KR20070037234A - Kit for detecting of hbv specific gene - Google Patents

Abstract

The present invention includes a PCR reaction mixture, HBsAg (surface antigen region) gene specific probe and primer mixture of HBV, internal control gene specific probe and primer mixture, positive control gene sample, internal control gene sample and sterile water for reaction. The present invention relates to a method for detecting HBV specific genes using an HBV specific gene detection kit and an electric kit. When using the HBV detection kit of the present invention, since the hepatitis B specific gene can be detected more easily and accurately, it can be widely used for accurate diagnosis of hepatitis B.

HBV specific gene, HBV specific gene detection kit

Description

Kit for Detecting of HBV Specific Gene

The present invention relates to an HBV specific gene detection kit. More specifically, the present invention relates to PCR reaction mixtures, HBsAg (surface antigen region) gene-specific probe and primer mixtures, internal control gene-specific probe and primer mixtures, positive control gene samples, internal control gene samples and reactions. The present invention relates to a method for detecting HBV-specific genes using an HBV-specific gene detection kit and an electric kit including sterile water.

Hepatitis B is a viral disease commonly occurring in tropical Africa, Southeast Asia and Far East Asia, and is known to be caused by hepatitis B virus (HBV). The previous HBV is a DNA virus with a coating belonging to the Hepadnavirus family, and serologically according to the virus's surface antigen (HBsAg), there are 8 subtypes, namely, ayw, ayw2, ayw3, ayr, adw, adw2, adw4 and adr. To date, hepatitis B is infected with about 300 million people worldwide, and it is known that infection causes hepatitis or cirrhosis and even chronic hepatitis (see Tiollais). and Buenda, Scientific American, 246, 48 (1991); Hu et al., PNAS, 87, 7140 (1990); Wei et al., J. Med. Virol., 45, 82 (1995).

Early detection of HBV is considered very important because hepatitis B can be treated easily in the early stages of the onset of disease. Therefore, studies on the method of early diagnosis of HBV infection are being actively conducted. have. Initially, immunological methods using antibodies capable of recognizing specific proteins of HBV have been intensively studied, but due to various shortcomings such as the large amount of samples required for testing, the high cost of antibody production, and the difficulty of storage, As a result, a method using PCR has been developed.

For example, Korean Patent Registration No. 41318 discloses a method for diagnosing hepatitis B by detecting a surface antigen gene of HBV from a patient's sample, and Korean Patent Registration No. 180987 discloses a method for detecting HBV from a patient's sample. A method for diagnosing hepatitis B by detecting a gene of X protein, which is a regulatory protein, is disclosed. Korean Patent Laid-Open Publication No. 2005-15407 includes a DNA chip containing an HBV-specific gene. A diagnostic kit for diagnosing infection is disclosed, and Korean Patent Publication No. 2003-47954 discloses a diagnostic kit for diagnosing HBV infection from a patient's sample using a multiplex PCR method. As such, a method using a PCR or a diagnostic kit may be used when the HBV gene is sufficiently present in a patient's sample. Therefore, a sample obtained from an initial patient who is not sufficiently infected with HBV may not be easily detected. It may be necessary, and the disadvantage is that even a small contamination of the sample shows a large error.

As a method for overcoming the above-mentioned disadvantages, recently, diagnostic kits using a real-time RT-PCR method capable of quantitating target RNA from a trace amount of sample have been developed and marketed. However, the diagnostic kits have problems of stability and reproducibility by providing RNA as a standard gene, or do not include reference gene quantification primers and probes used for standardization, and thus, accurate diagnosis cannot be performed. Therefore, efforts have been made to develop a method for more easily and accurately detecting HBV-specific genes infected with patients, but there are no results.

Therefore, there is a constant need to develop a method for detecting HBV-specific genes that are more easily and accurately infected with patients.

