Journal of VirologicalMethods, 13 (1986) 161-169
Elsevier
JVM 00483
161
DETECTION OF CITRUS EXOCORTIS VIROID IN CRUDE EXTRACTS BY
DOT-BLOT HYBRIDIZATION: CONDITIONS FOR REDUCING SPURIOUS
HYBRIDIZATION RESULTS AND FOR ENHANCING THE SENSITIVITY OF
THE TECHNIQUE
RICARDO FLORES
Institute de Agroquimica y Tecnologia de Alimentos (CSIC), Cal/e Jaime Roig 11, 46010 Valencia, Spain
(Accepted 13 December 1985)
Dot-blot assays to detect citrus exocortis viroid (CEV), in clarified sap and unfractionated total nucleic
acid preparations of CEV-infected Gynura aurantiaca and chrysanthemum, were impaired by the non-specif-
ic binding of the radioactive probe shown by the healthy controls. This non-specific background was
considerably reduced by the addition to the hybridization mixture, of the fraction of nucleic acids from
healthy plants which are insoluble in 2 M LiCl (containing mainly the large ribosomal RNAs). Sample
denaturation with formaldehyde was found to provide a high increase of hybridization, when compared
with samples either denatured with formamide or directly spotted. Nitrocellulose was observed to be a better
solid support than charge-modified nylon, in terms of the sensitivity of viroid detection by spot hybridiza-
tion.
viroids cDNA nucleic acid hybridization dot-blot assay
INTRODUCTION
The rapid detection and identification of viroids, a class of subviral pathogens
inducing several diseases in higher plants of economic importance, poses some specific
problems derived from the characteristic nature of these agents. Viroids are small
circular single-stranded RNAs not encapsidated by a coat protein (Diener, 1979)
which is the antigenic component of viruses. Therefore, techniques of the type of
enzyme-linked immunosorbent assays (ELISA) are not appropriate to viroids, for
which nucleic acid hybridization is a very interesting alternative. Mixed phase (or
dot-blot) hybridization was first applied to detect potato spindle tuber viroid (PSTV)
in clarified sap by means of a radioactive probe of DNA complementary to PSTV
(Owens and Diener, 1981). These successful results have not been easily extended to
other cases, as for example the detection of avocado sunblotch viroid (ASBV) in crude
extracts, due to the variable viroid titers, as well as to the presence of interfering
substances affecting the hybridization reaction (see for a review Owens and Diener,
1984). In these instances, it is necessary to work with partially purified and concentra-
0166-0934/86/$03.50 Q 1986 Elsevier Science Publishers B.V. (Biomedical Division)
162
ted preparations of nucleic acids instead of clarified sap, reducing the usefulness of the
technique as a rapid indexing method.
Similar problems were found in the detection of citrus exocortis viroid (CEV), in
crude preparations of Gynura aurantiaca and chrysanthemum, where high back-
grounds were observed in the healthy controls as a consequence of non-specific
hybridization. In this article conditions for reducing these spurious hybridization
results are reported, as well as a comparative analysis of different sample denaturation
procedures and solid supports for hybridization.
MATERIALS AND METHODS
Chemicals
Avian myeloblastosis virus reverse transcriptase and unlabeled deoxyribonucleo-
side triphosphates were obtained from Boehringer Mannheim; [a-32P]dCTP (specific
activity 3000 Ci mmol-‘) and human placental ribonuclease inhibitor from Amers-
ham International; Ficoll-400 and Sephadex G-50 fine from Pharmacia Fine Chemi-
cals; calf thymus DNA, DNase, DEAE-cellulose, formamide, polyvinylpyrrolidone
(PVP-lo), Triton X-100, 2-mercaptoethanol and dithiothreitol from Sigma Chemical
Company; CFll cellulose powder from Whatman; nitrocellulose membranes HAHY
(0.45 urn) from Millipore Corporation and charge-modified nylon membranes (Zeta-
Probe) from Bio-Rad Laboratories. All other chemicals were of reagent grade or the
best commercially available.
