JP2835855B2 - Human cytomegalovirus detection method - Google Patents

Description

The present invention relates to a method for detecting a specific DNA region of human cytomegalovirus (hereinafter abbreviated as HCMV).

The invention also relates to a detection kit for such a detection.

[Conventional technology]

HCMV is a virus of the genus Herpes, which is widespread worldwide. The majority of Japanese people are initially infected with HCMV due to birth canal infection at birth, post-natal trans-breast milk infection or horizontal infection, but in most cases it is subclinical and it is thought that the latent virus is retained throughout the body for life. Have been. HCMV
Demonstrates pathogenicity and causes clinical problems in cases of fetal / neonatal infection, iatrogenic infection due to blood transfusion / organ transplantation, or opportunistic infection in an immunodeficient state, sometimes leading to fatal progress. It is known to take.

In diagnosing HCMV infection, a cell culture method, an immunological method, a method for detecting viral DNA, and a histopathological diagnostic method are used.

[Problems to be solved by the invention]

Currently, the most reliable method of diagnosing HCMV infection is the isolation of virus by cell culture, but it may take up to two weeks depending on the sample. Other methods also have limitations in terms of detection sensitivity and detection sensitivity during the period required for diagnosis.

In recent years, genetic diagnosis technology has advanced, and HCMV DNA
Has been adopted as a rapid diagnostic method. However, the conventionally used Southern hybridization method cannot detect the virus DNA unless the DNA content is at least 0.2 to 1 copy per cell.
There is a limit as a detection method such as the necessity of a tissue sample of about 10 to 100 mg.

HCMV is found in blood and various organs, and is also excreted in urine, cervical secretions, breast milk, saliva, semen, tears, and stool. When detecting HCMV DNA from these samples, the copy number per cell is said to be quite low even when the virus is present in an activated state, and a highly sensitive detection method is required.

Recently, PCR [Polymerase Chain Reactio
n)] method was developed [Saiki et al .: Science, Vol. 230, pp. 1350-1354 (1985)]. PC
The R method is a method of specifically enzymatically amplifying only a target gene DNA or a part thereof and detecting the target gene from a small amount of sample with high sensitivity. By using this PCR method, it is possible to detect a viral gene in one out of 100,000 infected cells. Therefore, the PCR method is extremely effective for highly sensitive detection of HCMV DNA. Another advantage of this PCR method is that it is also effective against unpurified DNA. Therefore, it is possible to easily purify DNA from urine or blood samples and detect HCMV DNA.

Another problem with the detection of HCMV DNA is that HCMV DNA has high homology to other herpes virus DNAs, and thus there is a possibility that other herpes virus DNAs mixed in a sample may be erroneously detected. In the PCR method, the target gene region can be arbitrarily limited by a pair of primer DNAs. Therefore, regions with low homology to other herpes virus DNA
It can be set in CMV DNA.

An object of the present invention is to clarify a specific detection region in HCMV DNA by using a PCR method, and to provide a highly sensitive method for detecting HCMV and a kit for use in the method.

[Means for solving the problem]

In summary, the first invention of the present invention relates to a specific DNA region of HCMV, which is represented by the following formula II: And a method for detecting HCMV DNA, wherein the amplification is carried out using a pair of oligonucleotide primers represented by

Further, a second invention is a detection kit for performing detection using the method of the first invention, which comprises a pair of oligonucleotide primers represented by the above formula II. It relates to an HCMV DNA detection kit.

In order to solve the above problems, the present inventors have proposed a short unique region HindIII fragment V of HCMV strain AD169 [Journal of Molecular Biology (J. Mol. Biol.), Vol. 192, No. 1771]. ~ 208 pages (198
6)], a 305 bp region having relatively low homology to other herpes virus DNAs was set, and a pair of oligonucleotide primer DNAs required for amplification were synthesized. Next, HCMV
The present region was amplified by PCR using infected cells, and it was found that this region was efficiently amplified and detected with high sensitivity by an oligonucleotide probe.

 Hereinafter, the present invention will be specifically described step by step.

The short-chain specific region of the HCMV strain AD169 has already been cloned, and its nucleotide sequence is described in Journal of Molecular Biology, Vol. 192, pp. 177-208 (1986).