Accordingly, the present inventors have made efforts to develop a method for more easily and accurately detecting HBV-specific genes infected with patients. As a result, the present inventors include HBV-specific gene-specific primers and probes and internal control gene-specific primers and probes. When the HBV specific gene detection kit is used, it was confirmed that the HBV specific gene can be detected more simply and accurately, and thus the present invention has been completed.

After all, the main object of the present invention is to provide a kit capable of detecting HBV specific genes simply and accurately.

Another object of the present invention is to provide a method for detecting an HBV specific gene using an electric kit.

The HBV detection kit of the present invention comprises (i) a PCR reaction mixture comprising a buffer solution, DNA polymerase, KCl, MgCl ₂ , dATP, dCTP, dGTP, and dUTP; (Ii) a mixture of HBV probes and primers consisting of a sense primer (SEQ ID NO: 1), an antisense primer (SEQ ID NO: 2), and a probe (SEQ ID NO: 3), specific for the HBV surface antigen region (HBs); (Iii) an internal control probe and primer mixture consisting of an internal control gene specific, a sense primer (SEQ ID NO: 4), an antisense primer (SEQ ID NO: 5) and a TaqMan probe (SEQ ID NO: 6); (Iii) a positive control gene sample comprising all or part of the HBsAg gene of HBV; (Iii) an internal control gene sample comprising all or part of the BCR-ABL fusion gene in chronic myelogenous leukemia; And (iii) sterile water for reaction. At this time, the buffer solution is not particularly limited, but it is preferable to use a neutral buffer solution of pH 7 to 8, and (NH ₄ ) ₂ SO ₄ , calf serum albumin, EDTA, ROX (6-carboxy-X) in the PCR reaction mixture rhodamine) or mixtures thereof. In addition, the HBV specific probe is not particularly limited thereto, but the 6'carboxylate (6-carboxyfluorescein), hexachloro-6-carboxyfluorescein or tetrachloro-6 at the 5 'end It is preferable to use one labeled with tetrachloro-6-carboxyfluorescein and one labeled with 6-carboxytetramethyl-rhodamine at the 3 'end, and the internal control specific probe is Although not limited thereto, the 5 'end is labeled with 6-carboxyfluorescein as a fluorescent substance, the 19th thymine base from the 5' end is labeled with 6-carboxytetramethyl-rhodamine as a fluorescent substance, and the 3 'end is labeled. Preference is given to using this phosphorylated one.

The inventors of the present invention have sought to improve the conventional kits while researching various ways to detect HBV in a patient more effectively.

Among the kits for detecting HBV in patients used up to now, the kit using the real-time polymerase chain reaction device is known to have a relatively high accuracy. The technical principle is as follows: First, the HBV-specific gene in the sample As a template, when performing a polymerase chain reaction using an appropriate primer, a probe capable of binding to the HBV specific gene is added. In this case, the probe used is manufactured to label two different types of fluorescent materials at the 3 'end and the 5' end, respectively, and when the two used fluorescent materials exist within a predetermined interval, the fluorescence generated therein may cause mutual interference. In this case, a fluorescence does not develop. As a result, when the polymerase chain reaction is carried out, the polymerase chain reaction is sequentially performed by a DNA polymerase which binds a primer to one end of the template and recognizes it, and in the middle of the template, two kinds of fluorescent substances Labeled probes are bound. Subsequently, when the polymerase reaches the site to which the electric probe is bound, since the probe is sequentially decomposed from the 5 'end labeled with the fluorescent material, the polymerization reaction is continuously performed, and thus, the fluorescent material labeled at the 5' end is lost. Results. Accordingly, the interference phenomenon disappears, and the fluorescent substance labeled at the 3 'end undergoes color reaction, indicating that the HBV specific gene is amplified.

In addition, the present inventors used an internal control group to confirm whether PCR was normally performed. That is, if PCR was performed with the HBV-specific gene by using a gene that is normally expressed in a sample obtained from a patient regardless of whether HBV was infected as an internal control group, PCR was not normally performed if the internal control group was not amplified. Since it can be considered that it is not, it was possible to increase the reliability of the detected results.

