JPH04311400A - Probe for detecting human cytomegalovirous - Google Patents

Abstract

PURPOSE:To provide the subject new probe free from cross-hybridization with a relative virus such as varicella zoster virus, capable of specifically detecting human cytomegalovirous, readily synthesizable and having excellent reactivity, etc. CONSTITUTION:A probe for detecting human getomegalovirus(CMV), derived from D fragment of an EcoRI-cut segment in genom DNA of CMV, having a sequence specific to CMV and having a chain length composed of 20-70 bases, e.g. a probe having a base sequence of the formula. The above-mentioned probe is recommendably synthesized by beta-cyanoethylamidide method, etc., using a DNA synthesizer.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to human cytomegalovirus (Hum) by nucleic acid hybridization assay.
a Cytomegalo Virus, hereinafter referred to as CMV
The present invention relates to base sequences useful for the detection of nucleotide sequences (hereinafter abbreviated as PCR) and primers used for gene amplification by polymerase chain reaction (hereinafter abbreviated as PCR). CMV is 200
It is a virus classified in the genus Herpes with a genome size of over 1,000 kb. This virus exists widely among humans, and many humans are infected in the form of subclinical infection, and when the virus is activated due to organ transplantation or blood transfusion, or when an immunodeficiency state such as AIDS occurs. The disease develops and shows symptoms such as fever, pneumonia, and hepatitis. (Clinical Test MOOK, No. 28 "Clinical Test for Viruses" 1988, P194) Therefore, when transplanting kidney, liver, bone marrow, etc., it is essential to check for CMV infection.

0002

[Problems with the Prior Art] Immunological techniques such as ELISA and indirect hemagglutination are mainly used to check CMV infection, and most of these techniques are used to confirm the presence of antibodies against the virus. On the other hand, methods to directly prove the virus itself include virus isolation and culture using cultured human cells and methods using electron microscopy, but both methods are unsuitable for routine testing because they lack simplicity.

[0003] In recent years, methods for identifying or detecting viruses, bacteria, etc. at the genetic level based on the affinity of complementary nucleic acid sequences have become popular. According to this method, which is called a nucleic acid hybridization assay or the like, viral genes can be directly detected with relatively simple operations. CMV
Research is also underway at the genetic level, and the reactivity of fragments obtained by cutting DNA with restriction enzymes is being investigated. [Clin.Chem.
．． ) 31:P1514-, 1985, Journal of General Virology (J.Gen.Viro
l. )65:P1351-, 1984, etc.]

000
4] For example, among EcoRI cut fragments of genomic DNA,
It is known to use C, D, E, G, J fragments, etc. as probes. Among these EcoRI-cleaved fragments, the D fragment (17 Kb) can be used as a probe for Varicella zoster virus (Varicella zoster virus).
cella-zosterVirus (hereinafter abbreviated as VZV) may appear (clinical and viral,
15:P553.1987) was not satisfactory in terms of specificity. This phenomenon is due to the fact that hybridization occurs even if the base sequences do not match completely (cross hybridization).

[0005] On the other hand, the PCR method is attracting attention as a method that enables highly sensitive gene detection because it can generate many copy sequences from a small amount of DNA (or RNA). However, long sequences such as the D fragment are difficult to amplify by PCR.

[0006]

Purpose of the Invention The present invention aims to prevent cross-reactivity with closely related viruses such as VZV, which is observed when using the entire D fragment.
The purpose of this invention is to provide a probe that does not hybridize and a primer for amplifying the sequence to be detected.

[0007]

[Structure of the Invention] The present invention is directed to ``a human cytomegalovirus that is derived from the D fragment of the EcoRI-digested genomic DNA, has a sequence specific to the human cytomegalovirus, and has a chain length of 20 to 70 bases. A probe for detecting human cytomegalovirus characterized by
It is derived from the D fragment of the oRI cleavage fragment, has a sequence specific to human cytomegalovirus, and has a chain length of 20
15 to 3 near the 5' and 3' ends of the base sequence useful for detecting human cytomegalovirus, which is ~70 bases.
A primer for human cytomegalovirus gene amplification characterized by being composed of an oligonucleotide consisting of 0 bases.

[0008] In the present invention, CMV-specific and chain length 20
The probe consisting of ~70 bases is derived from the D fragment of the EcoRI-digested fragment, and specifically includes a sequence of at least 20 bases selected from, for example, the following base sequences. The sequence exemplified here is an approximately 1 kb fragment located at the most downstream (3' side) of the D fragment when the EcoRI cut fragment is further cut with HindIII.
HindIII-EcoRI fragment).

Base sequence: 5'-TTGTCCCGAA
ATGATATCCG TACTGGGTCC CA
TTTCGGGG CACGTGCTGA AAGCC
GTGTT TAGTCGCGGC-3' Of course, the probe according to the invention can also be selected from among the following base sequences which are complementary to the above sequence. Base sequence: 5'-GCCGCGACTA AACACGG
CTT TCAGCACGTG CCCCGAAATG
GGACCAGTA CGGATATCAT TT
CGGGACA-3'

[0010] The chain length of the probe needs to be at least 20 bases, preferably 30 bases or more to ensure specificity. On the other hand, if the length exceeds 70 bases, the reactivity decreases, so it is better to avoid increasing the chain length unnecessarily. These probes were prepared using the β-cyanoethyl amidide method [Nucleic Acid Research (Nucleic A
cid Res. ) 12: p4539-, 1984]
It may be synthesized by a method such as For synthesis, D
It is convenient to use an NA synthesizer.

