WO1988003956A1 - Procede pour detecter un virus dans une plante - Google Patents

Procede pour detecter un virus dans une plante Download PDF

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Publication number
WO1988003956A1
WO1988003956A1 PCT/SE1987/000558 SE8700558W WO8803956A1 WO 1988003956 A1 WO1988003956 A1 WO 1988003956A1 SE 8700558 W SE8700558 W SE 8700558W WO 8803956 A1 WO8803956 A1 WO 8803956A1
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WIPO (PCT)
Prior art keywords
virus
plant
rna
biotin
membrane
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Application number
PCT/SE1987/000558
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English (en)
Inventor
Anneli Tallberg
Original Assignee
Svalöf Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Svalöf Ab filed Critical Svalöf Ab
Publication of WO1988003956A1 publication Critical patent/WO1988003956A1/fr
Priority to DK382488A priority Critical patent/DK382488A/da

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the present invention relates to a process for detecting plant virus, more precisely by application of genetic engineering.
  • virus diagnostics is the specific instance of plant breeding where it has been found that molecular-biological tech ⁇ nology can be successfully used directly in practical breeding work.
  • E ramme usually involves measuring of the virus production after a test infection. This means that thousands of plants must be tested for the presence of virus in a relatively short time. Until some years ago, the most common method was simply to visually judge the plants for characteristic symptoms of a virus infection. The drawbacks of this form of selection are that an insigni ⁇ ficant virus content provides no visible symptoms, and at the same time a trained eye is required to make a fairly safe judgement.
  • ELISA ELISA
  • ELISA a serological method which is based on the antigenicity in the virus protein envelope and implies that the virus particles are detected by the reaction of antibodies with virus protein.
  • ELISA is the prevailing method for detecting plant virus and is routinely used for both seed control and breeding for resistance. The method has many advantages. It can be easily carried out, it is rapid and relatively sensi ⁇ tive, it may be used on a large scale and requires no expensive equipment. Unfortunately, also the ELISA test has its limitations by being unreliable at low virus frequencies, and when applied to e.g. barley yellow dwarf virus, it is difficult to obtain a pure virus preparation for the production of antibodies since the virus occurs merely in low frequencies.
  • the majority of plant viruses contains the nucleic acid RNA as the genetic material, enclosed by a protein envelope.
  • the ELISA test uses the antigenici ⁇ ty of this protein, but the antigenicity represents only 10% of the genoitiic information obtainable from the virus.
  • the object of the present invention is to provide a process for detecting plant virus.
  • the process is characterised in that plant sap is treated with a protein decomposing enzyme for removal of endogenously bound biotin, that virus RNA from the plant sap is bound to a filter membrane, and that prehybridisation is carried out, whereupon virus RNA is hybridised by incubation of the membrane with biotin-labelled DNA which is homo ⁇ logous to RNA in the virus to be detected, whereby the presence of the specific virus RNA becomes visually detectable through the appearance of a colour after addition of a biotin-specific enzyme.
  • the process according to the present invention is a non-radioactive molecular hybridising technique for detecting plant virus.
  • potatoes have been used as model crop, and the process has been effected for detecting the potato virus Y (PVY) in both leaf and tuber juice.
  • PVY potato virus Y
  • the process can of course also be used for detecting other plant viruses, e.g. barley yellow dwarf virus.
  • nucleic acid hybridisation for detecting PVY requires access to DNA complementary to the virus
  • the nucleic acid hybridisation technique therefore comprises the following steps:
  • PVY is produced in pure form from infected tobacco leaves according to Oxelfelt (pers. commun. ) .
  • the cell walls of the leaves are decomposed by homogenisation in a buffer, which prevents aggregation of the virus particles.
  • Both reducing and chelating substances are added to prevent/reduce the influence of polyphenol and enzyme activity, respectively.
  • Organelles, ribo- somes etc. are separated by centrif.ugation, whereupon the virus is concentrated by precipitation with poly ⁇ ethylene glycol, followed by purification by means of ultracentrifugation.
  • the purity of PVY is checked by measuring the absorption spectrum, while the infectivity is checked on tobacco, followed by an ELISA test. Isolation of RNA
  • RNA is isolated from PVY produced in pure form, with common phenol extraction for nucleic acids according to a modification by Clemens (1985). The purity of the RNA preparation is checked on the one hand by measuring the absorption and, on the other hand, by agarose electro- phoresis. Molecular cloning
  • DNA (cDNA) complementary to PVY-RNA is prepared by the RNase H method according to a modification by Gubler and Hoffman (1983).
  • the advantage of this tech ⁇ nique is that long, coherent DNA sequences are obtained, at the same time as the entire process takes place in a single test tube.
  • the concentration and amount of synthesised cDNA are estimated by labelling with radio- active phosphorus ( 32P).
