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Food Analytical Methods ISSN 1936-9751Volume 8Number 2 Food Anal. Methods (2015) 8:255-271DOI 10.1007/s12161-014-9915-6

Rapid Methods for Quality Assurance ofFoods: the Next Decade with PolymeraseChain Reaction (PCR)-Based FoodMonitoring

D. De Medici, T. Kuchta, R. Knutsson,A. Angelov, B. Auricchio, M. Barbanera,C. Diaz-Amigo, A. Fiore, E. Kudirkiene,A. Hohl, et al.

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Rapid Methods for Quality Assurance of Foods: the Next Decadewith Polymerase Chain Reaction (PCR)-Based Food Monitoring

D. De Medici & T. Kuchta & R. Knutsson & A. Angelov & B. Auricchio & M. Barbanera &

C. Diaz-Amigo & A. Fiore & E. Kudirkiene & A. Hohl & D. Horvatek Tomic & V. Gotcheva &

B. Popping & E. Prukner-Radovcic & S. Scaramaglia & P. Siekel & K. A. To & M. Wagner

Received: 25 February 2014 /Accepted: 10 June 2014 /Published online: 2 July 2014# Springer Science+Business Media New York 2014

Abstract Microbiological analysis is an integral part of foodquality control, as well as of the management of food chainsafety. Microbiological testing of foodstuffs complements thepreventive approach to food safety activities based mainly onimplementation and application of the concept of HazardAnalysis and Critical Control Points (HACCP). Traditionalmicrobiological methods are powerful but lengthy and cum-bersome and therefore not fully compatible with current re-quirements. Even more, pathogens exist that are fastidious tocultivate or uncultivable at all. Besides immunological tests,molecular methods, specifically those based on polymerasechain reaction (PCR), are available options to meet industryand enforcement needs. The clear advantage of PCR over allother rapid methods is the striking analytical principle that isbased on amplification of DNA, a molecule being present inevery cell prone to multiply. Just by changing primers and

probes, different genomes such as bacteria, viruses or parasitescan be detected. A second advantage is the ability to bothdetect and quantify a biotic contaminant. Some previouslyidentified obstacles of implementation of molecular methodshave already been overcome. Technical measures becameavailable that improved robustness of molecular methods,and equipment and biochemicals became much more afford-able. Unfortunately, molecular methods suffer from certaindrawbacks that hamper their full integration to food safetycontrol. Those encompass a suitable sample pre-treatmentespecially for a quantitative extraction of bacteria and virusesfrom solid foods, limited availability of appropriate controls toevaluate the effectiveness of the analytical procedure, thecurrent inability of molecular methods to distinguish DNAfrom viable cells and DNA from dead or non-cultivable cells,and the slow progress of international harmonisation and

D. De Medici (*) : B. Auricchio :A. FioreDepartment of Veterinary Public Health and Food Safety, IstitutoSuperiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italye-mail: [email protected]

T. Kuchta : P. SiekelDepartment of Microbiology and Molecular Biology,Food Research Institute, Priemyselná 4, P. O. Box 25,Bratislava 82475 26, Slovakia

R. KnutssonSecurity Department, SVA National Veterinary Institute, 75189 Uppsala, Sweden

A. Angelov :V. GotchevaDepartment of Biotechnology, University of Food Technologies, 26Maritza Blvd, 4002 Plovdiv, Bulgaria

M. Barbanera : S. ScaramagliaLaboratorio Coop Italia, 40033 Casalecchio di Reno, Bologna, Italy

C. Diaz-Amigo : B. PoppingEurofins CTC GmbH, Stenzelring 14b, 21107 Hamburg, Germany

E. KudirkieneDepartment of Food Safety and Quality, Lithuanian University ofHealth Sciences, Veterinary Academy Tilžės Str. 18,LT-47181 Kaunas, Lithuania

A. HohlDepartment of Food Science and Technology, Institute of FoodScience, University of Natural Resources and Life Sciences,Muthgasse 18, Vienna, Austria

D. Horvatek Tomic : E. Prukner-RadovcicDepartment of Poultry Diseases with Clinic, Faculty of VeterinaryMedicine, University of Zagreb, Heinzelova 55, 10000 Zagreb,Croatia

K. A. ToSchool of Biotechnology and Food Technology, Hanoi University ofScience and Technology, No. 1, Dai Co Viet, Hanoi, Vietnam

M. WagnerDepartment for FarmAnimals and Veterinary Public Health, Institutefor Milk Hygiene, Milk Technology and Food Science, Universityfor Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria

Food Anal. Methods (2015) 8:255–271DOI 10.1007/s12161-014-9915-6

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standardisation, which limit full acceptance of PCR-basedmethods in food control. The aim of this review is to describethe context and the prospects of PCR-based methods, as wellas trends in research and development aimed at solving thenext decade challenges in order to achieve full integration ofmolecular methods in food safety control.

Keywords Food safety . Rapid methods . Quality control .

PCR

Introduction

Microbiological analysis is an integral part of technologicalfood safety quality control and monitoring systems (Fig. 1).Microbiological testing of foodstuffs complements activitiesbased on implementation and application of the concept ofHazard Analysis and Critical Control Points (HACCP). Inaddition to at-plant-implemented self-control programs, defi-nition and application of microbiological criteria (MC) are asalient feature in food legislation and in providing inputs tomicrobiological risk assessment (MRA) (CodexAlimentarius2012; Hoorfar 2011).

The advances in MRA nowadays extrapolated as quantita-tive microbial risk assessment (QMRA), and the translation ofQMRA outcomes into risk management frameworks has ledto the establishment of a series of additional food safety riskmanagement metrics such as Food Safety Objectives (FSOs),Performance Objectives (POs) and Performance Criteria (PC).Where QMRA models are available or these metrics havebeen elaborated, those allow the establishment of a moredirect relationship between MC and public health outcomes(Manfreda and De Cesare 2014).

Whereas FSO is defined as “the maximum frequency and/or concentration of a (microbial) hazard in a food at the time ofconsumption that provides the appropriate level of protectionof health protection”, PO defines the maximum frequencyand/or concentration of a microbial hazard at a specific stepof food chain. The concentration of microbial hazard shouldbe lower than the defined FSO if micro-organisms can prolif-erate in the food product during its shelf life. As a directconsequence of the introduction of the concept of FSO/PO,the impact of assessing MC will increase, since these provideobjective means that PO or PC (or FSO) are being met at thedifferent steps of food chain. POs unlike FSOs are intended tobe easily measurable and verifiable.

FSO provides a recognised acceptable tolerance inmicrobiological hazards, and it also represents movingaway from the precautionary principle (“zero tolerance”concept) that was widely associated with microbiologi-cal end-product testing. A statement illustrating thischange in paradigm is moving test activities “from thepoint of sale” to the “point of risk”. These consider-ations, however, imply a move from qualitative datageneration based on detection of an organism to aquantitative approach. In addition, determination of POat different steps of food chain requires the definition ofa quantitative MC that might be sometime rather lowand should encompass the quantification of infectivemicro-organisms only.

This new risk-based metrics approach to food safety movesthe MC from qualitative to quantitative, and the microbiolog-ical methods have the main role to evaluate the effectivenessof the HACCP systems by a quantitative approach. Prolongedanalysis times typical of classical culture-based microbiolog-ical methods are incompatible with the HACCP systems.

