JP2001352989A - Dna, reagent for dna probe method and dna probe method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a DNA capable of carrying out a high sensitive assay when detecting HBV by the use of a DNA probe method and provide a reagent for DNA probe method using the above DNA and a DNA probe method. SOLUTION: This DNA is a DNA comprising at least 15 bases. The DNA is shown by the following (a) or (b): (a) A DNA successively selected from the base sequence represented by the sequence number 1 (see specification) or its complementary base sequence. (b) A DNA obtained by deleting, substituting or adding one or plural bases to the DNA successively selected from the base sequence represented by the sequence number 1 or its complementary base sequence. This DNA is hybridized with the DNA No.21-710 in the hepatitis B virus DNA under a stringent condition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA useful for the detection of hepatitis B virus using a DNA probe method, a DNA probe method reagent using the above DNA and D
It relates to the NA probe method.

[0002]

2. Description of the Related Art Hepatitis B is known as hepatitis B virus (HB).
V) is a disease that causes not only acute hepatitis but also chronic hepatitis, cirrhosis, and may lead to liver cancer. HBV is a particle also called a Dane particle, and the Dane particle has a double particle structure composed of an envelope and an inner nucleus-like structure. This coat is an HBs antigen, and the inner nucleus-like structure contains HBc antigen, HBe antigen, DNA polymerase and the like. HBs antigens have also been found in small spherical and tubular particles in the serum of infected patients.

[0003] Diagnosis of hepatitis B infection is based on the presence or absence of viral antigens or antibodies in human serum. Among such diagnostic methods, detection of HBV DNA by a nucleic acid probe method is known as a method that can detect viral infections at various clinical stages with high sensitivity.

Japanese Patent Application Laid-Open No. Sho 62-188970 discloses that a complex containing a single-stranded analyte, a labeling reagent set and a capture reagent set is reacted to form a complex, and the complex is bound to the formed complex. An assay method for measuring the amount of a label, wherein the labeling reagent set comprises a labeling reagent and a labeling probe having a sequence complementary to the labeling reagent recognition sequence and the analyte, wherein the capture reagent set comprises a capture reagent, A nucleic acid sandwich assay comprising a separation means and a capture probe having a sequence complementary to the capture reagent recognition sequence and the analyte is disclosed. In this publication, Bg of HBV DNA
A method is described for analyzing a fragment using a labeled probe and a capture probe each containing 30 DNA sequences of the II fragment.

[0005] JP-A-2-109999 discloses a sandwich hybridization analysis of HBV using a nucleic acid multimer having a branched structure. In addition, Japanese Patent Application Laid-Open No. 5-508862 further discloses that
For the purpose of high-sensitivity measurement, a comb-shaped branched polynucleotide is disclosed as a nucleic acid amplification multimer.

H using a hybridization method
Diagnostic kits are also commercially available for detecting BV DNA, for example, those using dot blot hybridization technology (Journal of Clini).
cal Microbiology, Jan. 199
0. p39-42); using antibody capture solution hybridization technology (Journal of Cl.)
initial Microbiology, Aug. 1
999. pp. 2461-2465). These methods involve adding an HBV-specific RNA probe to
This is for performing NA-DNA hybridization.

[0007] Further, by combining the sandwich hybridization analysis method and the PCR method, HBV DNA
There is also a report of a method for detecting (Journal o
f Clinical Microbiology, J
un. 1990. pp 1411-1416). This is H
The sample was amplified by a PCR method using primers specific for the BV polymerase gene and the X gene.
Sandwich hybridization is carried out using a BamHI fragment containing a gene encoding a Bs antigen bound to a solid phase as a capture probe, and a gene encoding a biotin-labeled HBc antigen as a detection probe.

US Pat. No. 5,780,219 discloses HBV
adw type DNA sequence at positions 403 to 427, 1522
The DNA sequence at positions １５1533, or 2367 to 239
A DNA probe that hybridizes to the second DNA,
Alternatively, a DNA probe complementary thereto is disclosed. The U.S. patent further includes 365 of the HBVadw type.
389, 455-479, 466-490, 1415
-1436, 1557-1587, 2301-233
Also disclosed are amplification primers that hybridize to the DNA sequence at positions 3,2418-2442 or 2421-2444. By using these techniques, hepatitis B virus infection can be distinguished from other virus infections with high sensitivity.

[0009]

SUMMARY OF THE INVENTION An object of the present invention is to provide a
It is an object of the present invention to provide a DNA capable of performing high-sensitivity measurement when detecting HBV using the A probe method, and a reagent for the DNA probe method and a DNA probe method using the DNA.

[0010]

Means for Solving the Problems The present inventors have made intensive efforts to obtain a highly sensitive detection method for examining the presence or absence of HBV infection. As a result, the DNA probe method using a DNA having a specific nucleotide sequence as a capture probe was used. By the D of HBV
Have found that high sensitivity measurement of NA can be performed,
The present invention has been completed. In addition, it was also found that higher sensitivity measurement can be performed by using a specific base sequence of the HBV DNA sequence as a target DNA.

That is, the present invention relates to a DNA comprising at least 15 bases, which is a DNA of the following (a) or (b): (a) a base sequence represented by SEQ ID NO: 1 or a base sequence complementary thereto; In a continuously selected DNA, or (b) a DNA continuously selected from the base sequence of SEQ ID NO: 1 or its complementary base sequence, one or several bases are deleted, substituted or added. And DNA that hybridizes under stringent conditions with DNAs 21-710 of hepatitis B virus DNA. The present invention also provides a reagent for a DNA probe method, wherein the above-mentioned DNA is used as a capture probe. The present invention is also a DNA probe method using the above DNA as a capture probe and hybridizing a target DNA with the capture probe. Hereinafter, the present invention will be described in detail.

The DNA of the present invention comprises at least 15 bases. If it is less than 15 bases, it is too short to be used as a capture probe in the DNA probe method, so that when hybridized with the target DNA, the hybridization efficiency does not increase,
Conversely, the background is increased, and as a result, high-sensitivity measurement cannot be performed. Preferably, 15 to 5
It is 0 bases, more preferably 25 to 35 bases.

In the present invention, the above (a) is a DNA continuously selected from the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence. The nucleotide sequence of SEQ ID NO: 1 is the HBVadr-type DNA of SEQ ID NO: 6.
The sequence corresponds to the sequence of the 150th to 290th sense strand, and its complementary base sequence corresponds to the sequence of the antisense strand (Nucleic Acids Research, 18 (15), 4).
587 (1990)).

(A) D which is continuously selected from the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence
When NA is used as a capture probe in the DNA probe method, the efficiency of hybridization with target DNA is high, and high sensitivity measurement can be performed. Preferably, in the nucleotide sequence of SEQ ID NO: 1, 60 to 110
It is selected continuously from the base sequence or its complementary base sequence. The nucleotide sequence of the 60th to 110th bases is 2 in the HBV DNA sequence of SEQ ID NO: 6.
It corresponds to the sequence of the 10th to 260th sense strand, and its complementary base sequence corresponds to the sequence of the antisense strand. In the present invention, among (a), those selected continuously from a base sequence complementary to the base sequence described in SEQ ID NO: 1 are preferable. A base sequence complementary to the base sequence described in SEQ ID NO: 1 can perform higher sensitivity measurement.