  • cDNA is inserted into a plasmi which is linearised by treatment with a restriction
  • SUBSTITUTE SHEET 5 enzyme The plasmid is transferred to and propagated in E. coli cells, whereupon colonies of plasmid + cDNA are identified on a selective medium. Bacteria and thus also plasmids from these colonies are propagated on a large scale, whereupon plasmid DNA is isolated by ultracentrifugation. The size of the plasmid and the inserted cDNA are determined by agarose electrophoresis.
  • RNA from plant sap (leaf or tuber juice in the case of potatoes) is bound to a cellulose nitrate membrane and is recognised by and hybridises with complementary DNA as added which is made detectable by labelling.
  • Such labelling can be made in different ways, most commonly by means of radioactive isotopes. Use is fre- quently made of 32P which, however, suffers from certain
  • the process according to the in- vention comprises labelling by means of nick translation with biotin derivatives of deoxyribonucleotides instead of 32 P.
  • Fig. 1 shows the result of a comparison between two different hybridisation and detection techniques using a probe labelled with photobiotin
  • Fig. 2 shows the result of a comparison between biotin labelling and labelling with 32P for detection of PVY in potato tuber juice.
  • a small amount of potato juice from leaves or tubers is applied to a cellulose nitrate filter, after the juice has been treated with a protein-decomposing enzyme (protease). This is necessary since biotin endogenously occurring in potato leaves is bonded to protein (Nikolau et al 1985) which, like nucleic acids, also binds to cellulose nitrate. If the protein is not decomposed before application, the endogenous biotin will be detected as if there were a virus.
  • a protein-decomposing enzyme protea protein-decomposing enzyme
  • the cellulose nitrate filter is "baked" at a high temperature in vacuum for binding the nucleic acids, int. al. virus RNA, whereupon a prehybridisation is made.
  • the prehybridising step is necessary so as to minimise the background disturbance in the filter owing to unspecific binding of ⁇ macromolecules. This is avoided by treating the filter with a solution whose ingredients have precisely the function of reducing or preventing an unspecific background.
  • the actual nucleic acid hybridisation begins (Gatti et al 1984), when the filter is incubated with biotin-labelled DNA which is homologous to PVY-RNA. If a sample is infected with PVY, virus RNA bound in the cellulose nitrate filter will be hybridised with a homologous cDNA strand and detected by an enzymatic reaction with biotin, which results in a coloured product clearly visible to the eye. Blue-coloured spots on the filter thus indicate that a hybridisation has occurred between virus RNA and complementarily prepared DNA, and consequently indicates a virus infection. Where nothing is to be seen, no hybridisation has occurred, and this implies a virus-free sample.
  • RNA are detected, which is the lower detecting limit
  • This method thus is very sensitive and clearly superior to ELISA for detecting small concentrations of virus.
  • the advantage of this test as compared to ELISA is also that the virus isolation need be made just once. With the molecular test, the bacteria are propagated by cDNA if required, but with ELISA, continuous virus purifications must be made for preparing antibodies. The capacity of the hybridising technique is very high and comparable to ELISA, i.e. the screening of thousands of plants takes only a few days.
  • the analysis can be carried out on potato tubers, which till now was not possible. This means that the presence of virus can be detected already in the growing crop, which is crucial to the grower, since he can judge whether the potatoes can be used as seed, or whether they are to be used as food- potatoes or industrial raw products. Except that the grower makes a direct economic profit by early planning, the method contributes to the national economy since perfect seed lots can always be selected.
  • the molecular hybridising test here described can be used for many other crops than potatoes. Thus, one may test, for example, the tolerance to barley yellow dwarf virus in pasture grass, which was previously not possible because laboratory methods were lacking.
  • the extract is left on ice for 2 hours, whereupon it is centrifuged for 3 min.
  • sample - A cellulose nitrate membrane (8 x 12 cm) is soaked in 80 ml H 2 0 for 10 min. and subsequently in 80 ml 20 x SSC for 10 min.
  • the membrane is allowed to dry before being mounted in a vacuum manifold so as to facilitate the applica- tion of samples
  • the prehybridising solution is treated at 100°C for 10 min. followed by rapid cooling on ice before being added to the hybridising pouch.
  • the cellulose nitrate membrane is incubated at 42 C in a shaking water bath overnight (at least 16 hours)
  • probe i.e. DNA complementary to virus RNA
  • This step is carried out according to the instruction issued by the supplier of the DNA detection kit used (BRL)
  • the membrane is dried between 2 Whatman 3MM papers in vacuum at 80°C for 10-20 min.
  • the membrane is incubated in the streptavidin solution for 10 min. during gentle shaking; the solution is pipetted over the membrane at intervals. The solution is decanted.
  • the membrane is washed in buffer (1) in a volume which is 10-20 times the volume of the poly(AP) solution for 2 min; repeated once
  • This step is carried out according to the descrip ⁇ tion issued by the supplier of the DNA detection kit used (BRL)

