If you’re a laboratory professional who thinks a programmable coffee maker is the greatest thing since sliced bread, you’re in for a pleasant surprise. Not only can you wake up to fresh brewed java at your prescribed time, you can now arrive at your workplace and find freshly made culture media ready and waiting to be used, thanks to the recent development of a new programmable media preparator.
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Explore This IssueApril/May 2017
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Say hello to the Masterclave 20 Automated Media Preparator, the brainchild of bioMérieux, Inc., Hazelwood, Mo., introduced in November 2016.
“With its automatic water-filling and autostart features, the Masterclave 20 has the ability to prepare fresh agar or broth that is ready when lab operations begin,” says J. Stan Bailey, PhD, director of scientific affairs for bioMérieux Industry.
With this preparator, one medium can be made at a time, during a cycle that takes less than 90 minutes.
“The Masterclave 20 is compact, mobile, and adaptable to any workspace,” Dr. Bailey relates. “A built-in cleaning gun provides fast and efficient cleaning. Our proprietary ‘I Connect’ technology offers fully automated traceability with built-in RFID (radio frequency identification), email alerts, and laboratory information management system (LIMS) connectivity capability. And the instrument is ISO 11133, ISO 7218, and GMP (good manufacturing practices) compliant.”
Another recent bioMérieux offering is GENE-UP, a proprietary real-time three-step polymerase chain reaction (PCR) pathogen detection system, which the company touts is fast, simple, accurate, and requires minimal training.
Dr. Bailey explains that GENE-UP’s first step, sample preparation and enrichment, includes a standardized protocol and workflow with single enrichment and incubation time between 8 and 24 hours. Step two consists of a simplified, generic 5-minute mechanical lysis. (What bioMérieux calls its Magic Cap eliminates the need to cap/decap tubes, so there is less hands-on time required, plus there is a decreased risk of cross-contamination, Dr. Bailey notes.)
The third step, amplification and detection, features the same PCR run for all parameters. “This allows for accurate results within one hour, a higher level of specificity than other molecular methods, and real-time PCR analysis coupled with end-point melt peak analysis,” Dr. Bailey says. “GENE-UP uses a different kind of FRET (fluorescence resonance energy transfer) technology with three levels of specificity, namely primers, FRET probes, and melt peak analysis. This offers an additional level of sensitivity valuable for detecting low level samples.”
Since GENE-UP was introduced in 2016, it has really taken off, Dr. Bailey boasts. “Laboratories are encountering workflow improvements in a molecular platform, equivalent or better data performance than their current method, and with the internal control and melt peak analysis, an immediate value and confidence in the results they are reporting,” he elaborates.
In July 2016, bioMérieux also introduced a new EHEC GENE-UP PCR Kit that combines stx and eae virulence genes, and the top six serogroups in one solution.
Specific protocols (which are simply enriched in prewarmed buffered peptone water) are available for raw milk and raw milk cheese (25 grams), raw beef and veal (25 grams and 50 to 375 grams), and produce (200 grams).
This kit’s EHEC/STEC (enterohemorrhagic Escherichia coli/Shiga toxin-producing E. coli) solution marries GENE-UP in combination with bioMérieux’s long-established VIDAS automated food pathogen detection system to provide what Dr. Bailey describes as “unrivaled specificity.”
“As a result, false positives are dramatically reduced,” Dr. Bailey emphasizes. “That makes this assay a real game-changer.”
BCN Research Laboratories, Inc., Rockford, Tenn., a commercial laboratory that tests food, ingredients, and environmental sponge samples for food manufacturers, has been using GENE-UP since July 2016.
“We typically run about 100 GENE-UP samples every day,” says Amy Pass, BCN’s senior lab technician. “However, one of our clients operates 20 manufacturing plants throughout the U.S. Twice a year, in March and September, that company conducts biannual heavy environmental testing at all of its facilities. So then we are evaluating an additional 150 Salmonella swabs and 150 Listeria swabs per month for each of those 20 plants, which means we are running an average of 400 tests, but up to about 900 to 1,000 tests, per day, during those two months.
