JP2632339B2 - How to detect potato and potato viroid group - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for detecting viroids belonging to the potato and bursal viroid (hereinafter referred to as PSTV) groups using a synthetic nucleotide probe having a specific base sequence.

[Conventional technology]

Viroid is a single-stranded cyclic RNA molecule, and is a pathogen that causes various symptoms such as atrophy, growth inhibition, and malformation when infected to plants. One of these is PSTV, which can cause spindles and cracks in tubers, reduce yields, and cause significant damage to its appearance.

Also, based on the nucleotide sequence homology of the RNA, PSTV (Note: PSTV itself, not a group), Citrus exocortis viroid (hereinafter referred to as CEV), kikuwa viroid (hereinafter referred to as CSV), tomato planta macho viroid ( Tomato top dwarf viroid (hereinafter referred to as TASV) is classified into the same group,
These are called PSTV groups.

On the other hand, in the production of aseptic seedlings by shoot apex culture and the like, which has been growing rapidly in recent years, it is essential to obtain viroid-free plants excluding such viroids. In particular, it should be noted that there is a report that viroid was detected even in a virus-free form (Academic Monthly, Vol. 40 (1987) P29)
~ P33).

Although the damage caused by PSTV has not yet been confirmed in Japan, measures must be taken to prevent infection from imported potatoes.

However, in the case of viroid, since it is made of only RNA as a self-component and has no protein, a conventional immunological detection method cannot be applied. Sap, etc.
Bioassays to detect the presence or absence of infection in the specimen depending on the growth of the specimen, methods for extracting RNA from the specimen and detecting it by electrophoresis, etc., and DNA complementary to total RNA of viroid ( Gene diagnosis using cDNA) as a probe has been performed.

[Problems to be solved by the invention]

However, bioassays to date require a great deal of time and effort, are not sufficiently sensitive, and the detection method using cDNA as a probe proves that this cDNA itself is infectious. However, this method also has a problem in terms of safety.

[Means for solving the problem]

The present inventors have solved the above problems, as a result of repeated studies to provide a more convenient and practical detection method,
A detection method using a particular synthetic oligonucleotide probe has now been established.

In other words, the present invention relates to a viroid belonging to the potato and bursal viroid group, wherein the nucleotide sequence having complementarity with the common sequence in the RNA is ^{5 ′} GTTTCCCCGGGG.
An object of the present invention is to provide a method for detecting potato and scleroderma viroids, which comprises using a synthetic oligonucleotide probe of ATCCCT ^{3 '} .

 Hereinafter, the present invention will be described in detail.

Nucleic acids have four bases (A-adenine, G-guanine,
A complex of C-cytosine, T-thymine (U-uracil in the case of RNA) and a sugar has a primary structure that is linearly arranged via phosphoric acid. A specific hydrogen bond combination is formed between AT (or U) and GC, and based on this complementarity, the single-stranded nucleic acid forms a double strand (hybrid) and stabilizes. . This affinity depends on the degree of complementarity between the two strands, which can be controlled by the temperature conditions. The present invention, utilizing the above points, was able to easily detect a target viroid by setting appropriate temperature conditions using a specific synthetic oligonucleotide probe.

For PSTV, CEV, CSV, TPMV, and TASV, the entire nucleotide sequence of the RNA has already been determined and is known to be the following sequence. (Fig. 1) The sequence of the synthetic oligonucleotide probe used here is complementary to the base sequence common to PSTVs.
Those with 15 to 25 bases were selected.

For example, when an 18mer (5′GTTTCCCCGGGGATCCCT3 ′) complementary to a part of the base sequence of the central conserved sequence portion (85-104 of PSTV) of the PSTV group viroid RNA is used as a probe, its hybrid melting temperature (Tm) Is 60
° C.
When reacted at 55 ° C., this probe reacts only with viroids of the PSTV group and does not react with viroids of other groups (eg, hop dwarf viroids (HSV)). That is, the viroid of the PSTV group can be specifically detected. It is desirable that the temperature conditions shown here be changed depending on the probe used.

The synthetic orthonucleotide used in the present invention can be synthesized by a known synthesis method such as the so-called phosphotriester method, and the detection after hybridization is performed as follows.
Any labeling method such as radioisotope ( ³² P) labeling, enzyme labeling, and fluorescent labeling may be used, and the labeling position is not particularly limited.

Regarding sample preparation for detection,
It may be carried out by a usual method of extracting the RNA from a plant, for example, the method of Japanese Society for Plant Pathology Vol. 50 (1984), pages 331 to 338.

〔Example〕

 Hereinafter, the present invention will be described specifically with reference to Examples.

Example 1 A small RNA fraction containing each viroid was extracted from tomatoes infected with PSTV, CEV, and CSV, respectively, and extracted with 4XSSC-7.5%
Heat denaturation treatment was performed at 60 ° C. for 15 minutes in formamide, and spotted on nitrocellulose paper. Spot size is 2μ
The stock solution was adjusted to contain 10 μg of small RNA. 6XSSC-0.1% SDS-5X Denhardt's reagent at 50 ° C, 1
After prehybridization for 6 hours, 6XSSC
Hybridization was performed in -0.1% SDS-5X Denhardt's reagent. The probe used was labeled 5 'end with ³² P complementary 18mer ^(5' GTTTCCCCGGGGATCCCT ^{3 ')} in 85 to 102-th sequence of PSTV. Specific activity is about 10 ⁸
dpm / μg DNA. Considering that the Tm of the probe is 60 ° C, the hybridization temperature condition was set to 55 ° C, so that the probe reacted with PSTV, CEV, and CSV,
It did not react with viroids of other groups or samples extracted from healthy leaves at all.