As the above (a), for example, SEQ ID NO: 6
In the HBV DNA sequence described in (1), the base sequences of the 221st to 250th sense strands and antisense strands (also referred to herein as Cap7 and AsCap7, respectively), and the 171st to 200th sense strands and antisense strands Base sequences (NH171 and AsNH17
1), the base sequences of the 181st to 210th sense strands and antisense strands (NH181 and AsNH
181), the nucleotide sequences of the 191st to 220th sense strands and antisense strands (NH191 and As
NH191), nucleotide sequences of the 201st to 230th sense strand and antisense strand (also referred to as NH201 and AsNH201), and nucleotide sequences of the 211st to 240th sense strand and antisense strand (NH211).
And AsNH211), from the 251st to 280th
The base sequence of the third sense strand and the antisense strand (Cap
8 and AsCap8). Preferably, it is AsCap7.

The above-mentioned (b) relates to a DNA selected from the base sequence of SEQ ID NO: 1 or its complementary base sequence in which one or several bases have been deleted, substituted or added. Which is a DNA that hybridizes with the 21st to 710th DNA of the HBV DNA under stringent conditions. Examples of the above (b) include those in which one or several bases are deleted, substituted or added in the base sequence exemplified in the above (a).

It has been reported that the HBV DNA sequence has a number of point mutations in the DNA sequence shown in SEQ ID NO: 6 (Scand. J. Infect. Dis., 28,
9 (1996); Hepato-Gastroenteorology, 45, 500 (199
8); J. Mol. Evol., 44, 583 (1997); FEBS Letters, 4
50, 66 (1999); Journal of Medical Virology, 46, 15
7 (1995)). In the present invention, in a DNA continuously selected from the nucleotide sequence of SEQ ID NO: 1 or its complementary nucleotide sequence, a DNA in which a previously reported point mutation site has been substituted with another nucleotide. Can be used. The point mutation site can be confirmed by comparison with a nucleotide sequence registered in GenBank or the like. For example, in the nucleotide sequence of HBV described in SEQ ID NO: 6, 159, 172, 174, 186, 187,
201, 208, 220, 223, 231, 235, 2
41st, 247th and the like. Examples of such DNA include an inosine-substituted probe described in SEQ ID NO: 9 (also referred to as AsCap7-Inosine); a DNA in which inosine is replaced by another base in the base sequence described in SEQ ID NO: 9; .

In the present invention, as (b), not only the point mutation site already reported above is substituted with another base, but also at other sites,
DNA in which one or several bases are deleted, substituted or added can be used. These are the HBV D
It must be one that hybridizes with the 21st to 710th DNA of the NA under stringent conditions. The 21st to 710th DN of the HBV DNA
As A, for example, DN of HBV described in SEQ ID NO: 6
DNAs at positions 21 to 710 in the A sequence can be mentioned. 21 of the DNA sequence of HBV described in SEQ ID NO: 6
The 710th to 710th DNAs can be suitably used as target probes described later.

As described above, it has been reported that the HBV DNA sequence has a point mutation in the nucleotide sequence of SEQ ID NO: 6, and therefore, the HBV DNA sequence has 21 to 710 positions. May have a point mutation in the nucleotide sequence of SEQ ID NO: 6.

A DNA having one or several bases deleted, substituted or added in the DNA continuously selected from the base sequence set forth in SEQ ID NO: 1 or its complementary base sequence (hereinafter referred to as the measurement target) ) Of the HBV DNA can be measured in the following manner. Maleimidized DNA to be measured is chemically bonded to a thiol-based solid phase and immobilized on a solid phase, which is the 21st to 710th dsDNA of HBV DNA and is fluorescently or radiolabeled. 1
The mixture was heated and denatured by heating at 00 ° C. for 5 minutes, and added at 42 ° C. in 0.1 M sodium citrate and 0.9 M NaCl (pH 7), which is a normal hybridization condition.
Perform hybridization for 1 hour. Thereafter, by washing the solid phase and measuring the fluorescence or radiation of the hybridized DNA, the DNA to be measured is
Hybridization with the 21st to 710th DNAs among the HBV DNAs can be confirmed. HBV D is immobilized on a solid phase on which 25 pmol of DNA to be measured is immobilized.
2 pmol of dsDNA at positions 21 to 710 of NA
It is preferable to use a DNA to be measured such that 30 fmol or more of the DNA hybridizes when hybridization is performed in 70 μl after addition. It is more preferably at least 50 fmol, even more preferably 70 fmol.
fmol or more.

The method for preparing the DNA of the present invention is not particularly limited. For example, solid phase synthesis using an automatic DNA synthesizer; synthesis by a combination of solid phase synthesis and PCR; It can be prepared by purification by column chromatography or the like.

Since the DNA of the present invention has high hybridization efficiency with HBV DNA, high sensitivity analysis can be performed when used as a capture probe in the DNA probe method. The reagent for a DNA probe method using the DNA of the present invention as a capture probe is also disclosed in the present invention.
One. The DNA of the present invention can be used not only for the DNA probe method but also for so-called western blotting, affinity chromatography and the like.

In the present invention, the term "capture probe" means a probe for capturing HBV DNA contained in a sample by hybridization. The form is not limited. Therefore, the reagent for DNA probe method of the present invention may be in the form of a solution, or
It may be bonded to various solid phases (insoluble carriers).

The above-described capture probe can be used to perform a highly sensitive DNA probe method by hybridizing with the target DNA. The above-mentioned target DNA is a target DNA to be captured by hybridization with the capture probe of the present invention, and means HBV-derived DNA contained in the sample. The target DNA may be the HBV DNA itself contained in the sample, may be a part of the DNA, or may be obtained by amplifying the HBV DNA contained in the sample. Is also good.

In the present invention, the target DNA is 60
HBV DN consisting of 0 to 1000 bases
DNA selected continuously from the base sequence at positions 2780 to 1250 in A is preferable. If the number of bases is less than 600 or more than 1000, the efficiency of hybridization with the capture probe is low, so that high-sensitivity analysis cannot be performed. Target DNA hybridized to solid phase
Is detected using a fluorescent or radioisotope label, the length of the target DNA may be as short as 600 bases or less. On the other hand, when measuring unlabeled target DNA hybridized to a solid phase by a method such as surface plasmon measurement or ellipsometry, a longer target DNA has a higher detection sensitivity and is advantageous. From 60
Zero or more bases are preferred.

Of the above HBV DNA, 2780-12
The 50th nucleotide sequence includes, for example, the nucleotide sequence at the 2780th to 1250th nucleotide in the DNA sequence shown in SEQ ID NO: 6; Preferably, the target DNA is selected continuously from the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence set forth in SEQ ID NO: 2 corresponds to 2780-10 of the HBV DNA of SEQ ID NO: 6.
It corresponds to the 00th base sequence. In the present invention, a base sequence consisting of 690 bases at positions 21 to 710 of HBV DNA can be suitably used as the target DNA.

