DNA Barcoding Meets Nanotechnology - Wiley Online Library

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International Edition: DOI: 10.1002/anie.201702120 German Edition: DOI: 10.1002/ange.201702120

Food Authentication

DNA Barcoding Meets Nanotechnology: Development of a Universal Colorimetric Test for Food Authentication Paola Valentini+, Andrea Galimberti+, Valerio Mezzasalma, Fabrizio De Mattia, Maurizio Casiraghi, Massimo Labra,* and Pier Paolo Pompa* Abstract: Food trade globalization and the growing demand for selected food varieties have led to the intensification of adulteration cases, especially in the form of species substitution and mixing with cheaper taxa. This phenomenon has huge economic impact and sometimes even public health implications. DNA barcoding represents a well-proven molecular approach to assess the authenticity of food items, although its use is hampered by analytical constraints and timeframes that are often prohibitive for the food market. To address such issues, we have introduced a new technology, named NanoTracer, that allows for rapid and naked-eye molecular traceability of any food and requires limited instrumentation and cost-effective reagents. Moreover, unlike sequencing, this method can be used to identify not only the substitution of a fine ingredient, but also its dilution with cheaper ones.

The exponential increase and diversification of the food

market are mirrored by a raise in authenticity/safety issues[1] associated with traded raw material and processed food. Consumers are susceptible to any form of food alteration (e.g., species substitution or mixing with other species) despite their growing attention to food ingredients in terms of nutritional and health effects.[2] Alteration is particularly relevant for certain market segments, such as seafood, spices, and herbal products, where the final foodstuffs are often processed in the form of slices or powder before sale, thus becoming morphologically unidentifiable.[3] For instance, up to 50 % of the commercialized fish products are mislabeled in the European market.[4] Moreover, it has recently been estimated that such frauds impair the global food market with losses of more than $10 billion per year.[5] Several studies and technical reports suggest the use of DNA barcoding[6] as [*] Dr. P. Valentini,[+] Dr. P. P. Pompa Nanobiointeractions & Nanodiagnostics Istituto Italiano di Tecnologia (IIT) Via Morego 30, 16163 Genoa (Italy) E-mail: [email protected] Dr. A. Galimberti,[+] V. Mezzasalma, Prof. M. Casiraghi, Prof. M. Labra Department of Biotechnology and Biosciences University of Milano-Bicocca P.za Della Scienza 2, 20126 Milan (Italy) E-mail: [email protected] Dr. F. De Mattia FEM2 Ambiente s.r.l. P.za della Scienza 2, 20126 Milan (Italy) [+] These authors contributed equally to this work. Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.org/10.1002/anie.201702120.

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a suitable approach to check for food identity and detect fraud.[3c, 7] This technique, which is based on sequencing, presents several important advantages, including the acquisition of molecular data with relatively low analysis costs and the availability of comprehensive reference sequence libraries, such as the barcode of life database BOLD (http://www. barcodeoflife.org). The success of DNA barcoding has been progressively recognized by government authorities, which have proposed its official adoption for the authentication of some food categories, as in the case of fish-based products by the US FDA.[8] Moreover, new regulatory directives concerning food labeling, such as the European Regulation No. 1169/ 2011[9] on the provision of food information to consumers, will inevitably drive national institutions towards the use of molecular DNA-based tools to address the issues of food authenticity and safety.[10] Nevertheless, the routine use of DNA barcoding in food authentication has technical drawbacks, including the need for high-quality DNA extracts from food matrices to allow for the amplification and sequencing of genomic fragments of at least 500 base pairs (bps), the long analytical timeframes required from DNA extraction to the final bioinformatics analysis, and the need for specialized laboratories and skilled personnel. These limitations make the method not suitable for the food market, particularly in the case of perishable items. In an attempt to overcome these drawbacks, we have developed NanoTracer, an innovative method for the rapid molecular authentication of any food by the naked eye, with simple and low-cost processing and limited instrumentation. The principle of NanoTracer is to simplify all of the analytical steps associated with standard DNA barcoding analysis (i.e., DNA extraction, barcode amplification, species identification), to make it sequencingfree and portable outside specialized laboratories. The simple workflow of NanoTracer is illustrated in Figure 1. In particular, we identified short subregions (about 40 bps long) of the barcode loci that display interspecies genetic divergence, owing to the highest frequency of variable nucleotide positions. The acceptance criterion was that such subregions displayed a minimum of four nucleotide differences among all of the species included in the dataset. Sequences that met this requisite were used as simplified barcodes (Figure 1 A), and were the target of asymmetric PCR-based amplification (Figure 1 C). In this reaction, the two primers are present in uneven concentrations, so that one of them is depleted during the amplification, yielding a singlestrand amplicon that is readily hybridizable without further processing. Moreover, in our assay, it includes a universal sequence that is attached during the amplification, which serves to prime the aggregation of (universal) DNA-function-