Fig. 1 The main purposes ofmicrobiological criteria accordingto “Proposed draft revision of theprinciples for the establishmentand application ofmicrobiological criteria for foods”Codex Committee on FoodHygiene Forty-fourth SessionNew Orleans, United States ofAmerica, 12–16 November 2012

256 Food Anal. Methods (2015) 8:255–271


These systems could benefit from the application of the so-called rapid methods, such as molecular–biological methods(Amagliani et al. 2012). The development of rapid, cost-effective and automated methods for the determination ofpathogenic bacteria, integrated with preventive strategies suchas HACCP, can significantly improve safety throughout thefood chain (Hofstra et al. 1994; Rodriguez-Lazaro et al. 2014).These considerations served for discussions in an expert groupon “rapid novel technologies, standardisation andharmonisation” of the European project “safe food forEurope” (acronym “FoodSeg”). The aim of this review is toreport the discussion outcome concerning the need for futureresearch activities at European level, in order to reduce oreliminate the drawbacks of molecular methods and to betterintegrate the use in food safety control.

Food Safety and Food Hygiene Criteria

The Regulation (EC) No. 2073/2005 and following amend-ments establish two types of criteria and require that foodbusiness operators take corrective action when these criteriaare not met:

1. Food safety criteria should be used to assess the safety of aproduct or batch of foodstuffs.

2. Process hygiene criteria should be used to ensure that theproduction processes are operating properly.

Food safety criteria are applicable throughout the shelf lifeof foodstuffs placed on the market. These criteria areestablished for micro-organisms (usually pathogenic micro-organisms), their toxins or metabolites in various food com-modities, e.g. Listeria monocytogenes in ready-to-eat foods,Salmonella in different foods of animal origin and vegetables,Staphylococcal enterotoxin in certain cheeses, milk powderand whey powder, Cronobacter spp. in infant formula foodsand histamine in fish. If a food safety criterion is not met, thisusually means that the food business operator will not be ableto place the foodstuff on the market or will need to remove thefood from the market (as stated in European CommissionRegulation 178/2002 laying down general food safety require-ments). The food business operator has furthermore to under-take steps that ensure that future production meets thecriterion.

Process hygiene criteria are used to assess the correctfunctioning of production processes with respect to hygiene.They are applicable to foodstuffs both during and at the end ofthe manufacturing process but before the commercialisationof the food products. Process hygiene criteria of a productionprocess are usually evaluated using indicator micro-organisms, which provide information on the hygienic status

of food production in the food chain and indicate thefollowing:

& The level of biological and faecal contamination (indirect-ly—the level of risk from intestinal pathogens)

& The effect of technological processes& Prognosis for the future spoilage of products stored

improperly& Epidemiological risks of potentially pathogenic micro-

organisms

Specific bacterial indicators are available for defined typesof food products and include Escherichia col i ,Enterobacteriaceae, staphylococci and enterococci (eitherstaphylococci and enterococci, or Staphylococcus sp. andEnterococcus sp.) (Kornacki and Johnson 2001). If a processhygiene criterion is not met, the product can be placed on themarket, but the food business operator must review the pro-duction processes and improve process hygiene to ensure thatfuture production will meet the criteria.

Food Safety and Food Analytics

The current legislation urges the food business operator(FBO) to take the responsibility for the production ofsafe food. FBOs establish self-control systems andHACCP concepts as backbones to reach this goal. It isessential to understand that the majority of food samplesare nowadays tested at FBO level, whereas the numberof food samples tested by public health authorities isdecreasing, in many cases due to cost-saving programs.In Austria, the public health sectors test around 35,000samples a year, which represent a small number com-pared to the samples tested at FBOs within in-houseroutines. The change in the legal framework segregatedfood testing into applications used at food businessoperations and those used by control authorities forfood control. FBO, however, can only take over thisresponsibility if a sample can be analysed within thetime the food is stored at a food enterprise (FE).Traditional microbiological methods are often consid-ered to be unsuitable for evaluating food safety criteriaat FEs due to their long duration, which may lead to asituation that the product is already supplied, or evensold, before data on its safety status are available. As aconsequence, most quality managers ensuring food safe-ty at food enterprises have moved into using rapidtechnologies, whereas the public health authorities stillwork with culture-dependent technology that is mostlystandardised by international standardisation bodies suchas the European Committee for Standardization (ComitéEuropéen de Normalisation (CEN)) or International

Food Anal. Methods (2015) 8:255–271 257


Organization for Standardization (ISO). Rapid, sensitiveand accurate detection methods are necessary to beintegrated with HACCP to improve safety of products.Therefore, research efforts devoted to the developmentof proprietary techniques (PTs) are still ongoing, and thecurrent literature contains many references to that ex-tent. A PT measures the same analyte as the corre-sponding reference (classical microbiological) methodand facilitates response through simple performanceand/or automation and better analytical performance (ac-curacy, sensitivity, specificity). If the time parameter isprioritised, the term “rapid methods” instead of PT isoften preferred.

Immunological and molecular biological nucleic acid-based methods are currently the best examples of PTcurrentlyimplemented into food testing. This review focuses in partic-ular on polymerase chain reaction (PCR)-based technologiessince immunological test was successfully introduced to foodtesting in the previous decade and not much progress withregard to the basic analytical principle that was achieved fromthereon. The introduction of amplification techniques (PCRand real-time PCR) in microbial diagnostics has beenestablished in research laboratories as a valuable alternativeto traditional detection methods. Compared to immunologicalmethods, PCR-based technologies have two essential advan-tages: (i) the biochemical principle of in vitro DNA amplifi-cation, since DNA is a suitable analyte in every organism andthousands of different organisms can be tested by very smallchanges in the analytical format; and (ii) the ability to quantifythe analyte. Speed, excellent selectivity, specificity, sensitivityand potential for automation are further important advantagesof amplification techniques. These advantages compared totraditional detection methods might well encourage end-usersto adopt amplification techniques in routine testing for food-borne pathogens (Rodriguez-Lazaro and Hernandez 2013).

Some previously identified obstacles of implementation ofmolecular methods have been already overcome. The robust-ness of PCR-basedmethods has been improved by developingreal-time PCR assays, and equipment and biochemical re-agents have become much more affordable (Rodriguez-Lazaro et al. 2013). Unfortunately, PCR-based methods stillsuffer from certain drawbacks that hamper their full integra-tion to food safety control, and those are the following:

& Lack of specific pre-treatments that allow quantitativeextraction of micro-organisms from foods, in particularsolid foods

& Inability of molecular method to distinguish DNA fromviable and dead cells

& Lack of definition of appropriate controls to evaluate theeffectiveness of the analytical procedure

& Slow progress of the international harmonisation andstandardisation of molecular methods

Molecular Methods in Food Analysis

At the beginning of the second millennium, molecularmethods were considered not suitable for routine testing offood products, because they looked good and worked wellonly if used in research laboratories with skilful technicians.However, in the last decade, nucleic acid-based methodsgradually started to replace or complement the culture-basedmethods in food control (Kuchta et al. 2014) and becameextensively utilised as alternative rapid methods for detectingpathogenic bacteria. For several pathogens, complete detec-tion procedures of different commercial and non-patentedmethods, including enrichment, were developed and validated(Rodriguez-Lazaro and Hernandez 2013; Rodríguez-Lázaroet al. 2007).

In this context, several international studies were per-formed in particular in the Framework of different EUfounded projects. Particularly, an interlaboratory trial, involv-ing 15 laboratories from 13 European countries, was conduct-ed to evaluate the performance of a non-proprietary gel-basedPCR method for the detection of Salmonella on artificiallycontaminated chicken rinse and pig swab samples (Malornyet al. 2004).

