Tag Archives: Focus Article

baby

Keeping Baby Food Safe: Sensitive Pesticide Residue Quantitation Beyond Maximum Residue Levels Using GC-MS/MS

By Paul Silcock
No Comments
baby

There are more than 1000 different pesticides in use around the world. While these chemicals are designed to target insects, weeds and other pests, residual amounts can remain on food that is subsequently eaten by consumers. The effects of pesticides on the population can be acute or chronic depending on the exposure. Acute over-exposure can cause poisoning and result in long-term effects such as cancer or reproductive issues. Chronic, lower dose exposure to pesticides has been associated with health issues such as respiratory problems, skin conditions, depression, birth defects, cancer and neurological disorders such as Parkinson’s disease.

People who face the greatest risk for adverse health outcomes from pesticide exposure are those in agricultural roles, who are more likely to come into direct contact with these chemicals. However, developing fetuses, infants and children, as well as pregnant and nursing mothers and women of childbearing age are at increased risk of experiencing negative health effects due to the presence of unsafe levels of pesticides in food. Exposure throughout a child’s development¬–including in the womb, infancy, early childhood, and puberty–can be particularly dangerous, affecting hormone regulation and brain development.

To minimize adverse health effects, the United States Environmental Protection Agency (EPA) and the European Union (EU) impose strict regulations on the amount of pesticides that can be applied to a crop, in order to limit the residue exposure downstream. Pesticides are assigned maximum residue levels (MRLs) depending on their toxicity, with the majority typically set at 10 µg/kg. However, due to the greater risk of certain compounds affecting the healthy development of infants and young children, some pesticides are controlled further: For instance, in the EU, specific pesticides are restricted in baby foods with MRLs of between 3–8 µg/kg.

Triple Quadrupole GC-MS/MS: Meeting the Needs of Pesticide Analysis

In order to test foods for pesticide residues at these very low levels, food safety laboratories require sophisticated analyte detection technologies. Gas chromatography-tandem mass spectrometry (GC-MS/MS) is a powerful analytical technique that offers the sensitivity and selectivity required to detect and identify pesticide residues at levels that often go beyond those mandated by regulatory authorities, even in complex sample matrices such as baby food. Indeed, GC-MS/MS can detect multiple residues within samples at levels as low as 0.025 µg/kg, much lower than the MRLs of regulated pesticides.

The sensitivity of the latest triple quadrupole GC-MS/MS systems is enabling levels of detection so low that many food testing laboratories have been able to adopt more efficient and universally-applicable sample preparation procedures based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) methods. Combining these modern GC-MS/MS systems with QuEChERS sample preparation techniques allows food samples to be analyzed directly, significantly reducing workflow complexity. Furthermore, the specificity of triple quadrupole GC-MS/MS can easily compensate for the additional matrix components or residual acetonitrile carried over from sample preparation.

EU SANTE Criteria for Pesticide Residue Quantitation

When it comes to the detection of pesticides in baby foods, workflows must comply with rigorous quality control and method validation standards. The EU SANTE/11813/2017 criteria outline three specific requirements that pesticide residue analysis methods must satisfy to achieve compliance.

Firstly, a minimum of two product ions must be detected for each pesticide with a peak signal-to-noise ratio greater than 3 (or in case noise is absent, a signal must be present in at least five subsequent scans), and the mass resolution for precursor ion isolation must be equal to or better than unit mass resolution. Secondly, the retention time of an analyte within a sample must not differ by more than 0.1 minutes compared with standards in the same sequence. Finally, the relative ion ratio for each analyte must remain within 30% of the average of calibration standards from the same sequence.

Fortunately, modern triple quadrupole GC-MS/MS systems are ensuring food safety testing laboratories comply with these criteria. In terms of peak detection and resolution, the specificities achieved using the latest triple quadrupole instruments meet or exceed the EU SANTE requirements by providing consistent data points regardless of sample preparation approach or matrix type. Precise detection at the ultra-low concentrations required for pesticide residue quantitation is routinely achieved using modern triple quadrupole GC-MS/MS systems, with analyses offering qualitative identification of each analyte among a large group of residues. Furthermore, the latest systems deliver stable ion ratios that are well within the required 30% range at the default 10 µg/kg MRL across multiple injections.

