Journal of Virological Method, 39 (1992) 291-298 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/%05.00
291
VIRMET 01387
A rapid chemiluminescent detection method for barley yellow dwarf virus Hanafy M. Foulya, Leslie L. Domierb and Cleora J. D’Arcya aDepartment of Plant Pathology and bUSDA-ARS Crop Protection Research Unit, University of Illinois, Urbana, IL (USA) (Accepted 24 April 1992)
Summary
Barley yellow dwarf virus (BYDV-PAV-IL) was detected with biotinylated in vitro transcript cDNA using a chemiluminescent substrate on nylon membranes. Signals were detected on X-ray film and quantified using either a densitometer or an ELISA plate reader. The time required for sample preparation was reduced so that the entire protocol could be completed in two days. The in vitro transcript probes could detect 1 ng of purified virus and as little as 1 ,ul of sap extracts prepared from infected oat shoots. Luteovirus; Dot blot; Hybridization
Introduction
Barley yellow dwarf viruses (BYDVs) are members of the luteovirus group (Francki et al., 1991; Waterhouse et al., 1988). BYDVs have isometric particles about 25 nm in diameter with a single capsid protein of approximately 22 kDa M, and a single-stranded RNA genome of approximately 5600 nucleotides. There are five well described strains of BYDVs which were initially distinguished by their principal aphid vector species (Gill, 1967; Rochow, 1969). Like all luteoviruses, BYDVs are restricted to phloem tissues so their overall concentrations in plants are very low. Visual assessment of BYD symptoms in field surveys and in evaluation of breeding lines under field conditions is inadequate for diagnosis and Correspondence to: Dr. Leslie L. Domier, USDA-ARS-MWA-CPRU, Department of Plant Pathology, University of Illinois, 1102 S. Goodwin Ave., Urbana, IL 61801, USA.
292
discrimination among strains of BYDVs (D’Arcy, 1984; Qualset, 1984). Consequently, it has been necessary to develop alternate techniques to assay plants for the presence of virus. The use of enzyme-linked immunosorbent assays (ELISA) has proven to be an efficient means to assess BYD infection (D’Arcy and Hewings, 1986; Rochow and Carmichael, 1979). More recently, techniques employing monoclonal antibodies, cDNA probes and polymerase chain reaction have been used to discriminate between strains of BYDVs and provide more sensitive probes for virus diagnosis (Robertson et al., 1991; D’Arcy et al., 1990; Lister et al., 1990; D’Arcy et al., 1989; Disco et al., 1986; Torrance et al., 1986; Waterhouse et al., 1986; Hsu et al., 1984). Nucleic acid hybridization is a powerful technique for detection of specific, complementary nucleic acid sequences and is being increasingly used for the detection of viruses (Feinman et al., 1984; Hull and Al-Hakim, 1988) and bacteria (Moseley et al., 1982) in plant extracts. The DNA probes are commonly labeled with radioisotopic labels such as phosphorus-32 or sulfur35. Various non-isotopic labels have been used as alternatives to radioisotopes including fluorophores (fluorescein), metal atoms (colloidal gold), enzymes (alkaline phosphatase, horseradish peroxidase), and biotin avidin enzymeconjugated systems (Mathews and Kricka, 1988; Wilchek and Bayer, 1988). Enzyme labels provide sensitivities nearly equal to the radioisotope probes without hazards of radioactivity. The sensitivity of these probes can be further enhanced by combining the enzyme label with an ultrasensitive luminescent detection method for the enzyme (Mathews et al., 1985). In this study, we used biotinylated in vitro transcripts made from cDNA of BYDV-PAV-IL to detect the homologous virus in extracts of oats using a biotin streptavidin alkaline phosphatase complex and a chemiluminescent assay (photographic detection of light emission).