Another assay for the detection of L. monocytogenes wasevaluated in a collaborative trial involving 12 European lab-oratories (D’Agostino et al. 2004).

The performance of a PCR-based method for the detectionof E. coli O157, previously validated on DNA extracted frompure cultures, was evaluated on spiked cattle swabs in aninterlaboratory trial, including 12 participating laboratoriesfrom 11 European countries (Abdulmawjood et al. 2004).

In addition, an ISO 6579 compatible enrichment coupled toan easy and inexpensive DNA extraction and a consolidatedreal-time PCR assay for detecting Salmonella in pork meatwas evaluated in a ring trial performed in 13 laboratories fromseven European countries (Delibato et al. 2014).

Twelve laboratories from six European countries partici-pated in a ring trial for evaluation of a real-time PCR-basedmethod for the detection of L. monocytogenes in soft cheese asfood model since representing a difficult matrix for bacterialDNA extraction and real-time PCR amplification(Gianfranceschi et al. 2014).

Also, a ring trial study for evaluating the performance of areal-time PCR-based method for the detection of botulinumneurotoxin producing clostridia in food was performed in fourdifferent European laboratories in Italy, France, theNetherlands and Sweden using 47 strains and 30 clinical andfood samples linked to botulism cases (Fenicia et al. 2011). Inaddition, this ring trail was performed in order to improvequality assurance also for the detection of deliberate contam-ination in the food chain (Knutsson et al. 2011).

A multi-centre collaborative trial involving eight laborato-ries in five European countries (two laboratories in France,



Italy and the Netherlands and one laboratory in Denmark andSweden) was also performed in order to validate a real-timePCR-basedmethod for the detection ofClostridium botulinumtypes C and D and their mosaic variants C–D and D–C(Woudstra et al. 2013).

Differently from the detection of bacteria in food whererapid method was used alternatively to the classical culturalmethod, real-time PCR is the exclusive method for the detec-tion of viruses in food.

Norovirus do not grow in any cell culture-based detectionsystem (Duizer et al. 2004), while human wild-type hepatitisA virus (HAV), even if rarely can grow in a cell culture, isgenetically unstable and requires several weeks to months inculture before it can be detected (Konduru and Kaplan 2006).Consequently, a real-time PCR was raised as an electivemethod for the detection of the presence of these viruses infood, water or environmental samples.

Since the presence of enteric viruses in food samples iswidely recognised (Koopmans et al. 2002), in particular inmussels (Suffredini et al. 2014; Pavoni et al. 2013) and infresh product as frozen soft fruits (Rizzo et al. 2014), differentprotocols were proposed for the detection of HAV (Sanchezet al. 2007) and noroviruses (Stals et al. 2013) in food usingreal-time PCR. Recently, a method for the detection ofnoroviruses was validated on naturally contaminated freshproduce samples (El-Senousy et al. 2013).

Advanced Sample Treatment and Nucleic Acid Extractionof Bacteria and Viruses

The pre-PCR processing step, including sample preparationand nucleic acid extraction, is one of the most critical steps inmolecular biological analysis. It has considerable influence onreproducibility of the analysis especially when quantitativedetection of a specific pathogen is needed. Most of the mo-lecular biological methods used are in vitro methods, whichwork perfectly in a standardised analytical environment that ispractically met only under perfect laboratory conditions. Thetheoretical or “in vitro” performance of these methods ischallenged when differences in analytical skills or sample-dependent inhomogeneity come into play. The sample prepa-ration and DNA extraction steps should aim at minimizinginhomogeneity through achievement of a certain level ofrobustness and analyte quality that should be mainlycharacterised by analyte purity and integrity. Moreover, forquantitative purposes, the amount of analyte initially presentin the food sample should not be changed through the se-quence of manipulations in the analytical chain. The holisticview on all steps in the analytical chain is unequivocal. This isof importance since extensive research on some steps has ledto an overwhelming number of papers (so more than 200papers have been published on PCRs for Salmonella),

whereas other steps remained widely out of focus. The ana-lytical chain is composed of sample preparation, nucleic acidextraction and application of the amplification or detectionplatform (Rossmanith and Wagner 2011). Unlike the clinicalmicrobiology that works on a limited number of types ofmostly liquid specimens, food samples are highly heteroge-neous in composition and physical state. The nucleic acidsneed to be purified from all other compounds that mightoriginate from the food and could influence the biochemicalkinetics of PCR. It has to be considered that pre-PCR process-ing step is often not included in validation. Therefore, it issuggested that standardised PCR methods also includemethods for sample processing in order to overcome someof the major problems associated with the sample by concen-trating the target organism, reducing the presence of inhibitorysubstances (collagen, polysaccharides, proteinases and calci-um) and converting a heterogeneous sample into a homoge-neous sample suitable for PCR (Postollec et al. 2011; Malornyet al. 2003; Hoorfar et al. 2004a; Radstrom et al. 2004;Radstrom et al. 2008). Clearly, it is required that PCR is evenmore adapted for “field use, which is quite far from perfectlaboratory conditions.

The procedures of sample preparation have gradually re-ceived increasing attention from both academia as well asindustry, mirrored by an increase in publications as well asapplications of such methods (Brehm-Stecher et al. 2009).However, so far, the topic remains a challenge for researchas there are many prerequisites for efficient sample prepara-tion methods (Rossmanith and Wagner 2011; Stevens andJaykus 2004; Rossmanith and Wagner 2010). The vast num-ber of procedures used for this purpose indicates that thischallenge is still a subject of intensive research. Most methodshave drawbacks, such as insufficient size of the processedsample, insufficient recovery, extensive labour requirementsor high costs. Particularly, promising approaches to overcomesample-related problems are separation using coated magneticbeads specific for the analyte, flotation or other physicalmethods and the lysis of whole sample matrices(Rossmanith et al. 2007).

Improved immunoseparation methods have become avail-able for rapid identification of cultivable and non-cultivablemicro-organisms (Olsvik et al. 1994). The principles andapplication of the method are simple but rely on antibodiesof suitable specificity under the conditions of use. Purifiedantigens are typically biotinylated and bound to streptavidin-coated paramagnetic particles. Immunomagnetic beads (IMB)have a potential to become convenient tools also for multi-pathogen detection. Tu et al. (2011) used streptavidin-coatedmagnetic beads conjugated with biotinylated capture antibod-ies to separate both E. coli O157:H7 and SalmonellaTyphimurium in a culture system. These were multi-pathogen capture IMB (IMB-M). The efficacy of these beadswas investigated in both pure and mixed culture suspensions,



as well as in inoculated spinach and ground beef. It was foundthat IMB-M were just as effective as the mixture of IMBagainst E. coli and Salmonella in capturing cells of bothorganisms. Using this approach, it was possible to detect1 CFU/g of E. coli and 100 CFU/g of Salmonella in a mixedculture, after 6-h enrichment at 37 °C.

Another approach combines cell capture using magneticparticles (MP) with multiplex PCR, offering an efficient,rapid, sensitive and inexpensive alternative for the routinedetection of food-borne pathogenic bacteria in food andstools. A recently published study demonstrated a novel mag-netic capture–multiplex PCR assay for the simultaneous de-tection of E. coli O157:H7, Salmonella and Shigella in stooland food samples (Zuo et al. 2011).

