Indicadores de resultados de mediano plazo

Nucleic acid methods that include an amplification step for the target DNA/RNA are now routinely employed in molecular biology. These methods, as outlined in the following, increase the target nucleic acid material by up to a millionfold and are particularly impor- tant in the arena of food microbiology, where one of the major hurdles is the recovery and detection of very low numbers of a particular pathogen. However, even with the incorporation of an amplification step, a liquid enrichment of the food sample (24 to 48 h) is generally still required to yield sufficient nucleic acid material from the target organism for the reaction.

Polymerase Chain Reaction (PCR)

The most popular method of amplification is the polymerase chain reaction (PCR) tech- nique. Since its development in 1983 (Mullis 1990) and the publication of the first experimental data on PCR (Saiki et al. 1985), this reaction has become an essential tool in molecular biology. In this technique, the DNA is extracted from the organism, and the double strands are denatured into single-stranded DNA. Short sequence DNA primers are annealed to the complementary DNA target in the organism. The primers are then extended across the target sequence using a heat-stable DNA polymerase (usually Taq polymerase, a thermostable and thermoactive enzyme from Thermus aquaticus) in the presence of free deoxynucleoside triphosphates (dNTPs), resulting in a double replication of the starting target material. Multiple repeats of the denaturation, annealing, and extension steps result in an exponential increase in the levels of the initial target DNA, thus greatly increasing the sensitivity of the method (Entis et al. 2001). Theoretically, a single gene target can be amplified a millionfold to allow detection in only a few hours. The sensitivity of PCR is limited, in part by the small sample volumes used in PCR and by the presence of inhibitory substances such as proteinases and collagen present in many foods (Powell et al. 1994; Kim et al. 2001). Sample preparation, including an enrichment step to increase the number of target cells, effective extraction of the pathogen from the enriched food, extraction of DNA, and the use of the appropriate DNA polymerases and primers are critical to achieve the desired sensitivity in PCR reactions (Rådström et al. 2003).

In conventional PCR, the steps in the PCR cycle are carried out in an automated, pro- grammable block heater. The PCR products are separated by gel electrophoresis, stained with ethidium bromide, and visualized using ultraviolet light (Olsen et al. 1995). A range of gene targets have been used in the PCR detection of pathogens. For Salmonella targets include species-specific genes (Kumar et al. 2003; Trkov and Avguštin 2003) and viru- lence genes (invA) (Rahn et al. 1992; C.-H. Chiu and Ou 1996). A number of suitable gene targets have been suggested for the detection and/or differentiation of enterohemorrhagic

E. coli. Species-specific targets include the rfbO157gene of the O157 antigen (Paton and

Paton 1998), wzx (O-antigen flippase) and wzy (O-antigen polymerase) for serogroups O26 and O113 (DebRoy et al. 2004), and virulence genes Vt1, Vt2, eaeA, and hlyA)

Molecular Technologies for Detecting and Characterizing Pathogens 159

(Paton and Paton 1998; Fagan et al. 1999). Targets for L. monocytogenes and Yersinia

enterocolitica include the listeriolysin O gene (Thomas et al. 1991) and the 16 S rRNA

gene (Wannet et al. 2001; Jaradat et al. 2002; Longhi et al. 2003; Kot and Trafny 2004). These targets have been widely employed for confirmation of pathogens from suspect colonies on agar plates and for direct detection from fecal samples. PCR methods have now been developed for detection of pathogens from foods, and table 8.1 outlines some recently reported conventional PCR methods for a range of food pathogens and identifies the target gene and food.