“We have a better work flow with GENE-UP compared to other lateral flow methods we have used in the past,” Pass adds. “Since we started using this technology, we have experienced a 25 percent increase in our sample load, but the amount of time our employees spend running the tests has stayed the same.”
Another benefit of GENE-UP, Pass mentions, is that it provides a definite positive or negative result. “So there is no subjective decision looking at the lateral flow strip to see one line or two,” she says.
Colony Tests and Counter
The instrument is designed to analyze a variety of Charm’s Peel Plate microbial tests, which were introduced in August 2015, according to Robert Salter, MS, the firm’s vice president of regulatory affairs.
The tests are prepared media in a shallow dish with an adhesive top. “They are aseptic ready-to-use tests that are simply rehydrated with the food or food dilution, and incubated at times and temperatures appropriate to the microbes being detected,” Salter explains. “Colonies appear as colored spots that are visually counted or automatically counted by the Peel Plate Counter. An air gap between the plate and cover allows colony quantitation, picking and determination of microbial morphology.”
Currently there are Peel Plate tests for aerobic bacteria (Peel Plate AC, introduced in August 2015), coliform bacteria (Peel Plate CC, introduced in 2016), Enterobacteriaceae (Peel Plate EB, introduced in 2017), yeast and mold (Peel Plate YM, introduced in 2016), heterotrophic bacteria in water (Peel Plate HET, introduced in January 2016), and coliforms/E. coli (Peel Plate EC, introduced in August 2015) for use in dairy products, ground meats, other foods, contact surfaces, and water.
“The Peel Plate AC uses standard plate count formulation with 48-hour incubation,” Salter relates. “Peel Plate EC and CC use coliform selective media with enzyme color substrates with 24-hour incubation. The Peel Plate YM (yeast and mold) tests use conventional potato dextrose formulation with three to five-day incubation. The Peel Plate EB (Enterobacteriaceae) tests use selective EB formulation with 24 to 48-hour incubation. And the Peel Plate HET (heterotrophic plate count) test uses R2A formulation for quantifying bacteria in water with three to five days incubation.”
The new Colony Counter reads all of the aforementioned Peel Plates, Salter says.
Additionally, Charm offers a high volume version of Peel Plates CC-HVS, YM-HVS, EB-HVS, and EC-HVS, designed for a 5 milliliter (ml) sample, typically used for food plant sponge sampling of the production environment, for greater sensitivity in ready to eat foods, and to test water. “These are viewed visually and not yet supported by the Peel Plate Counter,” Salter says.
Salter believes the Peel Plate offerings are an improvement on many existing simple-to-use microbial test products. “The Peel Plate provides a ready-to-use platform that is self-wicking, stackable, and resistant to sample pH/disinfectant effects,” he relates. “Charm Peel Plate tests are simple to interpret color spots that are specific without the need to confirm, but will allow traditional picking of colonies for additional microbial isolation, testing, and identification.
“Peel Plate CC and EC tests use traditional gram negative selective media with bacterial species specific color producing enzyme substrates and a 24-hour incubation,” Salter elaborates. “Coliforms produce easy to interpret red colonies, and E. coli blue colonies, that do not need additional confirmatory steps or difficult to interpret and time dependent gas production.”
The Peel Plate EC test holds Performance Test Method (PTM) Status 061501 with the AOAC Research Institute for total coliform in dairy products tested at 32 degrees Celsius and for E. coli and coliform detection in water, surface rinses, environmental sponges, and foods such as ground meats, eggs, chocolate, and dry dog food tested at 35 degrees Celsius.
Peel Plate AC uses a red color indicator for aerobic bacteria growth in a 48-hour incubation. It holds PTM certificate 071501 for dairy products at 32 degrees Celsius and ground meats, eggs, chocolate, environmental sponges, and dry dog food tested at 35 degrees Celsius.
