Molecular Microbial Diagnostic Methods: Pathways to Implementation for the Food and Water Industries

By Nigel Cook

Molecular Microbial Diagnostic Methods: Pathways to Implementation for the Food and Water Industry was developed by recognized and experienced highlevel scientists. It’s a comprehensive and detailed reference that uncovers industry needs for the use of molecular methods by providing a brief history of water and food analysis for the pathogens of concern. It also describes the potential impact of current and cutting-edge molecular methods. This book discusses the advantages of the implementation of molecular methods, describes information on when and how to use specific methods, and presents why one should utilize them for pathogen detection in the routine laboratory. The content is also pertinent for anyone carrying out microbiological analysis at the research level, and for scientists developing methods, as it focuses on the requirements of end-users.

• Includes information on how to introduce and implement molecular methods for routine monitoring in food and water laboratories
• Discusses the importance of robust validation of molecular methods as alternatives to existing standard methods to help ensure the production of defendable results
• Highlights potential issues with respect to successful implementation of these methods

• Language: English
• Publisher: Academic Press
• Release date: Oct 6, 2015
• ISBN: 9780124171701

Book preview

Molecular Microbial Diagnostic Methods

Pathways to Implementation for the Food and Water Industries

Edited by

Nigel Cook

Fera Science Ltd., York, UK

Martin D’Agostino

Fera Science Ltd., York, UK

K. Clive Thompson

ALcontrol Laboratories, Rotherham, UK

Table of Contents

Cover

Title page

Copyright

Contributors

Preface

Chapter 1: Food industry current status

Abstract

Introduction

Molecular methods currently used

Laboratory automation in molecular methods

Challenges

Acceptance of use by the food industry

Compliance with current legal requirements

Disclaimer

Chapter 2: Future directions for molecular microbial diagnostic methods for the food industry

Abstract

Introduction

Evolution of food microbiology diagnostics: from petri dishes to PCR

Why introduce an alternative molecular diagnostic method?

Characteristics of an ideal food molecular method

Past and current challenges

Current challenges

Concluding remarks

Chapter 3: Current status of molecular microbiological techniques for the analysis of drinking water

Abstract

Introduction and overview

The current state of play

The influence of standard and reference methods

Molecular techniques for testing potable water quality

Application of molecular techniques to wastewater

Conclusions

Chapter 4: What is now required for water?

Abstract

Introduction and overview

Indicator organisms

Pathogens in drinking water

Total heterotrophic bacteria

Feasibility of the use of routine rapid water molecular methods

Future predictions

Conclusions

Chapter 5: CEN/ISO standards for both culture and molecular methods

Abstract

Introduction

Standards, standardization bodies, and structures in the microbiology of the food chain

Standards developed in the microbiology of the food chain

Status of novel technologies

Conclusions

Chapter 6: Laboratory validation, verification, and accreditation of molecular methods

Abstract

Alternative methods

Foundations of an accredited laboratory

Quality systems

Cornerstones of accreditation

Maintaining accreditation

Customer education

Health and safety

Chapter 7: DNA extraction: finding the most suitable method

Abstract

Boiling method

Column extraction

Magnetic beads

FTA™ cards

RNA extraction methods

Conclusions

Chapter 8: Assessing organism viability and interpreting genomic unit versus colony forming unit data for water and food borne microorganisms, such as Legionella, Campylobacter, Salmonella, and Listeria

Abstract

Introduction

Polymerase chain reaction

Assessing microbial viability

Overcoming the viability hurdle

When to use viability discrimination

Standardization and quality assurance

Proficiency testing

Conclusions

Chapter 9: MALDI-TOF MS: a rapid microbiological confirmation technique for food and water analysis

Abstract

Introduction and overview

Current confirmation methods and advantages of MALDI-TOF MS

MALDI-TOF MS Listeria speciation validation

Chapter 10: Chapter highlights, future requirements, and conclusions

Chapter highlights

Future requirements and conclusions

Index

Copyright

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Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility.

