JPH03191784A - Probe for detection of tobacco necrosis virus, probe for detection of satellite tobacco necrosis virus, production of the probe and diagnosis of the virus - Google Patents

Abstract

PURPOSE:To obtain the subject probe useful for the detection of plant virus by extracting RNA from tobacco necrosis virus, forming a DNA complementary to the RNA, producing a double-stranded DNA, inserting the DNA into a host microbial cell through a plasmid and culturing the cell. CONSTITUTION:RNA is extracted from (satellite) tobacco necrosis virus and a DNA (cDNA) complementary to the extracted RNA is prepared. The prepared complementary DNA is converted to a double-stranded DNA (ds-cDNA) and integrated into a plasmid. The obtained recombinant DNA is inserted into a host microorganism and the transformed host microorganism is cultured. The objective probe for the detection of (satellite) tobacco necrosis virus can be produced by separating a nucleic acid probe complementary to the (satellite) tobacco necrosis virus accumulated in the culture liquid.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention provides a nucleic acid probe for detecting tobacco necrosis virus (hereinafter abbreviated as TNV) and satellite tobacco necrosis virus (hereinafter abbreviated as 5TNV); The present invention relates to a manufacturing method and a diagnostic method thereof. Nucleic acid probes prepared by this method can be used to infect plants such as tobacco, strawberries, and tulips with TNV and 5T.
It is useful as a diagnostic aid for confirmation of NV.

[Prior Art] Plant DNA viruses include cauliflower mosaic virus, carnation etching virus, etc.; plant RNA viruses include tobacco mosaic virus, alfalfa mosaic virus, and carabie mosaic virus; and purified viruses. It has been reported that it was possible to diagnose the presence or absence of a virus in a ground solution of a plant body by using the DNA itself or its cDNA labeled with 32p by nick translation as a probe (J. Virol.
Meth, 6, 215-224.1983)sTNV
(Strain 5V-1), its CDNA was cloned into plasmid pBR322, and the nucleotide sequence of 1239 was reported (J, Mo1. Biol, 143,
259-271, 273-287.1980) However, the genome nucleotide sequence of the 5 TNV strains that parasitize domestic TNVs has not yet been elucidated.

[Problems to be Solved by the Invention] As mentioned above, tobacco mosaic virus, alfalfa mosaic virus, and the like are known as DNA probes for detecting plant viruses. However, many plant viruses are known to cause harm to plants, and the reality is that these plant viruses cause severe damage to plants. For example, in the production of strawberries, strawberry seedlings are supplied by vegetative propagation of parent plants.

In this case, if the parent plant is contaminated with a virus,
All farmers who received seedlings derived from this parent plant will be affected. Therefore, it is desirable to have nucleic acid probes for various plant viruses as detection aids for early detection of plant virus infection.

The present invention aims to expand the types of nucleic acid probes for detecting plant viruses, and provides a nucleic acid probe that has not been previously reported, a method for producing the nucleic acid probe, and a method for producing a plant virus-infected probe using the nucleic acid probe. The present invention aims at diagnosing plants and provides nucleic acid probes complementary to tobacco necrosis virus and satellite tobacco necrosis virus, which have not been previously reported as nucleic acid probes.

[Means for solving the problem] In the TNV detection probe according to the first invention, TNV-
It is complementary to RNA.

In the TNV detection probe according to the second invention, in the TNV detection probe according to the first invention, the DNA probe represented by the base sequence of FIG.
This is an RNA probe corresponding to the base sequence shown in the figure.

In the TNV detection probe according to the third invention, in the TNV detection probe according to the first invention, the DNA probe represented by the base sequence of FIG.
This is an RNA probe corresponding to the base sequence shown in the figure.

In the 5TNV detection probe according to the fourth invention, the 5TNV detection probe
- It is complementary to RNA.

In the 5TNV detection probe according to the fifth invention, the fourth
In the probe for detecting 5TNV according to the invention, the DNA probe represented by the base sequence of FIG.
This is an RNA probe corresponding to the base sequence shown in the figure.

In the 5TNV detection probe according to the sixth invention, the fourth
In the probe for detecting 5TNV according to the invention, the DNA probe represented by the base sequence of FIG.
This is an RNA probe corresponding to the base sequence shown in the figure.

In the 5TNV detection probe according to the seventh invention, the fourth
In the probe for detecting 5TNV according to the invention, the DNA probe represented by the base sequence of FIG.
This is an RNA probe corresponding to the base sequence shown in the figure.