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Figure 1. NanoTracer strategy. A) Analysis of the barcode alignment to identify short polymorphic regions, B) rapid DNA extraction from raw food matrices, C) asymmetric PCR, D) colorimetric detection and sequencing-free species assignment.

alized gold nanoparticles (AuNPs), with a consequent red-toviolet color change, which is due to interparticle plasmon coupling[11] and detectable within few minutes by the naked eye (Figure 1 D).[12a] An element of major strength of the proposed method is that owing to their short lengths, these regions can be successfully amplified also starting from strongly processed food items where DNA is expected to be low in concentration and highly fragmented. Furthermore, it is possible to amplify such small targets through standard PCR even from raw DNA extracts without any purification steps. This is essential to reduce the analysis times and minimize the technical skills required for DNA manipulation. As a proof of concept, we applied NanoTracer on two model case studies of frequently reported food adulterations from the animal (seafood) and plant (spices) compartments. The selected case studies dealt with the authentication of the European perch, Perca fluviatilis, and saffron, Crocus sativus, as high-value food items to be checked for species substitution and mixing with cheaper food ingredients, respectively. In particular, P. fluviatilis represents one of the most frequent cases of mislabeling and species substitution in the Italian fish market, with almost 100 % of P. fluviatilis substituted with the less valuable Lates niloticus and Pangasius hypophthalmus.[13] Similarly, the expensive and highly requested spice saffron, C. sativus, is one of the most counterfeited food commodities in the world,[2c, 14] with more than ten plant species bearing portions with similar coloration being used to substitute or dilute real saffron. In detail, we assembled reference datasets of standard DNA barcoding loci, including the target of interest, the most frequently reported substituted species, and several related species. Nucleotide sequences were aligned using MUSCLE online[15] with default options. In the case of seafood, the short barcode polymorphic sequence was chosen from the Folmer region of the mitochondrial cytochrome c oxidase I gene (COI).[6a] P. fluviatilis-specific primers targeting the COI region were used to detect the authentic fish species, and primers specific for a short (35 bp) conserved region of the 16S ribosomal RNA gene were used for a positive control of the DNA extraction and amplification (datasets and primers are reported in the Supporting Information, Tables S1, S2, Angew. Chem. Int. Ed. 2017, 56, 8094 –8098

and S4). The target and control amplifications were performed under the same conditions, and they were run simultaneously in the same PCR run. The single-strand amplicon did not fold into any interfering secondary structure that could hamper the subsequent hybridization-based colorimetric detection, as predicted by the UNAfold DNAfolding algorithm. All of the reactions yielded a clear double strand and a single-strand amplicon of the expected size, as confirmed by control native polyacrylamide gel electrophoresis (Figure S1). We then directly analyzed these short polymorphic regions through the AuNP-based detection system to unequivocally assign species, with no need to perform sequencing or electrophoresis. After amplification, a small aliquot of the raw PCR products (2 mL) was directly added to the AuNP universal probe mix for colorimetric detection. As shown in the representative image in Figure 2, P. fluviatilis-containing samples, as well as the positive controls, clearly turned violet after 5 min of incubation at room temperature. All of the other samples, containing other fish

Figure 2. Application of NanoTracer to the authentication of P. fluviatilis samples. Top: Samples of four different species (PF = P. fluviatilis, SL = S. lucioperca, LN = L. niloticus, PH = P. hypophtalmus), amplified with P. fluviatilis-specific primers. Bottom: The same samples, amplified with control primers, targeting a conserved sequence in Osteichthyes. In both case studies, 2 mL of each PCR sample were added to the universal colorimetric detector. Photographs were taken after incubation for 5 min at room temperature.


