Detection of plant viruses and viroids by molecular hybridization

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G. Macquaire, T. Candresse and J. Dunez
Institut national de la recherche agronomique Station de pathologic végétale
33140 Pont de la Maye, France

PRINCIPLE

A viral particle is composed of nucleic acids (ribonucleic acid = RNA or deoxyribonucleic acid = DNA) and a capsid made up of several dozen to a thousand copies of coat protein subunit. In some cases the virus possesses an envelope composed of viral proteins integrated in membranes deriving from the host cell. Serological techniques detect the virus by specific recognition of the coat protein by specific antibodies developed in animals against this protein. Molecular hybridization techniques detect viral nucleic acids by specific recognition of their nucleotide sequence.

Nucleic acids are long, linear polymers of nucleotide molecules. Each nucleotide is in turn composed of several elements: a nitrogen containing base linked to a phosphate group and a sugar molecular (ribose for RNA and deoxyribose for DNA). DNA contains four different bases: Adenine (A), Guanine (G), Cytosine (C) and Thymine (T). In the case of RNA, Thymine is replaced by Uracil (U), the three other bases being the same.

DNA is usually found in a double-stranded configuration, i.e. two chains of DNA associate through specific base pairing (A pairs with T and C pairs with G). Base pairing is extremely specific and creates non-covalent hydrogen bonds that unite the molecules associated in this way. RNA is most commonly found in a single-stranded configuration but, like DNA, it possesses the capacity to form double-stranded structures through A-U and G-C pairing.

The specific pairing of the bases composing nucleic acids constitutes the basis for the formation of hybrids (double-stranded structure) between complementary molecules and, thus, for the use of molecular hybridization as a diagnostic technique.

Nucleic acid molecules differ from one another in the order and sequence of alignment of their nucleotides (= nucleotide sequence). The presence of two molecules of complementary sequences will lead to the formation of doublestranded hybrids under suitable conditions. For example, TCGGCGTAT will pair with AGCCGCATA to make a DNADNA hybrid.

A probe used for virus detection in molecular hybridization experiments is a single-stranded nucleic acid molecule prepared from a viral nucleic acid, single-stranded with a nucleotide sequence complementary to that of the target viral RNA molecule.

Thus a DNA probe with the sequence: TCGGCGTAT will specifically detect RNA and DNA molecules with the respective sequences AGCCGCAUA and AGCCGCATA. An RNA probe with the same specificity would be: UCGGCGUAU.

The molecular hybridization detection system presented here is based on a solid support hybridization, the samples being permanently immobilized on a nitrocellulose membrane (Figure 157). We will describe the technique using the two most frequently used types of probe:

• complementary DNA probes cloned in a plasmid vector;
• in vitro transcribed complementary RNA probes prepared from complementary DNA cloned into special purpose transcription plasmid vectors.

The probes can be labelled either radioactively or by incorporation of a non-radioactive marker such as biotin. The techniques for the determination of the probe-specific activity are described in Appendix 1.

EXPERIMENTAL SET-UP

Schematically the technique can be divided into five steps:

• Probe labelling. This is achieved by incorporation of a labelled (radioactive or biotinylated) nucleotide triphosphate precursor during in vitro reactions (nick translation for DNA probes or transcription for RNA probes).
• Sample preparation. The immobilization of the nucleic acids present in the plant extract on a nitrocellulose membrane by baking it for two hours at 80°C under vacuum.
• Hybridization. The labelled probe will form double-stranded structures under suitable conditions with the target nucleic acid immobilized on the membrane.
• Washing(s). Non-hybridized probe molecules are removed by successive washings of the membrane under stringent conditions.
• Hybrid detection. For radioactive probes, this is achieved by contact of an X-ray film with the membrane (autoradiography), usually for 24 hours. In the case of biotinylated probes, three additional steps are required (Figure 158):

1. Incubation in the presence of streptavidin which reacts specifically with the biotin molecules fixed on the probe;
2. Incubation in the presence of a biotinylated enzyme which will be trapped by the streptavidin already retained on the membrane;
3. Enzymatic reaction that results in the formation of a coloured product at the fixing point of the complex probe biotin-streptavidin-biotinylated enzyme.