Hi with low homology to other Hepulses in the short chain specific region
A 305 bp amplification region was selected within the range of the ndIII fragment V. It is represented by Formula I below.

To amplify this region, a pair of oligonucleotide primer DNAs, ie, 2
A primer DNA pair having a 0 base sense sequence and an acetylsense sequence of 20 bases from the 3 'end is required.

This primer may be any as long as it can anneal to the above-mentioned selected region. As an example, a primer DNA represented by the following formula II can be synthesized by a DNA synthesizer and HP
Can be purified by LC.

HCMV DNA to be detected is HCMV AD169 strain and Davis (Davi
s) It can be prepared from a human cell line infected with the strain, urine or the like.

For the preparation of DNA from cells, a known established method may be used.

For the PCR method, use Taq-Polyme
and a gene amplification kit containing the same are commercially available from Perkin Elmer Cetus, Inc., and a specific DNA region may be amplified using these and the primer pair of the present invention. For detection of HCMV DNA after amplification, for example, agarose gel electrophoresis and Dod hybridization can be used. At the time of dot hybridization, HCMV DNA can be specifically detected by using a region specific to HCMV in the amplification region, for example, an oligonucleotide probe DNA shown in Table 1 below.

This probe DNA can be synthesized and purified in the same manner as the above primer DNA.

By labeling the probe DNA, highly sensitive detection becomes possible. The labeling method is not limited to the radioisotope labeling method, and any known method such as enzyme labeling, fluorescent labeling, biotin / avidin labeling, or chemiprobe labeling may be used.

By using the selected primer pair and probe, a region that can be specifically and highly sensitively detected in the HCMV DNA sequence was clarified.

In fact, HCMV DNA could be detected from urine samples with a detection rate of 70%.

Also, HCMV DNA can be easily detected by preparing a kit with a primer pair for amplifying a specific DNA region of HCMV and a probe for detecting the amplified DNA region. The reagent used in the kit may be a solution or a lyophilized product.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Amplification and Detection of HCMV DNA by PCR (1-1) Preparation of DNA from HCMV-infected cells Human fetal lung cells (HEL cells) infected with HCMV AD169 strain or Davis strain were subjected to DME [Dulbecco's modified Eagle Medium (Dulbecco's Modified Eagle's Medium) Flow Laboratory, 10% FBS [Fetal Bovine Serum (Fatal Bovine Serum)
(Serum Flow Laboratory), 100 units / ml streptomycin (Meiji Seika) and 100 units / ml penicillin G (Banyu Pharmaceutical) in a 6 cm culture dish. Exit the culture where was approximately 10 ⁶ of the number of cells, the medium was removed. 5 containing TE buffer [10 mM Tris-HCl, pH 8.0, 1 mM EDTA], 0.1 M NaCl
After washing the cells with ml of the solution, 5 ml of a solution containing TE buffer and 0.5% SDS was added, the cells were left at room temperature for 20 minutes, the cells were peeled off the dish, and collected in a polystyrene tube. Add proteinase K to 100 μg / ml and add 70
C. for 2 hours. Then add 5 ml of an equal volume mixture of phenol and chloroform, gently stir, 12000 rpm,
After centrifugation for 5 minutes, the aqueous layer was separated. 5 ml of chloroform was added to the aqueous layer, stirred gently, and centrifuged similarly to separate the aqueous layer. To this aqueous layer, 0.5 ml of 3M sodium acetate and 10 ml of ethanol were added, and the mixture was thoroughly stirred, and then stirred at -70.
After standing at 15 ° C. for 15 minutes, the mixture was centrifuged at 12,000 rpm for 10 minutes to collect a precipitate of DNA. The precipitate was washed with 80% ethanol, dried, and dissolved in 1 ml of sterilized water. About 100 μg of DN
A could be recovered.

(1-2) Synthesis and Purification of Oligonucleotide Primer DNA and Probe DNA Primer DNA and probe DNA shown in Formula II and Table 1 were synthesized using a DNA synthesizer manufactured by Applied Biosystems, followed by deprotection and ion exchange. HPLC (TSK gel, DEAE-2SW
Column) and desalted with Sep-pak C ₁₈ (Waters) to obtain about 50 μg of each DNA.

