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Analyzing DNA in Food Samples

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Abstract

Over the past decade, qPCR has become the accepted method for DNA analysis of food items for identity, safety, and labeling accuracy. Methods to isolate DNA from food can be complicated because of carryover of food components that inhibit PCR, and because food preparation processes, such as canning or sterilization, can result in DNA fragmentation.  Methods to isolate DNA from foodstuffs need to be able to overcome these limitations. This article highlights the results of studies using the Maxwell® 16 DNA Purification System to detect species present in mixed meat samples, and the Wizard® Magnetic DNA Purification System for Food to purify contaminating ruminant DNA from animal feed.

Publication Date: 9/13

Methods for Extracting and Identifying DNA from Food

The availability of fast, accurate and convenient DNA amplification and sequencing methods has made DNA analysis a viable option for many types of investigation. In the field of food testing, qPCR and advanced sequencing methods have been applied to develop sensitive methods for accurate identification of food components and precise detection of contaminants. Advances in both techniques over the last decade have made it possible to identify ingredients at a molecular level—enabling precise characterization of any DNA-based item present in foodstuffs, from the combination of meats and plants present down to the spices used for flavoring.

Over the last few years, qPCR-based DNA analysis has been applied successfully to a wide range of food-testing scenarios including GMO testing (1) (2) , species identification in mixed meat products and animal feed (3) (4) , and detection of microbial contaminants (5) (6) . In March 2013, researchers at the Johannes Gutenberg University Mainz announced a new sequencing-based food testing method –“All food Seq”—which uses a whole genome sequencing/bioinformatics approach to attempt to identify all the DNA in a food item (7) . Compared to older methods such as microscopic examination, protein analysis and microbial culture, qPCR and sequencing-based food testing methods offer advantages such as time savings, specificity, sensitivity and suitability for automation.

Whole-genome sequencing approaches are made possible by the availability of next-generation sequencing technologies and the computing power to analyze the resulting data, compare it to sequences of known species and make potential matches. Using a sequencing-based method, you don’t need to know what you are looking for in order to find it. In theory, every DNA-based ingredient in a food item can be identified, provided that it has a match in your information database.

qPCR-based approaches are used to look for specific DNA sequences—either species specific sequences or, in the case of GMO testing, specific genetic sequences—in food items. In 2012, a paper published in Food Control (4) described a real-time PCR-based method for the identification of pork, chicken, turkey, beef and lamb in mixed meat and processed food samples. These authors used the Maxwell® 16 Tissue DNA Purification kit to automate the extraction of DNA from various meat mixtures, and then used PCR primers specific for each species to identify the component meats. They were able to detect individual species down to a level of 1% in mixtures spiked with various target meats. DNA-based methods for meat identification are more effective on processed and cooked meats than protein-based methods such as immunoassays, and are also more capable of distinguishing between similar species (4) (8) .

The isolation of DNA from food using traditional protocols can be a lengthy and potentially hazardous process. The availability of faster methods that avoid exposure to hazardous solvents and that are amenable to automation has enabled more efficient and successful DNA isolation from foods and contributed to successful qPCR analysis. DNA purification methods need to be able to purify DNA away from potential PCR inhibitors present in foodstuffs, and must also be able to purify DNA that may have been fragmented during food processing treatment, such as cooking, preservation or sterilization (9) (10) . Therefore, the choice of DNA purification method, as well as the choice of PCR targets can greatly influence success in food analysis, depending on the specific foodstuffs and target DNA involved. 

Species Identification: Food Identity

The method described by Camma et al illustrates one application of qPCR to precisely identify the species present in a processed meat mixture—an application relevant to food testing and labeling accuracy. This issue was brought to attention following the horsemeat scandal investigation in Europe in 2013, which uncovered mislabeling of products sold as beef to a large number of suppliers (11) . As a result, several supermarket chains recalled packaged frozen meals after DNA tests showed them to contain varying levels of undeclared horsemeat. The incident brought calls for more effective measures to ensure food labeling accuracy and for more testing standards that can verify the species content of meat products.

Species Identification: Food Safety

Ensuring food safety is one of the most important reasons for having accurate, reliable ways of detecting the precise composition of food items. For example, DNA-based methods are used to test animal feed for compliance with European regulations to minimize risk of bovine spongiform encephaopathy (BSE) transmission.