Extraction andfractionation of nucleic acids Apical leaves from non-infected Gynura
aurantiaca and chrysanthemum (Chrysanthemum morifolium Ramat, “Bonnie Jean”)
plants, were processed according to a method reported previously which includes
extraction with buffer-saturated phenol (Semancik and Weathers, 1972). Nucleic acids
were then fractionated into those soluble in 2 M LiCl (containing mainly DNA and
small RNAs) and the ones which are precipitated at this salt concentration (with the
large ribosomal RNAs, rRNAs, as the major components) (Semancik and Weathers,
1972).
Purification of CEV Young leaves with symptoms from G. aurantiaca plants infected
with a severe strain of CEV, were extracted as indicated in the previous paragraph. The
nucleic acids soluble in 2 M LiCl, were subjected to an additional fractionation either
by cellulose chromatography (Franklin, 1966; Semancik et al., 1975) or by ethanol
(Granell et al., 1983). CEV was subsequently purified by means of a bidirectional
electrophoretic technique (Schumacher et al., 1983), with the exception that the buffer
of the non-denaturing gel was Tris-acetate (Morris and Wright, 1975), and that the
segment of this gel containing the viroid band was applied directly on top of the
denaturing gel (Semancik and Harper, 1984).
163
Preparation of a complementary DNA probe to CEV Complementary DNA (cDNA)
was synthesized by the random primer method (Taylor et al., 1976) with some further
modifications (Maniatis et al., 1982). Reaction mixtures of a final volume of 50 ~1
contained: 100 mM Tris-HCl (pH 8.3), 10 mM MgCl,, 100 mM KCl, 10 mM dithio-
threitol, 25 U of human placental ribonuclease inhibitor, 1.2 mM each of dATP, dGTP
and dTTP, 50 uCi of [a-32P]dCTP, 1 ug of CEV-RNA, 35 ug of calf thymus deoxynu-
cleotide primers purified by passage through DEAE-cellulose (Maniatis et al., 1982),
and 32 U of reverse transcriptase. CEV-RNA and DNA primers were mixed, heated
for 1 min at lOO’C, and quenched in an ice water bath before addition to the synthesis
reaction (Owens, 1978). After adding the RNA-dependent DNA polymerase, the
reaction mixture was incubated at 37°C for 3 h and then stopped by the addition of 2 pl
of 0.5 M EDTA (pH 8) and 25 ~1 of 150 mM NaOH. During a subsequent incubation
for 1 h at 65°C the RNA template was hydrolyzed. The resulting solution was
neutralized with 25 pl of 1 M Tris-HCl (pH 8) and 25 ul of 1 M HCl, adjusted to a
volume of 0.5 ml with STE buffer (50 mM Tris-HCl, pH 7.2, 100 mM NaCl, 1 mM
EDTA) and applied on a small column (3 ml) of Sephadex G-50 fine, that was then
washed with the same buffer. After the void volume three fractions of 1 ml were
collected; the cDNA present in each of these fractions was recovered by ethanol
precipitation, using tRNA from yeast as a carrier, and resuspended in a small volume
of distilled water. Aliquots of these solutions were applied to glass fiber filters, that
were then washed with ice-cold 5% trichloroacetic acid containing 1% sodium pyro-
phosphate (three times), ethanol, and diethyl ether to determine the acid-insoluble
radioactive material with a Beckman LS 7500 liquid scintillation counter.
Preparation of samples for hybridization Young tissue from healthy and CEV-infect-
ed plants of G. aurantiaca and chrysanthemum was processed in two different ways.
