Instructor's manual and video

Introduction

NCM-ELISA (Enzyme Linked Immunosorbent Assay on Nitrocellulose Membrane) is an immuno-enzymatic assay. It uses a nitrocellulose membrane instead of a microtitre plate as support for the samples and reagents. NCM-ELISA after enrichment is as sensitive as the Double-Antibody Sandwich ELISA (DAS-ELISA), and it is easier and quicker. It has another important advantage: the nitrocellulose membrane with the samples can be stored for several weeks before continuing with the test. Therefore, the membrane can also be sent to another laboratory that can perform the assay.

The test consists of:

1. loading a very small amount of a plant extract (20 µl) on a nitrocellulose membrane  (=Dot-blotting),
2. blocking the area of the membrane that is free of samples (1 h incubation),
3. binding the samples with Ralstonia solanacearum (Rs)-specific rabbit antibodies (2 h incubation),
4. binding the Rs-antibodies complex with enzyme-labeled goat anti-rabbit antibodies (1 h incubation), and,
5. revealing the bound enzymes by adding the substrate leading to a coloration reaction (5 to 20 min.).

All of the steps are done at room temperature. The presence of Rs in the sample leads to the development of a purple coloration. The intensity of the coloration is proportional to the bacterial concentration. After the ELISA, the membrane is stable and can be stored for a long time.

Before performing the NCM-ELISA, an enrichment procedure must be carried out, by incubating the tuber extracts in a semi-selective broth (modified SMSA; Figure 1). This increases the sensitivity of the method by almost 1 million. As few as 10 bacteria per ml of tuber extract (bact./ml) can be detected instead of 10⁶ - 10⁷ bact./ml without enrichment. This allows the detection of Rs in potato tubers (or stems) latently infected, i.e., without visible symptoms.

All races, biovars and serotypes of Rs can be detected with the Rs-specific antibodies provided in the kit. Some saprophytic bacteria when in pure culture at concentrations equal to or higher than 10⁸ bact./ml may cross-react with the antibodies produced against Rs, therefore, when using the kit as a diagnostic tool (e.g. to confirm that isolated bacteria are Rs) the concentration of the suspected bacteria in the aqueous suspension must be equal to 10⁷ bact./ml. At this concentration Rs produces a positive reaction in ELISA but saprophytic bacteria do not. No cross-reaction has been detected by ELISA after enrichment of extracts prepared from tissues of the tuber vascular ring.

The CIP kit can be used for the monitoring of Rs in potato tubers essential for seed certification schemes and varietal evaluation for resistance to bacterial wilt. It can also be applied for the monitoring of Rs in stems of potato and other plants for research on the disease epidemiology (survival and spread of Rs). To detect Rs in plant roots it is preferable to use DAS-ELISA because the dark brown coloration of roots extracts may impede reading of the ELISA reaction on the membrane.

Number of samples to be analyzed by NCM-ELISA for the detection of latent infection by Ralstonia solanacearum in tubers

For the analysis of a basic seed lot

Each sample is a composite of 25 tuber fragments.

One seed lot = harvested tubers from the same field, of the same variety and originating from the same seed.

• 400 tubers per seed lot from a field of 0.5 to 2 hectares should be analyzed.
• Tubers should be selected at random, preferably at harvest, from heaps or from furrows, or from the stores at the beginning of the storage period. If tubers have to be taken before harvest (no earlier than two weeks before harvest), select plants in the field at random and then take two tubers per plant, also at random.
• Divide the 400 tubers into 16 sub-samples (25 tubers each), and take fragments from each tuber. Crush all the fragments from a sub-sample of 25 tubers in one bag, and from each bag take two 500 ml (0.5 ml) aliquots of tuber extract and mix each one with the same volume of SMSA broth for enrichment (incubation for 48 h at 30°C).
• For analysis by NCM-ELISA, each of these 32 sub-samples is dotted on the membrane (duplicates are not necessary unless the dotting was not satisfactory).

One kit is sufficient for analysis of 18 seed lots.

Important: In the case of international trade or litigation, confirm any positive results obtained from ELISA by (i) isolating R. solanacearum from the enriched extracts in Kelman’s or SMSA medium, and (ii) inoculating tomato plants with the isolates to confirm their pathogenicity.

For the analysis of pre-basic seed

Each sample is a composite of two tuber fragments.

• Analyze 0.5% of the plants from the same variety, same greenhouse and same multiplication lot.
• From each plant take two tubers at random, process them together and analyze them as one composite sample.
• Mix one 500 ml (0.5 ml) aliquot of tuber extract with the same volume of SMSA broth for enrichment.

For example, in a lot of 5,000 plants, 25 samples of two tubers will be analyzed (a total of 50 tubers). In this case one kit is sufficient for analysis of 23 pre-basic seed lots.

For germplasm evaluation

Each sample is a single tuber sample.

The purpose of the analysis is to assess latent infection in tubers of clones exhibiting no visible wilt in the field, or for quantitative comparisons between clones/varieties:

• For each clone, analyze 30 tubers individually.
• Select 30 tubers (of any size above 30 mm) at random in the experimental plots or take at random two tubers per plant from 15 plants.
• Process tubers individually and mix one 500 ml (0.5 ml) aliquot of tuber extract with the same volume of SMSA broth for enrichment.
• Convert the number (n) of infected tubers (positive ELISA) to a percentage per clone (n/30 x 100).

One kit is sufficient for analysis of 19 potato clones or varieties.