Example 2 A small RNA fraction containing each viroid was extracted from tomatoes infected with PSTV, CEV, and CSV, respectively, and 4XSSC-7.5%
Heat denaturation treatment was performed at 60 ° C. for 15 minutes in formamide, and spotted on nitrocellulose paper. Spot size is 2μ
The stock solution was adjusted to contain 10 μg of small RNA.

6XSSC-0.1% SDS-5 After performing prehybridization at 50 ° C for 1 hour in Denhardt's reagent, 6XSSC-
Hybridization was performed in 0.1% SDS-5X Denhardt's reagent. Probe is a complementary 18mer to 85-102 th sequence of ^{PSTV (5 'GTT * TCCCCGGGGATCCCT 3} ') *
The one in which the phosphate bond in the portion indicated by is labeled with biotin was used. This probe was prepared as follows.
That is, an aminoethylphosphonic acid type T * T dimer having a protecting group (Formula 1) After the synthesis of the oligomer by the phosphotriester method using the above method, the oligomer is removed, protected and purified.

Then BHSE (biotin-hydroxysuccinimide ester (formula 2)) Binds bioin to the amino group of the phosphonic acid moiety.
(Equation 3) The hybridization was carried out at a temperature of 55 ° C. For the detection after the reaction, an alkaline phosphatase conjugate of avidin which specifically binds to biotin was allowed to act, and BCIP (5-bromo-
4-chloro-3-indolylphosphate) and NBT (nitro-
blue tetrazolium) was used. In this case, the reaction between the probe and PSTV, CEV, and CSV was confirmed by color development, but did not react with various viroids or samples extracted from healthy leaves.

Example 3 A small RNA fraction containing each viroid was extracted from tomatoes infected with PSTV, CEV and CSV, respectively, and 4XSSC-7.5%
Heat denaturation treatment was performed at 60 ° C. for 15 minutes in formamide, and spotted on nitrocellulose paper. Spot size is 2μ
The stock solution was adjusted to contain 10 μg of small RNA.

6XSSC-0.1% SDS-5X After performing prehybridization at 50 ° C for 1 hour in Denhardt's reagent, 6XSSC-
Hybridization was performed in 0.1% SDS-5X Denhardt's reagent. The probe used was an 18-mer ( ^{5 ′} GTT * TCCCCGGGGATCCCT ^{3 ′} ) complementary to the 85-102nd sequence of PSTV, with the phosphate bond at the portion indicated by * being labeled with biotin. This probe was prepared as follows. That is, an aminoethylphosphonic acid type T * T dimer having a protecting group (Formula 1) Is deprotected and purified after synthesizing an oligomer by the phosphotriester method. Then a biotinylated reagent with an arm (Formula 4) Converts the amino group of the phosphonic acid moiety into a bioin.
(Equation 5) The hybridization was carried out at a temperature of 55 ° C. For the detection after the reaction, an alkaline phosphatase conjugate of avidin, which specifically binds to biotin, was allowed to act, and then BCIP (5-bromo-
4-chloro-3-indolylphosphate) and NBT (nitro-
blue tetrazolium) was used. In this case, the reaction between the probe and PSTV, CEV, or CSV was confirmed by color development, but did not react with other species of viroid or samples extracted from healthy leaves. Further, the detection sensitivity when the probe shown here was used was about 5 times or more that of the case of Example 2.

〔The invention's effect〕

As described above, by performing hybridization using the synthetic oligonucleotide probe of the present invention, viroids of the PSTV group can be detected extremely easily. Therefore, the present invention
It can be said that it exerts great effects on early diagnosis of plant diseases caused by group infection, prevention of viroid propagation, and production of aseptic seedlings.

[Brief description of the drawings]

FIG. 1 is a diagram showing the entire RNA base sequences of PSTV, TPMV, TASV, CEV, and CSV.

Continued on the front page (72) Inventor Takamitsu Shiba 1-19-13 Takashimadaira, Itabashi-ku, Tokyo Tosho 105 No. (56) Reference Chemistry and Biology, Vol. 25, No. 3 (1987) p. 154-163 Abstracts of 1987 Annual Meeting of the Japanese Society of Plant Pathology Abstracts (1987. Feb. 10) P. 245

Claims (2)

(57) [Claims] 1. A method for detecting viroids by hybridization using a synthetic oligonucleotide probe having complementarity with viroid RNA, the method comprising the steps of determining whether a viroid belonging to the potato or blast disease viroid group is complementary to a common sequence in the RNA. A method for detecting a potato or blast disease viroid group, comprising using a synthetic oligonucleotide probe having a base sequence of ^{5 ′} GTTTCCCCGGGGATCCCT ^{3 ′} . 2. The viroid belonging to the potato or bursal viroid group, wherein the viroid is a potato or bursal viroid, a citrus exocortis viroid, a chrysanthemum viroid, a tomato planta macho viroid and a tomato top vitrified viroid. Item 4. The detection method according to Item 1.