Next-Generation DNA Testing for Botanicals

Photo © Shutterstock.com/Sergey Nivens

Correction: This article was edited substantially on 10/4/16, 4 pm PST, to clarify that DNA testing is a qualitative test that can only identify the presence of a plant species' DNA. DNA testing does not quantify the amount of a plant species present and should be used in conjunction with other testing methodologies when testing for adulterants.

By Danica Harbaugh Reynaud, NSF AuthenTechnologies

DNA analysis is one way of testing botanical materials to determine whether other plant species are present in the sample. For instance, many don’t know that grasses such as oat grass and barley grass, as well as alfalfa, can sometimes contain other plants’ DNA. In fact, in a recent analysis, one of the top-selling herbs that can be found in kitchens around the world-oregano-was found to often contain DNA from other plants.

According to an NSF AuthenTechnologies annual review of over 1200 samples from nearly 250 different plant species, over 82% of the oregano samples we tested using DNA analysis were found to contain another plant species. (This review was conducted at our next-generation DNA testing lab in California.) Our results indicated that half of the oregano samples tested contained bindweed (Convolvulus arvensis), a potentially toxic common weed found on roadsides around the world. And, in fact, more than 5% of all botanical samples that we tested over the past year contained bindweed DNA.

In our annual review, NSF AuthenTechnologies found that more than 50% of the plant samples we tested contained DNA from other botanical species. In addition to bindweed, our annual botanical-testing review detected nearly 30 other potentially toxic plant species in nearly 150 plant samples, including several herbs used as substitutes for recreational drugs. One of these herbs, kratom (Mitragyna speciosa), was detected in an everyday cinnamon sample. In September, the U.S. Drug Enforcement Agency asked FDA to add kratom to the agency’s list of Schedule I drugs. On FDA’s import-alert website, FDA says, “there does not appear to be a history of use or other evidence of safety establishing that kratom will reasonably be expected to be safe as a dietary ingredient. In fact, the scientific literature disclosed serious concerns regarding the toxicity of kratom in multiple organ systems. Consumption of kratom can lead to a number of health impacts, including respiratory depression, nervousness, agitation, aggression, sleeplessness, hallucinations, delusions, tremors, loss of libido, constipation, skin hyperpigmentation, nausea, vomiting, and severe withdrawal signs and symptoms.”

DNA testing is a good first step toward identifying the presence of other botanical species. With DNA testing, we can identify which species are in the sample. However, chemical testing is needed to quantify the DNA in order to indicate how much of another botanical species is in the sample. Additional testing such as microscopy or organoleptic testing may also be warranted. If, after these tests, a problem is identified, it may alert a manufacturer or supplier of a potential issue with its supply chain.

The Value of Targeted Next-Generation DNA Sequencing

It is crucial to identify good tools to authenticate species in a sample. DNA testing is a method with a growing profile in recent years. When used in combination with other valid test methods, DNA testing can be a valuable component in the testing toolbox.

There is no denying that DNA testing has changed the landscape of numerous fields, such as forensics and medical diagnostics. Like computer microchips that get smaller, faster, and more powerful every year, DNA testing methodologies are becoming cheaper, faster, and more accurate.

Traditional DNA testing techniques have their limitations, however. DNA barcoding, for instance, which uses Sanger Sequencing and universal primers, falls short in authenticating samples that are processed, including impure samples of commercial products. Luckily, there’s a next generation of DNA sequencing that can help identify numerous species in a wide range of processed materials, and in only a few hours and for less, compared to many other traditional methods.

We call this technology Target-Specific DNA Sequencing (TSDS). TSDS detects and identifies the target species and potential other species including contaminants such as allergens. Because the test methods are unbiased, TSDS is also a great method for assessing current trends in impurities throughout the natural product supply chain, from seed to shelf.

Using the power of Next-Generation Sequencing (NGS) technology, TSDS applies specific tests that target unique regions of the genome and that can both authenticate the plant species and help to identify other species. Each specific test relies on a unique combination of polymerase chain reaction (PCR) primers, or probes, which produce millions of copies of each species in the material. Unlike traditional barcoding, which uses only long, universally available PCR primers for all species, the TSDS process also uses unique, short, genus-specific primers. These short primers allow the technology to test a much wider range of products than traditional DNA barcoding can handle, including products that are heated, processed, and extracted.

Once the DNA copies are produced, an NGS machine will elucidate the unique arrangement of bases (A, C, G, T) in the sequence. Each sequence is then compared to a validated reference DNA database for identification.

What Can TSDS Do?

In addition to identifying the target and other species and contaminants in a sample, TSDS can indicate the potential presence of an allergen or other potentially harmful species. In a recent study we conducted of ground black and white pepper samples purchased from multiple retailers around the world, we found that the white pepper samples frequently contained DNA of wheat or rice flour, posing a potentially serious health risk to those with related allergies. Even products labeled as “non-GMO” and “organic” were not immune to impurities by these fillers and allergens.

Novel methods such as TSDS are uncovering important trends in botanical impurities. This information can be useful to suppliers, manufacturers, and retailers as an early warning system for possible issues that could be detrimental to their businesses and may require further action in areas such as cleaning procedures, cross-contamination in the field or facility, or issues even further down the supply chain.

At SupplySide West on Friday, October 7^th, we will share more details about the results of our annual DNA-testing review. Don’t miss this presentation, entitled “DNA Reveals Unexpected Species in the Botanical Supply Chain.”

Danica Harbaugh Reynaud, PhD, is Global Director of Scientific Innovation for NSF International (Ann Arbor, MI) and co-founder of NSF AuthenTechnologies. NSF AuthenTechnologies is the first contract laboratory in the country to specialize in DNA authentication testing of natural products and their ingredients, offering ISO/IEC accredited next-generation DNA authentication testing services for natural products and their ingredients, including plants, animals, fungi, and probiotics. Danica is a botanical taxonomist and geneticist with over 15 years of research and testing experience. She completed postdoctoral work at the Smithsonian Institution and served as Director of QC Botany at Bionovo Inc. NSF AuthenTechnologies’ parent company, NSF International, offers a wide range of complementary chemical testing, including high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) for quality control of natural products. For more information, visit www.authentechnologies.com, write to info@authentechnologies.com, or call 510/914-3290.

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