Materials and Methods Virus strain
The virus used in this study was a vector nonspecific strain of BYDV from Illinois (BYDV-PAV-IL) transmitted by Rhopalosiphum padi L. Both the virus strain and aphids were maintained in/on barley (Hordeum vufgare L., cv. Hudson) kept in a growth chamber at 21°C with 16 h fluorescent and incandescent illumination. Probe preparation
BYDV-PAV-IL virions were purified from infected Coast Black described by D’Arcy et al. (1989). RNA was extracted by sucrose gradient centrifugation (Barnett et al., 1987). cDNA was synthesized synthetic oligodeoxynucleotide complementary to the 3’-terminal 20
oats as density using a nucleo-
293
tides of the BYDV-PAV-L genome (Miller et al., 1988) to prime first strand synthesis, followed by second-strand DNA synthesis and RNase H treatment (Gubler and Hoffman, 1983). Following the addition of EcoRI adapters, the cDNA was ligated into pBluescript KS (Stratagene, La Jolla, CA) and used to transform Escherichiu coli strain JM 109. The recombinant plasmid used in this study was designated pPAV410, and represents a nearly full-length copy of the BYDV-PAV-IL genome. Hybridization probes were prepared by in vitro transcription of pPAV 410 cDNA (Melton et al., 1984) in the presence of biotin-1 l-UTP (Sigma Chemical Co., St. Louis, MO). Sample preparation
Purified virus samples of known concentration (estimated spectrophotometrically) were diluted as required in 100 mM sodium phosphate buffer, pH 7.0. BYDV-PAV-IL infected and noninfected oat leaf samples were stored at -80°C. To prepare aqueous extracts, the tissues were squeezed between the rollers of a sap extractor (Erich Pollahne, Germany) washed with 50 mM phosphate buffer, pH 7.0 (1 g tissue/5 ml buffer). The extracts were centrifuged at 14 000 x g at 4°C for 15 min, then denatured with 3 vol of a solution of 33% formamide, 12% formaldehyde, 20 mM 3-[N-morpholinol-propane-sulphonic acid, 1 mM Na EDTA, 5 mM Na acetate, pH 7.0, incubated at 65°C for 5 min. The extracts were chilled on ice, diluted with 1 volume of cold 20 x SSC (1 x SSC = 150 mM NaCl, 15 mM Na-citrate, pH 7.0) and centrifuged again at 14 000 x g at 4°C for 15 .min. Samples were applied with aid of a Hybri-DOT manifold (Gibco-BRL) onto a Hybond-Nylon membrane (Amersham, Arlington Heights, IL) prewetted in 10 x SSC. After sample application, all wells were washed with 200 ~1 of 5 x SSC. The nylon membranes were then placed on a sheet of 3 MM Whatman filter, air dried for 1 hr, and UV crosslinked (Stratagene Stratalinker). Dot blot hybridization
Nylon membranes were prehybridized for 2 h at 42°C in 0.25 ml/cm2 membrane of 50% deionized formamide, 900 mM NaCl, 60 mM NaH2P04 aH20, 6 mM Na2 EDTA . H20, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), and 200 pg/ml yeast tRNA in a heat-sealable polyethylene bag. The membranes were subsequently hybridized with the biotinylated probes (in vitro transcripts) at 50 ng probe/ml in hybridization buffer overnight at 42°C. After hybridization, the membranes were washed twice for 5 min at 55°C in 5% SSC, 0.5% SDS; once for 30 min at 50°C in 0.1 x SSC, 1% SDS; and once for 5 min at room temperature in 2 x SSC. Nonspecific binding sites were blocked by incubating the membranes for 1 h at 60°C in 3% BSA in TBS-Tween 20 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). The membranes were incubated in streptavidin alkaline phosphatase conjugate diluted 1:1000 in
294
Virus
Fig. 1. Detection of purified BYDV-PAV-IL with biotinylated in vitro transcripts. The indicated amounts of purified BYDV-PAV-IL were denatured and spotted onto a nylon filter using a vacuum manifold. After hybridization, washing and application of AMPPD, the filter was exposed to X-ray film for 2 h.