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Contributors

Cristina Barbosa,     Biopremier – Innovation and Services in Biotechnology, SA, Lisbon, Portugal

Matteo Capocefalo,     ALcontrol Laboratories, Rotherham, UK

Sandra Chaves,     Biopremier – Innovation and Services in Biotechnology, SA, Lisbon, Portugal

Samuel Collins,     Public Health England, Biosafety Investigation Unit, National Infection Service, Salisbury, UK

Nigel Cook,     Fera Science Ltd., York, UK

Martin D’Agostino,     Fera Science Ltd., York, UK

Colin Fricker,     CRF Consulting Ltd, Reading, UK

Mário Gadanho,     Biopremier – Innovation and Services in Biotechnology, SA, Lisbon, Portugal

Simon Gillespie,     Scientific Services Department, Scottish Water, Edinburgh, UK

Gwénola Hardouin,     AFNOR (French Standardization Body) – Food Industry and Healthcare Department, Saint-Denis La Plaine, France

Marta Hernández,     Molecular Biology and Microbiology Laboratory, Instituto Tecnológico Agrario de Castilla y León, Valladolid, Spain

Mark Jones,     Global Technology Division, Hill Laboratories, Hamilton, New Zealand

Frieda Jorgensen,     Public Health England, Food Water and Environmental Microbiology Laboratory, National Infection Service, Salisbury, UK

Keith A. Lampel,     Division of Molecular Biology, Food and Drug Administration, Laurel, MD, USA

Alexandre Leclercq,     Institut Pasteur – National Reference Centre and Collaborative Centre for World Health, Biology of Infection Unit, Organization for Listeria, Paris, France

Bertrand Lombard,     ANSES-LSAl (French Agency for Laboratory of Food Safety, Food, Environmental and Occupational Health & Safety – Laboratory for Food Safety), Maisons-Alfort, France

Samanta Marengo,     Global Technology Division, Hill Laboratories, Hamilton, New Zealand

Sofia Nogueira,     Biopremier – Innovation and Services in Biotechnology, SA, Lisbon, Portugal

Emma V. Ridley,     ALcontrol Laboratories, Rotherham, UK

David Rodríguez-Lázaro,     Department of Biotechnology and Food Science, Microbiology Section, Faculty of Sciences, University of Burgos, Burgos, Spain

K. Clive Thompson,     ALcontrol Laboratories, Rotherham, UK

Erika Y. Tranfield,     Bruker UK Limited, Coventry, UK

Jimmy Walker,     Public Health England, Biosafety Investigation Unit, National Infection Service, Salisbury, UK

Caroline Willis,     Public Health England, Food Water and Environmental Microbiology Laboratory, National Infection Service, Salisbury, UK

George Wilson,     Division of Molecular Biology, Wilson & Associates, LLC, Timonium, MD, USA

Preface

The aim of this book is to address the shortage of guidance on the implementation of molecular-based methods for routine microbiological laboratories in the food and water industries. What laboratories and their clients can expect from routine use of these methods is discussed and outlined.

The main objective of this book is to provide clarification and encourage progress toward implementation of robust, sustainable, and fit for purpose molecular-based methods for food and water that can be employed by most routine microbiological laboratories with special emphasis on pathogens.

Most of these analytical laboratories will already have in place systems such as good laboratory practice, robust quality assurance/quality control programs, and some form of accreditation, which are essential requirements in ensuring the production of robust defendable results and many of these will also hold ISO 17025 accreditation for the tests that they carry out.

However, in the background there are many research laboratories and specialist companies continually developing novel molecular-diagnostic methods, with a large number having been published in the peer-reviewed scientific literature. Yet, very few of these methods have actually been fully implemented in end-user laboratories for high-throughput routine analysis. This is attributed to a lack of full validation (according to ISO 16140) of these published molecular-based methods.

The book uncovers industry needs for the use of molecular methods by providing a brief history of food and water analysis and the pathogens of most concern. It discusses the potential advantages of the implementation of molecular methods, describes information on when and how to use specific methods, how to interpret results, and presents why one should utilize them for pathogen detection in the routine laboratory. We envisage that the main readership will be laboratory managers; senior microbiologists in routine food and water microbiological laboratories, major clients of these laboratories, test kit manufacturers; regulators, food companies, water companies, water management companies, and academics.

