JP2000032992A - Detection of human cytomegalovirus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To rapidly and reliably detect human cytomegalovirus by treating a DNA primer pair containing a specific base sequence of oligonucleotide part, a DNA polymerase and an aqueous liquid test sample by DNA amplification followed by examination. SOLUTION: Human cytomegalovirus is rapidly, reliably and specifically detected by treating a mixture which includes the combination of the first DNA primer containing at least 15 bases of oligonucleotide part of the base sequence represented by formula I and the second DNA primer containing at least 15 bases of oligonucleotide of the base sequence represented by formula II, a DNA polymerase and an aqueous liquid test sample by DNA amplification process and then treating a reaction solution containing the resultant amplified DNA by DNA examination process. Further, this method is useful in the diagnosis or the like of cytomegalovirus infectious disease which shows various inapparent infections, causes a reactivation to malignant tumor, AIDS patients, organ transplantation patients, massive blood transfusion patients or pregnant women and is fatal as the case may be.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a human cytomegalovirus (Cytomegalovirus);
MV).

[0002]

2. Description of the Related Art Human cytomegalovirus (CMV)
Is a virus belonging to the family Herpesviridae, which is infected by the majority of adults. Many people are congenitally infected immediately after childbirth, but due to the large number of subclinical infections, no particular medical condition appears. Acquired are contact infections that originate from urine, saliva, breast milk or semen, and iatrogenic infections by blood transfusion or organ transplantation. CMV lurks in the living body for a lifetime, and when CMV is reactivated due to a host-side factor or re-infection with a different antigen CMV is observed. In particular,
Reactivation of CMV is seen in malignant tumors, AIDS patients, those who receive organ transplants, those who receive massive blood transfusions, and pregnant women,
The pathology varies from mild to fatal, such as infectious diseases such as liver disease, hepatoma, splenomegaly, and pneumonia, and diarrhea and vomiting. Definitive diagnosis of CMV infection is clinically extremely important. is there.

[0003] Conventionally, as a method for detecting CMV, a method of separating and identifying CMV from biological fluids (urine, saliva, etc.) and a neutralization test method have been performed.
It took weeks, and in times of urgency the results were not available for treatment. In addition, according to the serological detection method, it is not possible to collect serum in the acute or convalescent phase because CMV has many subclinical infections. Therefore, IgG and IgM can be collected using blood collected at appropriate intervals. There was no other way but to look at fate. Therefore, it was difficult to determine the presence or absence of the virus from the results. On the other hand, in the case of diagnosis of congenital infection of newborn infants, organ transplantation or reduction of immune function, etc., it is often required to judge the presence or absence of CMV in a short time. Heretofore, there has been no satisfactory method. Therefore,
There has been a demand for the development of a method and an in-vitro diagnostic agent that enable a highly accurate CMV diagnosis in a short time.

[0004]

SUMMARY OF THE INVENTION In order to detect CMV quickly and reliably, the present inventors searched for various base sequences specific to CMV and synthesized a DNA fragment complementary to that sequence. And CMV were evaluated as probes and primers, and found to be usable as probes and primers that specifically hybridize with CMV DNA and RNA. The present invention is based on this finding.

[0005]

Accordingly, the present invention relates to a first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by SEQ ID NO: 5 in the sequence listing and a sequence in the sequence listing. A mixture comprising a combination of a second DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the sequence of No. 6, a DNA polymerase, and an aqueous liquid test sample is subjected to a DNA amplification step, Subsequently, the present invention relates to a method for detecting human cytomegalovirus, which comprises subjecting the obtained reaction solution to a DNA test step. Further, the present invention relates to at least 10 nucleotides of the base sequence represented by the sequence of SEQ ID NO: 8 or the sequence of SEQ ID NO: 9 in the sequence listing.
The present invention also relates to a method for detecting human cytomegalovirus, which comprises contacting a probe containing a base with an oligonucleotide portion and carrying a label with a test sample, and detecting a signal from the label.

