“No amplifiable tuna DNA”—that was the result of a lab test ordered by a New York Times reporter looking into questions surrounding the content of Subway’s tuna salad, which has since made headlines globally in outlets including Fox BusinessThe Guardian, and Today. The paper’s investigation, published on June 19 in the Style section, involved collecting tuna salad samples from three Los Angeles–area Subway franchises, freezing them, and sending them on ice to an unnamed commercial food testing lab “across the country” for DNA analysis.

The piece followed a January 21 lawsuit levied against the sandwich giant alleging that the tuna salad their restaurants serve is “made from anything but tuna.” The plaintiffs, two residents of Alameda County, California, tell The Washington Post that the assertion is backed by “independent lab tests.” According to Subway, there is “simply is no truth to the allegations,” and a representative of the company tells People that the plaintiffs have walked back their claims, now alleging the meat isn’t 100 percent the sustainably sourced skipjack and yellowfin tuna Subway says it is.

On the surface, the lab test result reported by the Times might appear to support such an assertion. But scientists such as AJ Silva, a microbiologist and self-described food fraudist currently pursuing a doctorate at University of California, Riverside, say the finding could easily stem from the technical challenges involved in testing processed meats rather than from a lack of tuna in the restaurant’s sandwiches.

The Scientist spoke to Silva about the intricacies of seafood authentication and why it’s not always easy to determine what a restaurant is serving.

The Scientist: Why is it important to confirm that seafoods are what retailers say they are?

courtesy of AJ Silva

AJ Silva: Great question. So, thinking about our global seafood supply, we can see that over the last probably 20 to 30 years, we’ve seen this boom in the seafood industry, and in recognizing how complex the industry really is. When you go to the store, and you’re buying something at the store, you’re not really understanding the backstory of how that fish got to the market. . . [from] fishermen catching it out in the ocean to any of the processing steps that are involved.

It’s important, ultimately, for consumers to know what they’re buying is really what it’s labeled as, because of . . . one, health concerns—making sure that what’s being sold in the stores doesn’t have any concern about toxins. Or, maybe, kosher religious beliefs; for those individuals, that’s also a consideration. But I think the biggest concern is economic. . . . Substituting a less expensive fish for a more expensive fish can come at a serious cost in trust between the consumer and the industry.

TS: How do scientists like yourself go about authenticating what seafood is being served?

AJS: You receive, let’s say, a sample that is believed to be skipjack tuna. The main process that you go through is, first, you need to extract the DNA from that sample. There’s commercial kits to use. You’d undergo PCR to get millions of copies of DNA, and then the main method that’s used today is what’s called DNA barcoding.

You can think about [the DNA sequence of] each specific species of fish as almost like a QR code. . . . You’re ultimately able to identify, very specific to the species level, whatever type of seafood or fish that you’re looking into.

TS: What about cooked and processed seafoods, like canned tuna? Would you approach identifying species differently for them?

AJS: With any type of processing method, it has been found that DNA degradation is definitely an issue.

The full barcoding method, which is looking at the full 650-base-pair region of the cytochrome oxidase-1 gene, is the main way used. . . .  But in general, with authentication of these canned products, there’s now a new approach called mini barcoding, which is using small regions of that CO1 gene [and] allows us to ultimately be able to identify fragmented DNA pieces that have been broken down. Still, we might not get a full scope of the actual species that we’re looking at, especially if we’re looking at a mixture.

More advanced approaches, what are called high-throughput sequencing approaches, have been developed, which allow us to look into products that do maybe have multiple different tuna species or different seafood species within a single sample.

TS: How likely is it that DNA testing of tuna salad would be able to discern the species of fish involved?

AJS: If we’re looking at a traditional DNA barcoding–based approach, it would definitely be challenging, tuna salad being a mixed product that goes through various processing steps.

A negative test result here doesn’t mean that there’s no tuna in it.

The ingredients that you have in there might be affecting the ability to identify [the species], but in general, [DNA] degradation in processing is our main culprit.

I think in the broader scope of seafood identification, we’re challenged with being able to identify species after those really rigorous processing methods. There are a lot of changes that are happening chemically within that system—it’s not only physical degradation. . . . Adding a brine or oils, that’ll also affect the efficiency or yield that you can get of DNA.

The main takeaways, I would say, are that a negative test result here doesn’t mean that there’s no tuna in it, and that processing is definitely our major issue when it comes to being able to identify species.

TS: Are there any other analyses available to determine the species in heavily processed foods, or do you know of any under development?

AJS: Specifically for seafood authentication of canned or processed foods, our main method right now is definitely the mini barcoding method that has been developed and looked at through various studies. Protein analyses have been a common way in the past. But again, thinking about processing and canning, that definitely degrades protein. So that’s not an effective method. . . . [There are other] up and coming techniques that are being tested.

TS: Is it possible to determine from DNA testing where a fish was caught or whether it was caught using sustainable methods?

AJS: [Barcoding] doesn’t really give us that full picture of how is this caught, is this sustainably caught . . . unless you had knowledge of the source of where your product came from.

And that’s, again, going back to the global seafood chain, seeing the multiple steps—the tuna is being caught, it’s being sliced, it’s being distributed and different parts of that fish is going to different areas. . . . There are a lot of steps along the way that make it challenging for consumers to really know where their fish is coming from.

Editor’s note: This interview has been edited for brevity.