TBS-Tween for 10 min, then were washed twice with TBS-Tween for 15 min at room temperature, and once with 10 x surfactant wash solution (Gibco-BRL) for 1 h at room temperature. The alkaline phosphatase substrate (Cmethoxy-4(3-phosphatephenyl) spiro[ 1,2-dioxetan-3,2’-adamantane]) (AMPPD) was applied to the membranes, which were allowed to dry out of direct light. Light emission from the membranes was detected by exposing Kodak XAR Xray film to the membrane for 15 min to 16 h. Films were developed and signals were quantified by densitometry on a BioRad 610 video densitometer or a Biotek ELISA plate reader.
The biotinylated
in vitro transcript probes hybridized to preparations
of
295
purified BYDV-PAV-IL. The in vitro transcript probes could detect as little as 1 ng of purified virus (Fig. 1). The response was linear with the log of the amount of virus applied from 1 to 80 ng. The filter became saturated above 80 ng and it was not possible to discriminate among larger amounts of virus. The in vitro transcript probes could detect BYDV-PAV-IL in 1 ~1 of extract prepared from infected oat shoots (Fig. 2). This indicates that these extracts contained approximately 1 ng BYDV-PAV-IL (Table 1 and Fig. 2). The hybridization signal plateaued when greater than 60 ~1 was applied to the filter. When larger volumes of plant sap (>40 ~1) were applied, the nylon filter frequently became plugged before all of the sample had passed through the filter. After the denaturant and 10 x SSC were added to the 40 ~1 sample, the
Pl ;ap 50
00 80 60 40 20 10 8 6 4 2 1 Fig. 2. Detection of BYDV-PAV-IL in sap extracts prepared from infected and noninfected Coast Black oats with biotinylated in vitro transcripts. The indicated amounts of plant sap were denatured and spotted onto the nilon filter using a vacuum manifold. After hybridization, washing and application of AMPPD, the filter was exposed to X-ray film for 2 h.
296 TABLE 1 Mean optical density (OD)” values from dot blot hybridization of biotinylated BYDV-PAV-IL in vitro transcripts to extracts prepared from shoots of infected and noninfected Coast Black oats ~1 Sap
Infected
Noninfected
Infected/ Noninfected
150 100 80 60 40 20 10 8 6 4 2 1
1.235 1.471 1.295 1.791 1.376 1.205 1.556 1.263 1.481 1.181 0.765 0.620
0.706 0.900 1.132 1.556 1.122 0.188 0.092 0.109 0.081 0.083 0.079 0.096
1.75 1.63 1.14 1.15 1.22 6.40 16.91 11.64 18.40 14.31 9.74 6.49
aOptical density values were determined
using a Bio-Tek ELISA plate reader.
volume applied to the filter was 320 ~1, which was near the capacity of the well of the vacuum blotter. Samples containing more than 40 ~1 of plant sap required multiple applications. The ability to discriminate between infected and noninfected samples was also compromised at the larger volumes. The greatest differential in hybridization signals from infected and noninfected tissues was seen when 4 to 10 ~1 of denatured plant sap was applied to the filter. These smaller volumes rarely plugged the filter. Discussion
Chemiluminescent detection of nucleic acids provides a reliable method for detection and quantification of target RNA or DNA. The light emission produced from alkaline phosphatase cleaved AMPPD on nylon membranes can be detected on X-ray film and subsequently can be quantified by densitometric analysis. The chemiluminescent method described here can replace the use of isotopes in nucleic acid hybridization and hence in many molecular biology techniques. This would allow the use of nucleic acid probes in laboratories which do not have facilities for handling radioisotopes. Our data show that in vitro transcription probes are very sensitive and can be used routinely for the detection of BYDV-PAV-IL RNA. Similar results were reported by Varveri et al. (1988) and Lakshman et al. (1986), when they compared the sensitivity of DNA and in vitro transcript probes for detection of plum pox virus and potato spindle tuber viroid, respectively. The in vitro transcripts are single stranded, which precludes self-annealing of the probes. The signal is further enhanced because RNA/RNA duplexes are more stable than DNA/RNA duplexes. Moreover, the in vitro transcript probes are specific
297
for the nucleic acid sequence to be detected, allowing for detection of small quantities of BYDV RNAs in infected samples and eliminating the non-specific binding encountered with nick-translated cDNA probes. The assa described here detects quantities of purified BYDV-PAV-IL > 1 ng. Using K P probes in the dot blot hybridization, Waterhouse et al. (1986) were able to detect as little as 1 ng of purified BYDV-RPV from Australia. Therefore, the assay described here is at least as sensitive as the radioactive assay and the sample preparation is much simplified. In our procedure, the plant materials were extracted in a phosphate buffer, so the extracts can be used simultaneously for hybridization and enzyme-linked immunosorbent assays to detect BYDVs. A second advantage of the procedure is that we did not use chloroform and/or phenol extraction to clarify plant et al., 1986), thereby reducing the samples (Habili et al., 1987; Waterhouse possibility of loss of virus during extraction steps. A third advantage of our protocol is that the extraction conditions used in this assay reduced the viscosity of the plant extracts (Waterhouse et al., 1986). Finally, this method reduces the time for required sample preparation; 200 samples can be prepared in 2 h. With these improvements we believe the assay is suitable for large scale applications such as surveys or screening large numbers of cereal plants in breeding programs to detect BYDVs.