When carrying out hybridization assays, the probe according to the invention can be used, for example, with 32P-γ-A
It is preferable to radiolabel it using TP and use it. The sign has
In addition to radioactive isotopes such as 32P and 125I, enzymes such as alkaline phosphatase and peroxidase,
Known labeling substances such as fluorescent substances such as fluorescein and rhodamine and luminescent components such as luminol can be used.

[0012] Analytical targets include samples that may contain CMV, ie, urine, and infected cells such as white blood cells. These samples directly
Alternatively, nucleic acid components can be extracted and analyzed. If the amount of nucleic acid is large, it can be analyzed as is, but even more sensitive analysis is possible if it is amplified by PCR using primers described later.

Nucleic acids to be analyzed are prepared by blotting onto a nitrocellulose membrane, or by using the methods described in JP-A-1-2318.
After capture with a nucleic acid-adsorbing solid phase such as 98, if the probe is hybridized with the probe, it can be easily separated from unreacted probe. It is also possible to hybridize in a liquid phase system, and in this case, separation from unreacted probes is performed using a capture probe, various affinity chromatography, etc.

Next, the primers for CMV gene amplification according to the present invention will be described. The primer according to the present invention is composed of 15 to 30 bases near the 3' end and 5' end of the sequence to be detected by the probe, or a complementary sequence thereof. This sequence also preferably has a chain length of 15 to 30 bases, especially about 30 bases, taking into account specificity, reactivity, effort in synthesis, etc.

Taking as an example the sequence exemplified above as a probe, the sequence of the primer according to the present invention is, for example, 5'-GCGCGCCGCT CAGTCGC on the 5' side.
CTA CACCCGTACG-3', 3' side 5'
-CACGCGTACG TGGATACCCG TC
Each can be selected from among the sequences TGCAGGAG-3'. Of course, these complementary sequences 5
'-CGTACGGGTG TAGGCGACTG A
GCGGCGCGC-3' and 5'-CTCCT
GCAGA CGGGTATCCA CGTACGCG
TG-3' is also available. These primers can be produced using a DNA synthesizer and are used in accordance with, for example, "Science 23
0:P1350-, 1985''. Recently, kits and dedicated systems for PCR methods have become commercially available, and it is convenient to use them.

[0016]

[Operation] The CMV detection probe in the present invention is a VZV detection probe.
It specifically hybridizes to CMV DNA without cross-hybridizing with other closely related viruses. Furthermore, since the chain length is 30 to 70 bases, it is advantageous in terms of reactivity and the like. Furthermore, the CMV gene amplification primer of the present invention has the effect of specifically amplifying the complementary sequence of the probe by PCR method. In the present invention, since the chain length of the target sequence is set to 30 to 70 bases, rapid amplification is possible. The present invention will be explained in more detail below based on Examples.

[0017]

[Example] 1. Detection of CMV Using a probe with the sequence shown below, DNA of AD169 strain, which is a CMV standard strain, was detected.
We tried to detect. The probe used was synthesized with a DNA synthesizer (Cyclone, manufactured by Bioresearch). Probe: 5'-GCCGCGACTA AACACGG
CTT TCAGCACGTG CCCCGAAATG
GGACCAGTA CGGATATCAT TT
CGGGACA-3'

[0018] Ordinary method (J. Vir
The DNA of the AD169 strain was extracted in a serial manner and blotted onto a nylon membrane (Hybond-N, manufactured by Amersham). As a control, DNA extracted from two urine specimens positive for human herpes simplex virus type 1 (hereinafter abbreviated as HSV-1), VZV, and CMV was serially diluted and blotted in the same manner. The nylon membrane was immersed in 6x SSC for 5 minutes at room temperature, then 50mM Tris-1M NaCl, 0.
1% sodium dodecyl sulfate (hereinafter abbreviated as SDS), 1
It was immersed in mMEDTA solution for 1 hour at 42°C. then 50
% formamide, 5x Denhardt's solution, 5x SSPE,
Prehybridization was performed at 42° C. for 6 hours in a solution containing 0.1% SDS and 100 μg/ml salmon sperm DNA. The prehybridized nylon membrane was washed with 6x SCC and then subjected to the conventional method [Methods.
Methods in Enzymology
zymol. )65:P499-, 1980] by 3
In a hybridization solution (same composition as the prehybridization solution except containing 10 ng/ml labeled probe: 10 μCi instead of salmon sperm DNA) containing a probe labeled at the 5' end with 2P-γ-ATP,
Hybridization was performed at 2°C for 1 hour. After hybridization, 5 minutes at room temperature with 2x SSC, 0.1% SDS solution.
for 5 min at 65°C in 1x SSC, 0.1% SDS.
After washing for a minute, it was air-dried and autoradiography was taken. The results are shown in Figure 1. The labeled probe hybridized in proportion to the increase in the amount of CMV DNA, and no cross-hybridization with HSV-1 or VZV was confirmed. Furthermore, CMV-positive specimens can also be detected with sufficient sensitivity.

