The world is awash with new rapid testing technology that is enhancing food quality and safety knowledge for the global food industry and consumers alike.
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Explore This IssueDecember/January 2019
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Identifying Listeria Patterns
In July 2018, Rheonix Inc., Ithaca, N.Y., launched its Listeria PatternAlert assay, which the company calls a breakthrough method for rapidly identifying molecular patterns from Listeria strains.
“The method is designed to assist food producers in identifying harborage sites for persistent Listeria and in tracing back sources of contamination, says Brooke Schwartz, MBA, vice president for strategy and marketing, Rheonix.
According to Schwartz, feedback about the assay from food manufacturers and testing labs has been enthusiastic.
“We are supporting this technology because of the potential value that it would have for our clients,” says Timothy Freier, PhD, vice president of scientific affairs and microbiology, Mérieux NutriSciences, Chicago, Ill., Rheonix’s beta testing partner for the Listeria PatternAlert assay. “The ability to have Listeria tracking information within hours of a presumptive positive result would greatly enhance environmental contamination investigations, allowing manufacturers to find and fix issues before their product is impacted, saving costs and benefiting public health.”
Current strain typing methods take up to two weeks to complete and require an isolate in pure culture, Schwartz points out. “The Listeria PatternAlert assay, which is performed using the fully automated Rheonix Encompass Optimum workstation, enables users to detect molecular patterns in just six hours directly from a positive enriched sample, without the need for an isolate,” she relates. “Each result can be matched against a user’s specific PatternAlert database to identify their pattern matches across locations and time.”
Persistent Listeria strains that find a harborage site in a food facility can significantly increase the risk of broader contamination, and are a target of increasing regulatory scrutiny, Schwartz notes.
Schwartz explains that because the goal of the assay is to identify recurring patterns in a company’s facilities, its greatest value lies in the ability to match new patterns from a given user (i.e. company) to previously observed patterns. “However, even new users can see value in testing an initial group of samples, determining whether presumptive positives samples from a single day or week of testing reflect the same or different patterns,” she emphasizes. “The value increases as more samples are tested over time and across the user’s locations.”
According to Morgan Wallace, PhD, Rheonix’s scientific director for applied markets, the Listeria PatternAlert assay detects the presence or absence of independently occurring genetic targets that can sort Listeria into thousands of potential patterns. “Each pattern generated by the assay encompasses a group of strains and may include multiple species of Listeria,” he explains. “Our approach is to provide information directly from enriched samples that can help users identify recurring strains or populations. The discriminatory power of the PatternAlert assay was carefully calibrated to enable users to make informed decisions based on molecular patterns, without the assay providing a strain level characterization equivalent to whole genome sequencing (WGS) or pulsed-field gel electrophoresis.”
The assay, in combination with the PatternAlert analytical software, addresses the questions:
- Where and when have I seen this pattern before?
- Do I have a potential harborage site?
- Am I seeing the same pattern over time or across facilities?
WGS has a much finer level of discrimination, Dr. Wallace points out. With the ability to discriminate down to the single nucleotide level, WGS enables users to determine whether two strains are identical or very closely related to each other,” he says. “This level of differentiation may be desired when determining whether a specific strain is related to an outbreak or widespread food contamination.”
In some situations, the PatternAlert assay and WGS can be effectively used in combination, Dr. Wallace adds. “In a traceback situation, for example, many isolates might need to be sequenced to determine whether the outbreak strain is present,” he elaborates. “The isolates or positive enrichments can first be quickly screened for likely matches with the PatternAlert assay; only those with relevant patterns would go on to be sequenced.”
Three Tests in One
Thermo Fisher Scientific, Basingstoke, England, introduced its RapidFinder Salmonella Multiplex PCR (polymerase chain reaction) Workflow in November 2017. The technology is specifically designed to test raw, ready-to-eat, and ready-to-reheat poultry, as well as production environment samples and primary production samples, according to Cheryl Mooney, the firm’s global marketing and communications manager for food protection.
“What is particularly noteworthy is that with RapidFinder, laboratories performing tests for poultry producers can simultaneously screen samples for Salmonella (S.) species, S. Typhimurium (S. enterica subspecies 1 serovar Typhimurium), and S. Enteritidis (S. enterica serotype Enteritidis),” Mooney says. “We believe this is the first independently validated PCR assay of its kind. RapidFinder features simple sample preparation and provides combined Salmonella species and serovar results from a single test well in as few as 16 hours.”
The assay holds AOAC-Research Institute Performance Tested Methods program certification and has also been awarded the NF VALIDATION mark by AFNOR Certification for raw and ready-to-eat poultry meat and production environment samples. Certification for primary production samples is expected by the end of 2018.
“Many rapid methods, including other PCR tests, are available to indicate when Salmonella is present, but not many are capable of providing serovar identification at the same time,” Mooney points out. “To get that information, a laboratory must either run additional rapid tests, which can prove very expensive, or use traditional identification techniques that take several days to complete, thereby delaying the point at which product can be released or other actions taken. With its tri-fold detection capabilities, RapidFinder is a useful tool for Salmonella control programs in poultry production.”