Ultra-low-level Quantification of Pesticides Using Triple Quadrupole GC-MS/MS

In a recent study that put the capabilities of the latest triple quadrupole GC-MS/MS systems to the test, samples of baby food (carrot/potato and apple/pear/banana) spiked with a mixture of more than 200 pesticides at three concentrations (1.0, 2.5 and 10.0 μg/kg) were analyzed using the Thermo Scientific TSQ 9000 triple quadrupole GC-MS/MS system fitted with an Advanced Electron Ionization (AEI) source. Prior to injection into the instrument, the homogenized spiked samples were prepared for analysis using a QuEChERS method that included an acetonitrile extraction step, a clean-up step involving primary secondary amine (PSA) and dispersive solid phase extraction (dSPE), followed by acidification with 5% formic acid in acetonitrile.

The triple quadrupole GC-MS/MS system met all SANTE criteria at the three spiking concentrations in both food matrices. More than 97% of the target pesticide residues in the 1 μg/kg spiked sample had recoveries in the range of 70%–120%, highlighting the broad applicability of the method. The recoveries of the target pesticides from the apple/pear/banana sample spiked at 10 μg/kg are shown in Figure 1.

GC-MS/MS system, pesticide residue analysis
Figure 1. Recovery and precision data for apple/pear/banana extractions (n=6) at a concentration of 10 μg/kg, obtained using TSQ 9000 triple quadrupole GC-MS/MS system fitted with an advanced electron ionization (AEI) source.
GC-MS/MS system
(Figure 1 continued)

Triple Quadrupole GC-MS/MS: Supporting Exceptional Limits of Detection

To determine the limits of detection of the system, baby food samples prepared by the previously-described QuEChERS method were spiked with the same mixture of pesticides at 14 concentrations ranging from 0.025 to 250 μg/kg. Using the triple quadrupole GC-MS/MS system, the SANTE criteria were met for all of the pesticides targeted at the default MRL of 10 μg/kg. Additionally, more than 90% of the target compounds had a limit of identification (LOI) satisfying all SANTE requirements below 0.5 µg/kg, and more than 60% of the target residues met these criteria below 0.1 µg/kg (Figure 2).

Pesticide residue analysis
Figure 2. Number of target residues satisfying the EU SANTE requirements (carrot/potato sample matrix). IDL, instrumental detection limit; LOI, limit of identification.

Instrumental detection limits (IDLs) were also determined for each pesticide residue by performing 10 replicate injections of the lowest matrix-matched standard of carrot/potato that met all SANTE criteria. IDLs were then evaluated using one-tailed student t-tests, taking into account the concentration and absolute peak area %RSD for each compound. The evaluated IDLs ranged from approximately 5 fg (for chlorobenzilate) to 2.0 pg (for bioallethrin), with over 95% of the residues exhibiting an IDL of less than 500 fg on the column (equivalent to 0.5 µg/kg in each sample extract). These results highlight the exceptional performance of the system, offering quantitative analysis of more than 200 pesticides over up to five orders of magnitude.

Conclusion

Enforcing regulations on the amounts and types of pesticides used is essential to limit our exposure to safe levels. The latest GC-MS/MS systems are capable of detecting and identifying pesticide residues at levels far beyond those required under regulatory standards, helping food testing laboratories efficiently ensure the food our children eat is always safe to consume.

Karen Everstine, Decernis
Food Fraud Quick Bites

Recent Notable Incidents

By Karen Everstine, Ph.D.
No Comments
Karen Everstine, Decernis

We developed a system that tracks food fraud records using four categories: Incidents, inference records, surveillance records and method records. Food fraud incidents are documented occurrences of fraud that include contextual information about location, perpetrators, timeframe, geographic location and other characteristics. In many ways, incidents are the gold standard of food fraud records. However, there have been unsubstantiated reports of food fraud that were subsequently discredited (such as the “plastic rice” scandal of a few years ago). For this reason, for every incident we capture, we assign a “weight of evidence” classification to provide our assessment of the strength of the evidence. For example, incidents reported directly by regulatory agencies with supporting documentation will generally be assigned a “high” weight of evidence classification.