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Dans le procédé décrit, la sève d'une plante est traitée avec une enzyme décomposant les protéines, en vue de l'élimination de la biotine liée de manière endogène, et de l'ARN de virus provenant de la sève de la plante est lié à une membrane filtrante, et une préhybridation est effectuée, après quoi l'ARN du virus est hybridé par incubation de la membrane avec de l'ADN marqué par de la biotine, qui est homologue de l'ARN du virus à détecter, la présence de l'ARN du virus spécifique devenant visuellement détectable grâce à l'apparition d'une couleur après addition d'une enzyme spécifique de la biotine.
PCT/SE1987/000558 1986-11-27 1987-11-25 Procede pour detecter un virus dans une plante WO1988003956A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK382488A DK382488A (da) 1986-11-27 1988-07-08 Fremgangsmaade til paavisning af plantevirus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE8605095A SE455600B (sv) 1986-11-27 1986-11-27 Forfarande for detektering av vextvirus och dess tillempning for detektering av rodsotvirus och potatisvirus
SE8605095-2 1986-11-27

Publications (1)

Publication Number Publication Date
WO1988003956A1 true WO1988003956A1 (fr) 1988-06-02

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PCT/SE1987/000558 WO1988003956A1 (fr) 1986-11-27 1987-11-25 Procede pour detecter un virus dans une plante

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AU (1) AU8338287A (fr)
SE (1) SE455600B (fr)
WO (1) WO1988003956A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0370710A2 (fr) * 1988-11-19 1990-05-30 Scottish Crop Research Institute Séquence d'ADN codant le gène de la protéine de l'enveloppe du virus de l'enroulement des feuilles de pomme de terre
EP0444649A2 (fr) * 1990-02-27 1991-09-04 Agrilab Biotechnology Ltd. Procédé pour le dépistage des agents pathogènes des plantes, pouvant être utilisés sur le terrain et trousse diagnostique pour sa rÀ©alisation
CN1303423C (zh) * 2004-04-15 2007-03-07 云南省农业科学院 Pvy云南分离物tas-elisa检测试剂盒及其制备方法
CN1303422C (zh) * 2004-04-15 2007-03-07 云南省农业科学院 Pvx云南分离物tas-elisa检测试剂盒及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0133473A2 (fr) * 1983-07-05 1985-02-27 Enzo Biochem, Inc. Marquage in vivo de séquences nucléotides
EP0145356A2 (fr) * 1983-11-22 1985-06-19 National Research Development Corporation Méthode d'essai d'ADN ou ARN à partir de particules virales
WO1985004720A1 (fr) * 1984-04-05 1985-10-24 Howard Florey Institute Of Experimental Physiology Hystochimie d'hybridisation
EP0189280A2 (fr) * 1985-01-23 1986-07-30 Dekalb-Pfizer Genetics Essai qualitatif pour les acides nucléiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0133473A2 (fr) * 1983-07-05 1985-02-27 Enzo Biochem, Inc. Marquage in vivo de séquences nucléotides
EP0145356A2 (fr) * 1983-11-22 1985-06-19 National Research Development Corporation Méthode d'essai d'ADN ou ARN à partir de particules virales
WO1985004720A1 (fr) * 1984-04-05 1985-10-24 Howard Florey Institute Of Experimental Physiology Hystochimie d'hybridisation
EP0189280A2 (fr) * 1985-01-23 1986-07-30 Dekalb-Pfizer Genetics Essai qualitatif pour les acides nucléiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Methods in Enzymology, Vol. 118, pages 723-742, publ. 1986 (PETER PALUKAITIS), "Preparation and Use of cDNA Probes for Detection of Viral Genomes", (see pages 732-35). *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0370710A2 (fr) * 1988-11-19 1990-05-30 Scottish Crop Research Institute Séquence d'ADN codant le gène de la protéine de l'enveloppe du virus de l'enroulement des feuilles de pomme de terre
EP0370710A3 (fr) * 1988-11-19 1990-08-29 Scottish Crop Research Institute Séquence d'ADN codant le gène de la protéine de l'enveloppe du virus de l'enroulement des feuilles de pomme de terre
EP0444649A2 (fr) * 1990-02-27 1991-09-04 Agrilab Biotechnology Ltd. Procédé pour le dépistage des agents pathogènes des plantes, pouvant être utilisés sur le terrain et trousse diagnostique pour sa rÀ©alisation
EP0444649A3 (en) * 1990-02-27 1991-12-27 Agrilab Biotechnology Ltd. Procedure for the detection of plant pathogens under field conditions and a diagnostic kit for its application
CN1303423C (zh) * 2004-04-15 2007-03-07 云南省农业科学院 Pvy云南分离物tas-elisa检测试剂盒及其制备方法
CN1303422C (zh) * 2004-04-15 2007-03-07 云南省农业科学院 Pvx云南分离物tas-elisa检测试剂盒及其制备方法

Also Published As

Publication number Publication date
SE455600B (sv) 1988-07-25
SE8605095L (sv) 1988-05-28
SE8605095D0 (sv) 1986-11-27
AU8338287A (en) 1988-06-16

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