Why use Molecular Diagnostics in Agriculture?

Molecular diagnostics helps to answer the timeless question: “What the hell is that?!”

Here are few ways diagnostics can help:

  1. Disease confirmation
    • Many pathogens can cause similar symptoms or disease in their hosts. Identifying the causal agent (pathogen) of disease allows for more direct, targeted management approaches. Treatments or long-term management (crop rotations) have a greater chance of success.
  2. Environmental testing
    • Environmental testing of soil, air, water, or other non-host samples can help find where pathogens exist and monitor inoculum levels over time.
  3. Trait Confirmation
    • Many traits in plants are not obvious by looking at them, meaning the traits (beneficial or harmful) are hidden from our eyes. Molecular diagnostics can confirm whether certain traits are present or absent in a host (plant), pathogen (virus), or pest (weed).

Projects I work on at NAGC use new molecular diagnostic tests that focus on pathogen detection and trait confirmation. NAGC is a high-throughput testing lab that primarily uses quantitative Polymerase Chain Reaction (qPCR) for diagnostic testing.

At its core, qPCR diagnostics is a workflow that involves grabbing, smashing, extracting, and copying.

The first step of molecular diagnostics is to grab (or collect) a sample that will provide an answer to a particular question. The question could fall into the three bolded headers above. For our purposes let’s say the question is, “Is pathogen X in the soil?”.

The grabber will collect a small sample of soil. Next the soil will be violently smashed, also known as mechanical digestion which lyses or destroys microbial cells contained in the soil sample. Adding liquid reagents like detergents also chemically digests microbial cells.

After smashing, it is time to extract (pull out) and purify the genetic material (DNA and/or RNA) from the soil sample. This is a stepwise process that includes clean up reagents and methods (magnetic bead capture or series of filters). The final product is a solution containing a population of DNA or RNA from every microbe, plant, or animal cells within the original sample. If pathogen X is in the sample, its DNA/RNA will also be in this pool of purified genetic material.

Molecular diagnostics use PCR – a common biotechnology that makes copies of a genetic target of interest. We also call this amplification by PCR. The copying step increases the signal of the pathogen X’s DNA so we can find it among the background of the pool of DNAs in the sample. Importantly, the high specificity of PCR tests means that the target has to be present in the sample being tested, otherwise no amplification and, therefore, no PCR products will be created or detected.  

NAGC specializes in multiplexing, the ability to simultaneously amplify multiple targets (e.g., 3 pathogens) within a single PCR test. Multiplexing involves using different fluorescent probes that ‘light up’ during PCR when their targets are amplified in a test sample. For example, if two pathogens are found in the sample, one will produce a red signal, while the other produces an norange signal. 

The fluorescence of the probes is proportional to the starting levels of each pathogen in a sample. The higher the concentration of pathogen, the stronger the intensity of the probe during PCR. The machine performing the PCR also tracks the probe fluorescence during the cycling process. In the end, we examine amplification curves, to identify and quantify the target from a set of known standards.