It is preferable to use the target DNA amplified by the PCR method in order to perform a measurement with higher sensitivity. As the primers used in the PCR method, two types of primers consisting of 15 to 30 bases can be used. As the above primer, one in which one or several bases have been substituted can also be used, and the substitution position can be a point mutation site in HBV DNA. Examples of the base to be substituted include inosine. As such a primer replaced by inosine, a combination of the sense S-N1 / I of SEQ ID NO: 3 and the antisense AS-3 / I of SEQ ID NO: 4 is used, or
The target DNA can be obtained at a high amplification rate by using a combination of the sense side S-N1 / I described in SEQ ID NO: 3 and the antisense side AS-2 / I described in SEQ ID NO: 5. In addition, the replaced nucleotide itself after the point mutation can be used as a base to be substituted. In this case, it is used in combination with a primer having a normal nucleotide without mutation. By using a plurality of primers as primers on the sense or antisense side, a target DNA can be obtained at a high amplification rate.

The above PCR method is a NASBA method (Nucleic
Acid Sequence based Amplification), TMA method (Tra
nscription Mediated Amplification), strand
It can be performed by a replace method or the like. Alternatively, the reaction can be performed using a commercially available PCR kit and a PCR amplification device. When the target DNA is dsDNA, such as when amplified by the PCR method,
For hybridization with the capture probe, TE buffer (10 mM Tris-HCl buffer (pH 8), 1 mM ED
TA), etc., and thermally denatured at 4 ° C within at least 3 minutes
By cooling to ssDNA, ssDNA can be obtained stably. Cooling to 4 ° C. after standing for more than 3 minutes may cause ssDNA to reassociate.

In the present invention, the target DNA is hybridized with a capture probe, and the hybridized D
By analyzing the NA, the HBV DNA in the sample can be determined.
Can be detected. The conditions for the above hybridization are not particularly limited, and include, for example, the above-mentioned conditions of 42 ° C. for 1 hour in 0.1 M sodium citrate and 0.9 M NaCl (pH 7).

The above-mentioned capture probe can be prepared by a chemical binding method or a physical adsorption method using a microplate, a latex,
It can be used by binding to a solid phase such as gelatin particles, magnetic particles, a silicon chip, and a slide glass. Preferably, it is a method of bonding by a chemical bonding method. When the capture probe is not bound to the solid phase, a solid phase bound to avidin, a biotin-labeled capture probe, or the like can be used.

The above-mentioned target DNA can be labeled with a radioactive substance, an enzyme, a fluorescent substance, or the like for the purpose of easily performing the DNA probe method. After the hybridization, the label is analyzed, and the HBV in the specimen is analyzed. DNA of
Can be detected. Furthermore, in the measurement by the ellipsometry method in which the film thickness is measured by irradiating a laser beam, it is not necessary to label with a radioactive substance or the like, so that the measurement can be easily performed.

The sample which can be measured by the DNA probe method of the present invention is not particularly limited as long as it contains HBV, and includes, for example, human-derived serum, plasma, cell or tissue homogenate, and the like. By applying the DNA probe method of the present invention to various samples, a highly sensitive diagnostic method for HBV can be performed. In particular, by using a specific base sequence as a target DNA, it is possible to obtain an extremely useful effect such that detection of HBV can be performed easily and with high sensitivity.

[0033]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. The primers and probes used in the Examples are DNA automatic synthesizers (Abacus 6; SIGMA Geno
(manufactured by Sys Corporation).

Example 1 Design of Amplified Region of HBV Using the HBV described in SEQ ID NO: 6 as a template, DN was efficiently performed by PCR.
To amplify the A fragment, commercially available gene analysis software (Nation
al Biosciences) to select 8 gene regions (Table 1), and 8 pairs of sense and antisense primer pairs (Table 2) were prepared according to the design data. PCR kit (rT
aq DNA polymerase, Toyobo Co., Ltd.)
The NA fragment was amplified. The composition of the reaction solution was 50 pmol of primer and 2.
5 Unit Taq polymerase, 0.1 ng of pHB11 (plasmid containing the entire HBV gene described in SEQ ID NO: 6; HBV DNA was purified from serum of a Japanese patient, converted into circular double-stranded DNA with DNA polymerase, and cut with BamHI. , Also B
(Binded to pUC19 cleaved with amHI and named pHB11), 10 μl of 10 × Taq buffer, 8 μl of dNTP mix and finally made up to 100 μl with distilled water. A PCR reaction was performed using the amplification program shown in FIG. FIG. 2 shows the results of 2% agarose gel electrophoresis.

[0035]

[Table 1]

[0036]

[Table 2]

Nine capture probes (Table 3) were designed, targeting a total of seven regions of HBV671 bp and six high-amplification regions (5) other than the HBV 372 bp which had a large amount of amplified DNA.
Oligonucleotides aminated at the 5 ′ end (hereinafter NH ₂
-Probes) were prepared, and each probe-sensitized solid phase well was prepared according to Example 2.

[0038]

[Table 3]

Example 2 Immobilization of Oligo Probes on Microplate Wells Amination of Solid Phase Surface 40 μg / 150 μl of POLY (Lys-Phe) Hydrobromide dissolved in 6 × SSC [0.1 M sodium citrate, 0.9 M NaCl (pH 7)]
(Sigma; P2084) (hereinafter referred to as POLY (Lys-Phe)) was added to the wells of a microplate (Nunc, fluoro nunc module plate) and incubated at 37 ° C for 1 hour. Thereafter, the wells were washed with 50 mM NaHCO ₃ (pH 8.3),
Unadsorbed POLY (Lys-Phe) was removed. Thiol group formation of amino group sensitized to solid phase After washing, wells were reacted by adding 150 nmol / 200 μl 2-iminothiolane dissolved in 0.2 M NaHCO ₃ , 1 mM EDTA (pH 8), and incubated at room temperature for 1 hour. . 10mMKH ₂ P0 _4, 1
The wells were washed with mM EDTA (pH 7) to remove unreacted 2-iminothiolane.

Maleimidization of Oligonucleotide Probes Eleven kinds of NH ₂ -probes were chemically synthesized. N- (4-Maleimidobu
Tyryloxy) -succinimide (hereinafter referred to as GMBS) was dissolved in dimethylformamide, mixed with GMBS and NH ₂ -probe at a molar ratio of 300: 1, and incubated at 37 ° C. for 1 hour to prepare a maleimidated probe. Each maleimidated probe was separated and purified from unreacted GMBS by Sephadex G-25 (Pharmacia) distill column chromatography. Binding of maleimidated probe to thiol-based well Add each maleimidated probe in a volume of 25 pmol / 60 μl to thiol-based well, incubate at room temperature for 2 hours,
The maleimidated probe was chemically attached to the thiolated solid phase. Wells were washed with _{10mM KH 2 P0 4, 1mM EDTA} (pH7),
Unreacted probe was removed. Thiol group of excess on the solid phase, adding a 10 mM KH ₂ P0 _4, iodoacetamide dissolved in 1 mM EDTA (pH 7) at 200 nmol / 70 [mu] l, and incubated for 1 hour at room temperature, and blocked. _{10mM KH 2 P0 4, 1mM EDTA} (p
The wells were washed with H7), air-dried, and stored at room temperature in a dark place.