Innovative sample preparation strategies should also over-come the problem that food-borne pathogens are usuallypresent in food commodities at very low numbers.Therefore, novel sample preparation technologies processhigher sample volumes. A re-circulating automaticimmunoseparator Pathatrix® Auto has recently gainedAOAC-RI approval for its Salmonella, Listeria and E. coliO157 detection product range. This technology providesshortening of the time necessary for enrichment prior toPCR. The system is able to concentrate bacterial cells fromup to 60 ml of pre-enriched food samples, producing a con-centrate of 100 μl. The concentrate is processed by cell lysisand DNA extraction and further subjected to real-time PCR. Apooling option of the system is available for applicationswhere negative results prevail (Shields et al. 2012;Papafragkou et al. 2008; Lau et al. 2012; Zhao et al. 2012).

This re-circulating immune separation technology has beenalso applied to the concentration of HAV from food samples(Papafragkou et al. 2008). The hypothesis on the workingmechanism is that cationically charged magnetic particlescould be used in conjunction with this magnetic capturesystem for the concentration and purification of the virus fromfood matrices. The separation of the viral agent is facilitatedby binding the negatively charged proteins of the virus capsidto the positively charged magnetic particles (Papafragkouet al. 2008). A major advantage of the system is that largevolumes can be analysed (25 g of food, plus 225 ml of buffer),and the resulting sample is concentrated up to 500-fold.

An inexpensive sample preparation method for the separa-tion of gram-positive micro-organisms from various foodmatrices and blood was recently developed. This procedure,called “matrix lysis”, involves solubilisation of the food ma-trix followed by concentration of intact bacteria through cen-trifugation to detect <10 CFU/g target micro-organisms, whilefree DNA is eliminated by 5 log units (Mayrl et al. 2009;Rossmanith et al. 2007; Aprodu et al. 2011). This procedure isfriendly, specific and rapid, and high amounts of food samplescan be processed. So far, four different buffer systems havebeen successfully introduced, two of them exclusively for

real-time PCR quantification, while the latest two buffer sys-tems, based on a novel chemical substance class called “ionicliquids” (IL) or on MgCl2, also allowed for the application ofmicrobiological methods after matrix lysis (Mayrl et al. 2009;Mester et al. 2010b; Rossmanith et al. 2011; Mester et al.2010a).

Nucleic acid purification methods should be characterisedby a high efficiency (almost all DNA from the target cellspresent is harvestable), purity (low amounts of other macro-molecules are co-extracted) and integrity (the nucleic acidremains undegraded through the chemical and sometimesmechanical procedures applied). Nucleic acid extraction oftenutilises cell wall lysing chemistry (such as guanidine thiocy-anate to denature viral and bacterial coat proteins) in combi-nation with resins to bind the released nucleic acid, which isthen purified through successive washing steps before finalelution in a small volume (EFSA 2011). Nucleic acid extrac-tion should work efficiently either on DNA or RNA.

Particularly with enteric RNA viruses, for the detection ofwhich a reverse transcription stage is necessary, the capacityof an extractionmethod to obtain a nucleic acid sample as pureas possible is a particularly important point. Indeed, the highsusceptibility of reverse transcriptase to inhibitory substancesis a major limiting factor in such methods (Wilde et al. 1990).A lot of methods have been proposed for extracting viral RNAand simultaneously reducing the levels of inhibitors (Butotet al. 2007b). For bivalve molluscs, dissected digestive diver-ticulum (digestive gland) is used as the starting material withfurther enzymatic digestion using proteinase K (Jothikumaret al. 2005). For food contact materials, swabbing isemployed, followed by elution into buffer (Scherer et al.2008). For molecular biological analysis of water, includingbottled water, the established methods usually involve adsorp-tion of viruses on a positively or negatively charged mem-brane, and then the adsorbed viruses are eluted and concen-trated by ultrafiltration (Butot et al. 2007a). In order to eval-uate the efficiency of the entire analytical procedures to con-centrate enteric viruses in bottled waters, an ultrafiltrationmethod using charged filtration membrane was recently eval-uated step by step. The results showed that a considerablenumber of virus particles passed through the pores of themembranes instead of being trapped by the electrostaticcharges (Di Pasquale et al. 2010). A new method able toextract all the virus particles trapped by the membrane hasbeen developed (Perelle et al. 2009) and validated in aEuropean ring trial (Schultz et al. 2011). Recently, improve-ment in the release rate of virus particles from the membraneby the use of ultrasound prior to ultrafiltration was proposedfor detecting enteric viruses from different types of bottledwaters (Butot et al. 2013).

Detection of viruses in fruits and vegetables starts with theelution of the virus particles from the surface of the product(Sadovski et al. 1978) because it is assumed that naturally



contaminated samples carry virus particles only on the sur-face. However, HAV particles were found also trapped insidegrowing green onions probably after being taken up intracel-lularly through the roots (Chancellor et al. 2006). In lettuce,the viral contamination of the plants via roots cannot beexcluded but is apparently not an important transmission routefor viruses (Urbanucci et al. 2009).

Filtration through large porosity filters, previously treatedto reduce the adsorption of viruses, has also been used toremove food particles. This procedure was very useful alsoto concentrate viruses from raspberries and frozen fruits; inthis case, pectinase has to be added to prevent jelly formationin the eluate (Croci et al. 2008).

Immunochemical methods have also been applied to theseparation of viruses from food samples (Bidawid et al. 2000;Kobayashi et al. 2004; Shan et al. 2005; Tian et al. 2006; Tianand Mandrell 2006). However, the development of animmunoconcentration procedure for noroviruses has encoun-tered difficulties with obtaining antibodies and with theirvariability at the capsid level.

The discovery of human histo-blood group antigens(HBGA) on cells of the human gastrointestinal tract asnorovirus receptors has been used for developingimmunoconcentration procedures for selective binding ofNoV in faecal and food samples (Tan and Jiang 2005;Huang et al. 2005; Le Guyader et al. 2006).

Detection of Viable Cells or Infective Viruses

Traditionally, bacteria are considered viable when they can becultured. This concept is not applicable when the detection ofbacteria is performed by molecular methods, since the detec-tion is focused on bacterial DNA. Conclusively, an importantdrawback of nucleic acid-based methods is their inability todistinguish DNA from viable cells and DNA from dead (ornon-cultivable) cells. Different techniques were developed todetect only viable bacterial cells by molecular methods, suchas the detection of messenger RNA (mRNA) and the use ofDNA-intercalating dyes (propidium monoazide (PMA) andethidium monoazide (EMA)).

An appropriate viability criterion is the active metabolismof cells (Nocker and Camper 2009). Detection of mRNA bymolecular methods has been studied extensively as it isstrongly believed that its presence correlates with cell viability(Gonzalez-Escalona et al. 2009; Coutard et al. 2005; Hellyeret al. 1999; Liu et al. 2010). Different studies demonstratedthat targeting mRNA may reduce the possibility of falsepositive results at determination of viable cells, since thehalf-life of bacterial mRNA (hours) is much shorter than thatof DNA (days or months). In contrast, other studies showedthat both DNA and mRNA may persist in a detectable formfor many hours after cell death (Birch et al. 2001) and

demonstrated a potentially poor correlation between mRNAdetection and cell viability. Practical drawbacks are thatamplifiable template RNA may be difficult to extract fromfoods due to the presence of inhibitors, such as fat, proteinsand components from blood cells, the method being efficientonly if all the bacterial background DNA is removed enzy-matically by DNase treatment.