EXPERIMENTAL PROTOCOL

Probe labelling

DNA probes. Purified recombinant plasmid DNA is labelled (by incorporation of either ³²PsCTP, biotinylated dUTP or dCTP) by the technique of nick translation, using one of the several commercially available kits (e.g. BRL, Amersham).

RNA probes. After linearization of the purified recombinant plasmid downstream of the viral cDNA with a suitable restriction endonuclease, labelled RNA is produced by in vitro transcription using one of several commercially available kits (e.g. Promega, Biotec, Boehringer). Incorporation of either ³²P or biotinlabelled CTP is usually carried out.

Sample preparation

Many different plant samples can be used, consisting of leaves, stems, tubers, barks or fruits. It should be stressed that there is no standard protocol and that each protocol should be optimized for a given host/virus combinant. We will present here the technique we have developed for the detection of Plum Pox Virus, with additional advice on detection of other pathogens when appropriate.

Sample grinding. One gram of plant sample is ground in 4 ml of grinding buffer (Appendix 2) using a pestle and mortar (or other methods such as electric press or Polytron homogenizer when available). It is extremely important to use a buffer that will optimize the signal to noise ratio. The extract is then clarified by centrifugation for 10 min at 10 000 rpm. The samples can, if necessary, be deproteinized by including one volume of a 1:1 mixture of water-saturated phenol and chloroform during the grinding. This step is optional for the use of radioactive probes but necessary when using biotinylated probes.

Sample denaturation. If required, the nucleic acids contained in the supernatant are then denatured to ensure good binding of the nitrocellulose and availability of the sequences for hybridization. This step is important for the detection of viroids but often of no detriment in the case of most viruses. In a small microcentrifuge tube, 50 µl of sample are added to 50 µl of formaldehyde denaturation buffer (Appendix 2). The mixture is then incubated for 60 min at 60°C (the length of this incubation should be reduced for viruses). At this point, samples are ready for spotting on the membrane. They can also be stored for up to several months at -20°C. We have found that concentration of the nucleic acids present in the extract by ethanol precipitation is detrimental since it usually increases the non-specific background reactions and is therefore not recommended.

Nitrocellulose membrane preparation. Soaking of the membrane in a high-salt solution is required for proper binding of nucleic acids in the samples. The membrane is first soaked for 2 min in pure distilled water and then equilibrated for 10 min in 20XSSC buffer (Appendix 2).

Sample application and fixation. Twenty µl of sample are applied to the nitrocellulose membrane using a BRL "Hybri-dot" apparatus (Figures 167-169). Alternatively, 3-5 µl of sample can be applied directly (using a micropipette) to nitrocellulose that has been air-dried after soaking in 20XSSC. The membrane is then dried at room temperature and baked for a further 2 h at 80°C under vacuum to ensure stable binding of the nucleic acids to the nitrocellulose membrane. This can conveniently be achieved by using an electrophoresis slab gel drier or a vacuum oven. At this point, the membranes can be directly processed or sealed in a plastic bag and stored (at 4°C or -20°C) for up to several months.

Hybridization reaction

Pre-hybridization. In order to prevent nonspecific binding of the probe to the membranes, they are pre-incubated in the hybridization mixture (pre-hybridization). The membranes are sealed in a plastic bag in the presence of 1 ml of hybridization buffer (Appendix 2) for each 10 cm² of membrane, taking care to avoid trapping any air bubbles. The bag is then incubated for 2-3 h in a water bath at 42°C.

Probe denaturation. This step is included to remove any secondary structure of the probe and is especially important for DNA probes which are essentially double-stranded after the labelling reaction. A suitable quantity of probe is placed in a small disposable tube and incubated for 10 minutes (DNA probe) or 3 min (RNA probe) at 100°C in a bath of boiling water and then quickly chilled by placing the tube in an icebucket.

Hybridization. The pre-hybridization buffer is discarded and replaced by the hybridization buffer to which the denatured probe has been added. Use approximately 1 ml of buffer containing 1-2 x 10⁶ cpm/ml of radioactive probe or 200 ng/ml of biotinylated probe per 15 cm² of membrane (see Appendix 1 for probe specific activity determination). The plastic bag is then resealed and incubated in a water-bath overnight at 50°C.

Washing(s)

After hybridization is completed, the membrane is removed from the plastic bag and washed in a small plastic tray.

DNA probes.

Wash at room temperature for 5 min in three changes of Washing Buffer A, then proceed with two 15-min washes at 50°C in Washing Buffer B (see Appendix 2 for buffer composition).