References Barnett, O.W., Randles, J.W. and Burrows, P.M. (1987) Relationships among Australian and North American isolates of the bean yellow mosaic potyvirus subgroup. Phytopathology 77, 791-799. D’Arcy, C.J. (1984) Surveying for barley yellow dwarf. In: Barley yellow dwarf. CIMMYT, Mexico, pp. 4&42. D’Arcy, C.J. and Hewings, A.D. (1986) Enzyme linked immunosorbent assays for study of serological relationships and detection of three luteoviruses. Plant Pathol. 35, 288-293. D’Arcy, C.J., Martin, R.R. and Spiegel, S. (1989) A comparative study of luteovirus purification methods. Can. J. Plant Pathol. 11, 251-255. D’Arcy, C.J., Murphy, J.F. and Miklasz, S.D. (1990) Murine monoclonal antibodies produced against two Illinois strains of barley yellow dwarf virus: production and use for virus detection. Phytopathology 80, 377-381. D’Arcy, C.J., Torrance, L. and Martin, R.R. (1989) Discrimination among luteoviruses and their strains by monoclonal antibodies and identification of common epitopes. Phytopathology 79, 869-873. Disco, R., Lister, R.M., Hill, J.H. anh Durand, D.P. (1986) Demonstration of serological relationships among isolates of barley yellow dwarf virus by using polyclonal and monoclonal antibodies. J. Gen. Virol. 67, 353-362. Feinman, S.V., Berris, B., Guha, A., Suoknanan, R., Bradley, D.W., Bond, W.W. and Maynard, J.E. (1984) DNA:DNA hybridization method for the diagnosis of Hepatitis B infection. J. Virol. Methods 8, 199-206. Francki, R.I.B., Fauquet, C.M., Knudson, D.L. and Brown, F. (1991) Classification and Nomenclature of Viruses. Springer-Verlag, Vienna, pp. 309-3 11. Gill, CC. (1967) Transmission of barley yellow dwarf virus isolates from Manitoba by live species of aphids. Phytopathology 57, 7 13-7 18. Gubler, U. and Hoffman, B.J. (1983) A simple and very efficient method for generating cDNA
298 libraries. Gene 25, 263-269. Habili, N., McInnes, J.L. and Symons, R.H. (1987) Nonradioactive, photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus. J. Virol. Methods 16, 225-237. Hull, R. and Al-Hakim, A. (1988) Nucleic acid hybridization in plant virus diagnosis and characterization. Trends Biotechnol. 6, 213-218. Hsu, H.T., Aebig, J. and Rochow, W.F. (1984) Differences among monoclonal antibodies to barley yellow dwarf viruses. Phytopathology 74, 60&605. Lakshman, D.K., Hiruki, C., Wu, X.N. and Leung, W.C. (1986) Use of 32p RNA probes for the dothybridization detection of potato spindle tuber viroid. J. Virol. Methods 14, 309-319. Lister, R.M., Barbara, D.J., Kawata, E.E., Ueng, P.P., Fattouh, F. and Larkins, B.A. (1990) Development and use of cDNA probes in studies of barley yellow dwarf virus. In: World Perspectives on Barley Yellow Dwarf Virus. P.A. Burnet, ed. CIMMYT, Mexico, pp. 186-193. Mathews, J.A. and Kricka, L.J. (1988) Analytical strategies for the use of DNA probes. Anal. Biochem. 169, l-25. Mathews, J.A., Batki, A., Hynds, C. and Kricka, L.J. (1985) Enhanced chemiluminescent method for the detection of DNA dot hybridization assays. Anal. Biochem. 