Tag Archives: PCR

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PCR or LAMP: Food Safety Considerations when Choosing Molecular Detection Methods

By Joy Dell’Aringa, Vikrant Dutta, Ph.D.
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Food microbiology pathogen detection technology is constantly evolving and improving for fast, efficient and accurate analysis. Thanks to the wide commercialization of easy-to-use diagnostic kits, the end-user no longer needs a deep understanding of the intricacies of diagnostic chemistries to perform the analysis. However, when navigating the selection process in search of the technology that is best fit-for-purpose, it is critical to understand the key differences in principle of detection and how they can impact both operations and risk. Here, we will explore the difference between two broad categories of molecular pathogen detection: PCR and isothermal technologies such as LAMP.

PCR & LAMP Detection Chemistries: An Overview

PCR detection chemistries have come a long way from non-specific DNA-binding dyes like SYBR Green, to highly precise sequence-specific molecular probes. The efficiency of the real-time PCR reaction today allows for the use of a variety of detection probes, the most popular being Dual-Labeled Fluorescent Probes such as FRET, TaqMan probes, and Molecular Beacon probes.1 The precision of these probes is showcased in their ability to distinguish allelic single-nucleotide polymorphisms (SNPs).2,3 The most prevalent isothermal chemistry, Loop-Mediated Isothermal Amplification (LAMP), typically does not use molecular probes due to the lack of structure and formation consistency in its amplified products. As a result, LAMP mostly relies on detection through non-specific signal generation like ATP bioluminescence or non-specific dyes. In theory, this could come from specific and non-specific amplification events. This also makes LAMP inept to detect the allelic polymorphisms, which in some cases are critical to detecting crucial variations, like between close species, and within serotypes. In the end, the detection chemistries are only as good as the amplified products.

Key Takeaways:

  • PCR technology has improved greatly in detection efficiencies via target specific probes
  • LAMP technology typically does not utilize specific molecular probes, but instead relies on indirect signal generation
  • Target specific probes ensures signal from specific amplification events only
  • Indirect signal can come from specific and non-specific amplification events, which can lead to a reduced specificity and inability to detect in certain cases

PCR & LAMP: Amplification Strategies

Food safety pathogen detection protocols aim to find the single cell of a target organism lurking in a relatively large sample. In order to achieve detection, molecular technologies utilize amplification strategies to increase the concentration of target DNA to a detectable level. Nucleic acid amplifications in both PCR and isothermal technologies start by making a variety of amplified products. These products include non-specific amplifications (NSA), and specific (target) amplifications.4,5,6,7 Ideally, the concentration of the desired target amplified product increases over time to levels above NSA where the detection chemistries are able to provide a detectable signal from the desired amplified product (target). Various reaction components such as: Target DNA concentration, polymerase, buffers and primers play a defining role in maintaining the progressive amplification dynamics, and thereby act as core contributors to the robustness of the reaction. However, none play a more crucial contribution to the success of a reaction than temperature. Herein lies a key difference between the fundamentals of PCR and Isothermal amplification technologies.

Key Takeaways:

  • PCR and LAMP both make a variety of amplification products: Non-Specific (NSA) and Specific (target)
  • Ideally, target products increase above the levels of NSA to reach a reliable detectable signal
  • A variety of factors contribute to the overall robustness of the reaction

What Is the Difference between PCR and Isothermal Detection Technologies?

A key foundational difference between the two technologies lies in the utilization of the thermal profiles. PCR utilizes thermocycling, while isothermal does not. This difference is the tether around how the different amplification chemistries work. In PCR, the cyclical denaturation of DNA during thermocycling separates all dimers (specific and non-specific). As the reaction progresses, this leads to frequent correction of the amplification dynamics away from the NSA and favors amplification of the desired target amplifications. Isothermal chemistries do not have the ability to correct the NSA through thermocycling, so it must rely on alternate mechanisms to achieve the same result. For example, LAMP utilizes “nested” primers where the primer sequences outside the target region are used to create early amplification products. These are subsequently used as a template for the desired target amplifications. The presence of these extra primers, along with the diverse amplified structures formed during the LAMP reaction, creates many more opportunities for NSA production.5,8,9 This causes a less controlled and inefficient amplification, and is perhaps why the preheating of the DNA prior to the LAMP has shown to increase the LAMP sensitivity.10, 11 To the end user, this inefficiency can manifest itself in various ways such as restricted multiplexing, lack of internal amplification control, complex assay design, tedious sample prep methods, and increased chance for inaccurate results (i.e., false positives and false negatives).12 Scientific literature does provide a fair amount of evidence that, under controlled conditions, the isothermal amplification reaction can provide equivalent results to PCR. Isothermal chemistries also usually require simplified instruments and thereby can present interesting opportunities in non-conventional test environments with simple and predictable matrices. This likely explains the early footing of isothermal technologies in the clinical test environment as a “point of care test” (POCT) alternative. However, it must also be noted that recently PCR has also been adapted and successfully commercialized for the POCT format.13,14

