TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.5 DETECTION/Molecular Methods

Section 2.5.6
Non-radioctive PSTVd
Detection Using
Fluorescein-Labeled
Probes
The non-radioactive PSTVd detection protocol, based on the use of
fluorescein-labeled probes in Dot-blots, involves: labeling probe; solution
preparation; sample extraction and application onto Nylon membrane;
prehybridization; hybridization; washings; blockings; antibody incubation;
signal generation; and detection, using CDP-star as substrate.

The PSTVd probe is labeled by random priming system. Nonamers of

random sequence were used to prime DNA synthesis on a denatured
PSTVd-cDNA template, in a reaction catalyzed by the [exonuclease-free]
Klenow fragment of E. coli DNA polymerase l. Fluorescein-11-dUTP (FI-
UTP) partially replaces dTTP in the reaction so that a fluorescein-labeled
probe is generated.

Following hybridization, this protocol allows detection for hybrids (after a

blocking step) by incubation with an antifluorescein alkaline phosphatase
 (AP) conjugate. After washing off excess conjugate, probe-bound AP is
used to catalyze light production by enzymatic decomposition of a
stabilized CDP-Star (an aqueous solution of <1.5%(w/v)disodium 2-
'
chloro-5-(4-methoxyspiro(1,2-dioxetane- 3.2'-(5 -chloro)-tricyclo (3,3,1,1)
decan)-4yl)Phenyl phosphate). This substrate has a rapid, light output, so
exposure to film made in the first few hours after addition of the substrate
to a membrane will produce a strong signal.

Preparation of Fluorescein-labeled Probes by Random Priming

System

This protocol is based on the labeling of 50 ng of template DNA.

However, the standard reaction can be used to label 25 ng–2 mg of
template DNA.

1. Preparation of labeled probe

1.1 Dilute the DNA to be labeled to a concentration of 2–25 ng/µl in

either distilled water or TE buffer (10mM Tris-HCL, 1mMEDTA,
pH8.0).

1.2 Place the following tubes in an ice bath to thaw:

Nucleotide mix
Primers
Water
Leave the enzyme at –15°C to –30°C until required, and return it
to the freezer immediately after use.

1.3 Denature the DNA sample by heating for 5 minutes in a boiling

water bath; then chill on ice. It is advisable to denature in a
volume of at least 20 µl.

1.4 To a 1.5ml-microcentrifuge tube, placed in an ice bath, add the

appropriate volume of each reagent in the following order:

Nucleotide mix 10 µl
Primer 5 µl
Denatured DNA (50 mg) … µl *
Distilled water … µl **
Enzyme solution (Klenow) 5 units/ml 1 µl
Total 50 µl

*The volume depends on DNA concentration.

**Up to 50 µl.

1.5. Mix gently by pipetting up and down and cap the tube. Spin briefly
in a microcentrifuge to collect the contents at the bottom of the
tube.

1.6. Incubate the reaction mix at 37°C for 1 hour.

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 1.7. If any of the probe is be stored, rather than used in an immediate
hybridization, then the reaction should be terminated by adding
EDTA to a final concentration of 20mM. Probes can then be
stored in suitable aliquots in a freezer at –15°C to –30°C (in
darkness) for at least 6 months. Avoid repeated freezing and
thawing.

1.8. The yield of probe after labeling 25 or 50 ng of template will

typically be between 6–8 ng/µl. We recommend that you use the
rapid labeling assay as a check to ensure that you have
successfully labeled your probe.

2. Monitoring incorporation using the Rapid Labeling Assay

2.1. Prepare 1/5, 1/10, 1/25, 1/50, 1/100, 1/250 and 1/500 dilutions of
the 5X nucleotide mix in TE buffer.

2.2. Dot out 5 µl of your labeled probe and 5 µl of the 1/5 dilution of
nucleotide mix (negative control) onto a strip of Hybond-N+,
placed on nonabsorbent backing. Allow the aliquot to absorb (but
do not allow to dry) and wash the strip with gentle agitation in
excess pre-heated 2XSSC at 60°C for 15 minutes.