Further, the present invention relates to a first nucleotide containing at least a 15-base oligonucleotide portion of the nucleotide sequence represented by the formula (1a) (5 ')-TATAC CCAGA CGGAA GAGAA ATTCA- (3a) (1a) (Hereinafter, may be referred to as primer (1a)) or at least 15 bases of the nucleotide sequence represented by formula (1b) (5 ′)-ATGAA GTGTA TTGGG CTAAC TATGC- (3 ′) (1b) A first DNA primer containing an oligonucleotide portion [hereinafter sometimes referred to as primer (1b)] and represented by formula (2a) (5 ′)-TTCTC CTAAG TTCAT CCTTT TTAGC- (3 ′) (2a) A nucleotide sequence containing at least a 15-base oligonucleotide portion of the base sequence DNA primer [hereinafter sometimes referred to as primer (2a)] or at least 15 bases of the nucleotide sequence represented by the formula (2b) (5 ′)-ATAAG CCAT ATCTC ATCG GGGAG- (3 ′) (2b) A combination of a second DNA primer containing an oligonucleotide portion [hereinafter sometimes referred to as primer (2b)], or the formula (1c) (5 ′)-TGACCG CGCAT ACATC CCGAG TACAT- (3 ′) (1c (1) a first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by the following formula (hereinafter sometimes referred to as primer (1c)) and formula (2c): (5 ′)-ATGAC GTTGC TCCGT GGAAA GAGAC C- (3 ') (2c) A mixed solution containing a combination of a second DNA primer containing an oligonucleotide portion of at least 15 bases in the base sequence [hereinafter sometimes referred to as primer (2c)], a DNA polymerase, and an aqueous liquid test sample is prepared. The present invention relates to a method for detecting human cytomegalovirus, which is characterized in that it is subjected to a DNA amplification step, and subsequently to a DNA test step. In the base sequence herein, A is an adenine residue, C is a cytosine residue, G is a guanine residue, and T is a thymine residue.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The liquid test sample used in the method of the present invention is not particularly limited as long as it is a sample suspected of containing CMV. For example, urine, pharyngeal wipe, birth canal wipe,
Blood, immobilized free cells, tissue sections and the like can be used.

[0008] The CMV detection method according to the present invention mainly comprises (1) a DNA amplification step and (2) a DNA inspection step. In this DNA amplification step (1), PCR (P
oligomerase chain reaction)
Method can be used. By using the PCR method, it is possible to automatically amplify only a target DNA region up to about one million times from a small amount of DNA (Science
e, 239: 487-491, 1988). In the PCR method, a primer for a + strand (hereinafter, referred to as a first primer) and a primer for a − strand (hereinafter, referred to as a second primer) sandwich a DNA region to be amplified.
Seed DNA primers are used.

The combination of the first primer and the second primer used in the DNA amplification step (1) of the present invention includes (a) a first primer (1a) and a second primer (2a), and (b) a first primer. (1a) and second primer (2b), (c) first primer (1b) and second primer (2a), (d) first primer (1b) and second primer (2b), and (e) There are five combinations of one primer (1c) and the second primer (2c), and any one of these combinations can be used alone or two or more can be used simultaneously.

When the above-mentioned respective primers according to the present invention are used in the above-mentioned combinations (a) to (d), the total DN of CMV
In Region A, Immediate Early 1
(Hereinafter, referred to as IE1) can specifically amplify only a DNA region encoding a protein.
Specifically, 287 bp corresponding to a part of the IE1 gene by the combination (a), 426 bp corresponding to a part of the IE1 gene by the combination (b), and part of the IE1 gene by the combination (c) The corresponding 182 bp portion and the 321 bp portion corresponding to a part of the IE1 gene by combination (d),
Each is specifically amplified in large quantities. Further, when the above-mentioned combination (e) is used, the DNV encoding the phosphorylated protein (pp71) is contained in the entire DNA region of CMV.
Only the A region can be specifically amplified. Specifically, a 316 bp portion corresponding to a part of the pp71 gene is specifically and massively amplified. IE1 is 0. 0 on the genetic map.
75m. u. Pp71 is located on the genetic map at 0.53 m. u. Present nearby.

The first primer and the second primer may each have a length of 15 mer to 30 mer, but are generally preferably 20 mer to 25 mer. If it is less than 15 mer, the specificity during annealing decreases, and non-specific binding increases. Also, 30m
When the value exceeds er, it is not preferable because a secondary structure between primer molecules or within a molecule is easily obtained. Each base constituting each of the first primer and the second primer used in the method of the present invention may be modified (for example, biotinylated or labeled with a luminescent substance) in any known manner.

Each of the first and second primers according to the present invention is prepared by a known DNA synthesis method (for example, a phosphoramidite method) using a conventional automatic DNA synthesizer (for example, manufactured by Applied Biosystems). can do.