Key Takeaways:

  • PCR utilizes thermocycling, Isothermal does not
  • In PCR, thermocycling allows for the reaction to favor the target amplification over the NSA
  • LAMP must rely on alternate mechanisms to correct for NSA and these mechanisms lead to a less controlled and therefore inefficient amplification
  • Under controlled conditions, isothermal technology can provide equivalent results to PCR
  • Low instrumentation requirements make isothermal technologies interesting for non-conventional test environments (i.e. POCT); however, PCR has also been recently adapted as a POCT

Internal Amplification Controls in Molecular Pathogen Detection Technologies: The Value & The Challenges

The purpose of an internal amplification control (IAC) is to provide an indication of the efficacy of the test reaction chemistry. The closer the IAC is to the target DNA sequence, the better view into the inner workings of each reaction. For food microbiology testing, the role of the IAC is more important now than ever. Driven by regulations, industry self-accountability and brand protection initiatives, more food laboratories are testing diverse product types with novel and innovative formulations and ingredients. IAC capability not only helps with troubleshooting, but it also allows for a more confident adoption of the technology for new and diverse food and environmental matrices.

Over the years, PCR has progressively developed into a robust and efficient technology that can provide a dynamic IAC, giving the end user a direct look into the compatibility of the test matrix within the PCR reaction. From a single reaction, we can now make a qualitative assessment of whether the crude DNA prep from a matrix undergoing testing is working with this PCR or if it is inhibiting the reaction. With legacy technologies, including the older generation PCR’s, we were limited to an “it-did-not-work” scenario, leaving the end user blind to any insights into the reason. Since isothermal chemistries typically do not have an IAC, the end user is vulnerable to false results. Even when isothermal chemistries such as nicking enzyme amplification reaction (NEAR) can provide IAC, they typically do not mimic the target reaction and, therefore, are not a direct indicator of the reaction dynamics. This limits the end user back to the “it-did-not-work” scenario. LAMP technology attempts to mitigate the absence of IAC by performing a separate and external reaction with each test matrix. This strategy leaves the final result vulnerable to a number of factors that are otherwise non-existent for IAC: Sampling variations, reagent and machine anomalies, and user error. External control approaches also have a notable impact to the end user, as the burden to demonstrate fit-for-purpose of the method for even the smallest matrix composition change increases both validation and verification activities, which can have a notable financial impact to the laboratory.

There are a few reasons why IAC incorporation is not always plausible for isothermal technologies such as LAMP. First, inefficient, less-controlled amplification reactions leave little room for reliable and meaningful supplementary reactions, like the ones required for IAC. Second, the lack of consistent amplified products make it much more difficult to pinpoint a DNA structure that can be dependably used as an IAC. Third, lack of specific detection mechanisms makes it hard to distinguish signal from the target versus the IAC reaction.

Key Takeaways:

  • Internal amplification controls (IAC) are critical for the food industry due to complex and ever-changing matrix formulations
  • IAC is useful for troubleshooting, optimizing assay performance, and adapting test for novel matrices
  • PCR has evolved to provide dynamic IAC, leading to increased confidence in results
  • LAMP is not able to utilize IAC due to the nature of the amplification products, reaction efficiency, and lack of specific detection mechanisms

Follow the link to page 2 below.

Clear Labs Clear Safety

Will Next-Generation Sequencing Dethrone PCR?

By Maria Fontanazza
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Clear Labs Clear Safety

Today Clear Labs announced the availability of its next-generation sequencing (NGS) platform, Clear Safety, for pathogen testing. Competing head-to-head with PCR, the product intends to bring NGS into the routine production environment. Clear Labs is launching the product at the IAFP Annual Meeting this week in Salt Lake City.

