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).

165

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).

REFERENCES

Bar-Joseph, M., D. Segev, S. Twizer and A. Rosner, 1985, J. Viral. Methods 10, 69.

Barker, J.M., J.L. McInnes, P.J. Murphy and R.S. Symons, 1985, J. Viral. Methods 10, 87.

Diener, T.O., 1979, Viroids and Viroid Diseases (Wiley-Interscience, New York).

Franklin, R.M., 1966, Proc. Natl. Acad. Sci. U.S.A. 55, 1504.

Garger, S.J., T. Turpen, J.C. Carrington, T.J. Morris, J.A. Dodds and L.K. Grill, 1983, Plant Mol. Biol.

Rep. 1, 21.

Granell, A., R. Flares and V. Conejero, 1983, Anal. Biochem. 134, 479.

Macquaire, G., M. Monsion, C. Mouches, T. Candresse and J. Dunez, 1984, Ann. Virol. (Inst. Pasteur)

135E, 129.

Maniatis, T., E.F. Fritch and T. Sambrook, 1982, Molecular Cloning: A Laboratory Manual (Cold Spring

Harbor Laboratory, New York) pp. 129 and 230.

Maule, A.J., R. Hull and J. Donson, 1983, J. Virol. Methods 6, 215.

Meinkoth, J. and G. Wahl, 1984, Anal. Biochem. 138, 267.

Morris, T.J. and N.S. Wright, 1975, Am. Pot. J. 52, 57.

Owens, R.A., 1978, Virology 89, 380.

Owens, R.A. and T.0 Diener, 1981, Science 213, 670.

Owens, R.A. and T.O. Diener, 1984, in: Methods in Virology, Vol. VII, eds. K. Maramorosch and H.

Koprowski (Academic Press, New York) p. 173.

Schumacher, J., J.W. Randles and D. Riesner, 1983. Anal. Biochem. 135, 288.

Semancik, J.S. and L.G. Weathers, 1972, Virology 47, 456.

Semancik, J.S. and K.L. Harper, 1984, Proc. Natl. Acad. Sci. U.S.A. 81, 4429.

Semancik, J.S., T.J. Morris, L.G. Weathers, B.F. Rodorf and D.R. Kearns, 1975, Virology 63, 160.

Taylor, J.M., R. Illmensee and J. Summers, 1976, Biochim. Biophys. Acta 442, 324.

Thomas, P.S., 1983, in: Methods in Enzymology, Vol. 100, eds. R. Wu, L. Grossman and K. Moldave

(Academic Press, New York) p. 255.

White, B.A. and F.C. Bancroft, 1982, J. Biol. Chem. 257, 8569.