Clarified sap was obtained according to a published method (Owens and Diener,
1981), with some minor changes: 1 g of tissue was homogenized in a mortar with 1.5 ml
of 200 mM K,HPO,, containing 20 mM mercaptoethanol and 0.1% Triton X-100, and
the cellular debris removed by centrifugation (3000 rpm for 3 min in a clinical
centrifuge). Preparations of total nucleic acids were obtained by extracting the mate-
rial with buffer-saturated phenol (Semancik and Weathers, 1972) and precipitation
with ethanol of the nucleic acids, which were then resuspended in STE and dialyzed
against this buffer (final volume of 3 ml starting from 10 g of tissue). Unless otherwise
stated, samples were denatured with formaldehyde (White and Bancroft, 1982) and 5
ul spots with 2-fold dilutions, were applied to nitrocellulose membranes (2 X 6 cm)
that had been pre-treated with distilled water, equilibrated with 20 X SSC (3 M NaCl,
0.3 M trisodium citrate, pH 7.0), and dried with blotting paper (Whatman 3MM) and
placed under a lamp (Thomas, 1983). The spots were let to dry at room temperature
and after baking the membranes for 2 h at 80°C they were pre-hybridized for 2-3 hat
42°C with 1 ml of a solution consisting of 50% de-ionized formamide, 5 X SSPE (0.6 M
NaCl, 0.075 M trisodium citrate, 0.065 M NaH,PO,, pH 6.5, 0.01 M EDTA), 0.2%
164
Ficoll, 0.2% polyvinylpyrrolidone, 0.1% SDS and 100 ug . ml-’ of sonicated and
denatured calf thymus DNA (Garger et al., 1983). The membranes were then hybrid-
ized at 42°C with 0.5 ml of a solution containing the radioactive probe (1 to 1.5 X lo6
cpm), plus the same ingredients as in the previous one with the exception that the calf
thymus DNA was substituted by a preparation of nucleic acids from healthy plants as
indicated in Results. After hybridization the membranes were washed three times at
room temperature for 15 min in 2 X SSC, 0.1% SDS, once at 55°C for 15 min in 0.1 X
SSC, 0.1% SDS, once at 60°C for 15 min in 0.1 X SSC, 0.1% SDS, and exposed to
X-ray film at 4°C overnight (unless otherwise indicated) using DuPont Cronex Light-
ning Plus intensifying screens.
RESULTS
Properties of the synthesis reaction of CEV-cDNA Under the experimental conditions
stated in Materials and Methods, the specific radioactivity obtained was 5 X lo9 dpm .
ug-‘. A final MgCl, concentration of 10 mM in the reaction mixture gave the best
results, since when the concentration of this salt was 5 mM and 15 mM, the radioactivi-
ty incorporated was only 20% and 75% of the optimum respectively. It was also
observed that the amount of cDNA synthesized was two-fold higher at 37°C than at
42°C although the last value is the temperature of incubation that has been used in
some cases (Maniatis et al., 1982). When the three fractions of 1 ml collected after the
void volume of the Sephadex G-50 column were analysed, the maximum acid-precipi-
table radioactivity was found in the third fraction (containing the smallest fragments
of cDNA), whereas only 55% and 15% of this amount, was found in the first and
second fraction respectively. Nevertheless, the three fractions showed the same effi-
ciency in their ability to hybridize with CEV.
Reduction of non-specific hybridization background Figure 1A shows that when clari-
fied sap from CEV-infected and healthy G. aurantiaca was subjected to hybridization
with CEV-cDNA under standard conditions (Garger et al., 1983; Thomas, 1983)
unacceptably high backgrounds were observed in healthy samples. The presence ofcalf
thymus DNA or yeast tRNA in the hybridization mixture did not change the results,
and increasing the temperature of the last wash to 70°C caused a considerable loss of
the autoradiography signals corresponding to viroid-infected samples. In an attempt
to lower these spurious hybridization results, different nucleic acid fractions from
healthy plants were assayed. Figure 1B reveals that the additions to the hybridization
mixture of the fraction containing the nucleic acids insoluble in 2 M LiCl at a final
concentration of 250 pg. ml-‘, reduced to non-detectable levels the intensity of the
spots of healthy samples, leaving essentially unchanged that of CEV-infected ones.
The same effect could not be detected when the nucleic acids soluble in 2 M LiCl were
used (Fig. 1C). It was also noticed that the pre-hybridization step could be suppressed
without affecting the results (Fig. 1D and E).
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1 l/2 l/4 l/8 lA6CEV
Fig. 1. Autoradiograph of a spot hybridization of clarified sap of healthy (H) and CEV-infected (I) G.
auranriaca leaf tissue probed with CEV-cDNA. The hybridization mixture contained 250 pg ‘mlli of: calf
thymus DNA (A), fraction of nucleic acids from healthy plants insoluble (B) and soluble (C) in 2 M LiCl.