“Until the launch of Clear Safety, there was the duality between PCR and whole genome sequencing (WGS) where PCR was more applicable to routine testing and faster results,” says Mahni Ghorashi, co-founder of Clear Labs. “WGS is more expensive and slower, so the food industry has been using the technology as complementary until this time. This platform out competes PCR virtually on every level.”

Clear Safety was in the pilot phase only a couple of months ago when Ghorashi sat down with Food Safety Tech to give a brief overview of the technology. Now that the platform is officially out of pilot mode, it is accessible to all of the food industry, from third-party service labs to any food company that has an in-house lab. With less human labor involved, the platform reduces the potential for errors and does not require additional expertise. The process from sample to result has been simplified, and the bacterial enrichment and sample prep stages are identical to PCR, according to Ghorashi, who says that all a lab technician has to do is load the plates on the box and press “go”. Within 18 hours, test results are ready and can be accessed through a software platform.

Clear Labs, Clear Safety, PCR
Clear Safety is touted as the first NGS platform that can either match or outperform PCR systems as it relates to accuracy, turnaround time and cost. Chart courtesy of Clear Labs.

In discussing the capabilities of Clear Safety versus PCR, Ghorashi named a few other key differentiators:

  • Molecular profiling: The ability to drill down from species-level resolution to serotype to strain-level all in a single test within 24 hours (as opposed to today’s three-to-five-day timeframe)
  • Better accuracy and more automation, reducing human error
  • Multi-target analysis: The ability to run different kinds of pathogens at the same time
  • Software: LIMS built specifically for food safety testing

Clear Safety’s first area of focus is Salmonella. Ghorashi estimates that 90% of the poultry market, 80% of the pet food market and half of all contract service labs have piloted the platform. Next year E.coli and Listeria testing capabilities will be rolled out.

Martin Easter, Hygiena
In the Food Lab

The New Normal: Pinpointing Unusual Sources of Food Contamination

By Martin Easter, Ph.D.
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Martin Easter, Hygiena

Shiga toxin-producing E. coli in dry flour, and then romaine lettuce. E. coli O104 in fenugreek sprout seeds. Recent announcements of foodborne illness outbreaks have begun involving unusual combinations of bacteria and foods. These out-of-the-ordinary outbreaks and recalls are a small but growing part of the 600 million documented food poisonings that occur worldwide every year according to the World Health Organization. Preventing outbreaks from these new combinations of pathogen and food demand a range of accurate tests that can quickly identify these bacteria. Over the past several years, outbreaks from unusual sources included:

  • E. coli O121 (STEC) in flour: Last summer, at least 29 cases of a E. coli O121 infection were announced in six Canadian provinces. The source arose from uncooked flour, a rare source of such infections because typically flour is baked into final products. Eight people were hospitalized, and public health officials have now included raw, uncooked flour as well as raw batter and dough as a source of this type of infection.
  • E. coli O104:H4 in fenugreek sprouts: One of Europe’s biggest recent outbreaks (affecting more than 4,000 people in Germany in 2011, and killing more than 50 worldwide) was originally thought to be caused by a hemorrhagic (EHEC) E. coli strain that from cucumbers, but was but was later found to be from an enteroaggregative E. coli (EAEC) strain in imported fenugreek seeds—the strain had acquired the genes to produce Shiga toxins.
  • Mycoplasma in New Zealand dairy cows: While not unusual in cattle, the incident reported in August marks the pathogen’s first appearance in cows in New Zealand, a country known for strict standards on agricultural hygiene. The microorganism is not harmful to people, but can drastically impact livestock herds.
  • Listeria monocytogenes in food sources: Listeria monocytogenes causes fewer but more serious incidence of food poisoning due to a higher death rate compared to Salmonella and Campylobacter. Whereas Listeria has been historically associated with dairy and ready to eat cooked meat products, recent outbreaks have been associated with fruit, and the FDA, CDC and USDA are conducting a joint investigation of outbreaks in frozen as well as in fresh produce.
  • Listeria in cantaloupe: In 2011, one of the worst foodborne illnesses recorded in the United States killed 20 and sickened 147, from Listeria monocytogenes that was found in contaminated cantaloupes from a farm in Colorado. The outbreak bloomed when normal background levels of the bacteria grew to deadly concentrations in multiple locations, from transport trucks to a produce washer that was instead designed for potatoes.

The outbreaks underscore the fundamental need to have a robust food safety program. Bacteria can colonize many different locations and the opportunity is created by a change in processing methods and/or consumer use or misuse of products. So robust risk assessment and preventative QA procedures need to be frequently reviewed and supported by appropriate surveillance methods.