In the method for manufacturing a TNV detection probe and a 5TNV detection probe according to the eighth invention, the TNV or the 5TN
RNA is extracted from V, and DNA (cDNA) complementary to the extracted TNV-RNA or 5TNV-RNA is extracted from
), and the created complementary DNA was transformed into double-stranded 1 DNA (ds-
The two aRDNAs (ds-c DNA) are connected to a plasmid, inserted into a host bacterium, and the host bacterium is cultured to separate and generate a nucleic acid probe complementary to the accumulated TNV or 5TNV. It is something.

In the method for diagnosing TNV or 5TNV according to the ninth invention, one of the TNV detection probes or 5TNV detection probes according to the first to seventh inventions is used as a labeled probe by adding an enzyme or an isotope. , hybridizes the ground solution of the test plant with the labeled probe.

[The action TNV detection probe and the 5TNV detection probe are as follows:
You can do the following:

That is, strawberry TNV is inoculated into Nicotiana clevelandii, the virus is grown for 1 to 2 weeks, and then frozen. Crush the frozen contaminated leaves in an appropriate buffer using a Waring blender or the like.

The resulting crushed solution was centrifuged at 10,000 g for 10 minutes to remove residual material, and then centrifuged at low speed (Ho, ooog) and high speed (78,
000g) Repeat the centrifugation twice to obtain a fraction containing the virus as a precipitate. This precipitate was suspended in MIR solution, and TNV (from the middle layer) and 5TNV were separated by sucrose density gradient centrifugation.
(from the upper layer).

Each of the above viruses was treated with phenol extraction, ethanol precipitation, etc. to extract each RNA, and using this RNA as a template, cDNA was extracted with reverse transcriptase according to a conventional method.
Synthesize.

Since the resulting cDNA is single-stranded, this was used as a template to create two *Rc DNAs, and then the pUC strain (J,
Vieira, J., Messing, Gene, 19,
259 (1982)) and the M13mp strain (J, Mes
sing, Methods in Enzya+1o1
gy, volol, p2G, Acade+mic P
(1983)), and then transformed into Escherichia coli HBIOI, JM109, etc. and cloned, it is possible to easily obtain a clone having the cDNA of the desired viral RNA as an insert.

The resulting TNV clone or 5TNV clone was
Northern hybridization can be performed with NV-RNA or 5TNV-RNA to obtain hybridizable clones. Specifically, in this example, TNV
From the clone of plasmid pTNV36. pTNV8
Two clones were obtained in which 5TNV was inserted, and plasmid pYHMK1926. pYHMK
Three clones were obtained in which pYHMK1816 and pYHMK1802 were inserted.

For cloned cDNA, the dideoxy method (F, Sanger, 5th century, 214.1205
-1210 (1981); J, Messing, J, V
ieira, Gene, 19.269-276 (
(1982) ) allows the sequence to be performed.

The detection probe may be produced by the method described below.

■ Double-stranded DNA probe: A plasmid containing cDNA as an insert is purified using cesium chloride. The cDNA portion is cleaved with an appropriate restriction enzyme, and cDNA is obtained by electrophoresis. cDNA is labeled with 32p biotin or the like by nick translation or random primer method.

■ Single-stranded RNA probe: Double-stranded cDNA is recloned, for example, into transcription plasmid pGEM (T., Maniatis, et al.
Molecular Cloning (1982))
A single @74 RNA probe complementary to viral RNA can be obtained by sps or T7 RNA polymerase in the presence of 32P or biotin-labeled substrate.

■Single-stranded DNA probe: Based on the cDNA sequence results, a single-stranded DNA probe is produced by synthesizing DNA with an appropriate number of bases complementary to the viral RNA using a DNA synthesizer and labeling it with 32p-γ-ATP. can get.

Detection of virus infection can be easily carried out by plotting the ground liquid of leaves of the test plant on a nylon membrane or the like and hybridizing it with the above-mentioned probe.

In addition, the plasmids pTNV36 and pTNV36 obtained in this example
TNV8. pYHMK1926. pYHMK1816.
The Escherichia coli into which PYHMK1802 was inserted were Microtechnical Laboratory Bacteria No. 10708, Tetsukoken Bacteria No. 10707, Tekkoken Bacterial No. 10711, Fukken Bacteria No. 10710, and Chokoken Bacteria No. It has been deposited as No. 10709.

