AGDIA TESTING TECHNOLOGY

ELISA

(enzyme linked immunosorbent assay)

ELISA is a tried and true technology borrowed from clinical immunology where a combination of antibodies, chemicals, and samples can yield results visible by eye. There are a range of different ELISA formats and chemistries available which are used by Agdia Testing Services. The most common format used is what we call a DAS ELISA (double-antibody sandwich enzyme linked immunosorbent assay).

To perform this test, we add your sample to a specially prepared test well on a microtiter plate. The inside of the test well is coated with an antibody that captures and holds particles of the virus we're testing for.  After the sample has been incubated for a period of time, it will be washed out of the test well.  Any analyte captured by the antibodies coated to the well, will remain intact.

We then add an enzyme conjugate, which is a two-part substance made up of an antibody chemically joined to an enzyme. If any virus particles are present, held in place by the first antibody, some of the enzyme conjugate sticks to them and remains in the well when we wash it out.

Then we add a colorless substrate that the enzyme conjugate can break down into a colored one. If enzyme conjugate is present, attached to virus particles, color appears in the test wells.  Positive samples will vary in intensity depending on how much analyte or virus particles were present and captured. Negative wells will not react with the substrate, leaving the wells virtually clear.

After this substrate incubation the plate is read by a spectrophotometer and by eye. The spectrophotometer provides a numberic value that correlates with the amount of color in the well.  Higher values indicate more color and a higher analyte concentration.  Negative samples generate very low values that fall into a statistically different numeric range than positive samples.

Every time we perform a DAS ELISA test, we also test a positive control, a sample from a plant we know to be infected. Checking that the positive control well is colored helps us know the test worked properly.

PCR

(polymerase chain reaction)

Agdia has a rapidly expanding list of PCR tests. Some of these tests are designed to detect specific pathogens, such as bacterial blight of geranium (Xcp), whereas other PCR tests find entire families of viruses (tobamoviruses, carlaviruses, potexviruses, and geminiviruses) or phytoplasmas (formerly known as mycoplasma-like organisms). PCR tests are more sensitive than ELISA tests and are better suited to plant tissue that is likely to have lower numbers of pathogens, such as tissue culture.

PCR is able to detect pathogens by targeting their genetic material (DNA or RNA). The aim of the polymerase chain reaction is to make many copies of part of the pathogen's genetic material so there is enough to be detected. The PCR processs is made possible by an enzyme that can copy DNA. Since most plant viruses possess only RNA, we first must make a DNA copy of the RNA. In the case of DNA viruses, bacteria and phytoplasmas, their DNA is used directly. Because the genetic material of the pathogen is mixed with that of the plant, it is necessary to target only the genetic material to be copied. We do this by adding small pieces of DNA called primers, which stick only to borders of the region of genetic material that should be copied. These primers, then direct the enzyme where to start and end copying.

The PCR process uses a series of hot, cold, and warm cycles. Hot cycles split DNA into single strands. Cold cycles allow the primers to attach. During warm cycles, the enzyme makes a copy of each piece of primed DNA. If a phytoplasma is present, PCR will produce a large quantity of a certain piece of DNA. We use a process called electrophoresis that separates pieces of DNA by size to tell if that piece of DNA is present.

HYBRIDIZATION TEST

We can use antibodies to detect viruses because each virus has a unique protein coat that an antibody will stick to. But one group of pathogens, collectively called viroids, have no protein at all just a circular piece of the nucleic acid RNA. To detect viroids, we use a hybridization test, which can find this RNA. RNA "hybridizes" when two single strands come together like a zipper to form a double strand. To detect viroid RNA, we use a labeled piece of RNA called a probe that can hybridize with RNA from the sample.

First we extract the RNA from plant samples. Then we attach the RNA to a membrane. We apply the RNA probe, which is labeled with a chemical called DIG. If a viroid is present, the probe hybridizes with it. If not, the probe is lost when we wash the membrane.

To see the result of the test, we treat the membrane with a process that gives off light if DIG is present. We expose film to the membrane to detect the light. A spot on the film means that the viroid is present. Along with your samples, we also test samples from healthy and infected plants as controls.