When the HBV detection kit of the present invention is used, hepatitis B-specific genes can be detected more simply and accurately, and thus may be widely used for accurate diagnosis of hepatitis B: ie, genetic analysis samples and electric HBV detection. To the positive control gene sample and the internal control gene sample of the kit, the HBV probe and primer mixture, the internal control probe and primer mixture, and the PCR reaction mixture of the electric HBV detection kit were added and mixed, respectively, and then polymerase chain reaction (PCR) was performed. The HBV PCR product and the internal control PCR product of the genetic analysis sample and the positive control were obtained, respectively, and the amplification of the electric genetic analysis sample, the positive control gene sample and the internal control gene sample was confirmed. HBV specific genes can be detected more simply and accurately.

In addition, such a measuring method may be performed using a commercially available real-time polymerase chain reaction device, but the real-time polymerase chain reaction device is not particularly limited thereto, but LightCycler ^TM (Roche, Germany), ABI PRISM ^TM 7000/7700 (Applied Biosystems, USA), iCycler ^™ (Bio-Rad, USA), Rotor-Gene ^™ (Corbett, Australia), Opticon ^™ (PharmaTech, USA) and the like are preferably used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of HBV Detection Kit

Preparation of HBV detection kit consisting of 1.25 ml of PCR reaction mixture, 300 μl of HBV probe and primer mixture, 300 μl of internal control probe and primer mixture, 50 μl of positive control gene sample, 400 μl of internal control gene sample and 1 ml of sterile water for reaction It was.

Example 1-1 Preparation of PCR Reaction Mixtures, HBV Probe and Primer Mixtures, and Internal Control Probe and Primer Mixtures

First, PCR reaction mixtures were 100 mM Tris / HCl (pH 8.3), 20 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , ₇ mM MgCl ₂ , 1.625U DNA polymerase, 0.8 mM dUTP, 0.8 mM dATP, 0.8 mM dCTP and 0.8 Prepared by mixing mM dGTP.

Next, the HBV probe and primer mixture is prepared by mixing a sense primer (SEQ ID NO: 1), an antisense primer (SEQ ID NO: 2) and a probe (SEQ ID NO: 3) specific for the HBV specific gene having the following nucleotide sequence, Their 5 'ends were labeled with 6-carboxyfluorescein and the 3' ends were labeled with 6-carboxytetramethyl-rhodamine.

Sense primer: 5'-gccatttgttcagtggttcg-3 '(SEQ ID NO: 1)

Antisense Primer: 5'-aggtaaaaagggactcaagatgttgtac-3 '(SEQ ID NO: 2)

Probe: 5'-agggctttcccccactgtttggct-3 '(SEQ ID NO: 3)

Finally, the internal control probe and primer mixture was prepared by mixing the internal control gene specific, sense primer (SEQ ID NO: 4), antisense primer (SEQ ID NO: 5) and probe (SEQ ID NO: 6) having the following base sequence. At this time, the 5 'end of the probe was labeled with VIC, a fluorescent substance, and the 19th thymine base was labeled with 6-carboxytetramethyl-rhodamine, a fluorescent substance, from the 5' end, and the 3 'end was phosphorylated.

Sense primer: 5'-cagactgtccacagcattcc-3 '(SEQ ID NO: 4)

Antisense Primer: 5'-ccaacgagcggcttcact-3 '(SEQ ID NO: 5)

Probe: 5'-agaccctgaggctcaaagtcagatgctact-3 '(SEQ ID NO: 6)

Example 1-2 Preparation of Positive Control Gene Sample

As a positive control gene sample, a recombinant plasmid containing a 112 bp HBV surface antigen gene was used. Specifically, the DNA of the HBV surface antigen is isolated from ACCURUN 325 HBV DNA positive control (BBI Diagnostics, MA, USA), used as a template, and PCR is performed using the following primers to perform a portion of the 112 bp HBV surface antigen gene. The recombinant plasmid DNA prepared by inserting it into an expression vector (PCR 2.1 TOPO vector) was used as a positive control gene sample.