In the DNA amplification step (1) of the present invention, the first
The amplification cycle is repeated using a DNA polymerase, especially a thermostable DNA polymerase, along with a primer and a second primer. As a thermostable polymerase, DN which can maintain the activity particularly at a temperature of up to 95 ° C.
A polymerase, for example, a commercially available Taq polymerase can be used.

In the DNA amplification step of the present invention, the specific combination of the first primer and the second primer, DN
A mixed solution containing A polymerase and a liquid test sample is used. The amounts of the first primer, the second primer, and the DNA polymerase vary depending on the type of the liquid test sample, but can be easily determined within a range where the DNA amplification step by the PCR method can be performed. The mixture can optionally contain a buffer (eg, Tris-HCl buffer), a stabilizer (eg, gelatin), or salts (eg, sodium chloride).

In the method of the present invention, P
The amplification cycle of the CR method is performed. The amplification cycle includes: (i) a DNA denaturation step (about 90 ° C. to 95 ° C. for about 10
(Ii. Seconds to about 2 minutes) (ii) Step of annealing single-stranded DNA with the first primer and the second primer (at about 37 ° C. to 70 ° C. for about 3 minutes)
(0 seconds to about 3 minutes), and (iii) a DNA synthesis step with a DNA polymerase (about 65 to 80 ° C. for about 30 seconds to about 5 minutes).
The amount of DNA is doubled every cycle, and 2 ⁿ times after n cycles. In the present invention, the above-mentioned amplification cycle is repeated 10 to 60 times, preferably 20 to 40 times. In the last cycle, it is preferable to extend the heating time in step (iii) to about 5 to 10 minutes so that the DNA synthesis is completely performed.

When CMV is present in the test sample,
After completion of the amplification cycle, the D-value of about 50-500 bp
NA is synthesized in large quantities. This DNA is detected by the next DNA test step.

As the DNA test step, a method utilizing gel electrophoresis and ethidium bromide staining, a Southern blot hybrid method, a nucleotide sequence determination method using the dideoxy method, and the like can be used. When performing gel electrophoresis, for example, submarine electrophoresis using an agarose gel as a carrier or slab electrophoresis using acrylamide can be used.

Southern blot hybrid method, or i
When performing the n situ hybrid method, a non-radioactive probe (for example, an enzyme-labeled probe, a biotinylated probe, a digoxigeninated probe or a chemiluminescent substance,
A probe labeled with a fluorescent substance) can be used.

Further, when a nucleotide sequence determination method by the dideoxy method is used, a DNA autosequencer using a fluorescent label (Applied Biosystems) can be used.

The present invention further provides three types of DNA probes that can be used for specific detection of CMV-DNA. These DNA probes are probes containing at least a 10-nucleotide oligonucleotide portion of the base sequence represented by the formula (3) (5 ′)-CTCCT TAATA CAAGC CATCC ACATCT CCCG- (3 ′) (3) And probe A), and an oligonucleotide portion of at least 10 bases in the base sequence represented by the formula (4) (5 ′)-GCTGT CGGTG TTCCA AATGA ACGGCC TGAT- (3 ′) (4) A probe contained therein (hereinafter sometimes referred to as probe B), and at least 10 bases of the nucleotide sequence represented by the formula (5): (5 ′)-GACTT CTTCA CCCTG TTCTT CCTCG C TATC- (3 ′) (5) Probe containing an oligonucleotide portion of Be referred to as being there). These probes can be used alone or in combination of two or more, and three can be used simultaneously. In addition, the DNA testing step using these probes is performed alone or in combination with the aforementioned DNA amplification step using a combination of the primers (1a) to (1c) and the primers (2a) to (2c). be able to.

The probe A is specific to the exon 4 part of the gene encoding IE1. The length of the probe A differs depending on the type of pretreatment for the test sample.
When the test sample has undergone a DNA amplification step by the PCR method, DN amplified from 10 bp to the PCR method
It can be up to the size of the A fragment. When the test sample is not subjected to the DNA amplification step of the PCR method and the probe A is used for the in situ hybrid method, the length of the probe A is 10 bp to the size of the total DNA (1221 bp) of the exon 4 part. It is possible. A DNA probe having a nucleotide sequence represented by the formula (3) (30 me
r) is preferable because the test sample can be used for either a sample that has undergone a DNA amplification step by the PCR method or a case of the in situ hybrid method.