2.3. Prepare a reference strip by dotting 5 µl off each nucleotide mix

dilution, except the 1/5, onto a separate strip of Hybond -N+. This
reference strip can be re-used and stored, wrapped in Saran
Wrap, for several weeks at –15° to –30°C (in darkness).

2.4. Place reference and the washed strip on the piece of Whatman
3MM paper, lightly moistened with TE buffer, and take to the
darkroom. Visualize both strips (sample side down) on the UV
transilluminator. The closer the intensity of the labeled DNA to
the lower dilution (1/10), the more efficient the labeling reaction.
The washed negative control should retain little (or no)
fluorescence, indicating that the fluorescence on the probe is due
only to incorporated fluorescein and not to unincorporated FI-
dUTP. If significant fluorescein remains in the negative controls,
wash the filter for another 15 minutes. This is possible only if the
strip has not dried.

Sample preparation

When collecting the samples, always remember to identify them in the

plastic bags. Use a data sheet to identify the samples in the membrane.

a. Leaf and sprout samples

Collect the samples in the plastic bags (leaf samples from the top
of the plant, sprout samples from any part of the tuber).

Grind samples in the plastic bag together with the extraction buffer
(0.5 ml/0.5 g of tissue) and transfer sap to an Eppendorf tube.

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 Add Phenol/Chloraform (½ vol. of each). Mix well and centrifuge 5
minutes at 12,000 rpm.

Spot 4 µl of the clear supernatant onto the nylon membrane and

bake spotted membrane for 1h at 80°C (or expose for 5 minutes
on UV crosslinker).

b. Seed Samples:

Place adequate amount of seed (approx. 50) in the tube, and soak
in distilled water overnight. Drain water, and grind seeds in mortars
with 0.5 ml extraction buffer Transfer to an Eppendorf tube, and
add Phenol/chloroform (½ vol. of each). Mix well and centrifuge for
5 minutes at 12,000 rpm.

Spot 4 µl of the clear supernatant onto the nylon membrane and

bake the spotted membrane for 1h at 80°C (or expose for 5
minutes on UV crosslinker).

Extraction Buffer (80 ml)

Formaldehyde 37% 40 ml
20XSSC 20 ml
H2O 20 ml

Prehybridization and Hybridization

Put the membrane into the hybridization plastic bag (seal and cut a
corner of the bag)

Pre-heat the required volume of hybridization buffer (0.15ml/cm²) to

60°C; then put the buffer into the bag containing the membrane and
prehybridize for 1h at the same temperature.

Hybridization buffer (10ml)

Stock solution Amount Final concentration

20X SSC 2.5ml 5XSSC
Liquid Block 0.5ml 1/20 liquid block
10% SDS (w/v) 0.1ml 0.1%SDS
20% Dextrin sulfate 2.5ml 5% Dextrin sulfate

Transfer the required amount of probe to a clean Eppendorf tube (2µl per
ml of hybridization buffer) If the volume is less than 20µl make up to this
volume with distilled water. Denature the probe by boiling for 5 minutes,
and chill on ice.

Centrifuge the denatured probe briefly, then add to the prehybridization

buffer, (avoiding placing it directly onto the membrane). Seal the plastic
bag and mix gently.

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 Hybridize overnight at 60ºC with gentle agitation.

Washing, Blocking, and Antibody Incubation:

Perform washing and blocking steps using a plastic box.

a. Washings:

Prepare stringency wash solution I and pre-heat to 60°C. Transfer

the membrane to this solution (using 2–5 ml/cm² of membrane),
and wash for 15 minutes at this temperature with gentle agitation.
Carry out another wash at the same temperature as the first, but
using pre-heated solution ll, for 5 minutes.

Wash solution I (200ml)

Stock solution Amount Final concentration
20x SSC 10ml 1X SSC
10% SDS (w/v) 2ml 0.1% SDS
H2O up to 200ml

Wash solution II (200ml)

Stock solution Amount Final concentration
20x SSC 5ml 0.5x SSC
10% SDS (w/v) 2ml 0.1% SDS
H2O up to 200ml

b. Blocking:

Following the stringency washes, incubate blots with gentle

agitation for 1h at room temperature in 1 ml/cm² of a 1:10 dilution
of liquid blocking agent in Diluent buffer.

