TECHNIQUES IN PLANT VIROLOGY

CIP Training Manual

2.3 DETECTION/Serology

Section 2.3.5
Latex Test

The latex test is a modified version of the microprecipitation serological

technique. It is based on the formation of a visible precipitate between an
antigen (virus) and its specific antibody, which has been adsorbed to
latex polystyrene spheres.

Figure 1. Latex sensitization

 Latex is made up of polystyrene beads 810 nm in diameter (1 nm = 10–9
m). The polystyrene spheres are covered by the antibodies through a
process called latex sensitization.

Figure 2. Formation of precipitate in the latex test

The sensitized latex is mixed with sap or purified virus. As in

microprecipitation, an aggregate forms if the antigen is present in the
samples. Because the latex beads are larger than the antibodies, the
reaction can be seen by the naked eye when the petri dish is placed
against a dark background.

Materials

The following materials are necessary for the test:

− Plastic bags (10 x 15 cm)

− Test tubes (10 x 75 mm)
− Plastic petri dishes
− Hypodermic syringes (1 cm3 = 1 ml)
− Sensitized latex for each virus
− Distilled water
− 0.05 M Tris-HCl buffer pH 8 containing 0.01 M sodium bisulfite and
0.05% Tween-20
− Rotating mechanical shaker (optional)
− Automatic sap extractor (optional)

P.V. • Sec 2.3.5 – 99 • Page 2 - INTERNATIONAL POTATO CENTER

 Methods

The original latex test has been improved at CIP. The seven-step method
routinely used for leaf analysis is as follows:

Use a ruler and a crayon to draw parallel lines 0.8 cm from the inside
edge of a petri dish. Next, draw perpendicular lines to form squares.

Take leaves from various heights of the plant to be analyzed. Put

them into the plastic bag without allowing contact with hands. For
detecting PVS, it is essential to collect leaves from the middle or lower
third of the plant.

Always use a negative (healthy plant) and a positive (artificially infected

plant) as controls. For these controls, use healthy leaves from different
indicator plants and infected potato plant leaves from host plants.

Figure 3. Grid on petri dish for latex test.

Extract sap from each sample with an automatic juice extractor. If not
available, add 0.3 cm3 Tris-HCl buffer solution to a leaf sample in a
plastic bag. Place the bag on a table and press it several times with a
roller or another cylindrical object to squeeze sap from the leaves.

Collect the sap in a test tube and let stand for 2 hours, preferably at 4oC.

Add 0.45 cm3 of Tris-HCl buffer solution each in 2 test tubes (A) for
preparing the following dilutions:

− 1/10 dilution (first tube)

− 1/100 dilution (second tube)

Using a hypodermic syringe, transfer 0.05 cm3 sap from the top of the
collection tube. Avoid sediment (B).

Mix well by extracting all the mixture from the tube with the syringe and
then expelling it back into the test tube. The 1/10 dilution can also be
prepared by mixing 9 drops Tris-HCl buffer solution with one drop sap.

P.V. • Sec 2.3.5 – 99 • Page 3 - INTERNATIONAL POTATO CENTER

 Figure 4. Preparation of sap dilutions.

To prepare the 1/100 dilution, transfer 0.05 cm3 of the 1/10 dilution to the
second tube (C) and mix as before. The 1/100 dilution can also be
prepared by mixing 9 drops of the Tris-HCl buffer solution with one drop
of the 1/10 dilution.

Two dilutions are necessary for the test since reactions are not clearly
visible when the virus concentration is too high (D). Due to usually high
concentration of Andean Potato Latent Virus (APLV) in the plant, it is
advisable to use an additional 1/1000 dilution for detection.

Hold the syringe upright and let a drop of sensitized latex fall into each
square on the petri dish. Fill twice as many boxes as there are samples
(for APLV, fill 3 boxes). Prepare 4 additional squares for the controls
(negative and positive).

Place samples on the petri dish according to a recorded scheme so that

the location of the samples and controls can be easily identified.

Use another syringe to put a drop of the 1/100 dilution on a drop of latex.
Next, with the same syringe, put a drop of the 1/10 dilution on the next
latex drop.

Rinse the syringe 3 or 4 times with distilled water before using it for the
next sample.

Repeat the procedure for every sample and keep a record of the scheme
used. Next, cover the petri dish with a lid and place it in the shaker for
one hour at 110 rpm. Use a rotating shaker to induce visible positive
reactions. If not available, place the petri dishes on a piece of cardboard
and rotate them manually.

P.V. • Sec 2.3.5 – 99 • Page 4 - INTERNATIONAL POTATO CENTER

 Open the petri dish and observe the reactions against a dark
background. In positive reactions, the aggregation of latex particles will
be clearly seen. In negative reactions, the latex suspension will maintain
its milky appearance.

For detecting potato virus in tubers, break dormancy with Rindite and
store tubers at 20oC for two weeks. To extract tuber juice, cut thin slices
of the apical tip using a sterilized scalpel. Grind the slices in a juice
extractor with grooved rollers, or by using plastic bags.

Collect the extracted sap and centrifuge it at 5000 g for 15 minutes, or

stand in test tubes until starch grains precipitate. Remove supernatant
and use it to prepare 1:10 and 1:30 dilutions.

Figure 5. Results of a latex test. The two top rows show positive reactions
with formation of precipitate; the bottom row shows reaction-free
latex drops.

Recommended Literature
Fribourg, C.E. and J. Nakashima. 1984. An Improved Latex Agglutination
Test for Routine Detection of Potato Viruses. Potato Research
27:237–249.

P.V. • Sec 2.3.5 – 99 • Page 5 - INTERNATIONAL POTATO CENTER