RAPID DETECTION OF A PLANT-VIRUS BY SOLUTION HYBRIDIZATION USING OLIGONUCLEOTIDE PROBES

被引:6
作者
ROUHIAINEN, L
LAAKSONEN, M
KARJALAINEN, R
SODERLUND, H
机构
[1] UNIV HELSINKI,DEPT PLANT PATHOL,SF-00710 HELSINKI,FINLAND
[2] ORION CORP LTD,ORION GENET ENGN,HELSINKI,FINLAND
基金
芬兰科学院;
关键词
PLANT VIRUS DETECTION; SOLUTION HYBRIDIZATION; OLIGONUCLEOTIDE PROBE;
D O I
10.1016/0166-0934(91)90123-H
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A novel solution hybridization method for the diagnosis of a plant virus was evaluated. Synthetic oligonucleotide probes were used for the detection of potato virus X (PVX) in crude leaf sap extracts by hybridization in solution. Three 40-nucleotide-long oligonucleotide probes complementary to RNA sequences of potato virus X near the 3' end were synthesized. Two probes were P-32-labelled and one biotinylated. The three probes were allowed to form hybrids with the target viral nucleic acid in solution, and the formed hybrids were isolated with the aid of the biotinylated capture probe using avidin polystyrene beads after the reaction. Alternatively, hybrids were captured from the poly(A) tail of the viral RNA on oligo(dT) cellulose. The maximum signal was obtained after 4 h hybridization. About 70% of the maximum signal was obtained after 2 h hybridization. Sensitivity with the two P-32-labelled oligonucleotide probes was 1-5 x 10(7) molecules of PVX RNA. This corresponds to 0.6-3 ng of the virus. Crude leaf sap did not interfere with the detection of the virus. These results suggest that this solution hybridization method permits rapid detection of a plant virus in crude plant sap without sample pretreatment and may thus open new avenues for the development of a nucleic-acid-based ELISA-like diagnostic test for the detection of plant viruses.
引用
收藏
页码:81 / 90
页数:10
相关论文
共 35 条
[1]   DETECTION OF AVOCADO SUNBLOTCH VIROID BY HYBRIDIZATION WITH SYNTHETIC OLIGONUCLEOTIDE PROBES [J].
BARJOSEPH, M ;
SEGEV, D ;
TWIZER, S ;
ROSNER, A .
JOURNAL OF VIROLOGICAL METHODS, 1985, 10 (01) :69-73
[2]  
BARJOSEPH M, 1986, DEV APPLICATIONS VIR, P13
[3]  
Baulcombe D. C., 1984, 1984 British Crop Protection Conference. Pests and Diseases. Vol.1., P207
[4]   DEOXYNUCLEOSIDE PHOSPHORAMIDITES - A NEW CLASS OF KEY INTERMEDIATES FOR DEOXYPOLYNUCLEOTIDE SYNTHESIS [J].
BEAUCAGE, SL ;
CARUTHERS, MH .
TETRAHEDRON LETTERS, 1981, 22 (20) :1859-1862
[5]  
BERCKS R, 1970, CMI AAB DESCRIPTIONS, V4
[6]   CHARACTERISTICS OF MICROPLATE METHOD OF ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF PLANT-VIRUSES [J].
CLARK, MF ;
ADAMS, AN .
JOURNAL OF GENERAL VIROLOGY, 1977, 34 (MAR) :475-483
[7]   IMPROVED HYBRIDIZATION ASSAYS EMPLOYING TAILED OLIGONUCLEOTIDE PROBES - A DIRECT COMPARISON WITH 5'-END-LABELED OLIGONUCLEOTIDE PROBES AND NICK-TRANSLATED PLASMID PROBES [J].
COLLINS, ML ;
HUNSAKER, WR .
ANALYTICAL BIOCHEMISTRY, 1985, 151 (02) :211-224
[8]   NOVEL METHOD TO MAP TRANSCRIPTS - EVIDENCE FOR HOMOLOGY BETWEEN AN ADENOVIRUS MESSENGER-RNA AND DISCRETE MULTIPLE REGIONS OF VIRAL GENOME [J].
DUNN, AR ;
HASSELL, JA .
CELL, 1977, 12 (01) :23-36
[9]   NONRADIOACTIVE, PHOTOBIOTIN-LABELED DNA PROBES FOR THE ROUTINE DIAGNOSIS OF BARLEY YELLOW DWARF VIRUS [J].
HABILI, N ;
MCINNES, JL ;
SYMONS, RH .
JOURNAL OF VIROLOGICAL METHODS, 1987, 16 (03) :225-237
[10]   THE COMPLETE NUCLEOTIDE-SEQUENCE OF TOBACCO RATTLE VIRUS RNA-1 [J].
HAMILTON, WDO ;
BOCCARA, M ;
ROBINSON, DJ ;
BAULCOMBE, DC .
JOURNAL OF GENERAL VIROLOGY, 1987, 68 :2563-2575