How PCR amplification forms the foundation of DNA-based pathogen testing

The significant importance of the detection of pathogens, I will assume, is perfectly clear to anyone reading this blog. However, I believe it may make sense to discuss some of the general science that permits the declaration of foods as either safe or contaminated.   

Throughout my long, long career in bioanalytical science, I always felt like a detective trying to solve a case. Indeed, we have a lot in common with forensic scientists. We both search for “suspects”; we both need to identify those suspects with 100% confidence; and the price of being wrong could be pretty high in both cases as well. 

However, the major difference is, forensics uses a known crime scene to then hunt for suspects, while we hunt for suspects without knowing if a “crime” has even been committed. Methods used to identify suspects are another area in which we must use slightly different techniques, based on cost, time and confidence level. 

For example, a photo that implicates a suspect seems to offer a high level of confidence of proper identification. Yet, the photo needs to have been taken at a good resolution, the correct angle, etc. Otherwise, proper identification of the suspect cannot happen with 100 percent confidence. Forensics might increase the confidence level of suspect ID by matching fingerprints or DNA at the crime scene. In some ways, we do the same at our “crime scenes”! Yes, microbes don’t leave fingerprints (due to a lack of fingers!), however, microbes do have DNA. So, just like our forensics brethren, we can use a most reliable way to find and identify our suspect.

What is DNA?

Again, most anyone reading this blog post know the basics on what DNA is, but fewer people know the basics on its structure. 

DNA molecules are built of just four nucleobases: Adenine (A), Cytosine (C), Guanine (G), and Thymine (T). These four nucleobases code all genetic information through their sequence in a chain, in similar fashion to the way that computers code all information via sequences of “on” and “off” switches — at a very basic level. But it is made more complicated by the fact that DNA forms not just one but two chains that are connected two each other, forming a double helix.

The double helix is not just two random chains, however, because the chains can bind to one another only where A binds to T and C binds to G. 

DNA as an identification tool

DNA is a convenient object for pathogen detection based on some important properties of this substance. First, with some manipulations, DNA can form millions of copies out of one molecule. Moreover, one does not need copies of the whole molecules. One needs only copies of a part of DNA that codes a unique feature of an organism. For example, to detect Shiga toxin-producing E. coli (STEC), one needs to identify only small parts of DNA unique for coding E. coli itself and for coding a gene responsible for Shiga toxin. The rest of DNA molecule is not really needed.

The ability to create numerous copies of molecule fragments out of one molecule (known as “amplification”) is an extremely powerful tool as well, in that it solves the issue of sensitivity in a great way. Most DNA-based detection methods start with amplification through a method called Polymerase Chain Reaction (PCR).

PCR: how it works

PCR-based amplification employs a basic unit called a thermocycler. The machine can very quickly alternate the temperature of a low-volume reaction medium. A cycle starts by increasing the temperature to 95-98 C. Under these conditions, the two strands of the suspect double helix break apart. The temperature then is quickly lowered, allowing the DNA strands to rebind.

However, primers — short, chemically synthesized portions of DNA molecule that perfectly match the end of the “suspect” DNA fragment of interest — have been added to the solution in great numbers (think, billions of primers), so the chains typically rebind with two primers. Because of the extreme population of primers in the solution, there is a much higher chance the DNA strands will bind to primers rather than its original partner.

Along with primers, the reaction medium contains “building material” to complete the new double helixes: A, C, G, T, and polymerase enzyme. Primers binding to the chains trigger a polymerase chain reaction.

This reaction occurs only in the presence of a primer specifically attached to a chain. So, the second chain is quickly built over each of thermally separated chains. This forms two complete copies of the original double helix, doubling DNA material and creating two molecules out of one. The cycle restarts with the heating of the solution, and four molecular copies are made. Each subsequent cycle doubles number of copies.

We now have many millions of copies of DNA; not just any copies, but importantly, copies of the small DNA fragment that we are interested in. Proper selection of the primer pairs allows us to pick up only the part of DNA of interest, and amplification occurs only for that part of the molecule between forward and reversed primers. Thus, we narrowed down our area of interest and created a huge number of identical DNA pieces for further investigation.

How difficult is to run PCR?

Running PCR is an easy task nowadays, though it was a challenge to develop. A user receives a kit with everything that is needed in a tube. Users simply add a DNA sample into the tube, close the tube, set it into a PCR thermocycler, and press a start button. The process is usually completed in about one hour.

Does PCR always work properly?

Just like any process, PCR is not perfect: sometimes it fails. There are a variety of reasons why amplification might not occur or the wrong molecules were amplified, including simple operator error. However, PCR generally is a very robust, reliable technique with very low rate of failure.

What happens when PCR is done?

The final PCR product (called the amplicon) provides a fair amount of molecules for subsequent pathogen-detection steps, since PCR alone does not yet answer a question on presence/absence of a pathogen DNA in the sample. PCR simply provides the amplicon to more accurately determine a pathogen’s presence.

There are multiple methods — some more complicated, others less so — to answer this main question. Some only identify the presence/absence of any amplicon in PCR product. Certainly, if there is no amplicon, there is no pathogen in the sample. But these techniques may provide false positive data, particularly if the PCR process amplified the wrong molecules. Other techniques provide data on a structure of amplified molecule, and thus are pretty robust in terms of a rate false positives.

Nevertheless, when you’re looking for pathogen “suspects” via DNA-based methods, PCR amplification will be at the core of the process you’re going to use, and after reading this post, you will hopefully have a better grasp on how PCR works.