The probe B is specific to a gene encoding a phosphorylated protein (pp71). The length of the probe B also varies depending on the type of pretreatment for the test sample. When the test sample has undergone a DNA amplification step by the PCR method, the length is from 10 bp to the size of the DNA fragment amplified by the PCR method. be able to. When used in the in situ hybrid method,
Probe B is 10 bp to pp71 cDNA (1
680 bp). The DNA probe (30mer) of the base sequence represented by the formula (4) is used regardless of whether the test sample has undergone the DNA amplification step by the PCR method or the in situ hybrid method. Is also preferable because it can also be used.

The aforementioned probe C is specific to the exon 5 portion of the gene encoding IE1. Probe C is used for the in situ hybrid method. The length of probe C is 10 bp to 5 parts of exon (58%).
5 bp).

Probe A, Probe B and Probe C
Can be prepared by a method using succinoimide (for example, disuccinimidyl suberate), a maleimide method, and an active halogen (Active Halogen).
Method, a photoreaction using an azide, or a carbodiimide method.

Probe A, Probe B and Probe C
As the labeling substance, any conventionally known substance can be used. Preferably, a non-radioactive substance (enzyme, fluorescent dye, luminescent substance, biotin, etc.) is used. Labeled DNA
Can be roughly classified into (1) a method in which labeled DNA is directly prepared in the process of DNA synthesis, and (2) a linker-bound DNA is synthesized and then isolated, and a labeling reagent is acted upon. In particular, method (2) is preferable because the type of the label can be easily changed according to the purpose and is excellent in application. As a linker used in the method (2), 5′-dimethoxytrityl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimide] -2′-deoxyuridine-3 ′-[(2-cyanoethyl) -(N, N-diisopropyl]] phosphoramidite.

As a method for generating a signal from the label bound to the probe and further measuring the signal, any conventionally known method can be used.

[0027]

In the method of the present invention, only when CMV is present in the test sample, a base portion corresponding to a part of the IE1 gene or a part of the pp71 gene is determined by the combination of the first primer and the second primer. Is specifically amplified and synthesized in a large amount in a short time. Therefore, the presence of CMV in the test sample can be detected very specifically. Further, even if the amount of CMV in the test sample is very small, the DNA is amplified and synthesized, so that the sensitivity is high.

In the method of the present invention, since a labeled DNA probe specific to (optionally amplified) CMV-DNA is used, CMV can be detected very accurately. Furthermore, when the amplification step using the first and second primers and the test step using the probe are used together, the time required for the test is about 4 to 5 hours in the case of ethidium staining, and the time required for the Southern blot hybrid method is reduced. Even if it is performed, it is about 48 to 50 hours, and the result can be obtained quickly.

[0029]

The present invention will be described in more detail with reference to the following examples, which do not limit the scope of the present invention.

[0030]

Example 1 << Synthesis of primer >> Automatic DN 381A
An Adenine CPG column was attached to an A synthesizer (Applied Biosystems), and a primer consisting of 25 bases (1
a) was synthesized, and further, a cytosine CPG column was attached, and a primer (1b) consisting of 25 bases described in the sequence of SEQ.
Primer (2a) consisting of 25 bases described in the sequence
And a primer (2c) consisting of 26 bases described in the sequence of SEQ ID NO: 6 in the sequence listing, were synthesized.
A primer (2b) consisting of 25 bases described in the sequence of SEQ ID NO: 4 in the sequence listing was synthesized by attaching a G column, and attached to a sequence of SEQ ID NO: 5 in the sequence listing by attaching a thymine CPG column. A primer (1c) consisting of 25 bases was synthesized. Ammonia water (about 3
0%) disposable syringe containing 2.5 ml (2.5 ml)
Was connected to a CPG column in which the synthesis process was completed, and ammonia water was extruded into the column to elute the synthesized DNA fragment. The vial containing the recovered DNA ammonia solution was sealed and heated at 65 ° C. for 6 hours.
After cooling to room temperature, it was concentrated. Lyophilize the concentrate,
10 mM triethylammonium acetate (hereinafter referred to as T
EA-A) (pH 7.4), and after removing the precipitate, a separation column (YMC-Pack ODS) was applied to an L-6200 type high performance liquid chromatography apparatus (Hitachi, Ltd.) (hereinafter referred to as HPLC). -AM313: YMC
95% containing 5% acetonitrile
Purification was performed using a concentration gradient of TEA-A and acetonitrile, and the main peak was collected. 80% of the residue obtained
Acetic acid (adjusted with acetonitrile) was added to suspend the mixture, the mixture was kept at room temperature for 30 minutes, and dried under reduced pressure. 10m dry matter
It was dissolved in M-TEA-A, extracted with diethyl ether, and dried under reduced pressure. The dried DNA sample was dissolved in TEA-A, and after removing the precipitate, the second purification was performed by HPLC, and the main peak was collected. The purified DNA primer thus obtained was dried under reduced pressure and stored, and used in Example 3 described later.