151, 205-209. Melton, D.A., Kreig, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acid Res. 12, 7035-7036. Miller, W.A, Waterhouse, P.M. and Gerlach, W.L. (1988) Sequence and organization of barley yellow dwarf virus genomic RNA. Nucl. Acid Res. 16, 6097-6111. Moseley, S.L., Echeveria, P., Seriwatana, J., Tirapat, C., Chaicumpa, T., Sakuldaipeara, T. and Falkow, S. (1982) Identification of enterotoxigenic Escherichiu coli by colony hybridization using three enterotoxin gene probes. J. Infect. Dis. 145, 863-869. Qualset, C.O. (1984) Evaluation and breeding methods for barley yellow dwarf resistance. In: Barley Yellow Dwarf Virus. CIMMYT, Mexico, pp. 72-80. Robertson, N.L., French, R. and Gray, S.M. (1991) Use of group-specific primers and the polymerase chain reaction for the detection and identification of luteoviruses. J. Gen. Virol. 72, 1473-1477. Rochow, W.E. (1969) Biological properties of four isolates of barley yellow dwarf virus. Phytopathology 59, 158&1589. Rochow, W.F. and Carmichael, L.E. (1979) Specificity among barley yellow dwarf viruses by enzyme immunosorbent assays. Virology 95, 415420. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning. 2nd ed. Cold Spring Harbor Laboratory Press. Torrance, L., Pead, M.T., Larkins, A.P. and Butcher, G.W. (1986) Characterization of monoclonal antibodies to a UK isolate of barley yellow dwarf virus. J. Gen. Virol. 67, 549-555. Varveri, C., Candresse, T., Cugusi, M., Ravelonandro, M. and Dunez, J. (1988) Use of 32P-labelled transcribed RNA probe for dot-blot hybridization detection of plum pox virus. Phytopathology 78, 128&1283. Waterhouse, P.M., Gerlach, W.L. and Miller, W.A. (1986) Serotype-specific and general’luteovirus probes from cloned cDNA sequences of barley yellow dwarf virus. J. Gen. Virol. 67, 1273-1284. Waterhouse, P.M., Gildow, F.E. and Johnson, G.R. (1988) The luteovirus group. CMI/AAB Descriptions of plant viruses no. 339. Wilcheck, M. and Bayer, E.A. (1988) Review: the avidin-biotin complex in bioanalytical applications. Anal. Biochem. 171, l-32.
291
VIRMET 01387
A rapid chemiluminescent detection method for barley yellow dwarf virus Hanafy M. Foulya, Leslie L. Domierb and Cleora J. D’Arcya aDepartment of Plant Pathology and bUSDA-ARS Crop Protection Research Unit, University of Illinois, Urbana, IL (USA) (Accepted 24 April 1992)
Summary
Barley yellow dwarf virus (BYDV-PAV-IL) was detected with biotinylated in vitro transcript cDNA using a chemiluminescent substrate on nylon membranes. Signals were detected on X-ray film and quantified using either a densitometer or an ELISA plate reader. The time required for sample preparation was reduced so that the entire protocol could be completed in two days. The in vitro transcript probes could detect 1 ng of purified virus and as little as 1 ,ul of sap extracts prepared from infected oat shoots. Luteovirus; Dot blot; Hybridization
Introduction
Barley yellow dwarf viruses (BYDVs) are members of the luteovirus group (Francki et al., 1991; Waterhouse et al., 1988). BYDVs have isometric particles about 25 nm in diameter with a single capsid protein of approximately 22 kDa M, and a single-stranded RNA genome of approximately 5600 nucleotides. There are five well described strains of BYDVs which were initially distinguished by their principal aphid vector species (Gill, 1967; Rochow, 1969). Like all luteoviruses, BYDVs are restricted to phloem tissues so their overall concentrations in plants are very low. Visual assessment of BYD symptoms in field surveys and in evaluation of breeding lines under field conditions is inadequate for diagnosis and Correspondence to: Dr. Leslie L. Domier, USDA-ARS-MWA-CPRU, Department of Plant Pathology, University of Illinois, 1102 S. Goodwin Ave., Urbana, IL 61801, USA.