We also work diligently to avoid “double counting” food fraud incidents, although at times this can be challenging. Incidents may be reported in multiple media outlets and, at times, the reports may not include enough information to determine if it is a new report or related to an issue already reported. We cross-reference the dates and locations of reports, along with information about the ingredients and adulterants, to help ensure that isolated food fraud cases are reported as one incident.

Food fraud, Decernia
Food fraud incidents since 1980. (Source: Decernis Food Fraud Database)

Incidents we have captured in the past two months include $14 million of counterfeit wine discovered in China, which was reportedly based on a tip off. The Carabinieri and ICQRF in Italy discovered wines with added flavorings and other additives. The Guardia di Finanza in Italy also seized adulterated extra virgin olive oil. Counterfeit liquor was discovered in Russia and bootleg liquor containing methanol caused deaths in Malaysia.

Expired (possibly rotting) eggs intended to be powdered and used in food production were discarded by regulatory authorities in India. A customer in China reported that expired carrots were being re-labeled with new dates. Adulterated milk was discovered in Pakistan. In Kenya, reports surfaced of sodium metabisulfite being used on meat to enhance its appearance. Finally, in the United States, two companies were indicted for importing giant squid and selling it domestically as octopus (which usually has a higher retail price) over a period of three years. A review of these reports illustrates how challenging it can be to collect and standardize food fraud information, especially when it is reported in media sources.

Chelle Hartzer, Orkin
Bug Bytes

Not-So-Fantastic Pests and Where to Find Them

By Chelle Hartzer
No Comments
Chelle Hartzer, Orkin

Although no two food processing facilities face the exact same pest pressure, there are a few common pests you’re likely to spot. Depending on the type of pests in the surrounding area, different spaces in a building will be more at-risk for an infestation. Pests will feed on and contaminate product, and get into equipment, if undeterred. And considering many pests can be a potential health and safety threat, prevention is important to help protect your bottom line.

Instead of reacting to pest issues, adjust your integrated pest management (IPM) program to take a proactive approach to preventing the following six common pests.

Rodents

Some of the most clever and resilient creatures in the pest world, rodents are a versatile threat to food products. Usually brown or gray, mice and rats can compress their bodies to fit through holes and gaps the size of a dime and a quarter, respectively. Once inside, they reproduce rapidly. A single rat or mouse can produce more than 32 offspring per year. In addition, they can use their sharp teeth to gnaw through packaging and leave urine and feces droppings everywhere they go. All of this makes them an absolute terror once indoors. They’re smart enough to know hiding from humans is their best option, and they’ll even learn from unsuccessful trapping attempts.

Cockroaches

These notorious crawling insects are contaminators, too. Roaches love to get into dirty areas and run all over food contact surfaces, equipment and products, potentially dropping off disease-inducing pathogens on everything they touch. Cockroaches can fit through tiny gaps by flattening their bodies, making them tough (but not impossible!) to keep out. They tend to avoid coming out in the open during daytime hours, as they prefer to hide in the dark. If you spot one running around during the day, then it may be a sign of a larger infestation behind the scenes.

Flies

When flies detect a potential food source, they’re going to head straight for it. Stringent sanitation is the best way to minimize attractants, and keeping doors and other openings closed can help, too. Preventing flies is important, because they’re twice as filthy as cockroaches. In fact, more than 100 pathogens are associated with the housefly alone. These pathogens are transferred when the fly lands on a surface, contaminating the area. If flies are a threat, you should have fly lights placed strategically to reduce the population and monitor where they’re coming from.

Indian Meal Moths

This tiny insect feeds on a wide range of raw and finished goods, and leaves behind frass (insect droppings) that that can lead to major loss of products. If you don’t see the pest itself, which can be reddish-brown and silver-grey in color, you may notice the silk webbing spun by larvae. When someone notices this, immediate action is necessary, as it means the moths are reproducing and may be spreading amongst products in close proximity.