[0031]

Example 2 << Preparation of Enzyme-Labeled Probe >> The DNA probe was synthesized using the same apparatus as in Example 1 under the same conditions, except that the base described in the sequence of SEQ ID NO: 7 in the sequence listing was used. In the synthesis of 30 probes A, a guanine CPG column was used to synthesize the 18th T from the 5 'end.
Is the base 30 described in the sequence of SEQ ID NO: 8 in the sequence listing.
For the probe B consisting of 30 C-columns, the 18th T from the 5 'end was used using a thymine CPG column. And the 17th T from the 5'-terminal is 5'-dimethoxytrityl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimide] -2'-deoxyuridine-3 '-[(2- Cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite (Glen Research)
(Hereinafter, referred to as a linker). The position of the linker is not limited to this site, and it is also possible to replace another thymine site. Purification of the probe with a linker was also performed under the conditions described in Example 1.

3 nmoles of the purified probe with a linker were dissolved in 8 μl of sterile water. 0.2M-N
After addition of aHCO ₃ / 4mM-EDTA solution 8 [mu] l,
A solution of disuccinimidyl suberate (Pierce) (hereinafter referred to as DSS) in dimethyl sulfoxide (10
(mg / ml), and reacted at room temperature in the dark for 15 minutes. After completion of the reaction, 30 μl of sterilized water was added to the reaction solution, and the mixture was centrifuged. The supernatant was subjected to HPLC (Toso-G3 as a column).
2,000 PW, using water as the mobile phase) to give D
DNA to which SS was bound (hereinafter, referred to as decorative linker DNA) was fractionated and freeze-dried. Dry decorative linker D
Reagent prepared by concentrating NA for alkaline phosphatase EIA (Boehringer Mannheim) twice to 20 m
g / ml (hereinafter referred to as AP) (40 μl) was added, and the mixture was reacted at room temperature in a dark place for 16 hours. After completion of the reaction, 2.5 ml of 0.1 M Tris-HCl buffer (pH 8.4) was added, and HP
LC (column: Toso-DEAE-5PW) was used.
First, unreacted AP was removed with a 0.1 M Tris-HCl buffer (pH 8.4) containing 0.1 M-NaCl, and 0.33 M
-0.1 M Tris buffer containing NaCl (pH 8.4)
Eluted the target AP-labeled DNA. Fractionated purified AP
About 6 ml of the eluate of the labeled DNA was dialyzed against 0.1 M Tris buffer (pH 8.4) containing 3 M NaCl, and then concentrated to 1 ml. This concentrate was used as an AP-labeled probe in Example 4 described below.

[0033]

Example 3 << Amplification of CMV-DNA by PCR Method >>
2.5 ng / cytomegalovirus (Towne strain)
Prepare 30 μl of GeneAmp DNA
Amplification Kit (Takara Shuzo) 10
× 5 μl of a buffer for PCR and 8 μl of a dNTP mixed solution,
20-fold diluted Taq polymerase (AmpliTaq
5 μl of DNA polymerase (Cetus) and 1 μM of each of the primers prepared in Example 1 above were added, and the whole was adjusted to 50 μl with sterile water. (1) 1 minute at 94 ° C, (2) 6
Thirty cycles of a process consisting of treatment at 0 ° C. for 2 minutes and (3) treatment at 72 ° C. for 3 minutes were carried out, and finally DNA was amplified by a program carrying out treatment at 72 ° C. for 7 minutes.