Example 3 Test for Capture of Fluorescently Labeled HBV Double-Stranded DNA Using Probe-Immobilized Wells Seven kinds of fluorescent (FITC) -labeled double-stranded HBV DNAs selected as targets in Example 1 (on the side hybridizing with the capture probe) FITC label at the 5 'end) was prepared by PCR amplification. The 4 pmol was added to 70 μl of 10 mM Tris (pH 8), 1 mM EDTA
(Hereinafter, TE buffer) and incubated at 100 ° C. for 5 minutes. After rapid ice cooling, 35 μl of 18xSSC [0.3M sodium
citrate, 2.7 M NaCl (pH 7)] to give a sample having a final concentration of 6 × SSC. The sample was added to the probe-immobilized solid phase well and incubated at 42 ° C for 1 hour. After washing the wells with 1 × SSC, the fluorescence counts bound to the wells were measured. Table 4 shows the results of hybridization after adding 4 pmol of the fluorescent-labeled double-stranded HBV DNA.

[0042]

[Table 4]

AsCap. 7 (221-250) (HB shown in SEQ ID NO: 6)
The antisense strand corresponding to nucleotides 221 to 250 of the V gene sequence is named AsCap.7, and is referred to as such hereinafter) as a capture probe. HBV double-stranded D
NA hybridized at 80 fmol / well or more. The hybridized fluorescently labeled HBV double-stranded DNA is ds HB690 (21-710)
(Double-stranded HB corresponding to nucleotides 21 to 710 of the HBV gene sequence shown in SEQ ID NO: 6
DsHB671 (40-710), d
sHB960 (21-980) and dsHB682 (2889-355), and the gene region of HBV ranged from nucleotide 2889 to nucleotide 980. Also, dsHB682 (2889-3) of fluorescently labeled HBV double-stranded DNA
When 55) was used as the target DNA, 5 out of 7 capture probes having complementary sequences hybridized at 80 fmol / well or more. Ds HC5 used as a negative control
33 (1-533) (1 of the HCV gene sequence shown in SEQ ID NO: 10)
The double-stranded HCV DNA corresponding to the 533rd nucleotide to the 533rd nucleotide is hereinafter referred to as ``) is 17 fm.
ol / well.

Example 4 Method for Preparing Capture Probe-Immobilized Tip POLY (Lys-Phe) was dissolved at a concentration of 200 μg / ml in a 6XSSC (0.1 M sodium citrate, 0.9 M NaCl (pH 7.0)) solution to give a 6 mm square. Was dropped on a silicon chip of 35 μl. After incubation at 37 ° C. for 1 hour, the plate was sufficiently washed with distilled water and air-dried. 150 n in 0.2 M NaHCO ₃ , 1 mM EDTA (pH 8.3) buffer
mol of 2-iminothiolane in 70 μl of POLY (Lys-Ph
e) The chip was placed on the treated chip, reacted at room temperature for 1 hour, washed with distilled water to convert the amino group of Lys to -SH. The preparation of the maleimidated capture probe was performed using a 0.2 M phosphate buffer (pH 7.
0) 5 'terminal aminated capture probe and 300 times the amount
After adding GMBS (in moles) and reacting at room temperature for 1 hour, 1
Fractionation was performed with Sephadex G-25 (Pharmacia) using a phosphate buffer of 0 mM KH ₂ PO ₄ and 1 mM EDTA (pH 7.0). The maleimidated capture probe is 25 pmo on the -SH-modified chip
The mixture was added dropwise and reacted for 30-60 minutes. The chip on which the capture probe was fixed was thoroughly washed with distilled water, air-dried and stored.

Example 5 Hybridization of Target DNA with Capture Probe Immobilized on Chip As shown in Example 3, dsHB690 (21-710) target DNA and As Cap. 7 (221-250) of capture probe immobilized on a plate were used. )
Was found to increase the hybridization efficiency.
Then, using the chip, hybridization was attempted with the same target DNA and capture probe. Capture probes are Cap.1 (40-69) and Cap.7 (221-250) of the sense strand, and As of the antisense strand.
Cap.1 (40-69) and As Cap.7 (221-250) were fixed on a silicon chip (Table 5). DsH of three types of target DNA with different DNA length
B126 (177-302), ds HB346 (3170-3215, 1-299), ds HB69
0 (21-710) was PCR-amplified and purified, and both sense and antisense strands were labeled with ³² P at the 5 ′ end, and the negative control was similarly labeled with a 5 P end at ³² P ds HC533 (1-533) It was used. Hybridization is 6xS with 2pmol target DNA
70ul of the SC solution was placed on the chip, and the operation was performed at 42 ° C for 1 hour. Finally, after washing with 1xSSC, the amount of hybridized DNA was determined by liquid synth measurement. The results are shown in FIG.

[0046]

[Table 5]

The hybridization amount of ds HB690 (21-710) of the target DNA was smaller than that of Cap. 1 (40-69) capture probe compared to Cap.
1-250) The chip on which the capture probe was immobilized was higher, and the amount of hybridization of the antisense strand capture probe was higher than that of the sense strand.
9) and As Cap.7 (221-250) capture probe were reproduced. On the other hand, the target DNA length was ds HB690 (21
-710) is ds HB126 (177-302), ds HB346 (3170-3215, 1-29
It was found that the hybridization amount was higher than that obtained when 9) was hybridized. Target DN in this area under optimal conditions
In order to hybridize A, DNA was considered to require a certain length.

Example 6 Comparison of Hybridization Amount between Double-Stranded Target DNA and Chip-Immobilized Capture Probe of Antisense Strand Capture Probe The sequence of the 5'-side and 3'-side sequences of As Cap. 7 (221-250) capture probe 10 kinds of antisense strand capture probes (As NH1
51 (151-180), As NH161 (161-190), As NH171 (171-20
0), As NH181 (181-210), As NH191 (191-220), As NH201
(NH201-230), As NH211 (211-240), As Cap.7 (221-25
0), As NH231 (231-260), As NH241 (241-270), AsCap.8
(251-280)) was subjected to a combination test for hybridization with ds HB690 (21-710). The capture probe was immobilized on a silicon chip by the method described in
NA as ³² P-labeled ds HB690 (21-710), ds HC533 (1-5
33) was hybridized. Hybridization was carried out at 42 ° C. for 1 hour with 70 μl of a 6 × SSC solution containing 2 pmol of target DNA on a chip. After washing the chip with 1 × SSC, the amount of hybridized DNA was determined by liquid synthesizing. The results are shown in FIG.

The amount of hybridization of the capture probe As Cap. 7 was 100 fmol, but the amount of ds HB690 (21-710) that hybridized was further small on the 5 ′ side and 3 ′ side of As Cap.
Thus 181-150 including capture probe As Cap.7 (221-250)
Was considered to be a hot spot region with high hybridization efficiency to ds HB690 (21-710) which is a double-stranded target DNA.