Blocking solution (100ml)

Liquid block 10ml

Diluent buffer 90 ml

c. Antibody Incubation:

Prepare the conjugate solution, diluting the anti-fluorescein-AP

conjugate 5000-fold in freshly prepared 0.5% (w/v) BSA in Diluent
buffer. Incubate the membrane in conjugate solution (0.3 ml/cm² of
membrane) with gentle agitation at room temperature for 1h. For
this step, we recommend using a plastic plate.

Conjugate Solution

Diluent buffer 50 ml
Bovine Serum Albumin (BSA), fraction V 250 mg
Anti-fluorescein-AP conjugate 10 µl
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 d. Final Washing:

Remove unbound conjugate by washing for 3x10 minutes in

0.3%(v/v)Tween-20 in Diluent buffer at room temperature with
gentle agitation.

Signal Generation and Detection:

Drain off any excess washing buffer from the membrane (by
touching the corner of the membrane against the box used for
washing steps, or other convenient clean surface), and place it
(sample side up) on a sheet of Saran Wrap on a flat surface.

Pipette detection reagent (CDP-Star) onto the membrane (30–40

µl per cm²) and leave for 5 minutes.

Drain off excess detection reagent by touching the corner of the

membrane on to the Saran Wrap. The membrane must be
transferred to the development folder and the pipette must be
rolled over it to squeeze out any excess detection reagent or air
bubbles. Seal the edges using heat sealer.

Open the bag and, after draining, arrange the blots on one-half of
the open bag. Fold the bag over the blot. Ensure that the outside of
the bag is dry before exposing to film. Place the bag containing the
membrane (samples side up) in a film cassette, and take to a
darkroom.

Switch off the lights and place a sheet of Hyperfilm-MP or Kodak

X-OMAT XAR-2 on top of the bag. Close the cassette and expose
initially for 1h. Remove film and develop as follows:

Put the film in the developer solution for 2 minutes. Wash briefly in
water and place the film into the fixer solution for 2 minutes. Wash
again with water and let it dry.

Buffer preparation

a. 20X SSC: (50 ml)

0.3M Sodium Citrate dihydrate 4.42g
3M Sodium Chloride 8.76g

b. Diluent Buffer *600ml)

100 mM Tris-HCL 7.2g

300mM Sodium Chloride 10.51g
(adjusted to pH 9.5 with HCl)

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 *Use 90 ml for blocking step, 50 ml for antibody incubation, and 450 ml for the final
washings.

c. Developer solution
Dilute 50ml of Developer Kodak+175ml H2O

d. Fixer solution
Dilute 50ml of Fixer Kodak+175ml H2O

e. 10% SDS
Dissolve 10g of electrophoresis-grade SDS in 90ml of distilled
water. Heat to 68°C to assist dissolution. Adjust pH to 7.2 by
adding few drops of concentrated HCl. Adjust volume to 100 ml.
Wear a mask when weighting SDS. There is no need to sterilize
10% SDS. Store at room Temperature.

f. 50% Dextran Sulfate

Dissolve 50g Dextran sulfate water; adjust to 100-ml volume.

Heat in a water bath at 60°–70°C to dissolve completely (the

solution will be very dense). Store at –20°C.

Required solutions

• Labeling: Gene images Random Prime Labeling Module (Amersham

RPN3541).
• Hybridization: Hybridization buffer.
• Signal generation: Gene Images CDP-Star detection Module
(Amersham RPN3511)
• Development of photographic films: developer solution, fixer solution.

Recommended Literature
Salazar, L.F and M. Querci 1992. Detection of Viroid and Viruses by
Nucleic Acid Probes. In: Duncan, J.M and L. Torrance (eds.).
Techniques for the rapid detection of plant pathogens. pp. 129–144.
Blackwell Scientific Publications. Cambridge.
Kricka, J.L. 1992. Nonisotopic DNA probe techniques. United States. pp.
352.
Hames, B.D. and S.J. Higgins. 1985. Nucleic Acid Hybridization. A
Practical Approach. Oxford. p. 345.

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