RNA probes.

Four 20-min washes at 60°C in Washing Buffer C (see Appendix 2 for buffer composition). After the washes, the nitrocellulose membranes should be air-dried at room temperature.

Hybrid detection

Radioactive probes. An X-ray film (Kodak XAR or equivalent) is exposed to the membrane for 24 hours at -70°C using intensifying screens. After autoradiography, the film is developed using Kodak LX 24 developer and Ilford Hypam fixer. Within the limits of linearity of the response of the film, the intensity of the spots is proportional to the concentration of viral RNA present on the membrane. No non-specific signal should be obtained with healthy plant controls.

Biotinylated probes. The composition of the buffers is given in Appendix 2. Several commercially available kits can be used for the detection of biotinylated probes (e.g. BRL).

The membranes are first soaked for 1 min at room temperature in Buffer 1, then for 20 min at 42°C in Buffer 2 to saturate the protein fixing sites on the membrane. They are then dried and baked for 10-20 min at 80°C under vacuum.

Following the treatment, the membranes are rehydrated for 10 min in Buffer 2 and then incubated on a Petri dish in the streptavidin solution: 6 µl of a 1-mg/ml streptavidin solution diluted in 3 ml of Buffer 1. Incubate for 10 min at room temperature shaking occasionally.

The membranes are then washed well with at least three changes of buffer for 3 min each time. Incubate on a Petri dish with 3 ml of Buffer 1 containing 3 µl of a solution of biotinylated polymers of alkaline phosphatase (polyAP) at 1 mg/ml. Incubate for 10 min at room temperature with occasional shaking.

Wash abundantly with two changes of Buffer 1 and then with two changes of Buffer 3. The developing solution should be prepared at the last moment in the following way: add 33 µl of the Nitro-blue Tetrazolium solution to 7.5 ml of Buffer 3. Mix thoroughly, then add 25 µl of the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) solution mix. Incubate the membrane in this solution in a sealed plastic bag protected from light.

Maximum colour development is usually achieved within 4 h. To stop the development, simply wash the membrane in 20 mM Tris-HCl pH 7.5, 5 mM EDTA. The dried membranes can then be stored for several months in the dark to preserve the colour.

Appendix 1

DETERMINATION OF THE PROBE-SPECIFIC ACTIVITY

Following the labelling reaction, the radioactive DNA probe(1 µg) is precipitated with ethanol, freed from the unincorporated labelled nucleotides by several 70 percent ethanol washes, dried and finally taken up in 100 µl of sterilized distilled water. Two µl of the probe are then mixed with 3 ml of a 10 percent trichloroacetic acid (TCA) solution along with 10 µl of 3 mg/ml calf thymus DNA used as a carrier. The mixture is left for 30 min at 0°C and then filtered through a Whatman GF/C glassfibre filter. The filter is rinsed with 20 ml of a 5 percent TCA solution and then with 5 ml of ethanol before being dried. The radioactivity retained on the filter is then determined by liquid scintillation counting. The specific activity is given by:

Specific activity (cpm/ µg) = cpm x 100/2

Besides determining the specific activity of the probe, this technique can also help to calculate how much of the probe should be added to the hybridization reaction. Radioactive RNA probes can be counted in the same way.

For biotinylated probes, the result of the labelling reaction can be estimated by spotting dilutions of the probe on a membrane and comparing with a standard of known activity provided in the labelling kit.

Appendix 2

BUFFERS

Grinding buffer for viroids (GPS)

200 mN glycine
100 mM Na₂HPO₄
600 mM NaCl
1% SDS (sodium dodecyl sulphate)
Adjust pH to 9.5, autoclave 20 min at 120°C then add:
0. 1% DIECA (Na-diethyldithiocarbamate)
0.1 mM DTT (dithiothreitol)

Grinding buffer for viruses

50 mM Na-citrate, pH 8.3
2% PVP (polyvinylpyrrolidone)
Autoclave 20 min at 120°C then add:
1 mM EDTA
20 mM DIECA

Phenol/chloroform mixture

1 vol. water-saturated phenol
1 vol. chloroform/pentanol (24 /1)
20XSSC buffer
3 m NaCl 0,3 M Nacitrate pH adjusted to 7.0