[0034]

Example 4 << Confirmation of Amplified DNA by Electrophoresis >> The gel for electrophoresis was 5.4 g of Tris base and 2.75 g of boric acid.
And 2 ml of 0.5 M-EDTA (pH 8.
0) 1.8% or 0.7% of agarose gel was dissolved in 1 liter (hereinafter referred to as 0.5 × TBE), and the solution was prepared by flowing the gel on a Mupido (Advance) gel plate. Next, 0.25% bromophenol blue and 2 μl of a 15% aqueous solution of Ficoll Type 400 (Pharmacia) were mixed with 10 μl of the solution after the PCR reaction, and the entire mixture was placed in a sample well. 0.5 × TBE was placed in an electrode tank, and electrophoresis was performed at room temperature and 100 V for 45 minutes. After completion of the electrophoresis, the agarose gel was stained with ethidium bromide, and the result of the electrophoresis was confirmed under irradiation with a UV illuminator. The results are shown in FIG. In FIG. 1, is a DNA marker, and is a first primer (1
a) a combination of the second primer (2a) and (a),
Is the first primer (1b) and the second primer (2a)
(C) is a combination of the first primer (1b) and the second primer (2b) (d), is a combination of the first primer (1a) and the second primer (2b) (b), When the combination (e) of the first primer (1c) and the second primer (2c) and the combination (b) and the combination (e) were used simultaneously, the respective amplified bands (S in FIG. 1) region)
Is shown. The DNA marker is 250 bp from the bottom,
500bp, 650bp, 900bp, 1100bp,
They are 1410 bp, 2000 bp and 5000 bp. The band appearing in the R region is considered to be a primer and its dimer.

As is clear from FIG. 1, it is possible to amplify a target specific site of CMV by performing PCR using various primers according to the present invention. Further, even when a plurality of primers are mixed, each can be amplified.

After confirming the electrophoresis image, 1M-NaCl
The agarose gel is immersed in about 500 ml of a 0.5 N NaOH solution containing
1 in 0.5 M Tris-HCl buffer (pH 7.4)
Neutralized by soaking for hours. Add 2 to neutralized agarose gel
Nylon filter (Hybon) wet with 0xSSC
dN: Amersham) and blotted at room temperature for 16 hours. The nylon filter was dried under reduced pressure for 1 hour, and then baked at 80 ° C. for 1 hour. Next, 5 × S containing 0.1% of sarcosine, 0.2% of SDS, 5% of a blocking agent for nucleic acid hybridization (Boehringer Mannheim) and 30% of formamide.
The above-mentioned nylon filter was kept at 42 ° C. for 60 minutes in SC (hereinafter, referred to as hybridization buffer I), and then hybridized with the AP-labeled probe prepared in Example 2 (3 pmol). The above nylon filter was transferred to a buffer for isation I (5 ml),
The reaction was performed at 2 ° C. overnight. For the filter that has been subjected to the hybridization treatment with the AP-labeled probe, 1 m
2 × SSC containing M zinc chloride (hereinafter referred to as cleaning solution I)
(100 ml) for 5 minutes at room temperature was repeated three times. Next, a cleaning solution I (100%
ml) at 42 ° C for 45 minutes. Further, washing with washing solution I (100 ml) at room temperature for 5 minutes was repeated three times. Finally, 0.1 M NaCl and 10 m
0.1 M Tris-HCl buffer containing M-MgCl ₂ (pH
9.5) (hereinafter referred to as a coloring solution) for 30 seconds to 1 minute
The filter was soaked and blended.

75 mg of nitroblue tetrazolium (Boehringer Mannheim) was dissolved in 1 ml of a mixture of 30% sterilized water and 70% dimethylformamide (this solution is hereinafter referred to as an NBT solution). Also, 50 mg of 5-bromo-4-chloro-3-indolyl phosphoric acid (Boehringer Mannheim) was dissolved in 1 ml of dimethylformamide (hereinafter, this solution is referred to as BCIP solution). Coloring solution 1 containing 40 μl of NBT solution and 40 μl of BCIP solution
0 ml was placed in a plastic bag containing a nylon filter adapted to the above-mentioned color developing solution, and kept at room temperature for 5 minutes to 16 hours to develop color. Reaction with AP is 10m
10 mM Tris-HCl buffer containing M-EDTA (pH
7.5) Stopped by immersing the filter in 50 ml.

FIG. 2 shows the results of these experiments. 2 in FIG. 2 corresponds to in FIG. As is clear from FIG. 2, the probe of the present invention has complementarity to the amplified site, and in the CMV-containing sample, the amplified DNA
The band is specifically stained by the AP-labeled probe.
Therefore, the primers and probes of the present invention
It can be seen that V infection can be diagnosed.