Peel Plate YM uses a blue/green indicator for yeast and mold growth in a 3- to 5-day incubation. It holds PTM certificate 061601 for fruit, juices, dairy, flour, tortillas, hummus, and environmental sponges.
Based on additional multi-laboratory reference method comparative data, the Peel Plate EC test and the Peel Plate AC test were voted in the 2015 National Conference on Interstate Milk Shipments for inclusion into the Pasteurized Milk Ordinance governing U.S. milk testing requirements.
“Many of our customers are using Peel Plate tests to verify their sanitation and hygiene practices and to monitor and improve food product shelf life,” Salter notes. “They are competitively priced with other simple and ready to use microbial methods, saving time, skill, and labor required by the traditional microbial methods.”
The Colony Counter is a plug and play computer/camera/software that provides complete data management with a real-time print option, Salter points out. Date, time, operator, sample ID, test type, test matrix, count, sample dilution, calculated colonies/ml or gram product, raw plate image, and processed plate image are stored in folders and hyperlinked in a SQL database spreadsheet. Barcode scanning capability provides for a simple one button analysis.
“Onboard Ethernet enables real-time downloading to network SQL databases,” Salter says. “The images are saved as .jpeg files and are viewable at a later date. All data is reviewable by day, week, and month with the touch of a button. It provides for a simple integration with LIMS.
(Without the Peel Plate Counter, Peel Plate tests are scored based on visual counts of colonies, Salter notes. “Visual count is how the methods were compared to the reference methods—also visual—for the approvals,” he says.)
“Inasmuch as food companies are updating their microbial sanitation verification and their end product microbial monitoring programs to address the new Food Safety Modernization Act regulations, we believe the Peel Plate Counter is a valuable tool to assist in automated documentation and record keeping that will enable food production stakeholders to more easily meet audit and inspection requirements,” Salter adds.
Molecular Detection Chemistries
Roka Bioscience, Inc., Warren, N.J., stands out in many state-of-the-art food laboratories by offering differentiated molecular chemistries for pathogen detection. One such cutting-edge chemistry is target capture, Roka’s proprietary sample preparation method that is integrated into the company’s fully automated testing instrument called the Atlas System.
Simply stated, target capture uses highly specific nucleic acid hybridization to purify and concentrate only the target RNA of interest, according to W. Evan Chaney, PhD, Roka’s director of customer applications and microbiology.
“Roka’s target capture technology is the only fully integrated nucleic acid based sample preparation technology in the industry,” Dr. Chaney says. “This technology is the initial step in all of Roka’s automated assays and serves to not only add specificity, but to also clean up the sample prior to detection.”
The diversity of sample matrices in food related analyses results in very unique diagnostic application challenges, Dr. Chaney points out. “Our target capture technology helps to address these challenges by providing an ideal sample for downstream amplification and detection by molecular chemistries called transcription-mediated amplification (TMA) and hybridization protection assay (HPA),” he relates.
An RNA based amplification system, Roka’s TMA has been used in clinical diagnostics for many years and was first commercially introduced to the food industry in 2012.
“TMA is still novel within the food industry and many food safety professionals are not aware of the differences between it and incumbent testing methods, like PCR,” Dr. Chaney says.
TMA uses two enzymes to drive the reaction, RNA polymerase and reverse transcriptase, he explains. According to Dr. Chaney, TMA is very rapid, resulting in a billion-fold amplification of the target RNA within 15 to 30 minutes.
“One component of this efficiency is the greater abundance of RNA target in cells as compared to DNA,” he says. “TMA is different from older DNA based chemistries such as PCR in that it is isothermal and autocatalytic. The higher RNA copy number per cell, combined with TMA, results in a very robust amplification that may occur in a shorter timeframe as compared to PCR, which can translate into quicker turnaround times for results.”
All rapid methods have various analytical limits of detection in the enriched sample, Dr. Chaney points out.