292
discrimination among strains of BYDVs (D’Arcy, 1984; Qualset, 1984). Consequently, it has been necessary to develop alternate techniques to assay plants for the presence of virus. The use of enzyme-linked immunosorbent assays (ELISA) has proven to be an efficient means to assess BYD infection (D’Arcy and Hewings, 1986; Rochow and Carmichael, 1979). More recently, techniques employing monoclonal antibodies, cDNA probes and polymerase chain reaction have been used to discriminate between strains of BYDVs and provide more sensitive probes for virus diagnosis (Robertson et al., 1991; D’Arcy et al., 1990; Lister et al., 1990; D’Arcy et al., 1989; Disco et al., 1986; Torrance et al., 1986; Waterhouse et al., 1986; Hsu et al., 1984). Nucleic acid hybridization is a powerful technique for detection of specific, complementary nucleic acid sequences and is being increasingly used for the detection of viruses (Feinman et al., 1984; Hull and Al-Hakim, 1988) and bacteria (Moseley et al., 1982) in plant extracts. The DNA probes are commonly labeled with radioisotopic labels such as phosphorus-32 or sulfur35. Various non-isotopic labels have been used as alternatives to radioisotopes including fluorophores (fluorescein), metal atoms (colloidal gold), enzymes (alkaline phosphatase, horseradish peroxidase), and biotin avidin enzymeconjugated systems (Mathews and Kricka, 1988; Wilchek and Bayer, 1988). Enzyme labels provide sensitivities nearly equal to the radioisotope probes without hazards of radioactivity. The sensitivity of these probes can be further enhanced by combining the enzyme label with an ultrasensitive luminescent detection method for the enzyme (Mathews et al., 1985). In this study, we used biotinylated in vitro transcripts made from cDNA of BYDV-PAV-IL to detect the homologous virus in extracts of oats using a biotin streptavidin alkaline phosphatase complex and a chemiluminescent assay (photographic detection of light emission).
Materials and Methods Virus strain
The virus used in this study was a vector nonspecific strain of BYDV from Illinois (BYDV-PAV-IL) transmitted by Rhopalosiphum padi L. Both the virus strain and aphids were maintained in/on barley (Hordeum vufgare L., cv. Hudson) kept in a growth chamber at 21°C with 16 h fluorescent and incandescent illumination. Probe preparation
BYDV-PAV-IL virions were purified from infected Coast Black described by D’Arcy et al. (1989). RNA was extracted by sucrose gradient centrifugation (Barnett et al., 1987). cDNA was synthesized synthetic oligodeoxynucleotide complementary to the 3’-terminal 20
oats as density using a nucleo-
293
tides of the BYDV-PAV-L genome (Miller et al., 1988) to prime first strand synthesis, followed by second-strand DNA synthesis and RNase H treatment (Gubler and Hoffman, 1983). Following the addition of EcoRI adapters, the cDNA was ligated into pBluescript KS (Stratagene, La Jolla, CA) and used to transform Escherichiu coli strain JM 109. The recombinant plasmid used in this study was designated pPAV410, and represents a nearly full-length copy of the BYDV-PAV-IL genome. Hybridization probes were prepared by in vitro transcription of pPAV 410 cDNA (Melton et al., 1984) in the presence of biotin-1 l-UTP (Sigma Chemical Co., St. Louis, MO). Sample preparation