[0039]

Embodiment 5 << Detection of CMV by AP-Labeled Probe >>
HEL cells (Human Embryo Lung C
ell) was cultured on a chamber slide glass (Intermed: 21.3 mm x 20.0 mm) for 3 days, and then infected with CMV (Towne strain). Virus infected and uninfected HEL cells were cultured on glass slides for the same period (48 hours). After immersing the slide glass in 0.1 M sodium phosphate buffer (pH 7.2) containing 4% formaldehyde for 10 minutes,
Washing with a 0.1 M sodium phosphate buffer (pH 7.2) (hereinafter, referred to as NaPB) for 3 minutes was repeated three times. Next, after treating with 0.2N hydrochloric acid for 10 minutes,
Washed three times with aPB. In addition, 70%, 90% and 1
Each sample was dehydrated with 00% ethyl alcohol for 3 minutes and immersed in chloroform for 10 minutes. Finally, it was immersed twice in 100% ethyl alcohol for 3 minutes each, and then air-dried.

10% dextran sulfate sodium salt, 3
0% formamide, 0.025% herring sperm DNA and 1
The AP-labeled probe (2 picomoles) prepared in Example 2 was dissolved in 4 × SSC (100 μl) containing X Denhardt, spread on a slide glass, and kept in a 37 ° C. wet box for 16 hours. Next, take out the slide glass and 4 × S
After washing with SC for 10 minutes, the plate was washed in 1 × SSC containing 0.1 mM zinc chloride at 45 ° C. for 1.5 to 2 hours. Three
The washing solution was replaced every 0 minutes. Furthermore, after immersing in 1 × SSC containing 0.1 mM zinc chloride for 3 minutes,
0.1 M Tris-HCl buffer containing 5 M NaCl (pH
7.5) for 10 minutes. Finally, 0.1M-NaC
The mixture was equilibrated with 0.1 M Tris-HCl buffer (pH 9.5) containing 1 mM and 10 mM magnesium chloride (hereinafter referred to as AP buffer) for 3 minutes.

4 ml of NBT solution and B
4 μl of the CIP solution and 10 μl of a 5% sodium solution were added and mixed, and 300 μl of the mixed solution was placed on a slide glass previously prepared and reacted at 25 ° C. for 24 hours. Color development was stopped by treating with 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM-EDTA for 5 minutes.

FIGS. 3, 4 and 5 show the results of these experiments.
Shown in FIG. 3 shows infected cells using the probe (A), FIG.
Shows the results of infected cells using the probe (B), and FIG. 5 shows the results of infected cells using the probe (C). In addition,
When the probe (A), the probe (B) and the probe (C) were used for non-infected cells, no color developed. As is clear from FIGS. 3, 4 and 5, the probe (A), probe (B) and probe (C) used were (1) not cross-reactive with RNA and DNA of normal HEL cells, and (2) ) Hybridization with virus-specific RNA and DNA of CMV infected cells.

[0043]

Example 6 << Comparison of sensitivity with diagnostic method by virus isolation >> Using the culture supernatant of virus-infected HEL cells used in Example 5, the sensitivity by the conventional virus isolation method and the virus described in Example 3 were used. The sensitivity of the method of amplifying DNA by PCR was compared. According to ethidium bromide staining of Example 3, at least 0.1 PFU (Pl
aque Forming Unit). Furthermore, when Southern blot hybridization was performed using an AP-labeled probe under the conditions described in Example 3, at least 0.01 PFU could be detected.

[0044]

Example 7 << Primer specificity >> CM according to the present invention
The specificity of the V amplification primer was examined. As the virus, CMV (Towne strain), herpes simplex virus-
Type I (HSV-1: HF strain), herpes simplex virus-
Type II (HSV-2: UW268 strain), Varicella-zoster virus (VZV: H-N3 strain), EB virus (EB
V: B95-B strain) and HHV-6 (spontaneous rash disease isolate) under the conditions described in Example 3 and primer (1
b) and primer (2a), and primer (1c) and primer (2c),
A amplification was performed and the presence or absence of cross-reaction was examined.