Sawtoothed Grain Beetle

Unable to penetrate most packaging, sawtoothed grain beetles hunt for holes in packaging, which can be one millimeter in diameter, and lay eggs near the opening. Larvae then squeeze through the hole once hatched and begin feeding on product! Although they prefer processed food products like bran, chocolate and oatmeal, they’ll feed on just about anything they can get into. About three millimeters in length, these beetles love moldy, damp conditions, so minimize those attractants as much as possible.

Ants

Everybody has seen or been around ants before, but are you aware that they carry bacteria on their bodies capable of contaminating food? What starts with a few foragers can escalate quickly, as ants leave behind an invisible chemical trail leading other ants straight to a food source. Ants will feed on just about anything depending on the species, so identification is key. Generally only a few millimeters in length and ranging in color from black to red, ants can establish colonies under a building’s foundation, on lawns or in out-of-sight locations indoors.

Watch Out for High-Risk Areas

Understanding the biology of pests helps us to understand what they’re looking for and where they’re most likely to be hiding. Generally speaking, pests are attracted to places able to provide them with the three things they need to survive: Food, water and shelter.

Food doesn’t necessarily mean actual food products of course, as some pests—like cockroaches, flies and ants—will feed on any organic matter they can find. Remember, that includes garbage!

But taking out the trash and ensuring dumpsters are far away from the building aren’t the only ways to reduce pests. Quite the contrary, pests have a myriad of different hiding spots that should be checked by facility staff and a pest management professional regularly.

For starters, don’t overlook the break room. It’s easy to forget to take out the trash, which should be done at least daily depending on waste output. Break rooms also frequently have sinks with drains where food buildup can cause odors that are attractive to pests. Drain flies love this! Wipe down countertops and sweep/vacuum/mop daily to ensure larger food crumbs and debris are taken care of, and make sure your staff knows to clean up any spills immediately. Don’t forget those vending machines—when was the last time they were moved and cleaned underneath and behind?

Equipment can be a hot spot for pests, too. Insects, especially stored product pests, will hide beneath and behind heavy machinery. Pests don’t want to be exposed out in the open, so they’ll hide in small gaps and crevices. And if there is food waste or moisture present, watch out! Those attractants will prove irresistible if allowed to linger for too long, so make sure your cleaning schedule includes sanitation in and around equipment. Never overlook those hard-to-reach areas, or pests will make you pay.

Speaking of hard-to-reach areas, walls are often popular harborage areas for pests. Rodents are perhaps the most dangerous, as they pose a health and safety threat to employees and can contaminate product. Worse still, wiring in walls looks like roots to rodents. They’ll often chew through and create sparks—a potential fire hazard. Rodents are just one of many pests happy to live in your walls, so contact a professional if you notice activity.

Even once food is produced, packaged and stored, pests are still a threat! Stored product pests, like the Indian meal moth and sawtoothed grain beetle, can get into packaged products and live in it. They’ll feed and contaminate the product, then move onto the next, proving costly when large batches have to be thrown out. Thankfully, there are monitoring devices like pheromone traps to help identify where these begin to pop up, but again, you’ll want a professional’s help to ensure these tools are effective.

Don’t wait for pest sightings to occur before taking action. The best approach to pest prevention is a proactive one, and there’s not an insect or animal alive who can outsmart a trained pest management professional. Lingering issues will prove costly with time, as a product infestation or plant shutdown would be a painful hit on your business’s bottom line. Instead, create a plan that accounts for these pests and high-risk areas around your building, and you’ll be able to rest easier knowing you’re prepared for pest invaders.

Bryan Cohn, Foodlogiq

Managing Risk and Traceability in the Supply Chain

By Food Safety Tech Staff
No Comments
Bryan Cohn, Foodlogiq

Traceability and risk management go hand-in-hand. In a Q&A with Food Safety Tech, Bryan Cohn, food safety solution engineer at FoodLogiQ, shares his thoughts on risk and the critical role of communication.

Food Safety Tech: What does risk analysis mean in a complex supply chain?