One of the main disadvantages of conventional PCR is that laboratories must take spe- cific precautions to avoid amplicon carryover and consequently false-positive results. In addition, this technique is generally time-consuming and labor-intensive (Persing 1991). A limiting factor in the uptake of PCR methods, and indeed many other rapid pathogen methods by the food industry, relates to a lack of full validation against the accepted cultural method in accordance with the International Standards Organization (ISO). It is generally essential, when testing against microbial criteria set by regulatory authorities, to use a recognized ISO method or an equivalent rapid method. For Salmonella the procedure for determining equivalence is outlined in EN/ISO 16140: 2003. The lack of validation and ring trials is now being recognized as a shortfall, and research to fully validate methods is now being undertaken. D’Agostino et al. 2004 report a PCR assay for detection of

L. monocytogenes in raw milk that was evaluated in a collaborative trial involving 13

European laboratories. Similarly, Josefsen et al. 2004 have reported on the validation of a PCR-based method for detection of food-borne thermotolerant campylobacters from chicken in a multicenter collaborative trial. Malorny et al. 2004 have reported a multi- center validation of a PCR-based method for detection of Salmonella in chicken and pig samples, and Abdulmawjood et al. 2004 have reported on a multicenter interlaboratory trial for PCR-based detection of food-borne E. coli O157. PCR is commercially available as the BAX system (Dupont Qualicon) for a range of food-borne pathogens.

Real-Time PCR

Real-time PCR is now increasingly replacing conventional PCR as a rapid, sensitive, and specific molecular diagnostic technique (Bellin et al. 2001). Since its introduction, real-time PCR has revolutionized the field of molecular diagnostics, and the technique is

Pathogen Gene Target Food Reference

Salmonella 16S-23S rRNA Chicken and milk T. H. Chiu et al. 2005

Salmonella ogdH Chicken Jin et al. 2004

E. coli O157 eaeA Ground beef Uyttendaele et al. 1999

E. coli O157 eaeA, hlyA, vt1, vt2 Minced beef Fitzmaurice et al. 2004

L. monocytogenes actA Soft cheese Longhi et al. 2003

L. monocytogenes inlAB Frankfurters Jung et al. 2003

Campylobacter fl aA Chicken Oyofo et al. 1997

Campylobacter 16S rRNA Poultry Mateo et al. 2005

Mycobacterium 16S rRNA Milk Tasara et al. 2005

paratuberculosis

being used in a rapidly expanding number of applications (Arya et al. 2005). Real-time PCR allows continuous monitoring of amplification through the use of fluorescent double- stranded (ds) DNA intercalating dyes or sequence-specific probes (Wittner et al. 1997). Real-time PCR assays offer many advantages over traditional PCR methods. They are much quicker to perform, with the enhancements in speed attributed to reduced amplifica- tion time and the elimination of an additional step(s) needed for product detection. More- over, the use of a closed system for amplification and detection minimizes the potential for amplicon carryover contamination (Bankowski and Anderson 2004). Although expensive in capital terms, real-time PCR-based strategies are becoming more popular in research and public health laboratories and are being increasingly used for specific diagnostic applications and pathogen detection (Bellin et al. 2001; Taylor et al. 2001). A number of real-time PCR instruments are commercially available for use in PCR (table 8.2). Real- time PCR methods can be divided into those that are not sequence-specific, such as DNA minor groove binding dyes, and those that are sequence-specific and might even afford simultaneous detection and confirmation of the target amplicon during the PCR reaction (McKillip and Drake 2004). Currently, the method for nonspecific real-time detection of PCR amplicons employs fluorescent double-stranded DNA intercalating dyes such as SYBR Green I (McKillip and Drake 2004). SYBR Green I binds to the minor groove of ds DNA during the extension step of the PCR and falls off during the denaturation step (Bustin 2000; Lekanne Deprez et al. 2002). The specificity and sensitivity of SYBR Green I is limited as it binds to all ds DNA, including nonspecific PCR products and PCR primer dimers (Wittner et al. 1997; Bustin 2000). SYBR Green I has been successfully used in combination with melting curve analysis for mutation screening and allele discrimination (Lyon et al. 1998; Bennet et al. 2003), but it is also useful for food pathogen detection (table 8.3).

A diverse array of fluorescently labeled probes are in use for sequence-specific detec- tion of target DNA or RNA (McKillip and Drake 2004). They involve fluorescence reso- nance energy transfer (FRET) between fluorogenic labels or between one fluorophore and a quencher group (Didenko 2001).