Chapter 1

Food industry current status

Keith A. Lampel*

George Wilson†

*    Division of Molecular Biology, Food and Drug Administration, Laurel, MD, USA

†    Division of Molecular Biology, Wilson & Associates, LLC, Timonium, MD, USA

Abstract

To ensure a safe, global food supply, food testing has remained a staple within the food industry. Today’s industry is compelled to adhere to government regulations as well as the demand from the consumer for wholesome, nutritious, and safe foods. Over the past few decades, the emergence of newer techniques to analyze foods (in particular, the polymerase chain reaction) has included the refinement of molecular-based technology, and its transition from the research laboratory to the analytical laboratory. It is important to appreciate that such methods have their strengths and weaknesses. Molecular-based technology via its sensitivity and speed has facilitated the decision-making process, and has found a home in the food analytical laboratory. This chapter describes some of this technology and its application to the food industry, and also some of the future challenges that are on the horizon.

Keywords

nucleic acid technology

PCR

whole genome sequencing

food safety

Food Safety and Modernization Act

Introduction

A major concern of the international community is the production of a safe food supply. The United Nations has several separate global agencies such as the World Health Organization and Food and Agricultural Organization as well as regional agencies such as the European Food Safety Authority (EFSA), the US Food and Drug Administration (US FDA), and the US Department of Agriculture, Food Safety Inspection Service (USDA FSIS). These agencies act as impactful influences on the world stage, either in the capacity to support food safety or risk analysis programs. The underlying theme to all these activities is the integration of intervention and prevention of foodborne illnesses that account for millions of deaths and illnesses throughout the world.

The food industry is a complex industry with vast differences and challenges in its contribution to the global food supply. Some products, such as produce commodities, can be sold directly to the consumer or used as ingredients in other foods. In addition, other food products can be consumed directly, processed, or undergo further processing, for example, cooking either by the consumer or retailer/restaurant. Also, the range of commodity type, that is, matrix composition, spans from the simple to complex, and to the food industry, regulatory agencies, and the consumer, the issues of food safety and product integrity reflect this physical aspect. Furthermore, other noted concerns for the food manufacturers include facility contamination, and the means to ensure a safe environment for product processing.

One of the more dramatic changes in food safety is the means by which foods are tested, where a shift has taken place from complete dependency on bacteriological protocols to the integration of advanced test methods, and has changed laboratory analytics significantly. Inroads in technological advances, particularly in nucleic acid-based methods, over the past decades have influenced greatly different aspects of the food industry. The transition of microbial and food analyses from the traditional shake and plate approach to the emphasis on rapid diagnostic assays, primarily based on molecular methods, has made profound impacts in the farm-to-fork paradigm. This concept can be further expanded to encompass now the concept of the global food supply, and efforts to ensure that all food is safe to eat. The United States has made a significant foray into this realm by the passage of the Food Safety and Modernization Act (FSMA) in 2011, which shifts some of the burden of food safety onto the food industry, including both US domestic producers and international providers.

As part of the shake and plate approach (culture dependent), enrichment of a food sample was a critical step to first isolate the specific organism/pathogen, followed by a panel of biochemical and serological tests to identify the microbe. With the advent of specific molecular techniques, that is, the polymerase chain reaction (PCR), direct examination of foods (culture independent) was an idea that had a brief lifetime. Issues that were encountered included the presence of PCR inhibitors in foods, low numbers of targeted pathogens present, requirement to separate physically each step of sample preparation and PCR to minimize cross contamination. In the current realm of molecular techniques, specifically next-generation sequencing (NGS), some of the same concerns should also be addressed, including the determination of the real level of detection of which NGS is capable in regard to food analysis.

Another consideration of the culture-dependent/-independent approaches is that to date several microbial pathogens are not culturable (or culture independent), or are extremely difficult to culture, and require additional laboratory equipment and time (Stewart, 2012). These would include norovirus, human parasitic protozoa, for example, Cyclospora, and perhaps, those microbes that have been physiologically impaired or environmentally adapted that growth on artificial media does not happen. By environmentally adapted, this would mean that microbes, under certain conditions, such as low water activity as in powdered infant formula, can survive (and not necessarily grow), and pose a difficult challenge to isolate.