Membranes (D) and (E) were treated as (A) and (B) respectively, with the exception that the pre-hybridiza-
tion step was omitted. The spots of the last column on the right correspond to 0.5 ng (upper) and 5 ng (lower)
of purified CEV.
High backgrounds were also observed in healthy samples when total nucleic acid
preparations of G. auruntiaca, were probed with CEV-cDNA under standard condi-
tions (Fig. 2A). This non-specific hybridization was very much reduced, as in the case
of clarified sap, by incorporating in the hybridization mixture 250 pg. ml-’ of the
fraction of nucleic acids from healthy plants which is precipitated by 2 M LiCl (Fig.
2B).
Similar results were obtained from experiments where clarified sap and total nucleic
acid preparations of chrysanthemum, instead of G. auruntiaca, were used. Taking into
account the intensity of the spots corresponding to 0.5 ng of pure CEV, it was
estimated that the detection level of the method was of 50-100 pg per spot.
Effect of the sample denaturation procedure The experiments reported in the previous
166
1 l/2 l/4 l/8 l/16 CEV
Fig. 2. Autoradiograph of a spot hybridization of total nucleic preparations of healthy (H)and CEV-infect-
ed (I) G. auruntiara leaf tissue probed with CEV-cDNA. The hybridization mixture contained 25Oug. ml-r
of either calf thymus DNA (A) or the fraction of nucleic acids from healthy plants insoluble in 2 M LiCl (B).
Controls of purified CEV as in Fig. 1.
section were carried out with samples denatured with formaldehyde (White and
Bancroft, 1982). Figure 3 shows that formaldehyde denaturation provided a higher
sensitivity (specially in the case of total nucleic acid preparations), than spotting the
samples either directly without any pre-treatment (Owens and Diener, 1981) or after
heating them at 100°C for 3 min in 50% formamide followed by rapid cooling in ice
water (Garger et al., 1983). The non-specific binding of CEV-cDNA by healthy
samples, was also observed in the case of the two last procedures, and therefore, it was
not an effect associated with the denaturation with formaldehyde.
1 l/2 l/4 l/8 l/16 CEV
Fig. 3. Autoradiograph of a spot hybridization of total nucleic acid preparations of healthy (H) and
CEV-infected (I) chrysanthemum leaf tissue probed with CEV-cDNA. Samples were applied: after denatu-
ration with formaldehyde (A), directly without any pretreatment (B), and after denaturation with forma-
mide (C). The hybridization mixture contained 250 ug. ml-’ of the nucleic acid fraction from healthy plants
which is precipitated by 2 M LiCI. The spots of the last column on the right correspond to 5 ng (upper) and
0.5 ng (lower) of purified CEV. Exposure time: 72 h.
167
1 l/2 l/4 l/8 l/l6 CEV
Fig. 4. Autoradiograph of a spot hybridization of total nucleic acid preparations of healthy (H) and
CEV-infected (I) chrysanthemum leaf tissue probed with CEV-cDNA. Samples were applied to either
nitrocellulose (A) or charge-modified nylon membranes (B). The hybridization mixture contained 250
ug.ml-’ of the nucleic acid fraction from healthy plants which is precipitated by 2 M LiCI. Controls of
purified CEV as in Fig. 3. Exposure time: 48 h.
Comparison of solid supports for hybridization To perform an analysis of this kind
denatured samples were applied either to nitrocellulose membranes pre-wetted in 20 X SSC as indicated in Materials and Methods, or to charge-modified nylon membranes
that according to the supplier, do not need the high salt pre-treatment to bind the
nucleic acids. Figure 4 shows that an increased hybridization was observed in the case
of nitrocellulose membranes.