(1-3) Amplification of HCMV DNA by PCR method 0.5 μl each of 1 μg of HEL cell DNA infected with HCMV AD169 strain and Davis strain prepared in (1-1) and HEL cell DNA not infected with HCMV Take in a tube (Biovic) and heat-treat at 94 ° C for 10 minutes.
Gene Amp ^TM Kit PerkinElmer
10μs of 10 × amplification buffer [100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ,
0.1% (w / v) gelatin], 16 μl of 1.25 mM dNTP mixture (dATP, dGTP, dCTP, dTTP), 1 μm of 20 μM primer 1, 1 μm of 20 μM primer 2, 0.5 μ of 5 units / μ tack-polymerase Then, sterile water was further added to make a 100 µ solution.

After adding 100 μm of mineral oil (Sigma) to the upper layer of this reaction solution, an amplification reaction was carried out by an automatic gene amplifier Thermal-Cycler (Perkin-Elmer Cetus). The reaction conditions are 94 ° C,
30 cycles of denaturation for 1 minute → annealing of primer for 2 minutes at 55 ° C. → 2 minutes for synthesis reaction at 72 ° C.

After the reaction, after removing the mineral oil in the upper layer, 10 µ of the reaction solution was taken, and 3% NuSieve GTG agarose-1% Sea-Kem agarose (FMC)
Gel electrophoresis was performed, DNA was stained with ethidium bromide, and the amplified DNA was confirmed. A 305 bp band was confirmed from the DNA of HEL cells infected with the HCMV AD169 strain and the Davis strain. No DNA amplification was observed from HEL cell DNA not infected with HCMV.

(1-4) HCMV by dot hybridization
Detection of DNA The reaction solution obtained in (1-3) is treated at 94 ° C. for 10 minutes,
The DNA was denatured by rapid cooling in ice. 5 µ of the reaction solution was spotted on a nylon membrane brush (Shleicher & Shuell), irradiated with an ultraviolet transilluminator (254 nm) for 10 minutes, and the DNA was fixed to the membrane.

This membrane was prepared using a prehybridization buffer (5 × Denhardt's solution, 5 × SSC, 0.1% SDS, 100 μg / ml).
Prehybridization was performed in 10 ml of salmon sperm DNA) at 37 ° C. for 2 hours. Next, probe 1 shown in Table 1 labeled with less than 5 'with ³² P was added, and hybridization was carried out at 37 ° C. for 2 hours.

The ³² P-label of probe 1 is a Mega Label Kit (MEGAL
ABBL Kit) (Takara Shuzo). 10mo
le probe 1, 1μ 10 × phosphate buffer, 50μ
Ci of [.gamma. ³² P] ATP (Amersham), 10 units of T4
-Sterile water was added to the reaction solution containing the polynucleotide kinase to make 10 µ, and the reaction was carried out at 37 ° C for 30 minutes. After reaction 65
After treatment at 10 ° C. for 10 minutes, 2 μm (about 10 ⁸ cpm) of this reaction solution was used for hybridization.

After hybridization, the membrane was washed with 2xSSC, 0.1
Washing was carried out twice with a washing solution 1 containing% SDS at room temperature for 10 minutes, followed by washing twice with a washing solution 2 containing 2.0 × SSC and 0.1% SDS at 55 ° C. for 20 minutes. After the membrane was dried, it was exposed to -70 ° C. for 3 hours in a cassette containing an X-ray film (Fuji Film), and an autoradiograph was taken.

As a result, HCMV AD169 strain and Davis strain were infected.
Dots appeared from HEL cell DNA, but no dots were observed from HEL cell DNA not infected with HCMV.

These facts indicate that HCMV DNA was
Indicate that HCMV DNA can be specifically detected by hybridization with probe 1.

Example 2 Preparation of Kit for Amplifying and Detecting HCMV A kit for amplifying and detecting DNA of HCMV in a sample was prepared.

Primer 1 and Primer 2 were dissolved in 20 μl of TE buffer to give a 20 μM solution each as a primer for DNA amplification to prepare an HCMV primer solution (agent A).