[Example] The present invention will be specifically explained below with reference to experimental examples.

Experimental example (1) Purification of TNV and 5TNV Strawberry TNV collected in Kanagawa Prefecture was inoculated into Nicotiana Clevelandi species, and the virus was allowed to grow for one to two weeks.
Infected leaves with numerous netarosis were collected and frozen. This frozen infected leaf was treated with 3 containing 0.1% thioglymaric acid.
Double the amount of phosphate buffer (pH 7.0) was added and the mixture was ground using a Waring blender. Add 175 volumes of chloroform to the obtained crushed solution and stir at room temperature for 10 minutes.
The mixture was centrifuged at 00g for 10 minutes, and the aqueous layer was collected.

Low speed centrifugation (10,000g, 10 minutes) High speed centrifugation (
78,000g, 90 minutes) was repeated twice to obtain a precipitate fraction containing the virus.

After adding 2 ml of phosphate buffer to this precipitate obtained from 30 g of frozen infected leaves and keeping it at 4°C for 3 hours, 3.000 g of
Centrifugation was performed for 10 minutes, and the supernatant was further subjected to 10% to 40% sucrose density gradient centrifugation (Hitachi RPS OT rotor 25000
rpm, 6° C., 3 hours), and 5THV was separated and purified from the upper layer and TNV from the middle layer to obtain each virus.

After inoculating Nicotiana clevelandii with the virus, TNV was mainly obtained from early infected leaves, and 5TNV was mainly obtained from late infected leaves.

Using this method, 1 kg of infected leaves can be used to produce T N V lhg,
1 g of s T NV 1.3II was obtained. By electron microscopy, only virus particles with a diameter of 26 to 28 nm were observed in the TNV specimen, and only virus particles with a diameter of 17 nn+ were observed in the 5THV specimen.

Experimental Example (2) Synthesis of cDNA RNA was extracted from the virus obtained in Experimental Example (1) by the 5DS-phenol method. Based on the mobility by electrophoresis, TNV RNA (hereinafter abbreviated as TNV-RNA) and 5TNV RNA (hereinafter referred to as 5TNV-RN)
The number of bases of A) is 4.3, 1.
It was estimated to be 3.

First, polyA was added to the 3' end of the obtained viral RNA (J, S, Emtage et al., Nucl.
eic Ac1dResearch, 6.1221-1
239. (1979)), 1 ml of reaction solution (50a+M
/Tris-HCI, pH 8, 0, 0, 5mM/EDT
^, 12.5sM/MgCh.

2a+M/MnC1z, 200IIM/NaC1,1
mM dithiothreitol, 250 units/RNA5
in, 0.2mM/ATP, 10μCi
/'H-ATP.

Add 9 units of poly A polymerase to 50 μg/viral RNA), react for 15 minutes at 37°C, and then add 0.2
The reaction was stopped by adding 40 μm of 5M/EDTA. Addition of polyA to viral RNA was confirmed using a liquid scintillation counter.

Boehringer, Mannheim, and Yamanouchi (
A cDNA synthesis kit manufactured by BMY was used. pol
p as a primer to the viral RNA with yA added.
After associating Olyd T, complementary D
NA (cDNA) was synthesized.

After treating the obtained cDNA with ribonuclease H, E.
The DNA was reacted with E. coli DNA polymerase, T, and DNA polymerase to obtain double-stranded cDNA. The amounts of two [9c DNAs obtained from 20 μg of viral RNA were 4 μg (TNV) and 0.4 μg (s
TNV).

Two obtained & JI cDNA 2 μg (TNV), 0
．． EcoRI linkers were added to both ends of 2 μg (sTNV) using 74DN^ ligase (350 units), and both ends were made into sticky ends using the restriction enzyme EcoRI (20 units). That is, 2μm of 2Kizhen cDNA (TNV
(7) case), EcoRI linker-0-5ug-
Add T4 DNA ligase (350 units) and add 22
The reaction was carried out at ℃ for 4 hours. After adding 1 ml of 0.5M EDT^ to stop the reaction, cDNA with EcoRr linker was treated with phenol/chloroform and ethanol.
A9 was generated and dissolved in EcoRI buffer at 100 μC. Add 20 units of restriction enzyme EcoRI to this,
The reaction was carried out at 37°C for 2 hours.