FRET is a process by which energy is passed between molecules separated by 10–100 Å that have overlapping emission and absorption spectra (Stryer and Haugland 1967; Clegg

Instrument Manufacturer Location

ABI 7000 Applied Biosystems Foster City, California

ABI 7900HT Applied Biosystems Foster City, California

i-Cycler iQ Bio-Rad Hercules, California

LightCycler Roche Applied Science Indianapolis, Indiana

SmartCycler Cepheid Sunnyvale, California

Mx3000P Stratagene Cedar Creek, Texas

RotorGene Corbett Research Queensland, Australia

Apollo ATC 901 Apollo Instrumentation San Diego, California

BAX System Dupont/Qualicon Wilmington, Delaware

DNA Engine Opticon 2 MJ Research Waltham, Massachusetts

R.A.P.I.D. Idaho Technology Salt Lake City, Utah

Table 8.2. Some real-time PCR instruments currently available.

Molecular Technologies for Detecting and Characterizing Pathogens 161

1992). Regardless of the specific means in which the fluorophore-quenching pair is applied, these methods have the advantage of sequence specificity that ds DNA intercalating dyes do not offer (McKillip and Drake 2004). Sequence-specific detection can be performed using the linear oligoprobes such as the hybridization probes (HybProbes), dual-labeled oligoprobes (TaqMan probes), or hairpin oligonucleotides (molecular beacons) (Mackay, 2004). Several variations of the basic FRET chemistry exist, although many of these remain unproven in food systems (McKillip and Drake 2004). Real-time PCR technology has been used to detect a range of food-borne pathogens (table 8.3).

An advantage of real-time PCR assays over conventional PCR methods is that they can be used for quantification of initial target DNA. Accurate calculation of the initial amount of DNA and elimination of false-negative results can be obtained with the inclusion of an internal amplification control, which is now becoming mandatory (Hoorfar et al. 2004). It consists of an internal control amplified at the same time as the target gene but detected by a second fluorophore. Future applications of the real-time PCR technology may include the development of quantitative real-time PCR methods able to quantify the number of bacteria directly in complex materials such as food.

Real-Time PCR

Chemistry Pathogen Gene Target Food Reference

SYBR E. coli O157 stx1, stx2 — Jothikumar and

Green I Griffiths 2002

Salmonella fi mI — Jothikumar et al. 2003

Salmonella 16S rRNA Retail beef, Catarame et al. 2006 pork, turkey,

chicken

L. monocytogenes, HlyA, invA Sausage Wang et al. 2004

Salmonella

HybProbes E. coli O157 eaeA Beef products Ellingson et al. 2005 Campylobacter 16S rRNA Chicken Abu-Halaweh et al.

2005

Salmonella sipB, sipC Meat products Ellingson et al. 2004

Salmonella hylA Ground beef, Nguyen et al. 2004 beef hotdogs

E. coli O157 rfbE Ground beef, Nguyen et al. 2004 beef hotdogs

E. coli O157, O26, per, wzy, Minced beef O’Hanlon et al. 2004 O111 fliC-fl iA,

vt1, vt2

TaqMan E. coli O157 eaeA, stx1, Beef Sharma 2002

stx2

Salmonella Beef, shrimp Kimura et al. 1999

L. monocytogenes hlyA — Lunge et al. 2002 Molecular E. coli O157 rfbE Raw milk, Fortin et al. 2001

beacons apple juice

Salmonella himA — Chen et al. 2000

L. monocytogenes hlyA, iap Dried nonfat Koo and Jaykus 2003 milk

RNA-Based Amplifi cation Assays

While DNA is generally selected as a target molecule in designing PCR assays for food pathogens, a limitation of this approach is that it is not possible to distinguish between viable and nonviable bacteria, though this is somewhat overcome by sample enrichment that increases the numbers of viable cells and target DNA. mRNA, which has a short half-life, is a better target for the determination of viability (Sheridan et al. 1998). The isolation of RNA is an essential step in the analysis of patterns and mechanisms of gene expression (Surzycki 2000a). Moreover, the viruses of importance in food-borne illness have RNA, rather than DNA, as genomic material.