As a DNA detection probe, 2 μg of Probe 1 shown in Table 1 was dissolved in 20 μ of TE buffer to prepare an HCMV probe solution (agent B). Agent A and Agent B were combined into one set to form an HCMV amplification / detection kit (Table 2).

(3-1) Preparation of virus-infected cell DNA In order to confirm the specificity of HCMV DNA detection, the following 4
Amplification and detection were performed using the DNA of another species of Hepulse virus-infected cell as a template.

Epstein-Barr virus
rus) (EBV) infected Buckkit's lymphoma Tumor Ra
ji cells, Herpes Simplex Vir
us) 1 (HSV1) F strain, herpes simplex virus 2 (HSV2)
G strain and varicella-herpes zoster virus (Varicella-Zos)
HEL cells infected with O. ter Virus) (VZV) Oka strain were cultured by the method shown in (1-1), and the DNA was reduced to about 50% each.
μg was collected.

(3-2) Amplification by PCR and Dot Hybridization Using 1 μg of the cell DNA infected with EBV, HSV1, HSV2, and VZV prepared in (3-1), and using the method described in (1-3),
30 cycles of amplification reaction were performed using the MV amplification and detection kit A agent. After the reaction, dot hybridization was performed by the method shown in (1-4) using 10 μm after the reaction and the HCMV amplification / detection kit B agent. As a result of autoradiography, no dots were observed from cells infected with any herpes virus.

From these results, it was shown that by using the 305 bp region represented by the formula I in HCMV DNA for detection by PCR, it was possible to specifically detect only HCMV DNA without detecting any other herpes viruses. .

Example 4 Detection of HCMV DNA from urine sample (4-1) Preparation of DNA from urine sample A 5 ml urine sample was ultracentrifuged at 25,000 rpm for 1.5 hours, and the precipitate was collected. The precipitate was washed with TE buffer (10 mM Tris). -CHl, pH8.0, 1m
M EDTA) suspended in 0.5 ml solution containing 10.5% SDS, 100 μg
/ ml and treated at 70 ° C for 2 hours. Next, an equal volume mixture of phenol and chloroform
0.5 ml was added, the mixture was gently stirred, centrifuged at 12,000 rpm for 5 minutes, and the aqueous layer was separated. 0.5 ml of chloroform was added to the aqueous layer, stirred gently, and centrifuged similarly to separate the aqueous layer. To this aqueous layer, 50 µm of 3 M sodium acetate and 1 ml of ethanol were added, stirred sufficiently, left at -70 ° C for 15 minutes, and then centrifuged at 12,000 rpm for 10 minutes to collect a DNA precipitate. The precipitate was washed with 80% ethanol and dried. The DNA was dissolved in 50 μl of sterile water, and heat-treated at 94 ° C. for 10 minutes. Then, use Gene Amp ™ as described in (1-3).
An amplification reaction solution was prepared using the kit, HCMV amplification / detection kit A agent, etc., and 30 cycles of amplification reaction were performed. After amplification
Dot hybridization was performed using 5 μ of the reaction solution and the HCMV amplification and detection kit B by the method shown in (1-4).

As shown in Table 3, HCMV DNA was detected in 17 of the 24 cases.

In the cell culture method, the detection rate was 9 out of 24 cases. Therefore, the detection rate was significantly increased by using the PCR method as compared with the cell culture method.

[Effects of the Invention] As described in detail above, a specific region effective for highly sensitive detection of HCMV DNA using the PCR method according to the present invention is revealed, and a method and kit for highly sensitive detection of HCMV genome in a sample Was provided.

──────────────────────────────────────────────────続 き Continuation of front page (56) References Mol. Biol 192 p. 177-208 (1986) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12Q 1/68 C12N 15/38 CA (STN) DDBJ / EMBL / GenBank

Claims (2)

(57) [Claims] (1) The following formula I: A specific DNA region represented by the following formula II: A method for detecting human cytomegalovirus DNA, comprising amplifying using a pair of oligonucleotide primers represented by: 2. A detection kit for performing detection using the method according to claim 1, comprising a pair of oligonucleotide primers represented by the formula II according to claim 1. Human cytomegalovirus DNA detection kit.