Seph 10-s CL4B (column 16m1 diameter, 6hm length) with two sticky ends on both ends.
After gel filtration with ethanol and precipitation with ethanol, it was dissolved in 6 μl of distilled water. This is plasmid pUc118 or pUc
119 into the EcoR1 site. That is, the cDNA with sticky ends was transferred to Sepharose CL4B.
After gel filtration and ethanol precipitation, 7 μl of 74DN
It was dissolved in A ligase buffer. This solution 3.5I1
11, plasmid pU treated with restriction enzyme EcoRI
c11B or pUC1 90.5 μg% T4DN^ligase 200 units were added and reacted at 12.5° C. for 3 hours.

Experimental example (3) Cloning to E. coli 12. Transformation was performed using JM109. Add the above reaction completion solution to 2.5XIO' of JM109 competent cells, let stand in water for 15 minutes,
Heat treatment was performed at 2°C for 2 minutes. 50 μg/m of this E. coli
Agar medium containing ampicillin (Tryptone 100
g/l, NaCl sog/l, yeast extract sog/l, 2%-Xga in 1.5*agar)
ts.

μf, 0.2M-isopropylthiogalactoside 50μ
After culturing at 37°C overnight, white colonies were selected.

Among these, the boiling method (l(olmes, D, S, a
nd M.

Quigley, ^na1. Biochea+, 114
, 193. (1981)) conducted a mini-screening. In other words, 8% of the E. coli that showed white colonies
Sucrose, 0.5% tritium: /X-
100, 505M/EDT^, 10mM/
A Tris-0.1% lysozyme mixture was added and heated in boiling water for 60 seconds to recover the plasmid inside the bacterial cells. After the recovered plasmid was purified by phenol/chloroform/ethanol precipitation, restriction enzyme Ec
Process oRI't'. This was electrophoresed on a 1% agarose gel to determine whether ds-cDNA had been inserted into the EcoRI site of the plasmid of each colony.

Seven clones containing ds-cDNAs with different lengths of TNV, and 5 clones containing ds-cDNAs with different lengths of TNV.
Six clones were obtained as clones containing the following.

Furthermore, the seven clones of TNV obtained were subjected to Northern piperidization with TNV-RNA, and plasmid pTNV36. p
Two clones with TNV8 inserted were obtained.

In addition, among the 6 clones of 5TNV obtained in the same manner, 5T
As a loan that can hybridize with NV-RNA,
1926°PYHMK1816. to plasmid pYHM.
Three clones in which PYHMK1802 was inserted were obtained.

Experimental Example (4) Determination of the base sequence 2 O-ons of TNV into which the ds-cDNA obtained in Experimental Example (3) was inserted (pTNV36゜pTNV8)
and s T N V (D 3ri(1-:/(PYH
MK1926. pYHMK1816. pYHMK180
Regarding 2), the base sequence was determined by the dideoxy method using Takara Shuzo's M13 sequencing kit.

The orientation of the 10@jl DNA obtained by the M13 phage was determined from the results of hybridization between the viral RNA and the 10@jl DNA.

Figure 1 shows TN obtained with plasmid pTNV36.
Figure 2 is a base sequence diagram of a nucleic acid probe complementary to TNV obtained from plasmid pTNV8, Figure 3 is a base sequence diagram of a nucleic acid probe complementary to TNV obtained from plasmid pTNV8, and Figure 3 is a diagram of plasmid pYHM.
Base sequence diagram of a nucleic acid probe complementary to 5TNV obtained by K192B, Figure 4 shows plasmid pYHMK.
A nucleotide sequence diagram of a nucleic acid probe complementary to 5TNV obtained by 1816, Figure 5 shows plasmid pYHMK18.
FIG. 2 is a diagram of the base sequence of a nucleic acid probe complementary to 5TNV obtained by 02.

Experimental Example (5) Creation of Virus Detection S-t Probe Among the clones obtained in Example (3), 320 base pair TNV-RNA (7) CDNA was inserted) &:
Using a clone named PTNV36,
The method for creating two @l DNA probes is shown below.