Example 7 Hybridization of Inosine Displacement Capture Probe A point mutation is present in the adr-type HB sequence registered in GenBank. Since there are nucleotide positions corresponding to six point mutations in the sequence of the capture probe As Cap. 7 (221-250), the capture probe As Cap. 7-Inosine (221-250) ) (Table 5; SEQ ID NO: 9)
Was synthesized and fixed on a chip by the method described in Example 4. Hybridization was performed using 2 pmol of ³² as target DNA.
70 μl of a 6 × SSC solution containing P-labeled dsHB690 (21-710) or dsHC533 (1-533) was placed on the chip, and the reaction was performed at 42 ° C. for 1 hour. The chip was washed with 1 × SSC, and finally, a liquid synth was measured to determine the amount of hybridized DNA. The results are shown in FIG. Inosine-substituted capture probe As Cap.7-Inosi
ne (221-250) hybridized equivalently to As Cap.7 (221-250), and was considered to be compatible with the HB subtype adr.

Example 8 Selection of Primers with High Amplification Efficiency Based on the 690 bp DNA fragment selected in Example 1 and 2) in Table 1,
Furthermore, PCR primers with high amplification efficiency were selected. Six types of sense primers shown in Table 6 (HB sense, S
-N1, S-O1, S-O2, S-O3, S-O4) and 5 kinds of antisense primers (HB700AS3, AS-1, AS-2, AS-3, AS-4) HBs gene was amplified by PCR using a total of 30 primer sets. The composition of the reaction solution was 0.5 μl of 5 U / μl TaKaRa Ex Taq, 10 μl of 10 × Ex
Taq buffer, 8 μl dNTP mix, 1 μl 0 or 1 zepto
mol of pHB11, 100 pmol each of sense primer and antisense primer were added, and finally distilled water was added to make a total volume of 100 μl. After adding mineral oil,
Amplified with hermal cycler. The amplification program is shown in FIG. 5 μl of the amplified product was taken out and subjected to electrophoresis using 2% agarose. Table 7 shows the results. Especially S-N1 and AS
-1, S-N1 and AS-2, S-N1 and AS-3, S-O2 and AS-1, S-O2 and AS-
4, S-O3 and AS-1, S-O3 and AS-3, S-O3 and AS-4, S-O4 and AS-2
The amplification ratio was high with the combination of the primers. To confirm these combinations again, use pHB1
FIG. 7 shows the results of amplifying 1 and electrophoresis. It was confirmed that a sufficient amount of about 700b of DNA was amplified from 1zepto mol of pHB11 with this combination of primers.

[0052]

[Table 6]

[0053]

[Table 7]

Example 9 Selection of Enzyme with High Amplification Efficiency In Example 2, amplification was performed using Ex Taq manufactured by TaKaRa.
These comparisons were made because TaKaRa sells enzymes with several properties with different amplification efficiencies. TaKaRa
States that Z-Taq and Ex-Taq have the highest amplification efficiency.
Then, Taq, Z-Taq and Ex-Taq were compared. The combination of primers used in Example 8 was used. Taq
The composition of the reaction mixture is 0.5 μl 5 U / μl TaKaRa Taq, 10 μl
10 x Ex Taq buffer, 8 μl dNTP mix, 12 μl 25 mM
Add MgCl2, 1 μl of 0 or 1 zepto mol of pHB11, 100 pmol each of sense primer and antisense primer,
Finally, distilled water was added to bring the total volume to 100 μl. After addition of mineral oil, TaKaRa thermal cycler model TP20
Amplified at 00. The amplification program used was Ex-Taq of Example 8. Example 8: Ex-Taq PCR amplification
The same method was used. Z-Taq was reacted by the method recommended by TaKaRa. The composition of the reaction solution was 0.5 μl of 5 U / μl TaKaRa
Z-Taq, 5 μl 10 x Ex Taq buffer, 4 μl dNTP mi
x, 1 μl of 0 or 1 zeptomol of pHB11, 20 pmol each of a sense primer and an antisense primer were added, and finally distilled water was added to make a total volume of 50 μl. This is PE thermal
Amplified with cycler GeneAmp9700. Figure 8 shows the amplification program
Shown in 5 μl of the amplification product amplified by each enzyme was taken out and subjected to electrophoresis using 2% agarose. FIG. 9 shows the results.
Shown in The amplification efficiency was higher in the order of Ex-Taq>Taq> Z-Taq.

Example 10 Selection of Primers with High Amplification Efficiency Using Inosine-Substituted Primers The sequences of the 29 HBs gene sequences registered thus far were
When extracted from nk and compared, single base substitution was observed in some HBs genes. S-N1 and AS-1, S-N1 of the combination of the primers of high amplification efficiency selected in Example 8
And AS-2, S-N1 and AS-3, S-O2 and AS-1, S-O2 and AS-4, S-O3
In AS-1, S-O3 and AS-3, and S-O3 and AS-4, regions of the sequence containing this single nucleotide substitution are observed, so the nucleotide at the position corresponding to the single nucleotide substitution is substituted with inosine. The hybridization with the template HBs gene was not hindered. S-N1 / I and AS-1, S-N1 / I
And AS-2 / I, S-N1 / I and AS-3 / I, S-O2 / I and AS-1, S-O2 / I and AS
-4 / I, S-O3 / I and AS-1, S-O3 / I and AS-3 / I, S-O3 / I and AS-4 / I
And shown in Table 8. FIG. 10 shows the results of amplifying pHB11 of these combinations by the method of Example 8 and performing electrophoresis. In the past, Ex-Taq, Z-Taq and other enzymes having 3 '→ 5' exonuclease activity and proofreading function, despite primers having inosine being considered to have poor amplification efficiency, especially S -N1 / I and AS
High amplification efficiency with -2 / I, S-N1 / I and AS-3 / I, pH of 1zepto mol
A sufficient amount of about 700b of DNA could be amplified from B11.

[0056]

[Table 8]

Example 11 Inosine substituted primer and Ex
Examination of high-speed PCR using -Taq Using the inosine-substituted primer shown in Example 10,
We investigated a high-speed PCR method that can amplify template DNA in a short time. Primers are S-N1 / I and AS-1, S-N1 / I and AS-2 / I, S-N1
/ I and AS-3 / I, S-O2 / I and AS-1 / I, S-O2 / I and AS-4 / I, S-O3 / I
And AS-1 / I, S-O3 / I and AS-3 / I, and S-O3 / I and AS-4 / I. The composition of the reaction solution was 0.5 μl of 5 U / μl TaKaRaEx-Taq, 5 μl.
l 10 x Ex Taq buffer, 4 μl dNTP mix, 1 μl 0
Or 50 pmol each of pHB11 or sense primer and antisense primer of 1 zeptomol was added, and finally distilled water was added to make a total volume of 50 μl. This is PE thermal cycler GeneA
Amplified with mp9700. The amplification program is shown in FIG. 5 μl of the amplified product was taken out and subjected to electrophoresis using 2% agarose. The result is shown in FIG. At the annealing temperature of 55 ° C, sufficient amplification was observed with the combination of the primers S-N1 / I and AS-3 / I. On the other hand, annealing temperature
At 50 ° C., sufficient amplification was observed with the combination of the primers S-N1 / I and AS-2 / I and between S-N1 / I and AS-3 / I.