Bryan Cohn: Risk analysis means the same thing it has always meant. The concept of risk is elemental; it transcends all of humanity and is rooted deep within our very DNA. Sure, we’ve added tools and technology to help us, but we still can not see into the future; thus, there will always be a risk. The best way to perceive, evaluate and comprehend risk in a complex world is faster and more accurate communications.

FST: Why is communication critical to avoid or mitigate risks within the supply chain?

Cohn: Let’s use an analogy here. Nobody likes traffic, right? In the morning when you’re getting ready for work, you might turn on the local news or check your favorite navigation app to find out the traffic conditions along your commute. You know your commute like the back of your hand, and you’re aware of every potential trouble spot along the way. But like most of us, you probably rely on fast and accurate communication from either traffic cameras, local news reports, or navigation information on your phone to give you a real-time analysis of what is happening. So aside from the usual trouble spots, you are made aware of any unexpected traffic accidents, road construction, or weather delays, which allows you to make real-time, actionable decisions about your commute.

If we think ahead – the same way we do about our work commute – and re-evaluate our communication strategy around our supply chains, we can begin to take a much stronger proactive approach to risk analysis and mitigation. If we spot a trend within our supply chain that may increase risk, we can take action before a threat materializes or intensifies.

FST: Can your risk management plan create value in the company?

Cohn: Any time a good communications strategy is integrated into your risk management program, you create value. By soliciting, evaluating and responding to feedback, you will inherently mitigate risk by addressing potential problems before they become problems and identifying new threats in a fast moving complex supply chain.

Romaine Lettuce Outbreak: We Knew It Would Get Bad Quickly

By Maria Fontanazza
No Comments

This year’s multistate outbreak of E.coli O157:H7 infections linked to romaine lettuce affected 210 people, killing five. Although the outbreak was officially declared over by the end of June, questions still remain as to the exact source. Given the widespread nature of the outbreak and the speed with which illnesses occurred, there are many lessons to be learned from the case.

During last week’s annual Food Safety Consortium, industry stakeholders from the FDA, CDC and produce associations gathered to discuss agency action upon learning of the outbreak and where there is room for improvement.

The investigation began in April 2018 when the New Jersey Department of Health contacted the CDC about a cluster of E.coli O157:H7 illnesses from people who said they ate salads at various locations of the same restaurant chain. Three days later, the agency was able to confirm eight O157 isolates from six states with the same patterns using PulseNet. And five days after that, the CDC posted a notice on its website about the investigation of 17 cases across seven states.

“We knew right away that this was going to get bad and that it would get bad quickly,” said Matthew Wise, deputy branch chief for outbreak response at the Outbreak Response and Prevention Branch of the CDC. “We saw illnesses ramp up quickly.” He added that the agency saw a lot of illness subclusters, all with romaine lettuce as the common ingredient.

The epidemiological evidence clearly indicated chopped romaine lettuce, and it appeared that all the affected romaine was coming from the Yuma, Arizona growing region, noted Stic Harris, director of the Coordinated Outbreak Response & Evaluation Network at FDA. But then things got even more confusing, as an Alaskan correctional facility was also investigating a cluster of cases. This allowed the agency to trace the source directly back to Harrison Farms as the sole supplier to the correctional facility. However, as the multi-agency investigation continued, they uncovered that the source was not just one farm. “There were three dozen farms in the Yuma region that supplied romaine lettuce,” said Harris, adding that we may never know which exact farm, and even if it was one farm, that was the source of the outbreak.

(left to Right) Stic Harris, FDA; Matt Wise, CDC; Dan Sutton, Pismo Oceano Vegetable Exchange; Scott Horsfall, California Leafy Green Products Handler Marketing Agreement

During June, July and August, the FDA sent a multidisciplinary team of 16-18 people to conduct an environmental assessment of the affected area. Upon taking 111 samples, they found 13 different Shiga toxin-producing E. coli strains, but only three matched the strain of the outbreak. Water from 14 locations, including discharge, reservoir and canal water, was also tested. The environmental assessment found pervasive contamination in the water. But here was the big problem, said Harris: “There was no smoking gun. We don’t know how the E.coli got into the water, and we don’t know how the water got onto the lettuce.” He added that additional research is needed, and that government and non-government work must continue to identify the source.