DISCUSSION
Synthesis of a single-stranded probe of PSTV-cDNA by the random primer method,
has been reported previously (Owens, 1978). A similar approach has been followed in
the present work for the synthesis of CEV-cDNA, with some modifications which
improved the yield of the product. I have used this radioactive probe for the rapid
detection of CEV in crude extracts (clarified sap and total nucleic acid preparations
not subjected to further purification) of G. aurantiaca and chrysanthemum by spot
hybridization. In this regard, the high autoradiographic background shown by the
healthy controls represented an important problem. This difficulty has been also
found in the detection of PSTV and chrysanthemum stunt viroid (CSV), in non-frac-
tionated nucleic acid preparations of tomato and chrysanthemum respectively, using a
nick-translated probe of cloned PSTV (see Figs. 3 and 4 of Macquaire et al., 1984). The
problems that have emerged in the application of dot-blot procedures for investigating
the presence of ASBV in crude nucleic acid preparations, have been circumvented by
their more extensive purification (Barker et al., 1985). Other authors, also working in
the detection of ASBV by spot hybridization, have not reported these problems
(Bar-Joseph et al., 1984) although they have used diluted clarified sap and conse-
quently, the sensitivity of the method is lower.
As presented in this paper, the incorporation in the hybridization mixture of the
168
fraction of nucleic acids from healthy plants which are insoluble in 2 M LiCI, contain-
ing mostly ribosomal RNAs (rRNAs) of high molecular weight, reduced considerably
the non-specific background, even making unnecessary in some cases the pre-hybridi-
zation step. This effect cannot be attributed to the presence of rRNA sequences in the
CEV preparations that were used for synthesizing the cDNA, since after the final
purification steps in native and denaturing polyacrylamide gels, these preparations did
not have any contaminant RNA as revealed by a sensitive silver-based staining
procedure. Moreover, as stated in the previous paragraph, similar problems have been
observed using nick-translated probes of cloned viroids. It appears, therefore, that
substances present in crude extracts have the ability to bind non-specifically the probe,
and that this ability is blocked by the large rRNAs.
Proper denaturation is a critical step in order to ensure binding of RNAs to
nitrocellulose (Thomas, 1983). In this respect viroids, due to their high degree of
self-complementarity (Diener, 1979) could present some peculiarities. In the first
work in which dot-blot techniques were applied to viroid detection (Owens and
Diener, 1981), clarified sap of PSTV-infected tissue was applied directly to the nitrocel-
lulose. No pre-treatment of the preparations to be spotted on the solid support (Barker
et al., 1985), or heating at 100°C for 3 min (Bar-Joseph et al., 1985), have been used in
the case of ASBV. In a comparative study of several denaturation procedures applied
to purified PSTV and CSV samples, heating at 100°C for 5 min and then quick cooling,
was found to increase the sensitivity of the assay 5- to IO-fold in relation to untreated
samples (Macquaire et al., 1984). In the same study, denaturation with glyoxal or
formamide did not affect the extent of hybridization. From the results presented in
Fig. 3 it is clear that denaturation with formaldehyde increased dramatically the
sensitivity of CEV detection in total nucleic acid preparations, when this procedure
was compared with either formamide denaturation or direct spotting of the sample.
The differences resulting from the three denaturing methods were not so apparent in
the case of purified CEV. These observations could be interpreted by assuming that
pre-treatment with formaldehyde would break down any interaction of CEV with
other components of the total nucleic acid preparation, ensuring the complete denatu-
ration of the viroid molecules and their efficient binding to nitrocellulose.
The small size of viroids could also raise some specific problems regarding their
efficient binding to nitrocellulose, since it has been reported (see the review of Mein-
koth and Wahl, 1984), that fragments smaller than about 200-300 bp bind poorly to
this support. As the charge-modified nylon membranes do not have this limitation, a
comparative analysis between both types of membranes was carried out. The results of
this analysis indicated that nitrocellulose performed better with total nucleic acid
preparations as well as with purified CEV. Similar results have been obtainedin the case
of ASBV (Barker et al., 1985). Nevertheless, charge-modified nylon membranes could
still be of interest in those cases where sensitivity is not a crucial variable, since they
have a higher physical resistance and can be used without any pre-treatment.
The procedures reported here could be only relevant to viroids and not to plant
169
viruses, since with the latter spurious reactions seem not to be a problem and denatura-
tion with formaldehyde slightly increases the sensitivity of detection (Maule et al.,
1983).
ACKNOWLEDGMENTS
I acknowledge with thanks the technical assistance of V. Moncholi and M. Climent.
This work was partially supported by the Comision Asesora de Investigation Cientifi-
ca y TCcnica of Spain and by the Caja de Ahorros y Monte de Piedad de Valencia
(Spain).
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