Food safety and public health agencies like the European Food Safety Authority (EFSA) or the CDC have employed a wide range of detection and identification tests, ranging from pulse field gel electrophoresis (PFGE), traditional cell culture, enzyme immunoassay, and the polymerase chain reaction (PCR). In the case of Germany’s fenugreek-based E. coli outbreak, the CDC and EFSA used all these techniques to verify the source of the contamination.

These tests have certain advantages and disadvantages. Cell culture can be very accurate, but it depends on good technique and usually takes a long time to present results. PFGE provides an accurate DNA fingerprint of a target bacteria, but cannot identify all strains of certain microorganisms. Enzyme immunoassays are precise, but can produce false-positive results in certain circumstances and require microbiological laboratory expertise. PCR is very quick and accurate, but doesn’t preserve an isolate for physicians to test further for pathogenic properties.

Identification of the pathogens behind foodborne contamination is crucial for determining treatment of victims of the outbreak, and helps public health officials decide what tools are necessary to pinpoint the outbreak’s cause and prevent a recurrence. Rapid methods such as the polymerase chain reaction (PCR), which can quickly and accurately amplify DNA from a pathogen and make specific detection easier, are powerful tools in our efforts to maintain a safe food supply.

Recently, scientists and a third-party laboratory showed that real-time PCR assays for STEC and E. coli O157:H7 could detect E. coli O121, O26 and O157:H7 in 25-g samples of flour at levels satisfying AOAC method validation requirements. The results of the study demonstrated that real-time PCR could accurately detect stx, eae and the appropriate E. coli serotype (O121, O26 or O157:H7) with no statistical difference from the FDA’s Bacteriological Analytical Manual (BAM) cell culture method.

Agencies like the World Health Organization and CDC have repeatedly stated that historical records of food poisoning represent a very small percentage of true incidents occurring every year worldwide. Many of today’s most common food pathogens, like Listeria monocytogenes, E. coli O157:H7 or Campylobacter jejuni, were unknown 30 years ago. It’s not clear yet if unusual sources of contamination arise from increasing vigilance and food safety testing, or from an increasingly interdependent, globally complex food supply. No matter the reason, food producers, processors, manufacturers, distributors and retailers need to keep their guard up, using the optimum combination of tools to protect the public and fend off food pathogens.

Next-Generation Sequencing Targets GMOs

By Maria Fontanazza
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As the movement among consumers for more information about the products they’re purchasing and consuming continues to grow, the food industry will experience persistent pressure from both advocacy groups and the government on disclosure of product safety information and ingredients. Top of mind as of late has been the debate over GMOs. “Given all of the attention on GMOs on the legislative side, there is huge demand from consumers to have visibility and transparency into whether products have been genetically modified or not,” says Mahni Ghorashi, co-founder of Clear Labs.

Mahni Ghorashi, Clear Labs
Mahni Ghorashi, co-founder of Clear Labs

Today Clear Labs announced the availability of its comprehensive next-generation sequencing (NGS)-based GMO test. The release comes at an opportune time, as the GMO labeling bill, which was passed by the U.S. House of Representatives last week, heads to the desk of President Obama.

Clear Labs touts the technology as the first scalable, accurate and affordable GMO test. NGS enables the ability to simultaneously screen for multiple genes at one time, which could companies save time and money. “The advantage and novelty of this new test or assay is the ability to screen for all possible GMO genes in a single universal test, which is a huge change from the way GMO testing is conducted today,” says Ghorashi.

The PCR test method is currently the industry standard for GMO screening, according to the Non-GMO Project. “PCR tests narrowly target an individual gene, and they’re extremely costly—between $150–$275 per gene, per sample,” says Ghorashi. “Next-generation sequencing is leaps and bounds above PCR testing.” Although he won’t specify the cost of the Clear Labs assay (the company uses a tiered pricing structure based on sample volume), Ghorashi says it’s a fraction of the cost of traditional PCR tests.

The new assay screens for 85% of approved GMOs worldwide and targets four major genes used in manufacturing GMOs (detection based on methods of trait introduction and selection, and detection based on common plant traits), allowing companies to determine the presence and amount of GMOs within products or ingredient samples. “We see this test as a definitive scientific validation,” says Ghorashi. The company’s tests integrate software analytics to enable customers to verify GMO-free claims, screen suppliers, and rank suppliers based on risk.