Reverse Transcriptase PCR (RT-PCR)

Reverse transcriptase polymerase chain reaction (RT-PCR) is a variation of the PCR reac- tion and employs the enzyme reverse transcriptase to convert messenger RNA (mRNA) into complementary DNA (cDNA), which is subsequently amplified by DNA PCR (Sambrook and Russell 2001). Thus, this sensitive and powerful technique allows an exponential increase in the amount of mRNA in the form of cDNA copies. The benefits of this procedure include its sensitivity, its large dynamic range, the potential for high throughout, as well as accurate quantification. To achieve this, however, appropriate normalization strategies are required to control for experimental error introduced during the multistage process required to extract and process the RNA (Huggett et al. 2005). In fact, one of the main difficulties with this technique is that isolation of RNA is technically more difficult than DNA and is also less stable. RNA samples also can be contaminated with residual DNA, which makes it impossible to correctly determine RNA concentra- tion or perform RT-PCR, and thus removal of DNA is critical in this technique (Surzycki 2000b). RT-PCR has been used to monitor cell viability in bacteria of relevance for the food industry, such as VTEC (McIngvale et al. 2002), L. monocytogenes (Klein and Juneja 1997), and Salmonella (Szabo and Mackey 1999) and also parasites important in food-borne transmission such as Cryptosporidium and Giardia (Caccio 2004). RT-PCR has been used to identify viruses implicated in food-borne outbreaks in different countries (Sair et al. 2002; Di Pinto et al. 2003; Kobayashi et al. 2004). RT-PCR assays have also been used as part of a large surveillance study on the importance of enteric viruses as causes of illness across Europe (Koopmans et al. 2003).

Nucleic Acid Sequence-Based Amplification (NASBA)

An alternative to RT-PCR is the nucleic acid sequence-based amplification (NASBA). It is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets (Deiman et al. 2002). This method is reported to specifically amplify RNA but not DNA (Heim et al. 1998). This system selectively amplifies RNA through the concerted action of three enzymes: reverse transcriptase, RNAaseH, and RNA polymerase (Cook 2003). Since NASBA amplifies RNA using an RNA T7-polymerase promoter to generate multiple RNA products, ds DNA is not denatured and consequently not amplified (Chan and Fox 1999; Simpkins et al. 2000). NASBA has traditionally been used for the amplification of blood-borne viruses (Kievits et al. 1991; Van Gemen et al. 1993), but it was also optimized for detection of pathogenic bacteria such as L. monocy-

Molecular Technologies for Detecting and Characterizing Pathogens 163

(2003) report on the use of NASBA for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative inoculated foods (fresh meats, poultry, fish, ready-to-eat salads, and bakery products) following an 18 h pre-enrichment. The primer and probe set were based on mRNA sequences of the dnaK gene of Salmonella. NASBA has also been shown to be most useful for detection of organisms that are impossible or difficult to culture, such as viruses from sliced turkey and lettuce (Jean et al. 2004) and

Mycobacterium paratuberculosis from milk (Rodriguez-Lazaro et al. 2004).

Subtyping

Accurate typing and subtyping of pathogenic bacteria are essential if human cases of infection are to be linked within epidemiological investigations, and sources of infection traced (Thomson-Carter 2001). Typing methods can be classified as phenotypic (detecting characteristics expressed by the organism) or genotypic (directly examining the organism’s genetic content) (Maslow and Mulligan 1996). Phenotypic methods include phage typing, serotyping, and biotyping. A range of genotypic techniques for the detection of bacteria in food have been developed and applied as outlined in the following.

Restriction Fragment Length Polymorphism (RFLP)

Restriction fragment length polymorphism (RFLP) analysis investigates certain types of sequence polymorphisms, so-called point mutations that can be base exchanges, base dele- tions, or insertions (Meyer and Birchmeier 1995). The basic mechanism of RFLP analysis relies on the ability of restriction enzymes, the “endonucleases,” to cut double-stranded DNA according to a certain succession of bases in a process called digestion (Hummel 2003). RFLP can be performed by digestion of DNA samples followed by analysis using standard gel-transfer hybridization procedures. Another method is the restriction digestion of a PCR-amplified DNA segment that contains a variably present restriction site. The technique thus requires some knowledge of the DNA sequence flanking that restriction site (Dietrich et al. 1999).