FIG. 6 shows the results of these experiments. In FIG. 6, M is a DNA marker (the same marker as in FIG. 1), 1
And 7 are HSV-1, 2 and 8 are HSV-2, 3 and 9 are VZV, 4 and 10 are EBV, 5 and 11
Indicates the results of CMV, and 6 and 12 indicate the results of HHV-6. 1 to 6 are annealing temperatures of 55 ° C. and 7
-12 are results at an annealing temperature of 60 ° C. 182 and 316 bp only for samples 5 and 11 of FIG.
As apparent from the fact that bands appear in the order from the bottom (S and R are the same as in FIG. 1), according to the present invention, the CMV
Can be specifically detected.

[0046]

As described above, when various primers according to the present invention are used, specific sites of human cytomegalovirus (CMV) DNA contained in a small amount in a test sample are specifically and reliably amplified. Can be detected with high accuracy in a short time. In addition, when the probe of the present invention is used, CMV-DN
Since the specific site of A can be specifically and reliably detected, CMV can be detected in a short time with high accuracy.

[0047]

[Sequence List] <110> Iatron Laboratories, Inc. <110> Hirai, Kanji <120> A method for detecting human Cytomegalovirus <130> IATDIV <160> 9 <210> 1 <211> 25 <212> DNA <213> Cytomegalovirus <400> 1 TATACCCAGA CGGAAGAGAA ATTCA <210> 2 <211> 25 <212> DNA <213> Cytomegalovirus <400> 2 ATGAAGTGTA TTGGGCTAAC TATGC <210> 3 <211> 25 <212> DNA <213> Cytomegalovirus <400> 3 TTCTCCTAAG TTCATCCTTT TTAGC <210> 4 <211> 25 <212> DNA <213> Cytomegalovirus <400> 4 ATAAGCCATA ATCTCATCAG GGGAG <210> 5 <211> 25 <212> DNA <213> Cytomegalovirus <400> 5 TGACGCGCAT ACATCCCGAG TACAT <210> 6 <211> 26 <212> DNA <213> Cytomegalovirus <400> 6 ATGACGTTGC TCCGTGGAAA GAGACC <210> 7 <211> 30 <212> DNA <213> Cytomegalovirus <400> 7 CTCCTTAATA CAAGCCATCC ACATCTCCCG <210> 8 <211> 30 <212> DNA <213> Cytomegalovirus <400> 8 GCTGTCGGTG TTCCAAATGA ACGGCCTGAT <210> 9 <211> 30 <212> DNA <213> Cytomegalovirus <400> 9 GACTTCTTCA CCCTGTTCTT CCTCGCTATC

[Brief description of the drawings]

FIG. 1 is a photograph, instead of a drawing, showing the results of electrophoresis on DNA amplified by a PCR reaction using various primers of the present invention.

FIG. 2 is an explanatory diagram schematically showing the result of confirming the electrophoretic image of FIG. 1 by a Southern blot hybrid method using an enzyme-labeled probe.

FIG. 3 is an explanatory diagram schematically showing the results of performing in situ hybridization on HEL cells infected with CMV using the probe of the present invention.

FIG. 4 is an explanatory diagram schematically showing the results of performing in situ hybridization on HEL cells infected with CMV using another probe of the present invention.

FIG. 5 is an explanatory view schematically showing the results of in situ hybridization performed on HEL cells infected with CMV using still another probe of the present invention.

FIG. 6 is a photograph instead of a drawing, showing the results of electrophoresis on the specificity of the primer of the present invention for various viruses.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Takashi Hironaka 1-14-1 Higashikanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd. (72) Inventor Yuki Yamaguchi 1-14-1 Higashikanda, Chiyoda-ku, Tokyo (72) Inventor Hiroshi Kita 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Inside Yatron Co., Ltd.

Claims (2)

[Claims] 1. A first DNA primer containing an oligonucleotide portion of at least 15 bases of the base sequence represented by SEQ ID NO: 5 in the sequence listing, and a base sequence represented by SEQ ID NO: 6 in the sequence listing. A second D containing an oligonucleotide moiety of at least 15 bases of
A human cytomegalovirus comprising subjecting a mixed solution containing a combination of NA primers, a DNA polymerase, and an aqueous liquid test sample to a DNA amplification step, and then subjecting the resulting reaction solution to a DNA test step. Detection method. 2. A nucleotide sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9 in the sequence listing.
A method for detecting human cytomegalovirus, comprising: contacting a probe containing a base with an oligonucleotide and carrying a label with a test sample, and detecting a signal from the label.