1 E. coli with a 320 base pair cDNA in the insert
When cultured in broth containing 00μIL/■1 of ampicillin and the number of E. coli bacteria grew to 5 x 10'/1111 (Klett OD value 90), 170μg/m
1 of chloramphenicol was added and the mixture was further incubated day and night. After bacterial collection, DNA was extracted with lysozyme, and the plasmid was purified by cesium chloride density gradient centrifugation. That is, the collected E. coli was placed in 50mM Tris buffer (pH, o
) and washed thoroughly. Add 1.2 ml of 25% sucrose in 150 mM Tris buffer/0.1% lysozyme to the cells, leave at 0°C for 5 minutes, and then add 0.4 ml of 0.
．． 25M-EDTA was added. After 5 minutes, another 0.5m
1 of 5M NaCl and 0.2 ml of 10% SO3 were added and reacted at 0°C for 2 hours. 1 ml of 5hM-EDT
Add A, centrifuge at 3,6000 rpm for 40 minutes, and collect the supernatant.

Add 50a+M-EDTA to the collected supernatant; 3.
5111+, 3.2g of cesium chloride, 0.1
Add 4ml of 1% ethidium bromide to 3,600
The mixture was centrifuged at 0 rpm for 66 hours, and the plasmid fraction was collected under UV irradiation.

A 90 Mg plasmid was obtained by culturing 100+ nl of L broth. The obtained plasmid was treated with the restriction enzyme EcoR1.
and electrophoresis was performed on a 1.2% agarose gel. The c DNA part of TNV-RNA was exposed to long wavelength UV.
It was cut out at the bottom and purified by phenol extraction and ethanol precipitation.

This cDNA was labeled with 32p-α-dCTP using the Undambrimmer DNA labeling kit manufactured by Takara Shuzo Co., Ltd. to obtain a 32p-labeled 2-tZR DNA probe. A biotin-labeled double-stranded DNA probe was also obtained by labeling with biotin-11-dUTP.

5 μg of the 5-2 piperidization-infected leaf fragment was spotted on a nylon membrane, and plot hybridization with the 32p-labeled probe described above was performed according to a conventional method. That is, 1 g of the test plant was mixed with
Add 31 (5M sodium citrate) and grind. Gently centrifuge and spot 5 μk of the supernatant onto a nylon membrane. After UV irradiation for 3 minutes, incubate at 80°C for 1 hour and store at 4°C until use. Piperidization solution 10ml (X50 Denhardt solution 2ml
, IM-Tris buffer 0.51.5M-NaCl2
ml, 1 ml of 1% sodium phosphate.

lO%SD 31 ml, formamide 3.5+e
After incubating the membrane spotted with the coating solution at 65°C for 3 hours in inactivated bovine thymus DNA (I), the 32p-labeled probe was added, and the membrane was incubated at room temperature for 1 hour.
Allow to react for 4 hours. After washing this membrane with a washing solution containing 2% SDS, it was exposed to X-ray film to detect the presence or absence of viruses. In a test in which a known amount of virus was added to an uninfected homogenate, it was possible to detect 1 ng/spot of virus. The probe for TNV reacts only with TNV-RNA, 5TNV-RNA.

It did not react with TMV (tobacco mosaic virus)-RNA or ribosomal RNA. In addition, the 5TNV probe reacts only with 5TNV-RNA, and TNV-RNA,
It did not react with TMV-RNA or ribosomal RNA.

In the above examples, double-stranded DNA was used as the TNV detection probe and the 5TNV detection probe.
Although a (ds-cDNA) probe was obtained, it is also possible to create a single-stranded RNA probe or a single-tree MDNA probe from the obtained ds-cDNA.

That is, in the case of a single MRNA probe, double-stranded CDNA is isolated from pGEM, which is a transcription plasmid.
” (T. Maniatis).

et al, Moleculer Cloning (1
982)) In the presence of L, 32p or biotin-labeled substrate,
SF3 or T7 RNA polymerase generates a complementary single-tree RNA that is more sensitive than double-stranded DNA probes.
A probe can be obtained. Figure 6 is a diagram of the complementary RNA base sequence obtained from the nucleic acid probe shown in Figure 1, Figure 7 is a diagram of the complementary RNA base sequence obtained from the nucleic acid probe shown in Figure 2, and Figure 8 is a diagram of the complementary RNA base sequence obtained from the nucleic acid probe shown in Figure 2. The figure is a diagram of a complementary RNA base sequence obtained from the nucleic acid probe shown in FIG. 4, and FIG. 9 is a diagram of a complementary RNA base sequence obtained from the nucleic acid probe shown in FIG.

