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    Product Testing, Part 5: “Current testing methods: the positive-negative challenge

    Julia Stewart:
    Hello, this is PMA PR Director Julia Stewart, and welcome back to our continuing “Ask Dr. Bob” audio blog posts on product testing with PMA’s Chief Science &Technology Officer Dr. Bob Whitaker. In the last post we talked about the emergence of rapid, DNA-based testing to screen produce samples for the presence of possible pathogens, and spoke a little about the basic science behind them. Bob, today I want to get into the benefits and challenges we have with these tests and why it is important to be able to confirm positive tests. 

    Bob:
    Certainly, Julia. As we discussed before, these tests are relatively fast and our industry has adopted them because of our need to get results back in time to make harvest or shipping decisions. But, while these DNA-based tests can be very useful, they are also less than 100 percent conclusive. In other words, we have found over several years of testing and research that “positive” test results are not always in fact positive – that is, the product isn’t in fact contaminated – and “negatives” are not always negative. 

    First, let’s talk about the “positives” not always being so positive.  When thinking about product testing, keep in mind that we are talking about being able to detect a pathogen that may be present at a very low level among a very complex community of non-pathogenic bacteria living on the surfaces of fruits and vegetables. If you are testing for pathogens using a Polymerase Chain Reaction or PCR-based test, you have to be able to find those unique DNA pieces indicative of a specific pathogen from the DNA of perhaps 25-30 different strains of non-pathogenic bacteria.  And remember, some of those other strains may be very close relatives of your target and may only differ in a few DNA base pairs. 

    Julia:
    So how do you address that?

    Bob:
    That’s why you have to ask questions.  You have to work with your testing laboratory to understand exactly how they are using the PCR tests and why they feel the DNA primers they are using to identify pathogen DNA in the samples is both selective for the pathogens you wish to test for and sensitive enough to detect them even if they’re only there in very low levels. 

    The value of PCR testing methods basically comes down to the specificity of the DNA primers that are used.  In our last post we discussed primers and said they were small DNA fragments that match specific genes sequences unique to the target pathogen.  They bind to sample DNA and “prime” DNA replication thus enabling PCR testing.  Typically primers have been made to toxin genes like shigatoxin or genes that code proteins that permit binding of pathogens to the human intestine, in other words, genes that play a role in the virulence or pathogenicity of the bacteria.  It stands to reason that the more unique these primers are to the pathogen, the more sensitive and selective the test will be. Remember, these are only fragments or pieces of genes and sometimes the genetic difference between a pathogen and a non-pathogenic related species can be as little as just a few base pairs. So, if the primers are not selected very carefully, one can easily be fooled into thinking a pathogen is present while in reality it may only be a related strain or nothing at all. 

    We’ve seen this repeatedly in our industry, especially with Salmonella positives that with further analysis turn out not to be Salmonella at all, but closely related cousins like Klebsiella or Acetobacter.   

    Julia:
    So that’s where we hear about molecular positives or presumptive positives that were not confirmed in follow-up testing. 

    Bob:
    That’s right Julia. So it’s important to talk to your lab about the primers they are using.  You want to know how many primers they are using in the tests.  Obviously the more genes you test for the greater the selectivity of the test.  This approach is referred to as multiplex testing.  In general, you want to know that your lab is using methods recognized by FDA or analytical testing organizations like the AOAC.  You want to know how many of their “molecular positive” tests are actually confirmed by subsequent testing.  That will give you a good idea as to the value of the primers they are using in their PCR testing.  You also want to know if they participate in some type of accreditation or “check” program.  These programs are really a mechanism to check on the labs performance.  The accreditation body sends the lab samples, some with pathogens and others not and the lab has to identify the bacteria in the samples correctly to maintain their accredited status. 

    Julia:
    Useful advice Bob; and that’s just talking about the “positives” that may turn out not to be positive.  Next time, let’s get into when “negatives” are not negative.

    Listeners, thanks for joining us. We invite you to post your comments or feedback on this or other Ask Dr. Bob posts on the blog. Remember you can email Bob at AskDrBob@pma.com. In addition to listening to these and other Ask Dr. Bob blog posts, we invite PMA members to visit our new online Food Safety Resource Center on PMA.com and check out the lab testing white paper in the Education section. We are regularly posting new food safety content there to help you meet your company’s food safety needs.

    Until next time!

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