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Communications species or the negative control, remained red; thus the assay returned neither false positive nor false negative results (Figure 2). The second case study (saffron) represents an example of product adulteration rather than total ingredient substitution. Saffron is frequently mixed with other vegetable materials for economic gain (e.g., Curcuma longa, Carthamus tinctorius, and Calendula officinalis), while remaining morphologically undistinguishable from pure saffron. We chose the nuclear ITS2 genetic locus as a barcode for the application of NanoTracer, whilst the same universal AuNPs were used for the colorimetric detection. Primers specific for C. sativus and two possible diluting/substituting species, namely C. longa and C. officinalis, were designed and used for the asymmetric PCR amplification. An extensive in silico analysis demonstrated the specificity of the simplified barcode selected for C. sativus versus these two species, as well as versus an additional eight contaminant plants that are most commonly found in commercial saffron (Tables S3 and S4). As shown in Figure 3, the sample extracted from pure saffron pistils turned violet only when amplified with C. sativus-specific primers. The mixtures of powdered spices that were not authentic or pure saffron instead turned violet when amplified with the primers specific for the plants contained. Remarkably, in all cases, sequencing-free species assignment was therefore possible after a simple one-step, one-tube reaction, with readout by the naked eye. The applicability of NanoTracer in the food sector largely depends on the analytical simplification with respect to other

Figure 3. Application of NanoTracer to the authentication of saffron samples. Samples of pure or counterfeited saffron were amplified with primers specific for C. sativus (left), C. longa (middle), or C. officinalis (right). Top: Authentic C. sativus sample. The positive violet result is observed only when the sample is amplified with C. sativus-specific primers. Middle: Fraud by substitution of C. sativus with C. longa. Bottom: Fraud by dilution of C. sativus with C. longa and C. officinalis. The positive results with all three species-specific primers clearly indicate the presence of three different species in the spice mixture.

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DNA-based authentication methods. However, the replacement of DNA sequencing by a direct ON/OFF colorimetric response is not enough by itself to allow for rapid diagnostics as it is also necessary to simplify the other preparative steps, including DNA extraction. The reduction in size of the DNA markers may enable obtaining the amplified products also from fragmented DNA and raw extracts. In this context, we tested rapid DNA extraction procedures, based on a simple lysis step, that do not require any purification and/or instrumentation. Specifically, we evaluated the efficacy of the lysis buffer from the Phire Animal Tissue Direct PCR Kit (ThermoFisher Scientific, Bremen, Germany), which requires incubation at 96 8C for 2 min, and the buffer from the HelixAmpS Direct PCR [3G] Kit (Nano Helix, South Korea), which works at room temperature. The quality of DNA obtained with these two kits was very low, as demonstrated by spectrophotometric analysis showing several peaks from impurities, partially or totally overlapping with the peak at 260 nm (Figure S2 b). Therefore, these raw DNA extracts are not suitable as templates for conventional DNA barcoding analysis or real-time PCR amplification owing to the presence of fragmented DNA and interfering contaminants. Conversely, for NanoTracer, all DNA samples extracted with the different procedures can be used as templates, regardless of their purity. As a reference, the standard kit Qiagen, which employs column-based extraction and yields high-purity DNA, was also used (Figure S2 a). For comparison and validation, standard sequencingbased DNA barcoding analysis was performed on DNA extracted from fish and plant samples with the DNeasy Blood & Tissue Kit (Qiagen) and the DNeasy Plant Mini Kit (Qiagen), respectively. Notably, the concentration of purified genomic DNA was always > 5 ng mL@1 (Table S5). In fish COI barcode sequences, no insertions/deletions (indels) were detected, whereas 56 indels occurred in the plant ITS2 alignment (Table S6 and Alignment Files S7 and S8). In the fish case, the COI data, converted into a K2P distance matrix, gave the following results: mean nucleotide intraspecific distance: 0.1 % (standard error: 0.1 %; range: 0–0.2 %); mean nucleotide interspecific distance: 25.0 % (standard error: 3.0 %; range: 21.9–27.9 %). For the three tested fish species, the barcoding gap was large, with the target P. fluviatilis well differentiated from the two commonly mislabeled species L. niloticus and P. hypophthalmus. For the plant species, the ITS2 alignment (411 bps) showed high K2P distance (> 40 %) values separating C. sativus from the two adulterant species (C. longa and C. officinalis; see File S8). For both analyzed food items, the same pattern of K2P genetic divergence between the target species and their possible adulterants/ substituents was also confirmed when considering barcode sequences retrieved from international publicly available databases (see Tables S1 and S3). Notably, in contrast to NanoTracer, in the case of saffron, the identification of mixed samples containing diluting species was particularly challenging by traditional DNA barcode sequencing. Indeed, the latter technique could only identify the species present in larger amounts, failing in identifying minority species owing to the presence of overlapping peaks in the barcode electropherogram, low Phred