[0058]

Industrial Applicability The DNA of the present invention can detect HBV DNA with high sensitivity. Furthermore, HBV can be detected with higher sensitivity by using a DNA probe method using a specific base sequence as a target probe.

[0059]

[Sequence Table] <110> DNA, DNA probe reagent and DNA probe method <130> FR33 <160> 13 <210> 1 <211> 141 <212> DNA <213> Fujirebio Inc. <120> DNA > Hepatitis B virus, subtype adr <220> <223> Sequence Listing Free Text Sequence of the 150th to 290th sense strand in the HBVadr type DNA sequence <400> cggcgtttta 120 tcatattcct cttcatcctg c 141 <210> 2 <211> 1436 <212> DNA <213> Hepatitis B virus, subtype adr <220> <223> Sequence listing free text Base sequence from 2780th to 1000th base of HBV DNA < 400> 2 atcctctggg attctttccc gatcaccagt tggaccctgc attcggagcc aactcaaaca 60 atccagattg ggacttcaac cccaacaagg atcattggcc agaggcaaat caggtaggag 120 cgggagcatt tggg ttcactcgg tggactagcgggg cggacggcgg cggacggcgg cggacggg cag cagcgcctcc tcctgcctct accaatcggc 240 agtcaggaag acagcctact cccatctctc cacctctaag agacagtcat cctcaggcca 300 tgcagtggaa ctccacaaca ttccaccaag ctgtgctaga tcccagagtg aggggcctat 360 atcttcctgc tggtggctcc agttccggaa cagtgaaccc tgttccgact actgcctcac 420 ccatatcgtc aatcttctcg aggactgggg accctgcacc gaacatggag aacacaacat 480 caggattcct aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaagaatcc 540 tcacaatacc acagagtcta gactcgtggt ggacttctct caattttcta gggggagcac 600 ccacgtgtcc tggcccaaat tcgcagtccc caacctccaa tcactcacca acctcttgtc 660 ctccaatttg tcctggctat cgctggatgt gtctgcggcg ttttatcata ttcctcttca 720 tcctgctgct atgcctcatc ttcttgttgg ttcttctgga ctaccaaggt atgttgcccg 780 tttgtcctct acttccagga acatcaacta ccagcacggg accatgcaag acctgcacga 840 ttcctgctca aggaacctct atgtttccct cctgttgctg tacaaaacct tcggacggaa 900 actgcacttg tattcccatc ccatcatcct gggctttcgc aagattccta tgggagtggg 960 cctcagtccg tttctcctgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc 1020 tttcccccac tgtttggctt tcagttatat ggatgatgtg gtat tggggg ccaagtctgt 1080 acaacatctt gagtcccttt ttacctctat taccaatttt cttttgtctt tgggtataca 1140 tttgaaccct cataaaacca aacgttgggg ctactccctt aacttcatgg gatatgtaat 1200 tggaagttgg ggtactttac cacaggaaca tattgtacta aaaatcaagc aatgttttcg 1260 gaagctgcct gtaaatagac ctattgattg gaaagtatgt caaaggattg tgggtctttt 1320 gggctttgct gcccctttta cacaatgtgg ctatcctgcc ttgatgcctt tatatgcatg 1380 tatacaatct aagcaggctt tcactttctc gccaacttac aaggcctttc tgtgta 1436 <210> 3 < 211> 20 <212> DNA <213> Artificial Sequence <220> <221> modified base <222> 14 <223> i <220> <221> modified base <222> 18 <223> i <220> <221> modified base <222> 20 <223> i <220> <223> Sequence listing free text Inosine replacement primer, S-N1 / I <400> 3 accctgcacc gaanatgnan 20 <210> 4 <211> 24 <212> DNA <213 > Artificial Sequence <220> <221> modified base <222> 5 <223> i <220> <221> modified base <222> 19 <223> i <220> <221> modified base <222> 20 <223> i <220> <223> Sequence Listing Free Text by Inosine Primer, AS-3 / I <400> 4 cccancgttt ggttttatnn gggt 24 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> modified base <222> 9 < 223> i <220> <221> modified base <222> 10 <223> i <220> <221> modified base <222> 16 <223> i <220> <223> Sequence listing free text replaced by inosine Primer, AS-2 / I <400> 5 ggttttatnn gggttnaaat gtatac 26 <210> 6 <211> 3215 <212> DNA <213> Hepatitis B virus, subtype adr <220> <221> CDS <222> (29). . (709) <223> product = "surface antigen" <220> <221> CDS <222> (1775) .. (2326) <223> product = "core antigen" <300> <301> Ono Y., Onda H., Sasada R., Igarashi K., Sugino Y. and Nishioka K. <302> The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw <303> Nucleic Acids Res. <304> 11 < 305> 6 <306> 1747-1757 <307> 1983 <308> MEDLINE 83168919 <300> <301> Kobayashi M. and Koike K. <302> Complete nucleotide sequence of hepatitis B virus DNA of subtype a dr and its conserved gene organization <303> Gene <304> 30 <305> 1-3 <306> 227-232 <307> 1984 <308> MEDLINE 85077616 <300> <301> Takemura F., Ishii T ., Fujii N. and Uchida T. <302> Complete nucleotide sequence of hepatitis B virus <303> Nucleic Acids Res. <304> 18 <305> 15 <306> 4587 <307> 1990 <308> MEDLINE 90356395 <400> 6 ctcgaggact ggggaccctg caccgaac atg gag aac aca aca tca gga ttc 52 Met Glu Asn Thr Thr Ser Gly Phe 1 5 cta gga ccc ctg ctc gtg tta cag gcg ggg ttt ttc ttg ttg aca aga 100 Leu Gly Leu G Phe Phe Leu Leu Thr Arg 10 15 20 atc ctc aca ata cca cag agt cta gac tcg tgg tgg act tct ctc aat 148 Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn 25 30 35 40 ttt cta ggg gga gca ccc acg tgt cct ggc cca aat tcg cag tcc cca 196 Phe Leu Gly Gly Ala Pro Thr Cys Pro Gly Pro Asn Ser Gln Ser Pro 45 50 55 acc tcc aat cac tca cca acc tct tgt cct cca att tgt cct ggc tat 244 Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr 60 65 70 cgc tgg atg tgt ctg cgg cgt ttt atc ata ttc ctc ttc atc ctg ctg 292 Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Ple Leu Phe Ile Leu Leu 75 80 85 cta tgc ctc atc ttg ttg ttg ttg ttg ttg ttg ttg ttg ttg ttg ttg ttg tac caa ggt atg ttg 340 Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu 90 95 100 ccc gtt tgt cct cta ctt cca gga aca tca act acc agc acg gga cca 388 Pro Val Cys Pro Leu Leu Pro Gly Thr Ser Thr Thr Ser Thr Gly Pro 105 110 115 120 tgc aag acc tgc acg att cct gct caa gga acc tct atg ttt ccc tcc 436 Cys Lys Thr Cys Thr Ile Pro Ala Gln Gly Thr Ser Met Phe Pro Ser 125 130 135 tgt tgc tgt aca aaa cct tcg gac gga aac tgc act tgt att ccc atc 484 Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn Cys Thr Cys Ile Pro Ile 140 145 150 cca tca tcc tgg gct ttc gca aga ttc cta tgg gag tgg gcc tca gtc 532 Pro Ser Ser Trp Ala Phe Ala Arg Phe Leu Trp Glu Trp Ala Ser Val 155 160 165 cgt ttc tcc tgg ctc agt tta cta gtg cca ttt gtt cag tgg ttc gta 580 Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val 170 175 180 ggg ctt tcc ccc act gtt tgg ctt tca gtt ata tgg atg atg tgg tat 628 Gly Leu Ser Pro Thr Val Trp Leu Ser Val Ile Trp Met Met Trp Tyr 185 190 195 200 tgg ggg cca agt ctg tac aac atc ttg agt ccc ttt tta cct cta tta 676 Trp Gly Pro Ser Leu Tyr Asn Ile Leu Ser Pro Phe Leu Pro Leu Leu 205 210 215 cca att ttc ttt tgt ctt tgg gta tac att tgaaccctca taaaaccaaa 726 Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 220 225 cgttggggct actcccttaa cttcatggga tatgtaattg gaagttgggg tactttacca 786 caggaacata ttgtactaaa aatcaagcaa tgttttcgga agctgcctgt aaatagacct 846 attgattgga aagtatgtca aaggattgtg ggtcttttgg gctttgctgc cccttttaca 906 caatgtggct atcctgcctt gatgccttta tatgcatgta tacaatctaa gcaggctttc 966 actttctcgc caacttacaa ggcctttctg tgtaaacaat atctgcacct ttaccccgtt 1026 gcccggcaac ggtcaggtct ctgccaagtg tttgctgacg caacccccac tggatggggc 1086 ttggccattg gccaatcggg catgcgtgga acctttgtgg ctcctctgcc gatccatact 1146 gcggaactcc tagcagcttg ttttgctcgc agccggtctg gagcgaaact tatcgggact 1206 gacaactctg tt gtcctctc tcggaaatac