Clear Labs, GMO, testing
Screenshot of the Clear Labs GMO test, which is based on next-generation sequencing technology.

Clear Labs isn’t targeting food manufacturers of a specific size or sector within the food industry but anticipates that a growing number of leading brands will be investing in GMO testing technology. “We expect to see adoption across the board in terms of company size, related more to what their stance is on food transparency and making that information readily available to their end consumers,” says Ghorashi.

PCR Test, weighing milk powder

Spoil No More: Rapid Test for Dairy Products Goes Beyond Detecting Microbes

By Maria Fontanazza
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PCR Test, weighing milk powder

Detecting yeast and mold is one of the most time consuming parts of the testing process for dairy products. With more pressure to move products that have a short shelf life out the door as quickly as possible, time really is money. Having a rapid, real-time test that enables companies to make immediate production decisions can provide a significant advantage. “[This technology] brings test time within the same timeframe as other microbiology tests, so a test for yeast and mold is no longer the outlier. That’s a huge savings right there,” says Phil Coombs, product specialist at Weber Scientific.

biotecon_diagnostics_starprep
Weber Scientific was one of three recipients of the Food Expo Innovation Award on July 17, 2016 at the IFT Annual Meeting in Chicago.

Coombs is referring to Weber Scientific’s recently released PCR Yeast and Mold Quantitative Test, which has been validated for finished dairy products. The company was asked by Germany-based Biotecon Diagnostics, the creator of the newly developed PCR method, to be its partner in introducing the test to the U.S. market. The technology reduces testing time for yeasts and molds from five days to four hours or less—from sample prep to the time-to-result, with no pre-enrichment required. “We make a big deal out of this, because sometimes [companies] with a pathogen test will say they have a four-hour test but it’s not truly, from start-to-finish, a four-hour test—you have to do some form of pre-enrichment, and so it’s a 24–48 hour test,” says Coombs. “When looking at fermented milk product like yogurt, it might have a shelf life of about 50 days. There’s much more time for the yeast and mold (because they’re typically slower growing organisms) to get busy and spoil the product. Yeast and mold can tolerate the lower pH, so that’s been the biggest sector of interest so far.”

One of the features of the technology is its ability to protect against false-negative results from non-viable DNA and false-positives from previous PCR test runs, which greatly reduces the chances of cross-contamination as well.

PCR Test for dairy products
The PCR Yeast and Mold Quantitative Test conducts analysis on milk powder. Image courtesy of Weber Scientific.

Achieving a shorter time-to-result means that if a company uncovers an issue, it can take immediate remedial action rather than waiting several days. This can have a big economic impact on production and warehousing, along with releasing product into commerce and distribution, especially when dealing with products that require refrigeration. In addition, the PCR test goes beyond detecting microbes that will spoil fermented milk products and offers advantages in the broader context of reducing food waste and spoilage. “It will be attractive to many companies that are developing a broad range of sustainability measures,” says Fred Weber, president of Weber Scientific. “And to cut down on food waste at the consumer level is a big deal.”

The company expects AOAC approval next year.

Mislabeled Salmon

Rapid Salmon ID Test the Latest in Fraud Prevention

By Food Safety Tech Staff
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Mislabeled Salmon

Two rapid test kits have been launched for the identification of salmon species: Chinook (Oncorhynchus tshawytscha) and Sockeye (Oncorhynchus nerka). The tool kits were developed in collaboration with the University of Guelph and allow distributors, food processors and government regulators to positively identify the salmon species in less than two hours. 

Recent studies have revealed that a significant amount of the salmon sold in the United States is mislabeled.

The test kits are used in conjunction with a portable, real-time PCR system that provides DNA detection. The tools are part of the Instant ID Species product line from InstantLabs, which include seafood identification tests for Atlantic (Salmo salar) and Coho Salmon (Oncorhynchus kisutch as well as Atlantic Blue Crab (Callinectes sapidus) and U.S. Catfish (Ictalurus species).

Ravi Ramadhar, Food Safety Business Director for Life Sciences Solutions, Thermo Fisher Scientific
In the Food Lab

Molecular Diagnostics – Generation 3: 2005 to Present

By Ravi Ramadhar
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Ravi Ramadhar, Food Safety Business Director for Life Sciences Solutions, Thermo Fisher Scientific

In my previous blog, I covered the first two generations of Molecular Diagnostics: Generation one, was the advent of these tests prior to 1995, while the second generation saw the evolution of molecular diagnostics with the emergence of standardized food molecular and method workflow.