Molecular-based tools have been applied to segments of the food industry for nonpathogen identification, particularly for starter culture microbes, such as the lactic acid bacteria (LAB). Also, the increase of intentionally added live microbials such as probiotics has accelerated this use. In these instances, proper strain identification can be a critical aspect for the regulatory and good manufacturing practices for each food application. The current use of molecular analysis, PCR amplification, and amplicon sequence has focused on the 16S ribosomal DNA sequence (Amor et al., 2007). The next generation molecular tool, whole genome sequencing (WGS) can be used to analyze the microbial content of foods for nonpathogens such as probiotics (Patro et al., 2015), starter cultures (Cogan et al., 2006), and spoilage organisms. As noted in a review by Ercolini (2013), the use of high-throughput sequencing (e.g., WGS) from the developing stage to implementation in the food industry should take note of the strengths and weaknesses of this technology.

Since 1987, when the first tomato plants were modified and tested in field experiments, the food industry has invested significantly in the use of molecular-based technology to generate genetically modified organisms (GMOs). These are also known as genetically engineered plants. Initially instituted to provide a sufficient food supply worldwide with adequate nutritional quality, the first GMOs were genetically manipulated via recombinant DNA methods to generate plants that were resistant to specific herbicides and pesticides. For the GMO analysis of a food or a food ingredient, molecular-based methods have been used for a variety of plants, such as soy, corn, and other common components in many varieties of food. As a detection tool, molecular-based methods can provide information to the food analyst as to the presence and identity of the GMO. Last, from a historical perspective, the first technical application of genetic engineering was to produce enzymes in the production of foods, such as with chymosin in cheese manufacturing.

The objectives of this chapter are to attempt to answer the following questions:

1. What molecular methods are currently being used routinely?

2. What are the challenges that need to be overcome?

3. If new technology is not being widely used, why not?

4. Does information from the molecular methods allow compliance with current legal requirements?

As diverse as the food industry is, those molecular-based technologies commonly used are initially described as well as their acceptance and application within the farm-to-fork paradigm. The food industry, as well as government regulatory agencies, and the consumer, all are affected by the efforts to ensure a safe food supply. Rapid detection and identification of any microbial agent in food that has the potential to do harm are the key underlying basis for the utilization of molecular-based protocols, a significant addition to the conventional standards of bacteriology used for decades. An excellent review of the current status of molecular-based methods available for food safety and how data generated from this technology can be interpreted has been previously described (Ceuppens et al., 2014) and for the basis for molecular methods (Gorski and Csordas, 2010).

Molecular methods currently used

Since the days of DNA/colony hybridization, the rapid development of molecular-based methods to detect the presence of microbial pathogens has impacted significantly the ease of food analysis. Technologies such as PCR and WGS have revolutionized the field of food microbiology to permit the rapid and specific detection of pathogens in foods (Table 1.1). Molecular-based testing can be used as a verification that the food safety program implemented at a production facility is working, and, to demonstrate that a food meets a given specification. In addition, molecular-based tests can also be applied to identification strategies, from genus to strain-specific identification, thus serving as an important tool for differentiating and identifying strains used in food production, and with intentional addition of live microbes, such as with probiotics.

Table 1.1

Comparisons of Molecular Methods

WGS, whole genome sequencing; RE, restriction endonuclease; LOD, limit of detection.

¹ Addition of EMA or PMA to the PCR can be used to distinguish live and dead cells.

² N/A, not applicable; usually need isolated cells or viral particles.

³ Can be used directly on food sample preparations but is restricted in use due to LOD.