There are several challenges associated with the complexity of this type of produce outbreak, said Harris and Wise:

  • The production lot information disappears at the point of service
  • Having a commingled product hinders traceback
  • Records present a challenge because agencies try to look at each company and their individual records, and every company has their own way of doing things—this takes time
  • The breadth of the impacted area—trying to do an environmental assessment for that area was staggering work
  • People who eat lettuce eat it often
  • Many people don’t remember what type of lettuce they ate
  • The product has a short shelf life
  • Communication: The packaging isn’t transparent on where it’s grown

Scott Horsfall, CEO, California Leafy Green Products Handler Marketing Agreement, chimed in on the challenges posed by the complexity of the outbreak. “If you compare these numbers with the 2016 spinach outbreak…they’re very similar [in the] total number of illnesses [and] number of states involved. But in [the spinach outbreak], it led to a specific farm. What we saw this time was very different.”

TraceGains Sponsored Content
When it comes to food safety compliance, learning lessons the hard way is never a good practice. Violations, non-compliance and documentation mishaps put a major damper on your business. From delays in production to the dreaded recall, these mistakes can cost thousands, if not millions, of dollars. Don’t learn these lessons the hard way. See our Case Studies and learn how companies are avoiding these costly mistakes with TraceGains.

One of the large successes in dealing with the outbreak is that the agencies issued public warnings quickly, said Wise. The produce industry also came together to form the Leafy Greens Food Safety Task Force. In addition, FDA is expanding its sampling for the coming harvests, according to Harris. “I think that in terms of the speed of the environmental assessment, we need to be quicker with that. We apparently hadn’t done one in quite a long time at FDA,” he said.

Harris and Wise also stressed that for industry to work more effectively together, they need to work with the FDA and CDC before there is an outbreak.

“This outbreak was a frustrating experience for all of us,” said Horsfall. “We have to communicate more and better when we can. And as an industry, stop these outbreaks from happening.”

FDA: 172 Ill, 1 Death, Romaine Lettuce E. Coli Outbreak Likely Over

TG native ad

Food Safety Consortium

Were You There? Images from the 2018 Food Safety Consortium

By Food Safety Tech Staff
No Comments
Food Safety Consortium

 

Images credit: amybcreative

Alert

Outbreak of Salmonella linked to Raw Turkey Products Continues, USDA Facing Pressure to Name Brands

By Food Safety Tech Staff
No Comments
Alert

Just in time for Thanksgiving, consumers are worrying about whether the turkey they are buying for the holiday is contaminated with Salmonella. A multistate outbreak of drug-resistant Salmonella linked to raw turkey products has been going on for months, but now USDA is facing increasing pressure to name any associated turkey brands. According to the CDC, “a single, common supplier of raw turkey products or of live turkeys has not been identified.”

As of the agency’s last update (November 5), 164 people across 35 states have been infected with the outbreak stream of Salmonella Reading. 63 people have been hospitalized, and one death has been reported. Three people reported living in households where raw turkey pet food was given to pets.

Thus far the CDC isn’t advising retailers to stop sell raw turkey. It is stresses that consumers should follow the basic food safety steps to prevent Salmonella infections, including proper handwashing, cooking the turkey to the proper temperature (including reheating the meat), keeping food prep areas clean, proper thawing of turkey in the refrigerator and avoiding feeding pets raw food.

CDC states that if the information becomes available, it will provide notification related to the supplier(s) related to the outbreak.

John Besser, CDC, 2018 Food Safety Consortium

CDC: Quite a Year for Outbreaks, Exciting Time in Food Safety

By Maria Fontanazza
No Comments
John Besser, CDC, 2018 Food Safety Consortium

This year Salmonella outbreaks hit chicken, shell eggs, ground beef, pre-cut melon, dried and frozen coconut, pasta salad, chicken salad, turkey, ground beef, raw sprouts and breakfast cereal. There were also significant Cyclospora infections linked to salads sold at McDonalds as well as vegetable trays. For the first time in 10 years, a Listeria outbreak was linked to an FSIS regulated product (deli ham); ground beef was affected by E. coli O26. And perhaps the most notable outbreak of the year was the E.coli O157:H7 outbreak linked to romaine lettuce from the Yuma growing region.