“For example, most PCR methods require 104 or more cells per ml, whereas, TMA only requires 102 to 103,” he says. “This becomes quite important to prevent false negatives when considering industry’s move to larger sample sizes and reduced incubation times across an increasingly diverse and complex range of matrices.”
Post TMA, all Roka assays detect any amplified product utilizing HPA, which Dr. Chaney describes as a highly specific chemiluminescent reaction from which the intensity is measured by the Atlas instrument.
“In addition to the detection of any pathogen, each individual sample processed by Roka’s technology includes an internal amplification control, which ensures all reactions occurred,” he says, adding that all of these technologies are automated on the Atlas instrument.
“Our chemistries and controls, coupled with integrated sample preparation on a fully automated platform, translate into faster result times, laboratory efficiency, full traceability, and more accurate foodborne pathogen screening results, particularly for challenging sample matrices,” Dr. Chaney elaborates. “Roka’s technology is routinely utilized in many industry segments, including commercial laboratories, poultry, ready-to-eat meats, produce, dairy, confectionary, ingredients, cereals, multi-component foods, snack foods, and as a tool in pre-harvest food safety.”
TMA is routinely used by Marshfield Food Safety, LLC (MFS), Marshfield, Wis., a firm that specializes in providing customized, onsite process control laboratory services for U.S. food processing operations.
Holding accreditation to ISO 17025:2005 standards with the American Association for Laboratory Accreditation at all nine of its U.S. food testing laboratories, the MFS portfolio includes an extensive list of microbiology and chemistry laboratory test offerings.
“We have been using TMA for qualitative, semi-quantitative, and limits testing for Salmonella for four years,” says Roy Radcliff, PhD, chief executive officer, MFS. “We started using TMA for identifying Listeria species in early 2016, and we have been using it for L. monocytogenes since August 2016.”
Dr. Radcliff says that the ease of use and decreased hands on time are benefits of TMA enjoyed in MFS labs.
“Roka’s Atlas System integrates well with our LIMS,” Dr. Radcliff relates. “The automatic importation of results into the LIMS and tracking of TMA kit lot numbers simplifies the workflow and traceability, which in turn makes them easily auditable.”
Dr. Chaney mentions that, along with continually expanding its footprint by developing unique applications for its current food testing products, Roka endeavors to provide support to its customers, and also works with strategic partners. “We strive to partner with the industry we serve in collectively advancing food safety,” he emphasizes.
Roka’s menu of automated pathogen detection kits includes Listeria spp., L. monocytogenes, Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin-producing E. coli, as well as applications including semi-quantitative Salmonella. All of these kits utilize target capture, TMA, and HPA molecular chemistries, and these kits are utilized in Roka’s new applications, Dr. Chaney says.
“More recently, we have introduced a new kit for detection of Listeria spp. specifically in environmental samples, in addition to a novel workflow for use in mitigating the diagnostic challenges that may arise with use of industrial phage based processing aids,” Dr. Chaney adds. “We are currently validating some new and exciting options, such as media alternatives and new assay application parameters for our customers that we anticipate will confer a number of benefits and efficiencies to their operations.”
As general manager of Mérieux NutriSciences, Wendy McMahon, MS, CFS, oversees the company’s Silliker Food Science Center (SFSC) contract research laboratory, Crete, Ill.
McMahon believes matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectroscopy (MS) is an important tool for bacterial and fungal identification in food laboratories today. “It’s really used for determining unknown organisms, mostly spoilage and contaminations, with mold being a good example,” she points out.
Available commercially for less than 10 years, MALDI is a three-step soft ionization technique that allows the analysis of biopolymers such as DNA, proteins, peptides, and sugars, and also large organic molecules. The TOF is the type of mass spectrometer most widely used with MALDI, primarily because of its large mass range.
McMahon says it’s interesting that the microbiology world is using MS for bacterial identification, since MS is a tool used for chemical analysis. “Chemists get a kick out of this,” she quips.