In fact, the removal of DNA contamination in the RNAextract by DNase treatment has to be confirmed by PCRbefore the application of mRNA-based real-time (RT)-PCRin protocols for the detection of living cells. Repeated DNasetreatment may sometimes be necessary, since some bacterialDNA may be more resistant to DNase treatment (Kobayashiet al. 2009). This illustrates that the process is technicallyrather complicated and more time-consuming than DNA-based PCR, which limits the application of the mRNA-basedmethods in routine laboratory testing.

Another approach to a selective detection of living micro-bial cells is the use of EMA or PMA in combination withPCR-based molecular diagnostic techniques. This approachwas recently used in several studies and was reported to be aneasier-to-use alternative to mRNA detection. The use of EMAor PMA was effectively evaluated in different bacteria(Josefsen et al. 2010; Wolffs et al. 2001) and viruses(Parshionikar et al. 2010). PMA and EMA are two DNA-intercalating dyes that penetrate only to dead cells with com-promised cell membrane integrity but not to viable cells withintact cell membranes. The presence of an azide group isbelieved to permit cross-linking of the dye to DNA afterexposure to strong visible light. When exposed to light, pho-tolysis of EMA and PMA causes the azide group to beconverted to a nitrene radical, which covalently binds to anycarbon moiety in proximity, including both extracellular anddouble-stranded DNA contained in cells with compromisedmembranes, forming a carbon–nitrogen bond. This covalentlinking inhibits amplification by PCR. DNAwith bound EMAis insoluble and can be removed with cell debris during theDNA extraction process (Nocker et al. 2007).

However, it has been reported that EMA can also, to someextent, penetrate viable bacterial cells and there covalentlycross-link with DNA during photolysis (Rudi et al. 2005;Nogva et al. 2003). This may result in the loss of somepercentage of the genomic DNA of viable cells (Nockeret al. 2006). In Campylobacter, recent research has shownthat EMA has a toxic effect thus contributing to the load ofdead cells (Flekna et al. 2007). Recently, Minami et al. (2010)proposed treating bacteria before real-time PCR with a con-centration gradient of EMA. This approach may increase liveand dead distinction ability to assure that EMA destroyed onlyDNA derived from dead cells. This newly developed low-dose double-treated EMA-PCR was demonstrated to be veryeffective for viable Cronobacter sakazakii detection in pow-dered infant formula (PIF) (Minami et al. 2012). Interaction of



monoazide compounds with bacterial DNA is complex, and itmay depend on concentration of the dye, concentration of freeDNA, as well as on numbers of damaged and viable cellspresent in the sample.

Another way to improve this technique may be based onthe finding that long-amplicon PCR is more sensitive tomonoazide treatment of the template DNA. Long-ampliconPCR would require less monoazide treatment to block DNAfrom dead cells and thus reduce the loss in the detection of livecells (Soejima et al. 2011).

Further research should focus on the influx and efflux ofEMA or PMA to cells of different bacterial species and strainsat different conditions of incubation and the efficiency ofcross-linking via photo-activation to free DNA, DNA withindead and damaged cells and DNAwithin viable cells (Fleknaet al. 2007).

A physical separation approach that can be used to separateviable and dead cells is flotation, a technique that utilises theirdifferent densities. The cell densities of 12 different bacterialstrains, determined by centrifugation using a step densitygradient of Percoll, were between a very strictly defined range(1.031 and 1.120 g/ml) (Fukushima et al. 2007). This range ofdensities allowed the separation of living bacterial cells instationary phase from the food matrix and from dead bacterialcells.

The benefits of density gradient centrifugation as a samplepre-treatment method are well established and include (i) thepossibility of separating biological matrix particles and cellsof micro-organisms with different buoyant densities, (ii) main-taining cell viability, which allows isolation and analysis ofthe micro-organisms, and (iii) speed and easy handling. Todate, this technique has been used only for the detection ofYersinia enterocolitica in pork meat juice and for quantifica-tion of Campylobacter in chicken rinse samples (Wolffs et al.2004; Wolffs et al. 2005). Separation of food-borne pathogensis thought to be more feasible in liquid samples rather than insolid ones, and flotation may only be used on liquid samples.The aforementioned concept of matrix lysis does not impaircell viability when using ionic liquids and can be used bothwith PCR or conventional isolate identification (Mester et al.2010b). This concept was further researched by D’Urso et al.(2009), who described a new filtration-based method for thedetection of viable Listeria and Salmonella cells in foodsamples (D’Urso et al. 2009). In general, analytical problemsare caused by bacteria arrested in a viable but not cultivable(VBNC) state mostly after increased stress. VBNCmay causea deviation between quantities determined by classical micro-biology (as CFU) and data obtained by culture-independent,molecular quantification (Reichert-Schwillinsky et al. 2009).

In order to differentiate viable from non-viable cells, notonly in food where the bacteria are present on surface but alsoin the mass of the food matrix, the use of a short enrichmentstep has been also proposed (Kramer et al. 2011). The method

combines a short (8 h) non-selective pre-enrichment step ableto produce only viable cells in log-phase and a real-time PCRto evaluate quantitatively the number of living bacteria. Thisapproach was used to enumerate Salmonella in different eggproducts (Jakociune et al. 2013) and to detect verocytoxigenicE. coli (VTEC) in milk (Mancusi and Trevisani 2014).

However, the use of any non-selective cultivation maylead, in certain food matrices and in certain mixed culturesof micro-organisms, to a relative suppression of minor com-ponents of the microflora and thus affect the results of thedownstream analysis (Krascsenicsova et al. 2006).

What is true for bacteria constitutes an analytical problemin virus detection as well. The burden of infective entericviruses in a food is crucial to evaluate the real risk for humanhealth (Kim et al. 2011; Kim and Ko 2012). Unfortunately,molecular methods are unable to distinguish between infec-tious and non-infectious viruses, where upon the latter usuallyconsist of defective virus particles, containing differentamounts of intact or degraded viral RNA (Knight et al.2012). It was demonstrated that viral genomes may persist inthe environment for a longer time than the viral infectiousparticles (Richards 1999; Ogorzaly et al. 2010). For example,the HAV genome was detectable by RT-PCR for 232 days,while virus particles were detectable in cell culture for only35 days. This suggests that the detection of the HAV genomeby RT-PCR is not a reliable indicator of the presence of aviable virus (Koopmans et al. 2002).

Cell culture assay is the standard method for the detectionof viable human viruses (Nuanualsuwan et al. 2002), and theuse of a method integrating cell culture and PCR in order toconfirm the viability (infectivity) of the viruses proved effec-tive (Hyeon et al. 2011; De Medici et al. 2001; Reynolds2004). Unfortunately, the cell culture method is quite time-consuming, as up to 2 weeks is necessary to confirm theresults for HAV, and it is not applicable to norovirus, whichcannot grow outside of the human host. Analogically to bac-teria, the idea of using EMA or PMA could be a step forwardto identify infective enteric viruses, since the absence of a cellculture system for some food-borne viruses hampers the de-termination of infectivity of viruses when isolated from con-taminated foods. Again, the combination of dye and the targetorganism seems to be of great importance, as demonstrated ina study in which EMA failed to distinguish between viableand non-viable virions of avian influenza virus (AIV) (Graiveret al. 2010). The AIV envelope is similar but still differentcompared to a bacterial cell membrane, and it is possible thatEMA is unable to penetrate into membrane-compromisedAIV virions.