Fat in 30 Seconds
Soon after the ORACLE rapid fat analyzer was introduced by CEM, Corp., Mathews, N.C., in October 2016, the instrument was named one of the top new products at Pittcon 2017 by Instrument Business Outlook. Then it captured an IFT17 Food Expo Innovation Award in a field of some 40 entries.
“The ORACLE is the first instrument on the market that requires absolutely no method development for fat only analysis,” says Ian Olmsted, product manager of CEM’s Process Control Division. “At the touch of a button, ORACLE can analyze fat in any food sample with reference chemistry accuracy, without any prior knowledge of the sample matrix or composition. The instrument can analyze any sample containing from 0.05 percent to 100.00 percent fat with an exceptionally accurate and precise fat result in 30 seconds.”
According to Olmsted, the Oracle functions by providing direct isolation and measurement of hydrogen protons on fat molecules. “Repeatability with Oracle is better than with wet chemical extraction techniques,” he relates. “The instrument functions with newly developed nuclear magnetic resonance technology that completely isolates protons in fat from all other proton sources in food matrixes, such as carbohydrates and proteins. It is used in food processing labs for quality and process control and can now be used in high throughput central food testing labs with the addition of an automated robotic option. ORACLE can also be paired with CEM’s SMART 6 for combined rapid fat and moisture/solids analysis in less than 5 minutes.”
Droplet Digital Enhancements
In early 2019, Bio-Rad Laboratories, Hercules, Calif., plans to make available commercially its new dd-Check STEC (Shiga toxin-producing E. coli) solution that combines the company’s Droplet Digital PCR (ddPCR) technology and iQ-Check STEC real-time PCR assay.
“Employing the co-localization benefit of ddPCR, dd-Check STEC will reduce the number of false positive samples to quickly confirm the linkage of stx and eae,” says Mike Clark, MS, international PCR group manager of Bio-Rad’s Food Science Division.
“ddPCR technology is a method for performing digital PCR within several thousand water-oil emulsion droplets,” Clark relates. “The key to ddPCR is sample partitioning. In traditional PCR, a single measurement is performed on a single sample. In ddPCR, a single sample is partitioned into thousands of nano-sized droplets allowing thousands of independent, single amplification events within that sample.”
With a PCR reaction taking place in individual droplets, this technology brings several advantages and benefits to food safety testing. “These benefits include absolute quantification without the need for running a standard curve, greater tolerance to PCR inhibitors, and one-step unambiguous identification/confirmation of genomes bearing dependent markers (co-localization of markers),” Clark points out.
Co-localization ddPCR can detect true enterohemorrhagic E. coli (EHEC) positive samples in a variety of food matrices, Clark says. “Food matrices confirmed positive for EHEC, a highly pathogenic subset of STEC, results when two virulence factors, Shiga toxin (stx) and intimin (eae), are present together within one E. coli bacterium,” he elaborates. “The ddPCR technology makes it possible to detect both virulence markers in a single bacterium by observing the percent linkage of two markers (stx and eae) making it possible to discriminate bacterium containing both markers from multiple bacteria each carrying a single marker.”
The current method for detection of EHEC in food involves enriching a sample and using PCR to screen for the stx1/stx2 and eae genes.
“The challenge with this type of testing is that typical PCR cannot distinguish between bacteria carrying both virulence markers and mixed cultures in which these target genes are present in different cells,” Clark explains. “These presumptive samples must then go through a laborious confirmation process resulting in a high number of these samples confirming as negative for EHEC.”
NCBI Gene ID Tools
At the National Center for Biotechnology Information (NCBI), Bethesda, Md., involvement with rapid testing methods is limited to tools and databases used for rapid analysis of whole genome sequencing data, according to Michael Feldgarden, PhD, an NCBI staff scientist.
“We don’t have wet labs at NCBI, but instead collaborate with labs at public health agencies, such as the FDA, CDC, and USDA to analyze their whole genome sequencing data in real time,” Dr. Feldgarden points out. “Our tools are used by these collaborators to facilitate investigation of foodborne disease outbreaks and to track antimicrobial resistance genes.”
The NCBI Pathogen Detection pipeline currently has data on over 280,000 bacterial isolates, including the four major foodborne bacterial pathogens—Campylobacter, E. coli, Listeria, and Salmonella—as well as 18 other pathogens. “Within 24 hours of sequence data submission, the pipeline can identify closely related isolates, describe how they are related to each other, and provide different visualizations of these relationships and data in NCBI’s Isolate Browser,” Dr. Feldgarden says.
In 2018, for several of the foodborne pathogens, NCBI has added the capacity to identify a preliminary set of isolates related to a particular isolate within 60 minutes of the sequence data being uploaded to NCBI. “These tools provide a provisional set of isolates for our partner agencies and programs to investigate, helping them to focus their resources more effectively and determine cases for a full epidemiological analysis,” Dr. Feldgarden explains.
“In addition, NCBI recently developed AMRFinder, a publicly available software tool that uses a curated database of antimicrobial resistance genes we produced to identify antimicrobial resistance genes in genomes,” Dr. Feldgarden says. “All of the isolates in the Pathogen Detection system are screened for resistance genes through ARMFinder and these data are made available through the Isolates Browser.”