“It’s been quite a year for outbreaks,” said John Besser, Ph.D., deputy chief, enteric diseases laboratory branch, at CDC, referring to the pathogens that have plagued a variety of consumer products in 2018. “Out of this group, there are a lot of the things you’d expect, but also some brand new unexpected [products affected] like shredded coconut and Honey Smacks cereal.”

Despite the number of outbreaks that have hit the food industry in 2018, “this is a really exciting time to be in public health and food safety, because there are a lot of tools we can use to help make food safer,” said Besser. Most of the diseases that impact the food industry are preventable if their source can be identified, and using big data can have a tremendous impact on improving food safety.

Yesterday John Besser informed attendees at the 2018 Food Safety Consortium about CDC’s latest efforts in foodborne disease surveillance, which he defines as the
systematic collection, analysis and interpretation of health data. The agency is actively working to identify unrecognized gaps in the food supply chain and provide the industry with information it can use to make products safer. “The most important reason for detecting outbreaks is so we can identify the problem and fix it,” said Besser.

There are two ways that CDC detects outbreaks. The first is via the “citizen reporters” who are observant and alert the agency. (This is actually how E.coli O157 was discovered). The second is through pathogen-specific surveillance where CDC takes lab information and links cases that are geographically diverse. These cases are often widely dispersed and are the most effective way to find food production and distribution problems, and are often easier to address than local issues, according to Besser.

He went on to review the successes of PulseNet and the promise of whole genome sequencing (WGS) and metagenomics. The CDC’s PulseNet nationwide WGS implementation project is underway and will result in a “tsunami of data”, with the timeline as follows:

  • January 15, 2018: Listeria monocytogenes
  • October 15, 2018: Campylobacter jejuni/coli
  • January 15, 2019: Diarrheagenic E.coli (including STEC)
  • March 15, 2019: Salmonella enterica

Metagenomics will continue to play a large role in enabling unbiased sequencing of all nucleic acids in an environment. It will help to directly characterize sequences from samples, food and people (i.e., the gut), and could aid in pathogen discovery.

“I think within just a few years, it’s going to be the standard for tests,” said Besser. “My prediction is that you’ll be able to do this test in the production environment.”

Deadly Outbreaks and the Role of Metagenomics

magnifying glass

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

By Joy Dell’Aringa, Vikrant Dutta, Ph.D.
No Comments
magnifying glass

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.

Pages: 1 2
FDA

FDA Restaurant Study Finds Employees Not Properly Washing Hands or Keeping Foods to Temp

By Food Safety Tech Staff
No Comments
FDA

Yesterday FDA released the initial phase of its findings of a 10-year nationwide study that looks at the relationship between food safety management systems, certified food protection managers, and the occurrence of risk factors and food safety behaviors/practices, and how this contributes to foodborne illness outbreaks in retail establishments. This first phase collected data from 2013–2014; subsequent data collection will be from 2017 and 2021. The entire span of the study is 2013–2023.

The data collected and used in the 84-page “Report on the Occurrence of Foodborne Illness Risk Factors in Fast Food and Full Service Restaurants, 2013-2014” will be used as a baseline to evaluate trends in the occurrence of risk factors during the 2017 and 2021 data collection periods. Key findings in the report include the following:

  • Food safety management systems are important!
  • Restaurants had the most effective control over ensuring there is no bare hand contact with RTE foods as well as cooking raw animal foods (including meat, poultry and eggs) to the required temperature
  • Unsafe food behaviors in fast food and full-service restaurants. Improvement needed in:
    • Employee hand washing (knowing when and how to do it)
    • Proper temperature control of foods that require refrigeration to limit pathogen growth

Study results will be used to help advise retail food safety initiative and policies, industry partnerships and specific intervention strategies that target foodborne illness risk factors. It will also aid in providing technical assistance to state, local and other regulatory professionals. FDA put together a factsheet with highlights of the study.