To counteract risk of transmission of BSE through the food chain, the European Union banned the use of mammalian bone meal as a feed ingredient for farmed animals (12) , and also prohibited feeding of farmed animals with protein from the same species (13) . DNA testing using qPCR is the recommended method for testing animal feed for compliance with these regulations. The qPCR method provides huge advantages in terms of labor savings and sensitivity over the previous method—microscopic analysis of feed samples for bone fragments (14) (3) .

One of the major challenges using DNA analysis to detect bone meal in animal feed is that any DNA present in the feed is often damaged by sterilization of the feed during production (133°C for 20 minutes under steam pressure). Fumière et al (2006) described an effective qPCR method that overcame these limitations (3) . They detected ruminant DNA contamination in cattle and pig feed using a high-copy bovine mitochondrial DNA target sequence. The chosen sequences was short (68bp) and spanned more than one gene. The shorter target was more effective for detection because larger sequences are more likely to become fragmented during sterilization. Target DNA spanning two genes was used to try to decrease the likelihood of species cross reactivity. The method was able to detect bone meal-derived DNA in processed pig and cattle feed down to a limit of 0.1%.

Fumiere et al used the Wizard® Magnetic DNA Purification System for Food in a semi-automated process with a KingFisher® Magnetic Particle Processor (Labsystems) to extract DNA from 100mg of feed samples spiked with varying levels of mammalian bone meal. qPCR was performed on the isolated DNA in TaqMan® assays with specific primers for cattle or pig mtDNA targets.

A second study (14)  outlines the results of a blind, interlaboratory study to detect ruminant meat and bone meal in animal feed, also using qPCR.  In that study, each participating lab used different DNA extraction and qPCR methods, and the effectiveness of the methods was compared. All the labs were able to detect down to 0.1% of ruminant DNA in feeds mixed with varying amounts of bone meal contamination.  The Wizard® Magnetic System for Food along with the KingFisher® Magnetic Particle Processor was one of the extraction methods tested, another was the Wizard® DNA Clean up System, and a third was a manual chelex-based method. All of the DNA purification and PCR methods gave acceptable results.

In the EU, the Wizard® Magnetic DNA Purification System for Food is the recommended method for purification of DNA from animal feed for the purpose of qPCR-analysis for detection of  bone meal contamination with ruminant DNA. The extraction method was chosen based on the requirement to “ yield DNA of sufficient quality and quantity but is also to be suitable for routine use in terms of ease of operations, sample throughput and cost.” (15)  

Summary

Over the past decade, qPCR has become the accepted method for DNA analysis of food items for identity, safety, and labeling accuracy. Isolation of DNA from food can be complicated by carryover of food components that may be inhibitory to PCR, and because various treatments used in food preparation, such as canning or sterilization, can result in DNA fragmentation. Methods to isolate DNA from foodstuffs need to be able to overcome these limitations. Studies published in recent years have highlighted use of the Maxwell® 16 DNA Purification System and the Wizard® Magnetic DNA Purification System for Food in applications to identify contaminating DNA in food items.  In the EU, the Wizard® Magnetic DNA Purification System for Food is the recommended DNA extraction method for analysis of animal feed for contaminating bone meal from ruminants. In the future, the availability of whole genome sequencing methods that can be applied to food items may bring further options for the fast identification of DNA-based food ingredients. Because of challenges in the variety of sample types, levels of DNA degradation and stringency of testing requirements, isolation of DNA that is of sufficient purity and quality for the desired analysis method continues to be of critical importance to food testing applications.

References

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  3. Fumière, O. et al. (2006) Effective PCR detection of animal species in highly processed animal byproducts and compound feeds. Anal. Bioanal. Chem. 385, 1045–54.
  4. Cammà, C. et al. (2012) Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures. Food Control 23, 400-404.
  5. Malorny, B. et al. (2004) Diagnostic Real-Time PCR for Detection of Salmonella in Food. Appl. Env. Microbiol. 70, 7046–52.
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  11. Lawrence, F. (2013) Horsemeat scandal: The essential guide. The Guardian 15 February,
  12. European Union (2003) Commission regulation (EC) 1234/2003 amending Annexes I, IV and XI to EC Regulation 999/2001. Off. J. Europ. Union L173, 6-13.
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  15. (2013) EURL-AP Standard Operating Procedure: DNA extraction using the “Wizard® Magnetic DNA purification system for Food” kit.