Sense primer: 5'-gccatttgttcagtggttcg-3 '(SEQ ID NO: 7)

Antisense Primer: 5'-aggtaaaaagggactcaagatgttgtac-3 '(SEQ ID NO: 8)

Example 1-3 Preparation of Internal Control Gene Sample

As an internal control gene sample, a recombinant plasmid containing a b3a2 fusion gene, which is a BCR-ABL fusion gene of chronic myelogenous leukemia, was used. Specifically, using the RNA extraction kit (RNAqueous kit, Ambion, USA), K562 RNA was extracted from the K-562 cell line (ATCC No. CCL-243), 443bp b3a2 using cDNA synthesized from the electric K562 RNA A fusion gene was obtained and inserted into an expression vector (PCR 2.1 TOPO vector) to obtain a recombinant plasmid DNA, which was then diluted to 200 copies to prepare an internal control gene sample.

Example 1-4 : Preparation of HBV Detection Kit

1.25 ml of the PCR reaction mixture prepared in Example 1-1, 300 µl of the HBV probe and primer mixture prepared in Example 1-1, 300 µl of the internal control probe and primer mixture prepared in Example 1-1, An HBV detection kit consisting of 50 μl of the positive control gene sample prepared in Examples 1-2, 400 μl of the internal control gene sample prepared in Examples 1-3 and 1 ml of semi-application sterile water was prepared.

Example 2 Detection of HBV Specific Genes Using HBV Detection Kit

Using the HBV detection kit prepared in Example 1, HBV-specific genes were detected from samples of normal and hepatitis B patients, and it was confirmed whether the normal and hepatitis B patients could be distinguished.

Example 2-1 Preparation of Sample to be Measured

First, serum 1, 0.5 and 0.2 ml of treated hepatitis B patients (Experimental Groups 1-1 to 1-3) and serum 1, 0.5 and 0.2 ml of untreated hepatitis B patients (Experimental Groups 2-1 to 2-) 3) were obtained respectively, each serum was applied to a DNA purification kit (High pure nucleic acid purification kit, Roche, Germany) to extract the respective DNA, which was used as a sample.

Example 2-2 : Amplification of Samples and Analysis of Amplification Results

Prepare a reaction tube by dispensing 12.5 μl of the PCR reaction mixture prepared in Example 1-1, 3 μl of the HBV probe and primer mixture, 3 μl of the internal control probe and primer mixture, and 1.5 μl of sterile water. It was. Subsequently, the positive control gene sample prepared in Examples 1-2, the internal control gene sample prepared in Examples 1-3, and the experimental group 1- obtained in Example 2-1 were prepared in each of the reaction tubes. Dispense 5 μl of DNA from 1 to 2-3, apply it to ABI PRISM ^TM 7000/7700 (Applied Biosystems, USA), a real-time polymerase chain reaction device, and perform PCR to perform PCR with the HBV positive control PCR product. Sample PCR products were each obtained. In this case, PCR is denatured for 10 minutes at 95 ° C., and amplification is performed by repeating a series of 45 times of reaction for 20 seconds at 95 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C. It was. During the amplification, it was confirmed that fluorescence derived from 6-carboxytetramethyl-rhodamine labeled at the 3 'end was continuously developed, and thus amplification was normally performed. Subsequently, electro-amplified PCR fragments were obtained, followed by electrophoresis, and the presence or absence of the desired fragments was confirmed by O and X (see Table 1). In this case, O means that the target gene is amplified and confirmed as a band on the electrophoresis, X means that the target gene is not amplified and thus not identified as a band on the electrophoresis.