Sanchez et al. (2013) demonstrated that PMA treatmentprior to RT-quantitative PCR (qPCR) detection was a prom-ising method for assessing HAV infectivity. The use of PMAor EMA in conjunction with three surfactants (IGEPAL CA-630, Tween 20, Triton X-100) prior to RT-qPCR was also



shown to be effective to quantify the infectious particles ofHAV and of two strains of RV (SA11 and Wa), after heattreatment (Coudray-Meunier et al. 2013).

Owing to the importance of the viral capsid in infectivity,the measure of the capsid integrity or “virolysis” may repre-sent an alternative marker for virus infectivity in the absenceof a cultural model. Capsid integrity has been investigatedusing combined proteinase K and RNase treatment, in order tocorrelate PCR data with infectivity (Nuanualsuwan and Cliver2002). However, this treatment was difficult to control be-cause it depends on relative activities of individual enzymes,making the results difficult to reproduce. Assuming that non-intact capsids would not be able to bind to appropriate recep-tors, it was proposed to utilise the binding affinity of virusparticles to their natural receptors or other binding ligands(Nuanualsuwan and Cliver 2003; Ogorzaly et al. 2013). Forinstance, ligands, such as human histo-blood group antigens,demonstrated a high grade of affinity to noroviruses and thushave been considered as an alternative to antibodies (Mortonet al. 2009).

Appropriate PCR Control

PCR-based detection of micro-organisms in food is a compar-atively complicated process involving several steps, all ofthem being possible sources for errors. Those encompassimproper sample preparation (enrichment, cell lysis, DNAextraction, removal of inhibitors) or failure of PCR (compo-sition of reaction mixture, performance of the PCR cycler). Inorder to be able to control the PCR-based analysis, varioustypes of controls are recommended for use.

The correct performance of the reaction mixture can bechecked by the external (positive) control. However, it is moreimportant to control if PCRwas successful in each tube and ineach position of the cycler block. For this purpose, an internalamplification control (IAC) can be used, which is added toeach PCR tube. Many different formats have been describedso far, for example various plasmids or λ bacteriophage DNAmay be used as a template (Fricker et al. 2007). This templateis added at a low concentration, and it is detected by its ownprimers/probe designed to work at the same PCR temperatureprogramme. Theoretically, the addition of an exogenous IACshould not affect the main analytical reaction. However, thisinertness has to be checked for each application (main primer/probe system, reaction mixture composition, temperature pro-gramme) so as to avoid excessive primer–dimer formation.

Another technical alternative to IAC is the use of a specifictemplate that can be amplified with the same primers, so-called mimic, in the same reaction conditions (ThistedLambertz et al. 1998). In this setting, primers compete forthe main target and for the control target. The products of themain PCR and the control PCR are distinguished based on the

size of the amplified DNA fragment or using fluorogenicprobes labelled with different dyes. An advantage of thisapproach is a reduction in the number of types of oligonucle-otides present in PCR, which reduces the risk of their unwant-ed interaction. A drawback is that each diagnostic primer/probe system requires a new mimic molecule.

For the same purpose, an endogenous control, external orinternal, can be used. This is a separate primer/probe systemtargeting some universal DNA sequence present in any pro-karyotic (Greisen et al. 1994) or eukaryotic DNA (Breznaet al. 2006). A preferred format of this type of control isexternal, so as to avoid the situation that a strong controlsignal affects the signal from the main reaction.

A no-template control as a negative control should beincluded in each PCR-based method for food control. This isprepared in a separate tube and contains all PCR componentswith the exception of any DNA template. The results of thisreaction should be always negative. An accidental positiveresult would indicate contamination of working solutions,tubes or pipette tips by DNA.

The ideal type of control would, however, be the one thatcontrols the entire analytical procedure. This might be, forexample, a freeze-dried culture that would be added to thefood sample prior to enrichment, would pass all steps of theanalysis (enrichment, lysis, DNA extraction, PCR) and thenwould be selectively detected by PCR along with the target.The requirements suggest that it probably would be a micro-organism that is related to the target, cultures of which are ableto grow in all enrichment media, but not faster than the target.Either well-defined wild or specifically designed engineeredmicro-organisms may suit this purpose (Rossmanith et al.2011). Quantitative levels of such entire-process internal con-trol to be applied as well as their physiological state (e.g.mildly stressed) should be well studied so as to avoid thesuppression of growth of cultures of the target micro-organism. Phenotypic as well as genetic stability of controlmicro-organisms should be studied, in particular if engineeredmicro-organisms are intended to be used for this purpose.

The use of an appropriate process control has been alsodebated for the evaluation of the rate of recovery in the quanti-tative detection of viruses in different foodmatrices. Particularly,during the development of the method by the Tag 4 of CENworking group, a genetically modified strain of Mengo viruswas successfully utilised as a process control (Costafreda et al.2006). The use of feline calicivirus was also proposed as analternative process control (Di Pasquale et al. 2009).

Standardisation and Validation of Molecular MethodProtocols

According to the official definition of ISO, the term standarddescribes a “document established by consensus and approved



by recognised body that provides, for common and repeateduse, guidelines or characteristics for activities or their results,aimed at the achievement of the optimum degree of order in agiven context”(ISO/IEC 2004). A standard is of commoninterest and reflects the concerns of all stakeholders involvedby implementing the largest consensus and the broadest ap-plicability in food laboratories worldwide. Listing orrecommending commercial products in standard protocols isavoided as much as possible. Standards can be issued at thenational level (national standardisation bodies), the regionallevel (e.g. CEN) or the international level (ISO). Most stan-dards in food analysis are developed by CEN/TC275/WG6(CEN Working Group 6 “Microbial contaminants” ofTechnical Committee 275 “Food Analysis—Horizontalmethods of CEN) on the European level and by ISO/TC 34/SC 9 (ISO Sub-Committee 9 “Microbiology” of TechnicalCommittee 34 “Food Products”) on the international level. Asoutlined in the “Vienna Agreement”, usually, standards devel-oped by SC 9 are adopted also as CEN standards and viceversa (Lombard and Leclercq 2009).

Standardisation of modern technologies such as molecularmethods can be achieved either by the development of specificstandards for novel technologies or by elaborating standardslaying out the general guidelines for applying PCR in foodanalysis. Resolution No. 233 of the joint meetings of ISO/TC34/SC9 and CEN/TC275/WG6 states that novel technolo-gies can be introduced to a reference standard method if theperformance criteria of the reference method are not satisfyingor if the novel methodology targets not taxonomy (like theconventional method) but e.g. pathogenicity. Furthermore,resolution 233 stipulates that commercial products are not tobe mentioned in the standard but that their validity should bechecked against the standard reference method (Lombard andLeclercq 2009). This idea of validating data produced byculture-independent analysis against data of classical culture-dependent microbiology was well described in a recent paperwhere the authors suggested a novel strategy on how tovalidate molecular data by system analysis (Rossmanith andWagner 2011).

Use of PCR in food microbiology is covered by the Tag 3expert committee of CEN/TC/275/WG6. To date, only fewmethods based on the use of molecular techniques areharmonised and standardised by ISO/CEN. The majority ofpublished ISO/CEN norms deal with the general aspects ofmolecular methods, while other norms are already at draftlevel or in discussion (Table 1). In 2004, CEN initiated thedevelopment of a standard method for the detection ofnorovirus and HAV in foodstuffs based on PCR (Lees 2010),and this norm was recently published (Table 1). Specificnorms are missing for molecular detection of the most impor-tant food-borne micro-organisms.

Recently, norm ISO/TS 17919:2013 for the detection ofbotulinum type A, B, E and F neurotoxin-producing clostridia

using PCR was published. Some of the methods proposed inthe norm are evaluated in European collaborative works (DeMedici et al. 2009; Lindstrom et al. 2001; Fach et al. 2009).

