|
|
| Agdia home | Back to Testing Services | mail us | |
PCR and nucleic acid hybridization testing is available in Agdia Testing Services for the detection of plant pathogens.
For more information regarding PCR tests please contact Samantha Stahl in the Molecular Diagnostics department (1-800-62-AGDIA). For information regarding Hybridization tests please contact Bernard Kulemeka.
PCR Tests
PCR Viral Group TestsThe PCR group tests provide all the advantages of ELISA with the added advantages of broad detection and extreme sensitivity. Simplicity - A single test detects all members of a virus group. Speed -- About the same amount of time is required to run a PCR test as is required to run multiple ELISA tests. Cost effectivenesss -- Because multiple ELISA tests are necessary to screen plant material for multiple viruses, the PCR group test becomes an economical alternative for testing plants.
Nucleic Acid Hybridization
|
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.
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.