Purified virus samples of known concentration (estimated spectrophotometrically) were diluted as required in 100 mM sodium phosphate buffer, pH 7.0. BYDV-PAV-IL infected and noninfected oat leaf samples were stored at -80°C. To prepare aqueous extracts, the tissues were squeezed between the rollers of a sap extractor (Erich Pollahne, Germany) washed with 50 mM phosphate buffer, pH 7.0 (1 g tissue/5 ml buffer). The extracts were centrifuged at 14 000 x g at 4°C for 15 min, then denatured with 3 vol of a solution of 33% formamide, 12% formaldehyde, 20 mM 3-[N-morpholinol-propane-sulphonic acid, 1 mM Na EDTA, 5 mM Na acetate, pH 7.0, incubated at 65°C for 5 min. The extracts were chilled on ice, diluted with 1 volume of cold 20 x SSC (1 x SSC = 150 mM NaCl, 15 mM Na-citrate, pH 7.0) and centrifuged again at 14 000 x g at 4°C for 15 .min. Samples were applied with aid of a Hybri-DOT manifold (Gibco-BRL) onto a Hybond-Nylon membrane (Amersham, Arlington Heights, IL) prewetted in 10 x SSC. After sample application, all wells were washed with 200 ~1 of 5 x SSC. The nylon membranes were then placed on a sheet of 3 MM Whatman filter, air dried for 1 hr, and UV crosslinked (Stratagene Stratalinker). Dot blot hybridization
Nylon membranes were prehybridized for 2 h at 42°C in 0.25 ml/cm2 membrane of 50% deionized formamide, 900 mM NaCl, 60 mM NaH2P04 aH20, 6 mM Na2 EDTA . H20, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), and 200 pg/ml yeast tRNA in a heat-sealable polyethylene bag. The membranes were subsequently hybridized with the biotinylated probes (in vitro transcripts) at 50 ng probe/ml in hybridization buffer overnight at 42°C. After hybridization, the membranes were washed twice for 5 min at 55°C in 5% SSC, 0.5% SDS; once for 30 min at 50°C in 0.1 x SSC, 1% SDS; and once for 5 min at room temperature in 2 x SSC. Nonspecific binding sites were blocked by incubating the membranes for 1 h at 60°C in 3% BSA in TBS-Tween 20 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20). The membranes were incubated in streptavidin alkaline phosphatase conjugate diluted 1:1000 in
294
Virus
Fig. 1. Detection of purified BYDV-PAV-IL with biotinylated in vitro transcripts. The indicated amounts of purified BYDV-PAV-IL were denatured and spotted onto a nylon filter using a vacuum manifold. After hybridization, washing and application of AMPPD, the filter was exposed to X-ray film for 2 h.
TBS-Tween for 10 min, then were washed twice with TBS-Tween for 15 min at room temperature, and once with 10 x surfactant wash solution (Gibco-BRL) for 1 h at room temperature. The alkaline phosphatase substrate (Cmethoxy-4(3-phosphatephenyl) spiro[ 1,2-dioxetan-3,2’-adamantane]) (AMPPD) was applied to the membranes, which were allowed to dry out of direct light. Light emission from the membranes was detected by exposing Kodak XAR Xray film to the membrane for 15 min to 16 h. Films were developed and signals were quantified by densitometry on a BioRad 610 video densitometer or a Biotek ELISA plate reader.
The biotinylated
in vitro transcript probes hybridized to preparations
of
295
purified BYDV-PAV-IL. The in vitro transcript probes could detect as little as 1 ng of purified virus (Fig. 1). The response was linear with the log of the amount of virus applied from 1 to 80 ng. The filter became saturated above 80 ng and it was not possible to discriminate among larger amounts of virus. The in vitro transcript probes could detect BYDV-PAV-IL in 1 ~1 of extract prepared from infected oat shoots (Fig. 2). This indicates that these extracts contained approximately 1 ng BYDV-PAV-IL (Table 1 and Fig. 2). The hybridization signal plateaued when greater than 60 ~1 was applied to the filter. When larger volumes of plant sap (>40 ~1) were applied, the nylon filter frequently became plugged before all of the sample had passed through the filter. After the denaturant and 10 x SSC were added to the 40 ~1 sample, the
Pl ;ap 50
00 80 60 40 20 10 8 6 4 2 1 Fig. 2. Detection of BYDV-PAV-IL in sap extracts prepared from infected and noninfected Coast Black oats with biotinylated in vitro transcripts. The indicated amounts of plant sap were denatured and spotted onto the nilon filter using a vacuum manifold. After hybridization, washing and application of AMPPD, the filter was exposed to X-ray film for 2 h.