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Communications quality scores (< 40), or sequencing failure. This is clearly indicative of another remarkable advantage of NanoTracer versus standard sequencing-based methods, in that the first one is the only test suitable for the detection of food frauds by partial substitution of the authentic ingredient with cheaper diluents. Indeed, mixed samples of spices identified by NanoTracer included mixtures in which the percentage of the adulterant amounted to 10 % of the sample (Figure 3), and the system correctly detected the occurrence of adulteration. We also confirmed that contaminants present in as low as 1 % can be clearly detected (Figure S3). Our results confirm the high versatility of NanoTracer, which could be adopted under different circumstances to assess food frauds. As shown in the previous examples, it can be used to validate the identity of frequently substituted species.[16] Another field of application could be that of detecting new unregulated species, which, owing to a lack of toxicity assessments, may also raise public health concerns, as, for example, for the substitution of squid with Lagocephalus spp.,[4] a genus banned from the EU market owing to the high tetradotoxin content.[17] Finally, NanoTracer can easily cope with the increasing preference of customers for processed food, where the loss of morphological traits (e.g., filleted fish, powdered spices) favors deliberate or unintentional mislabeling.[16, 18] The EU has adopted specific regulations to make the identification and correct labeling of seafood mandatory to safeguard public health.[19] However, no guidelines with regard to the analytical techniques approved to confirm the correct labeling have ever been reported. Concerning herbal products and spices, the situation is even more complex. Both spices and herbal dietary supplements are affected by a very high level of counterfeiting, favored by the rapid increase in the number of products on the market with some regulatory gaps.[20] In this case, the standard DNA barcoding analysis is complicated by the fact that most frauds deal with dilutions with non-target plant species rather than with their total substitution, and by the vast diversity of commercialized items (e.g., extracts, powders, and shredded plant portions). Similarly to the case of seafood, government authorities, such as the US FDA, could benefit from the adoption of NanoTracer for large-scale screening. Future applications of this rapid test could also be developed towards the detection of trace contaminants, such as meat traces in vegan food, food allergens, or food-borne bacteria. Notably, unlike its high-tech counterparts, NanoTracer exploits robust reagents, a crucial aspect for working with complex raw matrices such as food items, which contain many interfering and inhibitor substances. We believe that the technical simplifications introduced by NanoTracer could provide a valid alternative for the quality assessment of food products. The test can be performed in less than 3 h, starting from the raw food matrix to the readout by the naked eye, it only requires a standard thermal cycler and inexpensive reagents (the total cost per assay is ! E 1), and it does not require particular technical skills. Importantly, the assay is highly parallelizable owing to the use of universal, targetindependent nanoparticle probes. In conclusion, we have developed a universal and inexpensive test for the genetic authentication of any food item Angew. Chem. Int. Ed. 2017, 56, 8094 –8098

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that can be used also in decentralized simple laboratories, both along the food supply chain and at the final points of retail, enabling large-scale implementation.

Acknowledgements This work was partially supported by the Italian Flagship Project NanoMax and by the Italian Ager 12 Project “Fine Feed For Fish—4F” (Grant 2016-0101). We thank F. Mussino for help during experiments.

Conflict of interest The authors declare no conflict of interest. Keywords: biosensors · colorimetric methods · DNA barcoding · food authentication · nanotechnology How to cite: Angew. Chem. Int. Ed. 2017, 56, 8094 – 8098 Angew. Chem. 2017, 129, 8206 – 8210

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[20] R. R. Starr, Am. J. Public Health 2015, 105, 478 – 485. Manuscript received: February 27, 2017 Revised manuscript received: April 5, 2017 Accepted manuscript online: May 23, 2017 Version of record online: June 12, 2017


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