Under McMahon’s leadership, the SFSC is launching the use of MALDI-TOF in the lab during the spring of 2017. “We expect hundreds of ID requests per month due to its quick time to result,” she predicts.
The SFSC is using bioMérieux’s VITEK MS to run its MALDI-TOF tests. “We made that decision based on the database,” McMahon relates. “Specifically, bioMérieux’s database has been established with an average of greater than 14 isolates per species and an average of 26 spectra per species, making it very specific. If an organism is not a part of the database (unidentifiable), then 16S ribosomal RNA (rRNA) gene sequencing can be used for identification.”
The time to result was also a deciding factor in selecting VITEK MS, McMahon adds, noting that it allows for faster investigations and decisions than getting identifications with gene sequencing affords.
“Microbiologists appreciate the quick turnaround time MALDI-TOF offers, less than 30 minutes once the isolate is ready, while requiring very little hands on time from a technician,” McMahon elaborates. “In contrast, the gold standard of 16S rRNA gene sequencing for bacterial identification takes a day of operations and a significant amount of hands on time.”
The SFSC has been using the 16S rRNA method for more than 10 years.
“MALDI-TOF is becoming more widely used throughout the food industry due to the quick results and ease of use,” McMahon says. “The clinical and pharmaceutical industries took to it first and the food industry is quickly catching on. MALDI-TOF’s use in food will increasingly provide companies with faster results when investigating spoiled product, mold contaminations or out of specification raw ingredient or finished product.”
Details to Work Out
There are details to work out in the increasingly more sophisticated world of food laboratory technology, especially with regard to the pathogen testing and detection end of things, says Lee-Ann Jaykus, PhD, the William Neal Reynolds Distinguished Professor in the Department of Food, Bioprocessing, and Nutrition Sciences at North Carolina State University, Raleigh, and also the scientific director of the USDA-NIFA Food Virology Collaborative.
“In recent years, several assays have been designed to meet the need of providing testing results in near real-time (same day), but by and large, they still require some cultural enrichment for pathogen detection, even though enrichment may be abbreviated,” Dr. Jaykus relates.
To get true real-time (in a matter of minutes) pathogen detection will require methods that are completely culture-independent, she says.
“Such pathogen detection will also require pre-analytical sample processing methods, also called ‘sample prep,’ to concentrate the organisms from the sample matrix, and remove matrix-associated inhibitory compounds,” she elaborates. “While some novel sample prep technologies have been launched in the past several years, no silver bullet has been found yet.”
Many groups, be they academic, industry, or government, are actively developing biosensor technologies, Dr. Jaykus points out. “Many of these technologies are novel and ‘sexy’ but still do not have the low detection limits necessary for pathogen detection in foods,” she says. “In addition, the sample matrix can be a significant impediment to analytical sensitivity. Another reason for sample prep, and a personal caution, is that without one (sample prep) we cannot have success in the other (biosensors).”
Dr. Jaykus believes that as detection become less dependent upon culture and more dependent upon nucleic acid sequence, the issue of bacterial cell or virus viability becomes more important. “Just because we can detect DNA does not mean that the organism is alive,” she notes. “This issue is of importance in making decisions about prevention and control in food safety, as well as management of recalls and outbreaks. It has not yet been resolved.”
Metagenomics, a term that reportedly first appeared in peer reviewed literature in 1998 (Handelsman et al), basically the study of genetic material recovered directly from environmental samples, promises to impact laboratory analysis with ever increasing significance. In 2005 Chen and Pactor defined metagenomics as “the application of modern genomics technique without the need for isolation and lab cultivation of individual species.”
What some scientists call the metagenomic revolution has resulted in a lot of DNA sequence data for various foodborne pathogens, Dr. Jaykus says, while emphasizing that, “relative to the volume of data available, we currently do not have the critical mass of scientists necessary to interpret it. We are also not entirely certain as to the practical use of those data in food safety management. This will become clearer in years to come, but the field is currently in its infancy stage.”