PCR amplification results for each sample Experimental group Positive control gene Internal control genes Sample genes Experimental Group 1-1 O O X Experimental group 1-2 O O X Experimental Group 1-3 O O X Experimental Group 2-1 O O O Experimental Group 2-2 O O O Experimental Group 2-3 O O O

As shown in Table 1, the sample genes of the experimental groups 1-1 to 1-3 for the normal group were not amplified, and the sample genes of the experimental groups 2-1 to 2-3 for the hepatitis B patients were Since it was amplified, it was found that when using the HBV detection kit of the present invention, hepatitis B patients can be distinguished.

On the other hand, it was found that both the positive control gene of 112bp and the internal control gene of 443bp were amplified in all experimental groups. At this time, the positive control gene was amplified by the sense primer (SEQ ID NO: 1), and the sample gene was not amplified nonspecifically from the amplified positive control gene, and it was analyzed that there was no error in the electric PCR amplification from the amplified internal control gene. .

As described and demonstrated in detail above, the present invention provides a PCR reaction mixture, a HBsAg (surface antigen region) gene-specific probe and primer mixture, an internal control gene-specific probe and primer mixture, a positive control gene sample, an internal control gene sample. And it provides a method for detecting the HBV-specific genes using the HBV-specific gene detection kit and electric kit comprising a sterile water for reaction. When using the HBV detection kit of the present invention, since the hepatitis B specific gene can be detected more easily and accurately, it can be widely used for accurate diagnosis of hepatitis B.

<110> BIOSEWOOM INC. <120> Kit for Detecting of HBV Specific Gene <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gccatttgtt cagtggttcg 20 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aggtaaaaag ggactcaaga tgttgtac 28 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 3 agggctttcc cccactgttt ggct 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cagactgtcc acagcattcc 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ccaacgagcg gcttcact 18 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 agaccctgag gctcaaagtc agatgctact 30 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gccatttgtt cagtggttcg 20 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aggtaaaaag ggactcaaga tgttgtac 28

Claims (3)

(Iii) a PCR reaction mixture comprising buffer, DNA polymerase, KCl, MgCl ₂ , dATP, dCTP, dGTP and dUTP; (Ii) a mixture of HBV probes and primers consisting of a sense primer (SEQ ID NO: 1), an antisense primer (SEQ ID NO: 2), and a probe (SEQ ID NO: 3), specific for the HBV surface antigen region (HBs); (Iii) an internal control probe and primer mixture consisting of an internal control gene specific, a sense primer (SEQ ID NO: 4), an antisense primer (SEQ ID NO: 5) and a TaqMan probe (SEQ ID NO: 6); (Iii) a positive control gene sample comprising all or part of the HBsAg gene of HBV; (Iii) an internal control gene sample comprising all or part of the BCR-ABL fusion gene in chronic myelogenous leukemia; And, (Iii) An HBV detection kit comprising sterile water for reaction. The method of claim 1, HBV specific probes have 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, or tetrachloro-6-carboxyfluorescein at the 5 'end. 6-carboxyfluorescein), thymine in the 10-20th base from 5 'is labeled with 6-carboxytetramethyl-rhodamine, the 3' end is phosphorylated, and internal control specific probe The 5 'end is labeled with 6-carboxyfluorescein as a fluorescent substance, the 19th thymine base from the 5' end is labeled with 6-carboxytetramethyl-rhodamine as a fluorescent substance, and the 3 'end is phosphorylated. By HBV detection kit. To the genetic analysis sample, the positive control gene sample of claim 1 and the internal control gene sample of claim 1, HBV probe and primer mixture, internal control probe and primer mixture and PCR reaction mixture of the HBV detection kit of claim 1 were respectively added and mixed. , PCR was performed to obtain the HBV PCR product and the internal control PCR product of the genetic analysis sample and the positive control, respectively, and the amplification of the electric genetic analysis sample, the positive control gene sample and the internal control gene sample Including the step of identifying, HBV specific gene detection method.