296 TABLE 1 Mean optical density (OD)” values from dot blot hybridization of biotinylated BYDV-PAV-IL in vitro transcripts to extracts prepared from shoots of infected and noninfected Coast Black oats ~1 Sap
Infected
Noninfected
Infected/ Noninfected
150 100 80 60 40 20 10 8 6 4 2 1
1.235 1.471 1.295 1.791 1.376 1.205 1.556 1.263 1.481 1.181 0.765 0.620
0.706 0.900 1.132 1.556 1.122 0.188 0.092 0.109 0.081 0.083 0.079 0.096
1.75 1.63 1.14 1.15 1.22 6.40 16.91 11.64 18.40 14.31 9.74 6.49
aOptical density values were determined
using a Bio-Tek ELISA plate reader.
volume applied to the filter was 320 ~1, which was near the capacity of the well of the vacuum blotter. Samples containing more than 40 ~1 of plant sap required multiple applications. The ability to discriminate between infected and noninfected samples was also compromised at the larger volumes. The greatest differential in hybridization signals from infected and noninfected tissues was seen when 4 to 10 ~1 of denatured plant sap was applied to the filter. These smaller volumes rarely plugged the filter. Discussion
Chemiluminescent detection of nucleic acids provides a reliable method for detection and quantification of target RNA or DNA. The light emission produced from alkaline phosphatase cleaved AMPPD on nylon membranes can be detected on X-ray film and subsequently can be quantified by densitometric analysis. The chemiluminescent method described here can replace the use of isotopes in nucleic acid hybridization and hence in many molecular biology techniques. This would allow the use of nucleic acid probes in laboratories which do not have facilities for handling radioisotopes. Our data show that in vitro transcription probes are very sensitive and can be used routinely for the detection of BYDV-PAV-IL RNA. Similar results were reported by Varveri et al. (1988) and Lakshman et al. (1986), when they compared the sensitivity of DNA and in vitro transcript probes for detection of plum pox virus and potato spindle tuber viroid, respectively. The in vitro transcripts are single stranded, which precludes self-annealing of the probes. The signal is further enhanced because RNA/RNA duplexes are more stable than DNA/RNA duplexes. Moreover, the in vitro transcript probes are specific
297
for the nucleic acid sequence to be detected, allowing for detection of small quantities of BYDV RNAs in infected samples and eliminating the non-specific binding encountered with nick-translated cDNA probes. The assa described here detects quantities of purified BYDV-PAV-IL > 1 ng. Using K P probes in the dot blot hybridization, Waterhouse et al. (1986) were able to detect as little as 1 ng of purified BYDV-RPV from Australia. Therefore, the assay described here is at least as sensitive as the radioactive assay and the sample preparation is much simplified. In our procedure, the plant materials were extracted in a phosphate buffer, so the extracts can be used simultaneously for hybridization and enzyme-linked immunosorbent assays to detect BYDVs. A second advantage of the procedure is that we did not use chloroform and/or phenol extraction to clarify plant et al., 1986), thereby reducing the samples (Habili et al., 1987; Waterhouse possibility of loss of virus during extraction steps. A third advantage of our protocol is that the extraction conditions used in this assay reduced the viscosity of the plant extracts (Waterhouse et al., 1986). Finally, this method reduces the time for required sample preparation; 200 samples can be prepared in 2 h. With these improvements we believe the assay is suitable for large scale applications such as surveys or screening large numbers of cereal plants in breeding programs to detect BYDVs.
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