“Like genomics itself, metagenomics is both a set of research techniques, comprising many related approaches and methods, and a research field. In Greek, meta means ‘transcendent.’”
So says the U.S. National Research Council (NRC) Committee on Metagenomics in its 2007 publication The New Science of Metagenomics: Revealing the Secrets of Our Microbial Planet.
“In its approaches and methods, metagenomics circumvents the unculturability and genomic diversity of most microbes, the biggest roadblocks to advances in clinical and environmental microbiology,” the NRC relates.
“Meta in the first context recognizes the need to develop computational methods that maximize understanding of the genetic composition and activities of communities so complex that they can only be sampled, never completely characterized,” the NRC continues. “In the second sense, that of a research field, meta means that this new science seeks to understand biology at the aggregate level, transcending the individual organism to focus on the genes in the community and how genes might influence each other’s activities in serving collective functions.
“Individual organisms remain the units of community activities, of course, and we anticipate that metagenomics will complement and stimulate research on individuals and their genomes,” the NRC predicts. “In the next decades, we expect that the top-down approach of metagenomics, the bottom-up approach of classical microbiology, and organism-level genomics will merge.”—L.L.L.
Pesticide Residue Analysis: European Perspective
Thermo Scientific’s Dionex Integrion HPIC (high pressure ion chromatography) system, introduced globally on Feb. 1, 2016, is making an impact in the food industry with its high-pressure capabilities that enable fast analysis without compromising data quality, according to Khalil Divan, PhD, director of marketing, food and beverage, for Thermo Fisher Scientific, Waltham, Mass.
“Ion chromatography offers targeted analysis and excels in analyzing ionic and polar pesticides, such as glyphosate, glufosinate, and chlorate, which are not amenable to common multi-residue gas and liquid chromatography methods,” Dr. Divan relates. “Developments in technology have enabled the use of IC-MS/MS (mass spectrometry/mass spectrometry) for pesticide analysis, specifically highly polar pesticides, thermally unstable compounds and low volatility compounds.”
“By coupling ion chromatography analysis on the Thermo Scientific Integrion HPIC system with the company’s Q Exactive Orbitrap mass spectrometer, we are able to perform multi-residue analysis of very polar pesticides without derivatization steps before the analysis,” says Amadeo Fernández-Alba, PhD, professor and director of the European Union Reference Laboratory for Pesticide Residues in Fruit & Vegetables at the University of Almeria, Spain. “Also, the combination of technologies means that no isotopically labelled standards are necessary for quantitation. Simultaneous MS and MS/MS analysis results in a sufficient number of ions for identification and quantitation with very stable retention times.”
Launched in the U.S. on June 1, 2015, the Thermo Scientific Q Exactive GC (gas chromatography) system brings Orbitrap GC-MS technology to the routine lab for the first time, Dr. Divan says. “It is an easy-to-use, dedicated GC-MS system that we believe provides an unprecedented level of highly sensitive, routine-grade performance for both targeted and non-targeted analysis, along with high confidence quantitation for the ultimate sample analysis workflow,” he mentions. “This is achieved through the superior resolving power, mass accuracy, linear dynamic range and sensitivity that Orbitrap technology delivers, combined with the intelligent data processing workflows provided by Thermo Scientific TraceFinder software.”
“At RIKILT, we have been developing and validating an Exactive GC method for analyzing a large number of pesticides in fruits, vegetables, cereals and feed ingredients,” says Hans Mol, PhD, natural toxins and pesticides group leader with the RIKILT Institute of Food Safety at Wageningen University & Research, Netherlands. “We have found the Exactive GC to be a suitable technology that can meet the requirements of the European SANTE guidelines for this type of analysis. Moreover, Orbitrap GC-MS allows for an easier way of screening and quantifying a large number of pesticides in a wide variety of food samples by offering a high-selectivity, non-targeted data acquisition workflow.”—L.L.L.