5×SSPE: 0.9M NaCl0
．． 05M NaH2 PO4 (pH7.4) 5m
M EDTA 100x Denhartz solution: Ficoll 400
20g polyvinyl pyrrolidone 20gBSA
10 with 20g purified water
Volume up to 00ml 2x SSC: 300mM NaCl 30mM Sodium Citrate

2. Amplification of CMV Amplification of the CMV gene was attempted using the primers according to the present invention. FIG. 3 shows the relationship between the complementary sequences of the primer and probe in this example. The sequences used as primers, the composition of the reaction solution, and the protocol of the PCR method are shown below. The samples used were the same CMV standard strain as in Example 1, one case of CMV-positive urine, and H
In addition to SV-1 and VZV, DNA of human herpes simplex virus type 2 (hereinafter abbreviated as HSV-2) was used. The primers used were synthesized using a DNA synthesizer (Cyclone, manufactured by Bioresearch).

Reaction solution: 1 ng of sample DNA extracted from cells (or 5 nl of boiled urine) 1 μM of 3'-side primer (sequence: 5
'-GCGCGCCGCT CAGTCGCCTA C
ACCCGTACG-3') 5' side primer
1 μM (sequence: 5'-CACGC
GTACG TGGATACCCG TCTGCAG
AG-3') 10x PCR buffer
10μl each dNTP
5 μl each Taq polymerase
0.5μl (final concentration 2.5U) (manufactured by BRL) Dilute to 100μl with purified water

[0022] PCR method protocol: “94°C/1.5
After 1 cycle of "94℃/1 minute → 60℃/2 minutes → 72℃/3 minutes" for 30 to 40 cycles (however, in the final cycle, ℃/3 minutes is 72℃/4 minutes.)

002
3] After the final cycle, collect 5 μl of the reaction solution,
When electrophoresis was performed on a 0.8% agarose gel, as shown in FIG. 2, a band of 190 bp (corresponding to the sequence in FIG. 3) was detected only for CMV. The molecular weight marker used was pBR322 DNA cut with HinfI. It can be seen that the CMV gene can be specifically amplified using the primers according to the present invention.

[0024]

[Effects of the Invention] The probe according to the present invention does not cross-hybridize with closely related viruses such as VZV, so it can specifically detect CMV, and since the chain length is appropriate, it is easy to synthesize and has excellent reactivity. Further, the complementary sequence of the probe according to the present invention has a size that can be amplified by PCR method. On the other hand, the primer of the present invention has the effect of specifically amplifying the complementary sequence of the probe.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing the results of autoradiography in Example 1. The numbers on the right are CMV-
The amount of DNA (pg) of the AD169 strain is shown.

FIG. 2 is a schematic diagram showing the results of gel electrophoresis in Example 2. The band corresponding to 190 bp is indicated by an arrow.

FIG. 3 is a sequence diagram showing the relationship between complementary sequences of primers and probes in Examples. The underlined portions are the complementary sequences of the primer and probe.

[Explanation of code] CMV+1: CMV positive urine specimen 1C
MV+2: CMV positive urine specimen 2 VZV: VZV standard strain HSV-1: HSV-1 standard strain CMV-AD: CMV-AD169 strain PBR322/HinfI: HinfI cleavage fragment of PBR322 DNA HSV-2
:HSV-2 standard strain

Claims (4)

[Claims] [Claim 1] Human cytomegalovirus genome D
A probe for detecting human cytomegalovirus, which is derived from the D fragment of the EcoRI cleavage fragment in NA, has a sequence specific to human cytomegalovirus, and has a chain length of 20 to 70 bases. [Claim 2] Base sequence 5'-TTGTCCCCGA
A ATGATATCCG TACTGGGTCC C
ATTTCGGGG CACGTGCTGA AAGC
The human cytomegalovirus detection probe according to claim 1, which has a sequence with a chain length of 20 bases or more selected from CGTGTT TAGTCGCGGC-3', or a complementary sequence thereof. [Claim 3] Human cytomegalovirus genome D
The 5' end of a base sequence useful for detecting human cytomegalovirus, which is derived from the D fragment of the EcoRI cleavage fragment in NA, has a sequence specific to human cytomegalovirus, and has a chain length of 20 to 70 bases. and a human cytomegalovirus gene amplification primer characterized by being composed of an oligonucleotide consisting of 15 to 30 bases near the 3' end. [Claim 4] Base sequence 5'-GCGCGCCGC
T CAGTCGCCTA CACCCGTACG-3
' and 5'-CACGCGTACG TGGATA
The human cytomegalovirus gene amplification primer according to claim 3, which has a sequence with a chain length of 15 bases or more selected from CCCG TCTGCAGGAG-3', or a complementary sequence thereof.