acctccttcc catggctgct cgggtgtgct 1266 gccaactgga tcctgcgcgg gacgtccttt gtctacgtcc cgtcggcgct gaatcccgcg 1326 gacgacccgt ctcggggccg tttgggcctc taccgtcccc ttcttcatct gccgttccgg 1386 ccgaccacgg ggcgcgcctc tctttacgcg gtctccccgt ctgtgccttc tcatctgccg 1446 gtccgtgtgc acttcgcttc acctctgcac gtcgcatgga gaccaccgtg aacgcccacc 1506 aggtcttgcc caaggtctta cataagagga ctcttggact ctcagcgatg tcaacgaccg 1566 accttgaggc atatttcaaa gactgtttgt ttaaagactg ggaggagttg ggggaggaga 1626 ttaggttaat ggtctttgta ctaggaggct gtaggcataa attggtctgt tcaccagcac 1686 catgcaactt tttcacctct gcctaatcat ctcctgttca tgtcctactg ttcaagcctc 1746 caagctgtgc cttgggtggc ttaggggc atg gac att gac acg tat aaa gaa 1798 Met Asp Ile Asp Thr Tyr Lys Glu 230 ttt gga gct tct gtg gag tta ctc tct ttt ttg cct tct gac ttc ttt 1846 Phe Gly Ala Ser Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe 235 240 245 250 cct tct att cga gat ctc ctc gac acc gcc ttt gct ctg cat cgg gag 1894 Pro Ser Ile Arg Asp Leu Leu Asp Thr Ala Phe Ala LeuHis Arg Glu 255 260 265 gcc tta gag tct ccg gaa cat tgt tca cct cac cat aca gca ctc agg 1942 Ala Leu Glu Ser Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg 270 275 280 caa gct att gtg tgt tgg ggt gag ttg atg aat ctg gcc acc tgg gtg 1990 Gln Ala Ile Val Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val 285 290 295 gga agt aat ttg gaa gac cca gca tcc agg gaa ttg gta gtc agc tat 2038 Gly Ser Asp Pro Ala Ser Arg Glu Leu Val Val Ser Tyr 300 305 310 gtc aat gtt aat atg ggc cta aaa atc aga caa cta ttg tgg ttt cac 2086 Val Asn Val Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His 315 320 325 330 att tcc tgt ctt act ttt gga aga gaa acg gtt ctt gag tat ttg gta 2134 Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val 335 340 345 tct gtt gga gtg tgg att cgc act cct caa gcc tac aga cca cca aat 2182 Ser Val Gly Val Trp Ile Arg Thr Pro Gln Ala Tyr Arg Pro Pro Asn 350 355 360 gcc cct atc tta tca aca ctt ccg gaa act act gtt gtt aga cga cga 2230 Ala Pro Ile Leu Ser Thr Leu Pro G lu Thr Thr Val Val Arg Arg Arg 365 370 375 ggc agg tcc cct aga aga aga act ccc tcg cct cgc aga cga agg tct 2278 Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser 380 385 390 aaa tcg ccg cgt cgc aga aga tct caa tct cgg gaa tct caa tgt 2323 Lys Ser Pro Arg Arg Arg Arg Ser Gln Ser Arg Glu Ser Gln Cys 395 400 405 tagtatccct tggactcata aggtgggaaa ctttactggt ctctattctt ctactgtacc 2383 tgtctttaat cctgagtggc aaactccctc ctttcctaat attcatttac aggaggatat 2443 tattaataga tgtcaacaat atgtaggccc tcttacagtt aatgaaaaaa ggagattaaa 2503 attaattatg cctgctaggt tctatcctaa ccttaccaaa tatttgccct tggataaagg 2563 tattaaacct tattatcctg aacatgcagt taatcattat ttcaaaacta ggcattattt 2623 acatactctg tggaaggctg gcattctata taagagagaa actacacgta gtgcctcatt 2683 ttgtgggtca ccatattctt gggaacaaga gctacagcat gggaggttgg tcttccaaac 2743 ctcgacaagg catggggacg aatctttctg ttcccaatcc tctgggattc tttcccgatc 2803 accagttgga ccctgcattc ggagccaact caaacaatcc agattgggac ttcaacccca 2863 acaaggatca ttggccagag gcaaatcagg tag gagcggg agcatttggg ccagggttca 2923 ctccaccaca cggcggtctt ttggggtgga gccctcaggc tcagggcata ttgacaacag 2983 tgccagcagc gcctcctcct gcctctacca atcggcagtc aggaagacag cctactccca 3043 tctctccacc tctaagagac agtcatcctc aggccatgca gtggaactcc acaacattcc 3103 accaagctgt gctagatccc agagtgaggg gcctatatct tcctgctggt ggctccagtt 3163 ccggaacagt gaaccctgtt ccgactactg cctcacccat atcgtcaatc tt 3215 <210> 7 <211> 226 <212> PRT <213> Hepatitis B virus, subtype adr <400> 7 Met Glu Asn Thr Thr Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln 1 5 10 15 Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu 20 25 30 Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly Ala Pro Thr Cys 35 40 45 Pro Gly Pro Asn Ser Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser 50 55 60 Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe 65 70 75 80 Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val 85 90 95 Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly 100 105 110 Thr Ser Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Ile Pro Ala 115 120 125 Gln Gly Thr Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp 130 135 140 Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg 145 150 155 160 Phe Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu 165 170 175 Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu 180 185 190 Ser Val Ile Trp Met Met Trp Tyr Trp Gly Pro Ser Leu Tyr Asn Ile 195 200 205 Leu Ser Pro Phe Leu Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val 210 215 220 Tyr Ile 225 <210> 8 <211> 183 <212> PRT <213> Hepatitis B virus, subtype adr <400> 8 Met Asp Ile Asp Thr Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp 20 25 30 Thr Ala Phe Ala Leu His Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Val Cys Trp Gly Glu 50 55 60 Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Glu Leu Val Val Ser Tyr Val A sn Val Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Val Gly Val Trp Ile Arg Thr 115 120 125 Pro Gln Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr 145 150 155 160 Pro Ser Pro Arg Arg Arg Arg Ser Lys Ser Pro Arg Arg Arg Arg Ser 165 170 175 Gln Ser Arg Glu Ser Gln Cys 180 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> modified base <222> 4 <223> i <220> <221> modified base <222> 10 <223> i <220> <221> modified base <222> 11 <223> i <220> <221> modified base <222> 16 <223> i <220> <221 > modified base <222> 20 <223> i <220> <221> modified base <222> 28 <223> i <220> <223> Sequence Listing Free Text Inosine Substitution Probe, AsCap7-Inosine <400> 9 ccancgatan ncaggncaan ttggaggnca 30 <210> 10 <211> 533 <212> DNA <213> Hepatitis C virus <400> 10 aggaagatag ag aaagagca accgggtaaa ttccctgttg catagttcac gccgtcctcc 60 agaacccgga caccatgtgc cagggccctg gcagcgcccc ctaggggggc gccgacaagc 120 ggaatgtacc ccatgaggtc ggcgaagccg catgtgaggg tatcgatgac cttacccaaa 180 ttgcgtgacc tacgccgggg gtccgtgggg ccccaactag gccgggagcc acggggtgac 240 aggagccatc ctgcccaccc cataccctcg ttgccataga ggggccaagg gtacccgggc 300 tgagcccagg tcctaccctc gggccggcga gccttgggga taggttgtcg ccttccacga 360 ggttgcgacc gctcggaagt cttcctagtc gcgcgcacac ccaacctggg gcccctgcgc 420 ggcaacaggt aaactccacc aacgatctga ccaccgcccg ggaatttaac gtcccgtggg 480 cggcggttgg tgttacgttt ggtttttctt tgaggtttag gatttgtgct cat 533 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> modified base <222> 11 <223> i <220 > Sequence Listing Free Text Inosine Substitution Primer, S-02 / I <400> 11 ccctgctcgt nttacaggcg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> modified base <222> 1 <223> i <220> <223> Sequence Listing Free Text Inosine Substitution Immer, S-03 / I <400> 12 nttacaggcg gggtttttct 20 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <221> modified base <222> 8 <223> i <220> <221> modified base <222> 17 <223> i <220> <223> Sequence Listing Free Text Inosine Substitution Primer, AS-4 / I <400> 13 taaggganta gccccancgt ttggt 25