Validation protocols on the national and international levelhave been developed by many bodies. On the internationallevel, one of the most recognised and hence important proto-cols is the ISO standard 16140:2003 “Microbiology of foodand animal feeding stuffs—Protocol for the validation ofalternative methods”. This standard outlines general principlesand the technical protocols for the validation of alternativemicrobiological methods. Details about method comparisonstudies and interlaboratory studies are stated here for bothqualitative and quantitative methods. An overview of therequired parameters is given in Table 2.

Another key organisation in validation and standardisationof methods for microbiological testing of food is theAssociation of Official Analytical Chemists (AOAC)International. AOAC currently run two validationprogrammes: the Official Methods of Analysis (OMA) andthe Performance Tested Methods Programme (PTM). A thirdprogramme, the peer-verified methods, was disbanded in July2008. AOAC recently added a third programme of FirstAction methods to facilitate rapid adoption of new methods,which includes also microbiological methods based on PCR.

According to the MoniQA European project workinggroup (www.moniqa.eu) when comparing the protocols ofthe main validation organisations, there is a clear need foruniformity in different areas (Table 3). One such area is astandard strain collection used for validating methods. Forexample, AOAC specify that isolates should be obtainedfrom ATCC, while other validating organisations will acceptstrains acquired from regional culture collections or “in-house” collections. There is a wide variation in both the typeof enrichment methods and the primary enrichment conditionsemployed in various validation studies. For example, withsome pathogens, different enrichment methods are chosen tovalidate different methods, and different enrichment methodscan be used to validate the same methods with different foodmatrices.

Certain changes would be also useful regarding the widevariety of food matrix materials used for detection and enu-meration in validation studies. Such variety may be useful atassessing repeatability of the methods but not for comparingmethods between studies. Contra productivity of the currentapproach may be illustrated by an example of moleculardetection of thermo-tolerant Campylobacter, for which anunexpected matrix effect was observed, leading to underesti-mation of the bacteria in milk (Rosenquist et al. 2007).

With molecular methods, where reproducibility, reliabilityand accuracy may depend on a broad range of variables, thereis a need to precisely detail all relevant information whenconducting collaborative trials. For example, with PCR-based methods, the type or brand of thermo-cycler (Schoder



http://www.moniqa.eu/

et al. 2003) and the working temperature of the lab mayinfluence the outcome of the experiment (Anonymous 2010).

It is necessary that entire analytical methods are validated.In many cases, the validation study was focused on one steponly (e.g. PCR conditions) and thus gave no information on

how the validated PCR protocol would work in routine anal-ysis. Therefore, it is recommended to select a certain sample,an appropriate sample processing method and the most suit-able PCR reagents and amplification parameters and thenperform validation of the whole process instead of individualprocess steps (Hoorfar et al. 2004b).

It is necessary that the analytical methods are validated withreal-world samples. Many validation studies do not providesufficient information on the efficacy of the method becauseonly spiked samples are used, although there is growing concernon the comparability of results obtained by spike experiments

Table 1 The status of the standard references on use of molecular methods for detecting pathogenic micro-organisms in food and water

Standard reference Title Status

EN ISO 20837:2006 Microbiology of food and animal feeding stuffs—polymerase chain reaction (PCR) for the detection offood-borne pathogens—requirements for sample preparation for qualitative detection

published

EN ISO 20838:2006 Microbiology of food and animal feeding stuffs—polymerase chain reaction (PCR) for the detection offood-borne pathogens—requirements for amplification and detection for qualitative methods

published

EN ISO 22118:2011 Microbiology of food and animal feeding stuffs—polymerase chain reaction (PCR) for the detection andquantification of food-borne pathogens—performance characteristics

published

EN ISO 22119:2011 Microbiology of food and animal feeding stuffs—real-time polymerase chain reaction (PCR) for thedetection of food-borne pathogens—general requirements and definitions

published

EN ISO 22174:2005 Microbiology of food and animal feeding stuffs—polymerase chain reaction (PCR) for the detection offood-borne pathogens—general requirements and definitions

published

CEN ISO/TS 13136:2012 Microbiology of food and animal feed—real-time polymerase chain reaction (PCR)-based method for thedetection of food-borne pathogens—horizontal method for the detection of Shiga toxin-producingEscherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups

published

ISO/TS 15216-1:2013 Microbiology of food and animal feeding stuffs—horizontal method for detection of hepatitis A virusand norovirus in food using real-time RT-PCR—Part 1: method for quantitative determination

published

ISO/TS 15216 -2:2013 Microbiology of food and animal feeding stuffs—horizontal method for detection of hepatitis A virusand norovirus in food using real-time RT-PCR—Part 1: method for qualitative determination

published

ISO/TS 17919:2013 “Microbiology of food, feeding stuffs and environmental samples—polymerase chain reaction (PCR) forthe detection of food-borne pathogens—detection of botulinum type A, B, E and F neurotoxinproducing clostridia”

published

ISO/CD TS 18867 “General requirements relating to molecular methods for detection and quantification ofmicro-organisms”—draft standard of PCR detection of enteropathogenic Yersinia spp.

in discussion

Microbiology of food and animal feeding stuffs—detection of Vibrio parahaemolyticus in seafoods: Part1—quantitative determination of total, thermostable direct haemolysin (TDH) and thermostabledirect-related haemolysin (TRH) positive Vibrio parahaemolyticus using nucleic acid hybridisation

in discussion

Table 2 Performance characteristics to be evaluated during validation ofqualitative and quantitative alternative methods by comparison studies(A) and interlaboratory studies (B) in accordance with ISO standard16140:2003 “Microbiology of food and animal feeding stuffs—protocolfor the validation of alternative methods”

Qualitative methods Quantitative methods

A

Relative accuracy Relative accuracy

Relative specificity Specifity

Relative sensitivity Relative sensitivity

Relative detection level Detection and quantification limits

Inclusivity and exclusivity(selectivity)

Inclusivity and exclusivity (selectivity)

– Linearity

B

Relative accuracy Relative accuracy

Percentage specificity Repeatibility (repeatability limit)

Percentage sensitivity Reproducibility (reproducibility limit)

Accordance –

Concordance –

Concordance odds ratio –

Table 3 Areas of uniformity in the protocols used for comparing Micro-biological methods in the different validation methods according toMoniQA European project working group coordinated by Paulin S(Anonymous 2010)

• Reference method (no particular method suggested by any validationorganisation for each pathogen)

• Relevant food categories for each target pathogen (AOAC hashighlighted more categories than ISO or NordVal)

•Number of required inocula levels (this ranges from 3 to 5 depending onthe organisation)

• Number of strains required for sensitivity experiments (this ranges from1 to 2 depending on the organisation)

• Number of test portions

• Performance indicators

• Number of laboratories required to participate in interlaboratory andcollaborative trials



(healthy cultures) and those obtained in routine analysis(stressed cultures) (Hoorfar et al. 2004b; Havelaar et al. 2010).

A lack of information also exists when it comes to quanti-tative real-time PCR and reverse transcription real-time PCR.Many publications do not provide sufficient detail on theexperimental conditions, and hence, reliable and unequivocalinterpretation is hardly possible. A set of guidelines describingthe “Minimum Information for Publication of QuantitativeReal-Time PCR Experiments” (MIQE) aims to overcome thisshortage by an increased consensus on the performance andinterpretation of real-time PCR protocols (Bustin et al. 2009).For standardisation purposes, four areas (study design, tech-nical detail, analysis methods and statistics) have to be definedfor any real-time PCRmethod. TheMIQE guidelines considerall of them, asking for details on the experimental design,sample (processing, storage), nucleic acid extraction, reversetranscription, the real-time PCR step and data analysis (Bustin2010; Bustin et al. 2009).