Pulse Field Gel Electrophoresis (PFGE)

Pulse field gel electrophoresis (PFGE) employs restriction enzymes (endonucleases) to make a limited number of cuts in bacterial chromosomes to provide chromosomal restric- tion patterns that form a “fingerprint” for each organism (Méndez-Álvarez et al. 1995). In this technique, DNA is first prepared and encapsulated in agarose plugs, digested with appropriate restriction enzymes depending on the target organism, and subjected to electro- phoresis in which an electric field periodically changes direction and/or intensity relative to the agarose gel (Maule 1998). It is important that the test organisms are embedded in agarose plugs and that the DNA is released in situ because this minimizes shearing of the DNA before it is digested with restriction enzymes (Maslow and Mulligan 1996). The periodical reorientation of the electric field allows the separation of large DNA fragments, which would not be adequately separated by conventional agarose gel electrophoresis using a constant (direction) electric field (Maslow and Mulligan 1996). In PFGE, each time the field is switched, larger molecules take longer to change direction and have less

time to move during each pulse, so they migrate slower than smaller molecules, leading to overall separation (Basim and Basim 2001). In this way, while conventional agarose gel electrophoresis may achieve a maximum resolution of 50 kb, PFGE is able to separate molecules as large as 12 Mb (Maule 1998). The concept of a contour-clamped homogene- ous electric field to separate large DNA molecules was introduced by Chu and coworkers (1986). PFGE requires highly technically skilled personnel but is very reproducible and has the advantage of generating genetic data that can be statistically analyzed. Using standardized protocols, PFGE is used to share genetic information on food pathogens in national and international networks such as PulseNet (a U.S. national network of public health and food regulatory agency laboratories coordinated by the Centers for Disease Control and Prevention) and the Europe International surveillance network (ENTER-NET) for the enteric infections.

PFGE has been used extensively in the investigation of sporadic cases and outbreaks of food-borne microbial infection and has also proved invaluable in molecular characteriza- tion and epidemiology of infection related to verotoxigenic E. coli (Thomson-Carter 2001),

Salmonella (Ross and Heuzenroeder 2005), L. monocytogenes (Okwumabua et al. 2005),

and Clostridium botulinum (E. A. Johnson et al. 2005).

Amplifi cation-Based Typing Methods

Various typing systems that use PCR-amplification-based methods have been described. One of the main disadvantages of amplification-based methods is that many factors can affect reliability and reproducibility (Tyler et al. 1997).

Random Amplifi cation of Polymorphic DNA (RAPD)

Random amplification of polymorphic DNA (RAPD) is a PCR-based method in which the primers are chosen arbitrarily rather than based on knowledge of the sequence to be ampli- fied. The stringency of primer annealing is low to allow priming of imperfectly matched sequences. PCR performed under these conditions generates a complex pattern of PCR products that is, at least in theory, unique to a particular bacterial strain. This method is distinct from the classic PCR in its use of a single primer instead of two and a low-strin- gency annealing temperature (Williams et al. 1990). Cocolin et al. (2005) have reported on the benefits of a RADP-PCR as an epidemiological tool for L. monocytogenes.

Amplifi ed Fragment Length Polymorphism (AFLP)

The amplified fragment length polymorphism (AFLP) technique is based on the selec- tive PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves the digestion of DNA with restriction enzymes and then the ligation of the fragments’ ends to nucleotide adapters that are designed in such a way that the initial restriction site is not restored after ligation. This allows simultaneous restriction and liga- tion, while religated original fragments are cleaved again. Finally PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. Visualization of the amplified fragments is then performed by gel electrophoresis (Vos et al. 1995). Two restriction enzymes are used, one with an

Molecular Technologies for Detecting and Characterizing Pathogens 165

average cutting frequency (like EcoRI) and a second one with a higher cutting frequency (like MseI) (Sharbel 1999). The vast majority of bands detected on AFLP gels are frag- ments flanked by both enzyme recognition sites. Like RAPD, AFLP analysis is applicable