[Brief description of the drawings]

FIG. 1 is a PCR amplification program in Example 1.

FIG. 2 shows a PC of HBV DNA fragment in Example 1.
It is a photograph of 2% agarose gel electrophoresis of the amplified product by R.

FIG. 3 is a graph showing evaluation results of capture probes with different target DNAs in Example 5.

FIG. 4 is a graph showing the evaluation results of antisense strand capture probes in Example 6.

FIG. 5 is a graph showing evaluation results of an inosine-substituted capture probe in Example 7.

FIG. 6 is a PCR amplification program in Example 8.

FIG. 7: PC of HBV DNA fragment in Example 8
It is a photograph of 2% agarose gel electrophoresis of the amplified product by R.

FIG. 8 shows a PCR amplification program in Example 9.

FIG. 9 shows a PC of HBV DNA fragment in Example 9.
It is a photograph of 2% agarose gel electrophoresis of the amplified product by R.

FIG. 10 is a photograph of a 2% agarose gel electrophoresis of an amplified product of an HBV DNA fragment by PCR in Example 10.

FIG. 11 shows a PCR amplification program in Example 11.

FIG. 12 is a photograph of a 2% agarose gel electrophoresis of an amplified product of a HBV DNA fragment by PCR in Example 11.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court ゛ (Reference) // C12M 1/00 C12N 15/00 ZNAA (72) Inventor Ken Ando 2-62 Nihonbashihamacho, Chuo-ku, Tokyo No. 5 Inside Fujirebio Co., Ltd. (72) Inventor Tetsu Ito 2-62-5 Nihonbashi Hamacho, Chuo-ku, Tokyo F-term inside Fujirebio Co., Ltd. 4B024 AA14 CA01 CA09 HA14 4B029 AA07 AA23 BB20 CC03 CC08 FA03 4B063 QA01 QA07 QA18 QA19 QQ03 QQ08 QQ10 QQ42 QR32 QR55 QR56 QR83 QS25 QS34 QX07

Claims (11)

[Claims] 1. A DNA consisting of at least 15 bases, which is a DNA of the following (a) or (b): (a) continuously selected from the base sequence of SEQ ID NO: 1 or a complementary base sequence thereof DNA, (b) a DNA continuously selected from the base sequence of SEQ ID NO: 1 or its complementary base sequence, wherein one or several bases are deleted, substituted or added, DNA that hybridizes under stringent conditions with the 21st to 710th DNAs of the hepatitis B virus DNA. 2. The nucleotide sequence of SEQ ID NO: 1
In the DNA continuously selected from the base sequence complementary to the base sequence at positions 0 to 110, or the base sequence complementary to the base sequence at positions 60 to 110 in the base sequence described in SEQ ID NO: 1, 1 Is a DNA in which one or several bases have been deleted, substituted or added, and
2. A DNA that hybridizes under stringent conditions to DNAs 21 to 710 of NA.
The described DNA. 3. The DNA according to claim 1, which comprises 15 to 50 bases. 4. The DNA according to claim 3, comprising 25 to 35 bases. 5. A reagent for a DNA probe method, wherein the DNA according to claim 1, 2, 3 or 4 is used as a capture probe. 6. A DNA probe method, comprising using the DNA according to claim 1, 2, 3, or 4 as a capture probe, and hybridizing a target DNA with the capture probe. 7. The target DNA is composed of 600 to 1000 bases and comprises 2 of hepatitis B virus DNA.
The DNA probe method according to claim 6, which is a DNA continuously selected from the nucleotide sequence at positions 780 to 1250. 8. The DNA probe method according to claim 7, wherein the target DNA has been amplified by a PCR method. 9. The target DNA is a primer consisting of 15 to 30 bases and amplified by using two kinds of primers in which one or several bases may be replaced by inosine. The DNA probe method as described above. 10. The primer is used in combination with the sense S-N1 / I of SEQ ID NO: 3 and the antisense AS-3 / I of SEQ ID NO: 4. The DNA probe method as described above. 11. The primer is used in combination with the sense S-N1 / I of SEQ ID NO: 3 and the antisense AS-2 / I of SEQ ID NO: 5. The DNA probe method as described above.