— Andrei Gindilis, senior scientist, andrei@werfoodsafety.com

Scientists toilet-training cows: crazy or smart?

I recently read an article published by Bloomberg that caught my eye: “Scientists Are Toilet-Training Baby Cows to Cut Emissions”. I’m including the link in case you haven’t seen it.

A couple of my colleagues here at We R Food Safety! asked me, the official, professional scientist on staff, if I had any comments on the research, and if I’d write a blog about the craziness of the whole thing. Regarding the research, what more can anyone say? The title speaks for itself. My reaction to the studies, however, requires more effort.

In short, to me the toilet-training experiment does not make any sense — not scientific, economic, environmental or any other. There is a Russian witticism: “If you want your cow to eat less and give more milk, just feed it less and milk it more.” Sounds logical, no? So, basically, these scientists are at approximately the same level of pragmatism as that Russian witticism. 

That portion answered, are they crazy? The answer — maybe surprising to you — is: Not at all. They are perfectly sane, and simply taking an advantage of today’s situation, which is: Any studies done under an umbrella of saving planet Earth get funded and get public attention. Public attention is then converted to funds again. And the show goes on. 

With this in mind, I would suggest (free of charge!) a few similar scientific topics that these researchers might use as a follow-up to their toilet-training study: 

1. Scientists Train Baby Cows to Lift Weights to Improve Digestion and to Cut Emissions. (for bonus fun, replace weightlifting with dancing, singing, hopping, etc.) 
2. Scientists Are Sauna-Training Baby Cows to Sweat More and Urinate Less to Cut Emissions.
3. Scientists Teach Yoga to Baby Cows So They Breathe Less to Cut Emissions.

If you haven’t figured out by now, the algorithm is clear. The key for researchers is to Cut Emissions from cows. I patiently await the mainstream media articles covering these crucial, potential, Earth-saving studies.

To return to the matter at hand, however: The idea to help the environment and save the planet remains important, for certain. However, I wonder how much funding that could be used for real research is wasted on studies like these, which appear crazy or ridiculous, but are a product of the publicity and funding mechanisms in place?

— Andrei Gindilis, senior scientist, andrei@werfoodsafety.com

Salmonellosis outbreak from ’95 does NOT support humidity as a critical parameter in jerky processing

I just had a rather enlightening conversation with a senior USDA-FSIS official about the need for humidity during jerky manufacturing.

I asked, “How does humidity destroy pathogens?”

The official really didn’t know the answer, but he did state that, without humidity, pathogens can become heat-resistant, and Salmonella can grow and survive normal lethality.

When I asked for the studies that backed up these statements, the official pointed to the same studies they always do. I then explained how those studies didn’t actually take product to lethality (i.e., what was surviving was only surviving longer than the non-treated Salmonella). The official countered by citing the salmonellosis outbreak from 1995 in New Mexico — as is typical of this conversation.

Here, too, the problem with citing that case is lethality, as the product implicated in that outbreak should be considered a raw beef jerky, not fully cooked.

Per the published outbreak report by the CDC (https://www.cdc.gov/mmwr/preview/mmwrhtml/00039423.htm), partially frozen product was placed on racks and was pushed into a drying room. The chamber temperature (not the product’s internal temperature) was set at 140 F for three hours, then reduced to 115 F for approximately 19 hours. Therefore, the product was never exposed to a lethality treatment, and hence, by definition, it was a raw product. The report also states that no product temperatures were ever recorded.  

I promised to send the official the CDC report to remind them (or make them aware) that the production technique that was used resulted in a raw product that clearly wasn’t ready to eat (and that doesn’t even factor in other inputs or lack of them, such as sanitation, equipment, plant elevation, etc.).

It’s a shame this conversation even needed to happen, and that we have to point this out about a case more than 25 years in the past. However, it’s not the only time we’ve had these conversations lately — we have been seeing more and more actions by USDA-FSIS around humidity requirements based on these incorrect assumptions and faulty arguments. When we bring up actual science around heat resistance and pathogen growth, little discussion occurs, and FSIS inevitably falls back on the New Mexico outbreak as their “final answer” for why humidity is needed in jerky processing.

Is humidity during processing a control factor? Yes, as it assists in heat transfer. When you are measuring the internal temperature of a product, you are, in fact, measuring the transfer of energy into the product. One way to increase the rate of transfer is to increase the humidity in your cooking vessel.  At home, when you boil something, it cooks faster than it would if you cooked it in a pan, given the same energy input. But that doesn’t make humidity a critical parameter, it makes it a supporting parameter. The critical parameter is the rapid transfer of energy into the product to denature the proteins of the pathogens, rendering them non-viable (killing them). You measure this energy transfer by using internal product temperature monitoring devices.  

The entire paradigm of “chamber” temperature and “chamber” humidity now falls on a very niche part of the industry (who implement and know their process — and make some great products!). It doesn’t apply to the vast majority of the industry making jerky or other shelf-stable products.

HACCP is about analyzing for hazards, not creating a plan and then making products. Know your process and apply your controls, and you’ll be in great shape.

However, unless you want to spend a lot of time arguing with regulators, point to the sentence in the 1999 version of Appendix A, which allows you to rely on a sealed oven during cooking as the evidence of how you deal with heat transfer. I promise you that it will be a lot less frustrating!

— Andrew Lorenz, president, We R Food Safety!, andrew@werfoodsafety.com