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. We’re in the middle of a continuing series on the challenges of pathogen testing in produce. In the last post we talked about how a positive is not always a positive and the importance of looking at the selectivity and sensitivity of tests.

Bob, in the last post you also said that a negative test result might not actually be a negative. That seems even more worrisome to me. I mean if you test a product and the result is negative and you ship it out only to find later that it was positive, that can be a big problem.  Can you elaborate on that?

Bob:
Sure, Julia.  There are basically three issues to look at here: enrichment, interference and sampling issues.  Let’s take enrichment first.  Enrichment is simply the method used to permit any bacteria that might be in a sample to grow and multiply so you can get enough DNA to be able to isolate it and start the process of determining if any of it comes from a pathogen.

Since pathogens are typically present only in very low levels, when present at all, this process of enrichment can be critical.  You also have to appreciate that these pathogens thrive in warm, humid, high nutrient environments — in other words our intestinal tracts and the surface of a fruit or vegetable is not necessarily the best environment for them.  This enrichment method permits these bacteria to recover, if you will, and begin growing again.  So, it’s important that conditions and times are maximized to permit pathogen growth.  This typically takes 12 to 18 hours.  Failure to use proper enrichment methods can result in pathogens going undetected even though they may have been present in the original sample.

If that wasn’t enough of a challenge, we’ve got the issue of interference. Fruits and vegetables are extremely complex in terms of their chemistry. They’re made up of thousands of chemicals, and that composition can sometimes change dramatically based on variety or growing conditions.  Some of these chemical constituents can actually interfere with pathogen detection by interfering with DNA isolation, purification or detection.  It seems like it is almost necessary to develop specific isolation protocols for every commodity to be sure the tests are selective and sensitive enough to find pathogens if they are present.

Lastly, we have the sampling issues. If your sampling program is not sufficient, it is quite possible that if there is a low level or non-uniform contamination event, like we typically see in produce, that you could simply miss including contaminated fruits or leaves in your sample.  We’ll talk about this more in a future post.  In fact, I think this is actually a more important consideration in product testing than even the tests themselves so it warrants a more extensive discussion.   But in the end, if you don’t have a dynamic sampling program, you can easily miss contamination and get a “negative” result for a field or batch of finished product that might indeed be contaminated.

Julia:
Thanks Bob.  You’ve raised some good points here.  After hearing this, I can see why you always say that food safety programs should first focus on preventing contamination from ever happening.  Detecting it after the fact is not a simple issue.  I can also see why you repeatedly emphasize how important it is for those doing testing to ask questions and fully understand what their labs are doing, and what the data may or may not mean. 

Next time, let’s dive into this issue of confirmation testing and how that can be used to verify these initial or PCR-based screening test results.  Listeners, thanks for tuning us in today and we look forward to speaking with you further on this issue of product testing.

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!

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!