG. 2, and columns 4 to 12 are HBV positive or negative serum, resulting from contamination of the primers BCO1 and BCO2 from the results of FIG. HBV DNA can be detected without false positives.

Claims (11)

In the hepatitis B virus DNA detection kit comprising a primer for PCR, a 10-fold concentration PCR buffer, dNTP (dATP, dCTP, dTTP and dGTP), and a heat resistant polymerase, (A) the primary PCR primer is BSO1 (5 The primers for '-GACAAGAATCCTCACAATACC-3') and BSO2 (5'-ACCACATCATCCATATAACTGAAAG-3 '), and (b) secondary PCR are BSI1 (5'-ACCTCCAATCACTCACCAACC-3) and BSI2 (5'-AGGATGATGGGATGGGAATA-3'), And (c) the 10-fold concentration PCR buffer contains 200-500 mM Tris / HCl, pH8.3, 100-500 mM KCl, 15-40 mM MgCl ₂ , 3-6% dimethyl sulfoxide and 1.0-5.0 mg / Hepatitis B virus DNA detection kit for a two-step PCR, characterized in that the buffer solution consisting of ml of bovine serum albumin. (A) Boil the serum, dilute it with distilled water, and centrifuge to obtain a supernatant. (B) Perform single-chain polymerase chain reaction (PCR) using the supernatant, and (C) a part of the PCR product. Amplifying the DNA of the hepatitis B virus present in the serum by performing a double-winding PCR using, (D) checking the amplified DNA, hepatitis B virus DNA detection method in the serum. The detection method according to claim 2, wherein in step (a), the serum is boiled at 94 ° C for 4 minutes. The method of claim 2, wherein the primary and secondary PCR are performed using a capillary thermal cycler. According to claim 2, wherein the first PCR reaction solution of step (b) is the supernatant of step (a), the primers BSO1 and BSP2 and 10-fold concentration PCR primer for the first step, and each dNTR (dGTP) , dATP, dTTP, dCTP) and Taq DNA polymerase. The total volume of the first PCR reaction solution of step (b) is 10 μl, and 1 μl of the supernatant of step (a), 1 μM of primers BSO1 and BSO2, and 1 μl of 10-fold concentration PCR buffer. And a dNTP concentration of 50 μM and 0.4 unit of Taq DNA polymerase. According to claim 2, wherein the (b) the first PCR reaction of 95 ℃, 5 seconds; 55 ° C., 10 seconds; And 72 cycles of 15 seconds at 72 ° C. According to claim 2, wherein the second PCR reaction solution of step (c) is a part of the reaction product of step (b), primers BSI1 and BSI2 and 10-fold PCR buffer of claim 1, and each dNTP and Tap DNA polymerase Detection method comprising a. The method according to claim 8, wherein the total volume of the secondary PCR reaction solution of step (c) is 10 μl, and 1 μl of the reaction product of step (b), 1 μM each of primers BSI1 and BSI2, and 1 μl of 10-fold concentration PCR buffer. And a dNTP concentration of 50 μM and 0.4 unit of Taq DNA polymerase. The detection method according to claim 1, wherein the 10-fold concentration PCR buffer solution of (C) consists of 500 mM Tris / HCl, pH8.3, 200 mM KCl, 30 mM MgCl ₂ , 5% dimethylsulfoxide and 2.5 mg / ml bovine serum albumin. Kit. The detection method according to claim 2, wherein the secondary PCR reaction of step (c) is performed 30 times under the same conditions as the primary.