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    Product Testing, Part 4: “Current Testing Methods Explained


    Julia Stewart:
    Hello, this is PMA PR Director Julia Stewart, and welcome back to PMA’s audio blog, “Ask Dr. Bob” with PMA’s Chief Science & Technology Officer Dr. Bob Whitaker. This post is part of a continuing series on product testing. Bob, today I want to follow up on some things you mentioned in your last post. You spoke about doing initial screening tests and then confirming those results.  Can you explain what all these tests are?

    Bob:
    Yes Julia, I agree we need to spend a little more time on this subject, and I’m going to start with an apology. It’s hard to find the sweet spot in describing some pretty complex science in terms that are both accurate and understandable. So, I may take some liberties here for the sake of clarity for the majority of our listeners, and I apologize if I rile up some scientific colleagues.

    In recent years, the industry has begun using rapid, DNA-based tests to reduce testing time.  These rapid tests are derived from molecular biology methods that permit a lab technician to identify small pieces of pathogen DNA and make many copies of these pieces so they can be visualized and measured. 

    Remember, there is a lot of DNA in a typical testing sample. You have DNA from the plant itself, as well as from all the different types of bacteria that may live on the surface of that fruit or vegetable.  On top of that, the amount of DNA from a possible pathogen isn’t expected to be present in very high amounts because we know that contamination frequencies are very low and don’t seem to occur uniformly. You are literally looking for a needle in a haystack. 

    Therefore, scientists needed a way to be able to rapidly detect unique pieces of pathogen DNA.  One solution was to use DNA replication to make a large number of copies of these small, pathogen-specific DNA fragments so they can fish them out and detect them. The method used to amplify or make a lot of copies of DNA fragments is called PCR or polymerase chain reaction technology.  Polymerase is simply an enzyme that every living cell has.  Its job in nature is to copy DNA so that cells can grow and divide.  PCR is based on using alternating heat cycles to cause sample DNA to “melt” or open up.  Once the DNA opens up, it can be copied.  High heat causes the double stranded DNA helix to unwind and open.  As part of the reaction, a primer DNA is added to the mixture.  A primer is simply a fragment or piece of DNA made from a known pathogen gene.  This primer essentially seeks out and attaches to the opened sample DNA if that sample has the exact same structure.  If the primer attaches, it “primes” or promotes replication of the pathogen gene sequence if present and using this open or exposed DNA template, make a copy of the target pathogen DNA fragment.  Cooling stops replication and closes the sample DNA.  Subsequent heat cycles open the sample DNA for additional replication until virtually millions of copies are made.  With PCR, we are able to copy DNA fragments at incredibly fast rates.  In fact, the replication is so fast that within a matter of a few minutes, many millions of copies of a specific gene fragment, say a pathogen gene fragment, can be produced. Once you have these fragments amplified you can detect specific gene fragments by several different techniques and confirm the potential presence of pathogen DNA thus indicating the sample was contaminated with a pathogen. 

    The amplified DNA fragments are like all biological materials in that they have a size and an electrical charge.  Therefore, when a scientist subjects a literal soup of multiple DNA fragments to an outside electrical current, they arrange themselves in a pattern based on their size and electrical properties.  This pattern looks like a series of dark bands lined up in a gel matrix that physically resembles jello.  This technique is called pulse field gel electrophoresis or PFGE.  The patterns derived from PFGE can be very consistent and used to identify specific gene fragments from specific types of bacteria; it is essentially a fingerprint.  All of you fans of the CSI crime dramas or Court TV know about DNA fingerprints and how they can be used to find the bad guys.  Well, this is basically the same thing.

    Alternatively, you can also detect the presence of pathogen genes directly during the PCR reaction.  The pieces of DNA that serve to catalyze DNA replication in PCR, or “primers” have been engineered in some cases to emit light when they permit DNA replication.  The light emitted from these “beacons” can be measured and reflects the replication of pathogen DNA.  

    Julia:
    Bob thanks, that was an awesome layperson’s description. Ok, so I get it now. That’s why we hear about samples being “PCR positives” or “molecular positives”.  So, we have a rapid test that can find out if a sample has a pathogen present. How long does this test take?

    Bob:
    Well each lab is a little different, but you need to grow out the bacteria in a process called enrichment and then do the actual DNA isolations and detection work. It can run anywhere from 16 to 36 hours based on specific methods used and the logistics of getting a sample to a lab.

    Julia:
    This sounds too perfect Bob.  Something tells me there’s more to this than meets the eye.  Next time, let’s explore the other half of this testing issue and see if there are limitations of this type of rapid, DNA-based testing.

    Thanks to all our listeners and join us next time!

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