PCR

The underlying basis of PCR (Saiki et al., 1985) is the in vitro amplification of targeted sites in either DNA or RNA molecules by mimicking the steps of in vivo DNA replication to yield amplicons (PCR products) of sufficient/detectable quantity for downstream manipulation or detection. Theoretically, one genome in a microbial cell or virus particle can act as a suitable template to generate detectable amplified products. Improvements in PCR chemistry, enrichment methods, the availability of kits, and instrumentation since PCR came onto the scene have now enabled this technology to become a common tool in analytical laboratories as well as a research and development application. Its potential as a powerful tool for microbial diagnostics as well as a means to evaluate genomes has been well-established.

The advantages of PCR for the detection of specific microbes in food samples lie within its inherent specificity. It can detect nonculturable organisms and identify atypical colony formers, such as sorbitol-fermenting Escherichia coli O157:H7, rapid analysis, and provide critical information on the serotype, genotype, or pathotype. Conversely, as compared with culture-based methods, PCR

1. Can carry a higher cost regarding technical operators, reagents, and instruments.

2. Is adversely affected by food-derived inhibitory substances, and thus requires stringent controls to ensure that the lack of amplification is due to actual absence of target, and not by reaction inhibition (Rossen et al., 1992).

3. If it has a poor LOD, its use may lead to false negative results.

4. Does not distinguish between live and dead cells or infectious or noninfectious viral particles.

Real-time PCR

Real-time PCR enables concurrent amplification and quantitation of nucleic acid targets, with real-time data readout facilitated by the incorporation of DNA-intercalating dyes and/or fluorescent probes added to the PCR mixture prior to amplification. The power of real-time PCR techniques lies in the ability simultaneously to detect and quantify during DNA amplification. As a result, real-time assays can have an improved LOD, and can provide results faster than conventional assays that depend on some form of gel electrophoresis for fragment size confirmation. Also, there is less potential for cross contamination with real-time PCR, as there is no need to open the reaction tube during the method. Moreover, the availability of multiple dyes and probes that fluoresce at different wavelengths allows for the development of multiplex assays using this rapid detection method. Moreover, the number of samples that can be analyzed at the same time can be expanded to hundreds. In addition, the time of analysis, previously 2–4 h, has been reduced to less than 1 h in a number of different commercially available instruments.

Digital droplet PCR

A recently developed technology, digital PCR, can be performed on several different platforms, such as microfluidic or droplet systems. One of the key features of droplet PCR is that template DNA molecules are separated on droplets (typically ∼20,000 nL) that are formed in a water–oil emulsion. In this platform, digital PCR partitions nucleic acids (template) onto hundreds to thousands of these individual droplets; partitioned templates on droplets will be amplified whereas droplets without template will not. Incorporation of dye-labeled probes into amplified products generate signals that are displayed subsequently on the instrument. Digital PCR can also quantify the amount of template initially analyzed. Another advantage to this technology is that it can be less sensitive to PCR inhibitors that confront classical and other means of real-time PCR amplification. This promising technique is described in Chapter 2.

Isothermal techniques

The high demand for handheld diagnostic devices that can be used to detect pathogens in the field has led to the development of a number of isothermal amplification methods (reviewed by Gill and Ghaemi, 2008). Unlike amplification reactions such as PCR that require several reaction temperatures, isothermal reactions can be performed at a uniform temperature, thereby eliminating the need for expensive equipment. Several variations of this technology exist, and some are discussed in later sections. Variations of isothermal amplification techniques include transcription-mediated amplification, nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification of DNA (LAMP), isothermal multiple displacement amplification, and helicase-dependent amplification (HDA). These techniques use DNA or RNA as target molecules for amplification, and have been successfully used in diagnostic applications.

Sequencing

WGS, as its name implies, is one of the most significant technologies at hand today. Briefly, it is a means to determine the entire nucleotide sequence of a microbial or viral genome and can be accomplished in a relatively short period of time, taking only a few days from sample preparation to the sequence readout. Analysis of generated sequence data requires powerful computer programming to assemble the overlapping fragments generated from the sequence reactions. The amount of coverage, that is, the number of times that a particular nucleotide has been sequenced is critical to both the assembly and accuracy of the final genome sequence information. Metagenomics can enable genome sequencing of the entire microbial population present in a sample, particularly in situations when microbes, such as some viruses, are not culturable (Handelsman, 2004). As a note, in addition to DNA, total RNA can also be sequenced.