Formaldehyde denaturation buffer

5XSSC
25 mN Na₂HPO₄
5x Denhart (0.1% each of bovine serum albumin (BSA), Ficoll and PVP 360)
50% deionized formamide
200 µg/ml of denatured calf thymus DNA

DNA probes hybridization buffer

4 vol. DNA probes pre-hybridization buffer
1 vol. 50% dextran sulphate

RNA probes pre-hybridization and hybridization buffer

50% formamide
50 mM phosphate buffer pH 6.5
5XSSC
0.1% SDS
1 mM EDTA
0.05% Ficoll
0.05% PVP 360
200 µg/ml of denatured salmon sperm DNA

Washing buffers

2XSSC
0.1% SDS

Washing buffer B

0.2XSSC
0.1% SDS

Washing buffer C

0.1 XSSC
0.1% SDS

Biotinylated probes development buffers

Buffer 1

100 mM Tris-HCl pH 7.5
100 mM NaCl
2 mM MgCl2
0.05C% Triton X100

Buffer 2

Buffer 1 plus 3% BSA

Buffer 3

100 mM Tris-HCl pH 9.5
100 mM NaCl

Commercial source of chemical

FIGURE157 Hybridization on nitrocellulose (nc) membrane

Hybridization on nitrocellulose (nc) membrane (left) Infected sample containing normal plant cell RNAs and viral RNA (the target sequence to which the RNA probe hybridizes). Because of this hybridization the labelled probe will be retained on the membrane (right)Healthy sample containing only normal plant cell RNAs. The probe cannot hybridize with any sequence: it will not be retained on the membrane and will be eliminated upon washing

FIGURE 158.Schematic representation of the system used for the detection of biotinylated probes

FIGURE 159 Plant sample consisting of leaves, bark, roots, tubers, fruits etc.

FIGURE 160 Plant sap is extracted using an electric press

FIGURE 161 A drop of sap is added to a grinding buffer contained in the microcentrifuge tube

FIGURE 162 Sap and grinding buffer are mixed using a vortex

FIGURE 163 Low-speed centrifugation (10 000 rpm for 10 min) is carried out to pellet the plant cell debris and to separate the phases it a phenol deproteinization step is included

FIGURE 164 For viroids, the supernatants are supplemented with formaldehyde and incubated for 60 min at 60°C (omit this step for most viruses)

FIGURE 165 In the meantime, one nitrocellulose membrane and three sheets of Whatman 3MM paper are cut, soaked in water and equibilitrated in 20XSCC buffer

FIGURE 166 One 3MM filter and the nitrocellulose are placed in the BRL Hybri-dot system (or equivalent)

FIGURE 167 One 3MM filter and the nitrocellulose are placed in the BRL Hybri-dot system (or equivalent)

FIGURE 168 Twenty microlitres of extract are spotted on the membrane while gentle vacuum is applied

FIGURE 169 Twenty microlitres of extract are spotted on the membrane while gentle vacuum is applied

FIGURE 170 The membrane is taken out of the blotting apparatus and air-dried

FIGURE 171 The membrane is sandwiched between the two remaining filters and baked in vacuum for two hours in a gel dryer

FIGURE 172 The membrane is sandwiched between the two remaining filters and baked in vacuum for two hours in a gel dryer

FIGURE 173 After drying, the membrane is placed in a plastic bag which is sealed on three sides

FIGURE 174 After drying, the membrane is placed in a plastic bag which is sealed on three sides

FIGURE 175 The pre-hybridization buffer is added to the plastic bag and the bag is completely sealed

FIGURE 176 Pre-hybridization is carried out by incubating the bags for 2-4 hours in a water-bath under suitable conditions

FIGURE 177 The probe is denatured for a few minutes in a bath of boiling water

FIGURE 178 The bag is cut open, the pre-hybridization buffer discarded and replaced by the hybridization buffer containing the denatured probe. The bag is then resealed

FIGURE 179 Hybridization is carried out by incubating the bag overnight at the desired temperature in a water-bath

FIGURE 180 After hybridization, the membrane is removed from the bag and washed in several changes of washing buffer

FIGURE 181 Afler washing, the membrane is air-dried

FIGURE 182 An X-ray film is exposed to the membrane

FIGURE 183 An X-ray film is exposed to the membrane

FIGURE 184 After autoradiography is completed, the X-ray film is developed and

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