Introduction

DAS-ELISA, or double antibody sandwich enzyme-linked immunosorbent assay, is an immuno-enzymatic assay that uses a microtitration plate as a base for the samples and reagents employed.

The procedure used to detect Ralstonia solanacearum bacteria in potato stems is summarized in Figure 1. It consists of the following:

• The coating of a microtitration plate with Ralstonia solanacearum (Rs)-specific rabbit immunoglobulins (IgG)
• The addition of solutions prepared from stem samples to the wells of the microtitration plate
• The addition of enzyme-labeled Rs-specific rabbit immunoglobulins (conjugated IgG)
• The addition of enzyme substrate – if Rs is present in the stem extract this produces a color reaction, the intensity of which is proportional to the concentration of bacteria.

Ralstonia solanacearum may be present in the stems of asymptomatic plants at low concentrations. Therefore, before performing the DAS-ELISA, an enrichment procedure must be carried out to allow the bacteria to multiply to detectable levels. This is done by incubating the stem extract in an enrichment broth at 30°C for 48 hours with constant agitation. Use of this procedure means that the DAS-ELISA test is capable of detecting the bacteria from original bacterial concentrations of as low as 20 bacteria per milliliter of extract (instead of the 10⁶  bacteria per gram needed if enrichment is not undertaken).

All races, biovars and serotypes of Rs can be detected using the Rs-specific immunoglobulins provided in the kit. Some saprophytic bacteria may cross-react with the antibodies produced against Rs when in pure culture at concentrations equal to or higher than 10⁸  bacteria/ml. However, after enrichment of the stem extract containing saprophytic and endophytic bacteria that are usually in the vascular ring, no cross-reactions have been detected in DAS-ELISA.

This kit can be used to detect Rs in the stem of potato two to four weeks before harvest. Monitoring for Rs in this way is essential for potato certification schemes. The kit can also be used to detect Rs in the tubers and roots of potato or other hosts, in order to study the epidemiology of the disease.

The meaning of this sentence isn’t quite clear. Do you mean ‘Saprophytic and endophytic bacteria usually occur in the vascular ring. However, no cross-reactions have been found to occur in the DAS-ELISA after enrichment of the stem extract.’? If not, please explain your meaning and I will provide a grammatical rewrite.

Sample collection and sample size

Collection method and sample size used for the analysis of a basic seed lot

• One seed lot = a batch of harvested stems from one field. All harvested stems must be of the same variety and grown from the same batch of seed.
• It is recommended that, from a field of 0.5 to 2 hectares, 400 stems per seed lot should be analyzed. Plants should be selected at random by following a zig-zag pattern through the field.
• Using a pair of horticultural scissors, cut a fragment 10 cm long from the base of the plant, above soil level. One principal stem must be collected from each of the 400 plants.
• The scissors must be disinfected between the cutting of each composite sample (25 stem fragments) by dipping them in a 1% sodium hypochlorite solution.
• Wrap each stem in paper and put it inside a paper bag.
• Analyze the samples the same day or, if necessary, keep them in a cool place (around 15°C) for no more than one day. Do not keep the samples in the refrigerator.

Collection method and sample size used for the analysis of pre-basic seed

• Sample and analyze 0.5% of plants from the same greenhouse. Plants must be of the same variety and must come from the same multiplication lot.
• Each ‘sample’ must consist of ten stem fragments (thus forming a composite sample).
• Analyze the samples on the same day or, if necessary, keep them in a cool place (around 15°C) for no more than one day. Do not keep the samples in the refrigerator.

If you have to test 25 clones (100 plants per clone), take a sample of each of the first 10 plants, and then amalgamate them to make a composite sample. Repeat this process for every 10 plants. This will give 10 composite samples per clone. In total, you will obtain 250 composite samples (10 samples for each of the 25 clones). The kit contains enough material to analyze all these samples. If even one sample is positive the entire lot should be discarded.

• What about the 10 cm fragments? Or, do you mean 'wrap each 10-cm stem sample in a piece of paper and place it inside a paper bag". But, how do you know which samples make up which composite sample? This is confusing. Alternatively, do you mean "wrap each composite sample of 25 stem fragments in paper and put them inside a paper bag'? Also, should the bag be marked in any way? You mention marker pens above, but don't actually mention using them.