Microarrays

DNA microarray technology has evolved directly from conventional DNA–DNA or DNA–RNA hybridization techniques, such as Southern blotting. Microarrays consist of an arrayed series of defined oligonucleotides, referred commonly to as probes that are covalently bound to a solid support surface, such as glass, silicon chips, or even to microscopic beads. Collections of spots (probes) are arranged in an orderly prescribed landscape so that capture of the probe–target hybridization, usually detected by means of fluorescence, can be automatically scored. In each well, the target is usually a specific gene or unique region of a microbial chromosome or plasmid. These hybridizations can be monitored and perhaps quantified, based on the amount of fluorescence generated by the number of probe–target bound molecules. Microarray technology is revolutionary because unlike traditional DNA hybridization that works with one probe at a time, arrays can handle tens of thousands to millions of probes all placed onto one chip. This technology has been routinely used in clinical laboratories and its transformation to the food industry has been limited to date.

Restriction enzyme-based methods

PFGE has been a gold standard for the characterization of microbes, and has been the backbone for the Center for Disease Control and Prevention’s PulseNet Laboratory Network. PFGE utilizes restriction enzymes, particularly low cutting frequency enzymes, such as SmaI and NotI, to generate a DNA fingerprint based on the resultant pattern of the genome digestion. PFGE is highly discriminatory, successfully differentiating at the strain level, is reproducible, and generates a banding pattern that is easy to interpret. The DNA fingerprint is analyzed by specific computer programs so that the pattern can be given a specific profile number and databases with PFGE patterns can be easily searched to find matching isolates. In food safety systems, the information generated from bacterial colonies can be used in different programs, such as surveillance, outbreak investigations, as well as provide regulatory agencies and the food industry data that can be used to trends of specific isolates.

Ribotyping

Ribotyping is based on the inherent ability of single-stranded DNA molecules to hybridize with one another. Isolated microbial DNA is digested with restriction enzymes, and the cut DNA is then analyzed by agarose gel electrophoresis. The DNA is then transferred to a nitrocellulose or nylon membrane for hybridization with labeled 16S, 23S, or 5S rRNA gene probes. Because bacteria have multiple copies of rRNA operons in their chromosome, several fragments in the restriction digest mixture hybridize with the probe, resulting in a microbial fingerprint. Ribotyping, in general, has a greater discriminatory power at the species level than at the strain level. One commercially available system is based on restriction patterns of digested genomes with specific rRNA coding regions, as indicated earlier. The restriction fragment length polymorphism (RFLP) patterns generated, in digitized from, are unique for each pathogen tested; and therefore, data generated during analysis of foods or the environment can be compared to a reference database available by the manufacturer. These unique strain-level fingerprint patterns have been used for starter culture characterization, identification of cross contamination, and trace the source of contamination.

Laboratory automation in molecular methods

Traditional microbiology laboratories, especially those with large test volumes, are evolving toward laboratory automation to improve operational efficiencies, cost effectiveness, and reduced time to results, while at the same time maintaining a high degree of accuracy in the results being reported. Automation of molecular methods is taking place through the use of robotics, coupled with computer programming, which are the corner stone of commercially available instrumentation. Today, some companies offer walk-away automated systems. The various technologies described in this chapter take the food sample from the front end of sample preparation, through the pipetting and dispensing of test reagents, the incubation and detection, and the reporting of test results. Readers can visit laboratory information websites, www.rapidmicrobiology.com, that identify commercially available automated systems for food microbiology.

Challenges

Sample preparation

One of the critical components to any successful analytical protocol that should be carefully considered is the preparation of food samples for downstream analyses. Inhibition by compounds/chemicals found in food was a major hurdle in the initial application of earlier molecular-based techniques (e.g., PCR) employed by laboratories years ago. Today, with the advent of better commercially available extraction/DNA or RNA isolation kits, most of the inhibition issues have been resolved.