A specific problem regards validation of reverse transcrip-tion real-time PCR. The method is currently the gold standardfor the detection of (small) amounts of mRNA due to themethod’s unchallenged simplicity, cost efficiency, accuracyand availability (Pfaffl et al. 2002; Pugniere et al. 2011). Asthis method combines reverse transcription with subsequentreal-time PCR, variations in results stem not only from real-time PCR but also from RT yields. RT yield is influenced bytotal RNA concentration, target template quantity and back-ground DNA, isolation method and inhibitory substances likemyoglobin, collagen or lipids (Pugniere et al. 2011).

Once a standardised PCR/real-time PCR protocol is ready,it cannot be considered as final but has to be continuouslyrevalidated in order to assess its sensitivity against newlyarising strains or to consider enhanced detection methods(Malorny et al. 2003).

A new initiative was started in 2011 by AOAC, theInternational Stakeholder Panel for Alternative Methods(ISPAM). The purpose of this initiative is to developharmonised, internationally accepted standard validation guide-lines for alternative (rapid) chemical and microbiologicalmethods, by leveraging global networks of experts to reachconsensus on an analytical validation protocol. Under thisinitiative, AOAC has called together representatives from otherorganisations that operate in the same fields, including ISO,CEN, NordVal (Nordic Committee on Food Analysis, Oslo,Norway), MicroVal (MicroVal, Delft, the Netherlands) andAFNOR (French Standardization Organization, Paris, France).In addition to the organisations, the panels also include repre-sentatives from food and feed industry and kit and equipmentmanufacturers as well as governments. All these stakeholdersuse this type of methodology or have to base risk assessment onthe results obtained by using these methods on their samples.

One of the major goals of the panel is to come to aharmonised approach on how to reduce the validation effort

(e.g. fewer laboratories required or fewer samples required)without significantly sacrificing the quality of the study,allowing on one hand validation to take place faster, beforethe methods are already obsolete, and, on the other hand, tohave mutual recognition of methods validated under the newscheme, i.e. all organisations will accept the successful vali-dation study without the need for repeating it for their organi-sations’ approval.

ISPAM formed two working groups: a microbiology groupand a qualitative chemistry working group. The ISPAMmicro-biology group identified five high-priority areas for their futurework: reference methods, selection of food/category (samplematrix), number of levels/samples/fractional positive, resultsanalysis and criteria/statistical analysis, number of samples/replicates/method comparison and collaborative phase.

Cost of Molecular Methods for Food Analysis

Molecular methods for food analysis are currently mostlyavailable as kits, but the situation will probably change whenthe necessary international norms are published. Laboratoriescan then make use of the fact that molecular biological equip-ment and biochemicals have becomemuch more affordable inrecent years.

Currently, the use of kits or automated systems may seemexpensive, with prices as high as $10–$15/7 EUR–10 EURper test, sometimes over $25/18 EUR per test. The cost ofreagents and instruments used in rapid assays is very high,$30,000–$50,000/20,000 EUR–35,000 EUR. Such costs areaffordable only for large companies. When it comes to detect-ing micro-organisms by alternative or innovative rapidmethods, capital costs for automated PCR systems are rela-tively high compared to those for some other rapid methods.Consumable costs are also higher in comparison to rapidculture-based techniques. However, there are considerablecost benefits in terms of reduced technician labour time andsavings on training. There is a clear cost benefit in rapid testresults allowing faster HACCP verification and positive re-lease of finished food products. A recent study carried out atthe German Federal Institute for Risk Assessment (BfR) con-cluded that, taking all costs into account, real-time PCR couldbe significantly less expensive than a conventional ISO cul-ture method (http://www.rapidmicrobiology.com/test-method/pcr-for-food-microbiology/).

Conclusion

In order to achieve full integration of molecular methods infood safety control, certain drawbacks of PCR-methods stillneed to be addressed and clarified, particularly the access tothe target cell by advanced sample treatment concepts and



http://www.rapidmicrobiology.com/test-method/pcr-for-food-microbiology/

http://www.rapidmicrobiology.com/test-method/pcr-for-food-microbiology/

improved strategies to distinguish DNA of viable cells fromboth DNA of dead or non-cultivable cells. In Europe, molec-ular methods, either immunological or nucleic acid-based, arecurrently used for preliminary and rapid screening of thepresence of pathogenic micro-organisms in food. The positiveresults are considered presumptive and require further confir-mation by traditional culture-based methods (Hoorfar 2011).Also in USA, AOAC-approved rapid methods are used asscreening tools. Using first-line rapid testing and second-lineculture-dependent confirmations makes food testing costly,consuming time and labour (Mandal et al. 2011). It has fur-thermore led to the fact that molecular tests are used more andmore by FBOs but not by public health authorities. It would bea clear step forward to a more efficient food safety system ifresponse actions could be accelerated up by accepting PCR-based tests as reliable and definitive.

The slow implementation of novel technologies in foodmicrobiology is even more remarkable since a lot of compar-isons between molecular and traditional cultural methodsdemonstrated the equivalence of the data obtained from bothapproaches to date (Gianfranceschi et al. 2014; Lofstrom andHoorfar 2012; Schultz et al. 2010; Zhang et al. 2011; Josefsenet al. 2004; Abdulmawjood et al. 2003; Lubeck et al. 2003a;Lubeck et al. 2003b; Josefsen et al. 2003; Fenicia et al. 2011;Delibato et al. 2014). In order to support the implementationof molecular methods, their standardisation through the re-sponsible standardisation bodies should be prioritised, whichwould facilitate the acceptance of nucleic acid-based foodanalytics. It must by the ultimate goal that, when using mo-lecular methods, the need to confirm positive results should belimited to cases where a physical isolate is needed for epide-miological or research purposes; if the physical isolate is notrequired for subsequent studies, the positive result obtained bymolecular method should not require confirmation anymore.

Acknowledgments This research was supported by the framework ofthe EU project funded by the 7th Framework Programme of the EuropeanUnion “Safe Food for Europe—Coordination of research activities andDissemination of research results of EC funded research on food safety(project acronym: FOODSEG) Grant agreement no. 266061. This publi-cation reflects the views only of the authors, and the European Commis-sion cannot be held responsible for any use which may be made of theinformation contained therein.

Conflict of Interest No financial relationship with other institutions orprivate industry has influenced the results of this study. D. De Medici hasno conflict of interest, T. Kuchta has no conflict of interest, R. Knutssonhas no conflict of interest, A. Angelov has no conflict of interest, B.Auricchio has no conflict of interest, M. Barbanera has no conflict ofinterest, C. Diaz-Amigo has no conflict of interest, A. Fiore has noconflict of interest, E. Kudirkiene has no conflict of interest, A. Hohlhas no conflict of interest, D. Horvatek Tomic has no conflict of interest,V. Gotcheva has no conflict of interest, B. Popping has no conflict ofinterest, E. Prukner-Radovcic has no conflict of interest, S. Scaramagliahas no conflict of interest, P. Siekel has no conflict of interest, K.A. To hasno conflict of interest and M. Wagner has no conflict of interest. Thisarticle does not contain any studies with human or animal subjects.

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