In addition, in the 1-proposed DNA probe, based on the cDNA sequence results, DNA with an appropriate number of bases complementary to the viral RNA is synthesized using a DNA synthesizer and labeled with Imp-γ-ATP, etc., to generate 1-wooden DNA. Probes can be obtained in large quantities and at low cost.

[Effects of the Invention] As explained above, the first to third inventions have specificity for TNV-RNA, and the fourth to seventh inventions are nucleic acid probes that are complementary to TNV-RNA. This is a nucleic acid probe that has specificity for and is complementary to the 5TNV-RNA. In the eighth invention, the nucleic acid probes having specificity for TNv and 5TNV can be easily obtained by culturing host bacteria. The obtained nucleic acid probe is
It has the base sequence as described in the invention and the fifth to seventh inventions, and has the specificity to hybridize with TNV-RNA and 5TNV-RNA. Further, as described in the ninth invention, even a minute amount of virus can be easily detected by using the nucleic acid probes according to the first to seventh inventions as detection probes. The TNV detection probe and 5TNV detection probe produced according to the present invention described above are useful for TNV infection and 5TNV infection of plants such as tobacco, strawberries, and tulips.
It is very useful as a detection aid for determining the presence or absence of NV infection at an early stage.

[Brief explanation of drawings]

Figure 1 shows TN obtained with plasmid pTNV36.
Figure 20 is a base sequence diagram of a nucleic acid probe complementary to TNV obtained using plasmid pTNV8.
A nucleotide sequence diagram of a nucleic acid probe complementary to 5TNV obtained by K 1926, Figure 4 shows the plasmid pYHMK.
A nucleotide sequence diagram of a nucleic acid probe complementary to 5TNV obtained by 1816, Figure 5 shows the plasmid pYHMK1.
Figure 6 is a diagram of the complementary RNA base sequence obtained from the nucleic acid probe shown in Figure 1, and Figure 7 is shown in Figure 2. Complementary RN obtained from the nucleic acid probe
A base sequence diagram, 's8 diagram is a complementary RNA base sequence diagram obtained from the nucleic acid probe shown in Figure 4, and Figure 9 is a complementary RNA base sequence diagram obtained from the nucleic acid probe shown in Figure 5.
A base sequence diagram.

Claims (9)

[Claims] (1) A probe for detecting tobacco necrosis virus that is complementary to the RNA of tobacco necrosis virus. (2) The tobacco necrosis virus detection probe according to claim 1, which is a DNA probe represented by the following base sequence or an RNA probe corresponding to the following base sequence. . [There is a gene sequence. ] (3) The tobacco necrosis virus detection probe according to claim 1, which is a DNA probe represented by the following base sequence or an RNA probe corresponding to the following base sequence. . [There is a gene sequence. ] (4) Satellite tobacco necrosis virus RNA
Satellite tobacco necrosis virus detection probe complementary to . (5) The satellite tobacco necrosis virus detection probe according to claim 4, wherein the probe is a DNA probe represented by the following base sequence or an RNA probe corresponding to the following base sequence. Probe for. [There is a gene sequence. ] (6) The satellite tobacco necrosis virus detection probe according to claim 4, wherein the probe is a DNA probe represented by the following base sequence or an RNA probe corresponding to the following base sequence. Probe for. [There is a gene sequence. ] (7) The satellite tobacco necrosis virus detection probe according to claim 4, wherein the probe is a DNA probe represented by the following base sequence or an RNA probe corresponding to the following base sequence. Probe for. [There is a gene sequence. ] (8) The tobacco necrosis virus (TNV) or the satellite tobacco necrosis virus (sTNV)
), extract DNA (cDNA) complementary to the extracted tobacco necrosis virus RNA or satellite tobacco necrosis virus RNA, and convert the created complementary DNA into double-stranded DNA (ds- c.
The double-stranded DNA (ds-cDNA) is ligated to a plasmid, inserted into a host bacterium, and the host bacterium is cultured to obtain a nucleic acid complementary to the accumulated tobacco necrosis virus or satellite tobacco necrosis virus. A method for producing a tobacco necrosis virus detection probe and a satellite tobacco necrosis virus detection probe, which comprises separating and producing the probe. (9) One of the probes for detecting tobacco necrosis virus or the probe for detecting satellite tobacco necrosis virus according to claims 1 to 7 is added with an enzyme or an isotope to make a labeled probe, and the ground liquid of the test plant and the A method for diagnosing tobacco necrosis virus or satellite tobacco necrosis virus, which comprises hybridizing with a labeled probe.