In the current use of molecular-based technology, this becomes even more evident when considering what microbes are targeted for detection/identification. NGS, an upcoming science for different aspects in the food industry, faces similar challenges. For example, are all extraction kits equally useful for Gram positive or negative bacteria, yeasts/fungi, viruses, or parasitic protozoa? In one study, several commercial extraction kits for bacterial genomic DNA were evaluated and, not surprisingly, not all were equivalent in regard to quantitative DNA yield and PCR amplification (Irwin et al., 2014).

In regard to culturable versus nonculturable microbes, the efficiency of extraction methods becomes the critical point for accurate analysis of food samples. Several key facets should be noted with both sets of microbial populations. For microbes that can be grown in liquid media, the number of targeted organisms, after enrichment, will depend on the microbial load of indigenous populations found in each food as well as its physiological state, for example, stressed cells. In addition, the inherent bias toward one type of bacteria, for example, Gram negative or Gram positive, can affect the final size of the targeted population. Therefore, the requisite number of genomes required for any molecular-based method may be influenced by several factors.

The situation with nonculturables is more tenuous since these organisms cannot be grown in vitro; and therefore, the number of targeted cells is fixed. Therefore, the extraction process should be efficient for both Gram positive and negative bacteria, as well as, other nonculturable microbes, such as some viruses and parasitic protozoa. With respect to NGS, similar to PCR, the LOD (discussed later) becomes a significant factor to yield an accurate result, otherwise, false negative data can be generated that may affect human health (foodborne illnesses) or loss of product integrity (spoilage).

Limit of detection

As with any method, the ability to detect low numbers of targeted microbes would be an ideal asset to verify that a food commodity is safe to consume – an important factor for the consumer. In addition, food that is free of microbes that can affect product integrity (a positive aspect for the food industry) will pose no concerns for the regulatory agencies. As stated earlier, PCR techniques are theoretically capable of detecting one target molecule per reaction. To achieve an optimal LOD, enrichment media formulations, incubation time, and temperatures should be optimized in the development phase of molecular methods.

Some methods incorporate the use of immunomagnetic beads to capture and concentrate the target bacteria after only a few hours of incubation. The laboratory total test time to results is critical to the producers of food commodities that have a short shelf life, that is, fish, raw meats, and poultry.

In regard to NGS, the same standards that are applied to PCR (e.g., live vs. dead cells distinction) should be incorporated into any risk management scheme as well as product security. Although the strength of NGS lies in the ease of microbial identity, with appropriate computer power and software, a comprehensive and widely accepted protocol should be established that incorporates a validated method for specific uses. The challenge ahead for designing a validation plan for NGS is several fold; technological advancements may utilize different chemistries and a validation study for each new method may be required; standard criteria need to be established to be used in strain identification, for example, how many single nucleotide polymorphism (SNPs) constitute a new isolate from the original strain? In addition, since each food commodity carries its own background bacterial populations, their effect on the recovery or identification of the targeted microbe may significantly be impacted. The LOD from foods may be quite different from pure cultures or from exponentially grown cells.

Live versus dead/infectious versus noninfectious

One of the major drawbacks with the use of PCR is its inability to distinguish between live and dead cells, or in the case of viruses, infectious, and noninfectious particles. Some adaptations to PCR, such as the addition of ethidium monoazide or propidium monoazide to the reaction mix, have been reported to differentiate live and dead cells (Nocker et al., 2006) as well as infectious and noninfectious viruses (Fittipaldi et al., 2010; Parshionikar et al., 2010). The same issue also applies to NGS, specifically when used for direct examination of food samples. At this time, no distinction has been made between live and dead cells or infectious and noninfectious agents with any molecular-based method used to directly examine foods. Consequences of these results reverberate with the consumer and the food industry. Specifically, a positive match of whole genome sequence data to a known pathogen can indicate the presence of a live/infectious agent or a moribund or noninfectious microbe.

Interpretation of results (PCR+/culture−)

Situations can arise in the analysis of food samples that require a careful review of the data to make a critical decision as to the acceptability of that food. One such anomaly would be a PCR positive result yet the targeted organism was not able to be cultured. Would this indicate that the targeted cells were dead and could not be isolated but that their genome was amplifiable by