The advent of automated DNA sequencing and use of multiple fluorescent dyes by companies like Applied Biosystems and Roche led to the development of multiple fluorescent dyes and real- time quantitative PCR systems (qPCR). At first these qPCR systems were only used in the research environment, but quickly found their way to the food industry.

Applications such as quantitation of GMOs and multiple pathogen targets became common. Real-time PCR systems permitted users to visualize amplification as it happened and enabled simultaneous detection of multiple targets. With the use of newer chemistries and improved enzymes, shorter amplification cycles – sometimes as low as 40 minutes – could be achieved. The real-time systems offered faster time-to-result with additional target probes and thus higher target specificity. As with most molecular methods, the workflow was sensitive to food matrix inhibition and required alternative sample preparation methods to meet the wide variety of food matrixes.

Within this generation of solutions, alternatives were introduced, that promised faster, easier or more sensitive results. These included alternative to either the detection method or enzymes utilized Iisothermal amplification, for example without need for multiplexing capability of qPCR or internal controls, as well as targeting alternative nucleic acid such as RNA were introduced to the food market. These incremental improvements did not lead to any significant new paradigms or improvements to the food testing workflow. Their emergence instead led to an explosion of additional and alternative molecular platforms for food, without any real innovation. Within this, solutions introduced to the food industry eventually brought us to where we are today.

Directly taking systems from the clinical diagnostics workflow and introducing these platforms and systems as food solutions. While these systems automate the entire workflow or automate the PCR setup it remains to be seen if with their higher complexity and high maintenance these systems can survive the food industry. The basic molecular workflow for food has remained intact since its introduction in the late 1990s with innovation more or less stagnant. What’s needed is for someone to truly develop a platform from the ground up with the food laboratory in mind.

Today’s landscape and what’s next

Today, there are some early signals of where innovations and changes for food labs will emerge. A recent poster by Nestle, for example, highlighted the uses of next-generation sequencing (NGS) and DNA sequencing to develop a DNA method to allow the identification of coffee varieties through the value chain, from the field to the finished product. The method is applied on routine basis to guarantee the purity and authenticity of raw material used by Nespresso.

Applications of NGS in outbreak response and trace back investigations are being used in parallel with existing technologies. Finally, availability of new sequencing data enables better assay design and development of adjacent technologies.

NGS was preceded by emulsion amplification and sequencing by synthesis. These developments led to the development and introduction of digital PCR. Within a digital PCR reaction, millions of simultaneous reactions from one sample occur. The advantages of dPCR include lower and absolute, not relative gene copy number. The data has high precision and has better tolerance to inhibitors. These characteristics can lead to better and more precise molecular tests in food. , Before dPCR wide spread adoption is seen, however, the limitations of high cost and limited dynamic range must be addressed.

It’s not only in the testing labs and adjacent technologies that NGS is having an impact. In the labs driving innovation in food and food ingredient development, applications of NGS are being used to develop targeted food ingredients.

Nestle is the leader in this convergence of food, health and nutrition and over the last three years, the company has acquired and formed partnerships targeting the space. In its formation of the Nestle Institute of Health Sciences, Emmanuel Baetge, head of NHIS, emphasized NHIS expertise and research capabilities using systems biology, next generation sequencing, and human genetics.

The world of food safety is as dynamic as the natural flora of food itself. Changing regulations, evolving organisms, technological change and consumers’ changing tastes require new solutions. The requirements of the food laboratory have not changed. They are the protectors of brands and the teams we trust to deliver safe and quality foods. However, how they do that has and will continue to change.

Next time… molecular serotyping.

References:

  1. Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) Available at: www.genome.gov/sequencingcosts. Accessed 1/13/2014 [DOA 1/13/12014].
  2. Beilei Ge and Jianghong Meng , 2009 14: 235 Advanced Technologies for Pathogen and Toxin Detection in Foods: Current Applications and Future Journal of Laboratory Automation DOI: 10.1016/j.jala.2008.12.012.
  3. Morisset D, Sˇ tebih D, Milavec M, Gruden K, Zˇ el J (2013) Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR. PLoS ONE 8(5):e62583. doi:10.1371/journal.pone.0062583.
  4. http://www.nestle-nespresso.com/asset-libraries/Related%20documents%20not%20indexed/Nespresso%20poster%20ASIC2012%20DNA%20traceability.pdf