DNA based typing, identification and detection systems...

ELSEVIER International Journal of

Food Microbiology 33 (1996) 35-49

htematianal Journal ofFoodh4icmbidagy

DNA based typing, identification and detection systems for food spoilage microorganisms:

development and implementation

Abstract

The rapid identification of spoilage microorganisms is of eminent importance to the food industry. It provides the food industry with the opportunity to reduce economical losses by designing adequate intervention measures. The use of identification systems based on biochemical and physiological characteristics resulted often in disappointing identification results and misidentifications. This will inevitably lead to inappropriate strategies to prevent spoilage. This review discusses the potential of the DNA based identification technology including the polymerase chain reaction (PCR) for the identification and specific detection of microorganisms. Fingerprinting methods based on the DNA-probe technology enable a clear insight in the identity of microorganisms on different levels, varying from genus to strain level depending on the systems used. Discrimination between subspecies and strain level is shown to be helpful for investigating routes and sources of contamination. Differentiation at the species level is demonstrated to be essential in order to design a highly specific detection system enabling to signalize a microorganism that belongs to a particular species. Also indicated in this review is the necessity and the technical approach to detect microorganisms that display a particular undesirable trait.

*Corresponding author. Tel.: + 31 030 6944720; fax: + 31 030 6954186; e-mail: vander- vossen~voeding.tno.nl

016%1605/96~$15.00 Q 1996 Elsevier Science B.V. All rights reserved

PIISO168-1605(96)01136-I

1. Introduction

Generally, food spoilage has often been recognized as inconvenient but was not considered to be as serious as the contamination of food with pathogens. A lot of attention has been paid to food-borne pathogens. Together with the increased i~~plement~tion of hazard analysis critical control point (HACCP) progr~lrnllle~. this resulted in a reduced prevalence of food-borne infe~tiolls from a number of end-products. Food spoilage is becoming a major problem to the food industry because the scale in which food and beverages are currently produced has increased considerably during the last decades. This implies that the economical impact of spoilage problems is usually very high. In addition, the consumer demands fbt

milder processing, preservation and storage conditions (fresh products. less salt etc.) contribute also to the increase of spoilage problems in industry. A clear insight in the range and extent of the problem remains however limited.

Spoilage of foods and beverages is the result of microbial activity of a variety of microorganisms. The microbial flora which will develop during storage depends highly on intrinsic parameters (e.g. water activity. pH, redox potential, nutrients. antimicrobial compounds etc.) extrinsic parameters (e.g. temperature, humidity. atmosphere etc.), modes of processing and preservation, and implicit parameters (e.g. direct and indirect microbial interactions) (Delik, 199 I ; Mossel, 1982).

In addition to this basic knowledge, it could be advantageous to identify the ~~~i~r~~org~~~~is~~s that are able to proliferate in the product. It offcrs the opportunity to take pre~~~uti~)t~s to ~il-culnv~i~t spoilage by the nii~l-oor~~~lli~rns identitied. This could be established either by tuning the conditions of processing and storage or by blocking the entrance of the undesired microorganisms into the production chain. This underlines the necessity to have tools available to identify the microorganisms causing problems and to recognize the origins and the routes of contaminations.

Unfortunately, traditional identification methods which arc based on laborious morphological and physiological tests often fail in this respect. They lack discrimi- natory power and as a consequence, lnisi~~e~~titi~~tion occurs frequently. Routine detections and eliunier~tion of ~li~roorg~~~~isn~s by tr~lditi~~n~~l plating techniques and subsequent identification, results in the slow and incomplete release of data which, as a consequence, can be used for retrospective evaluation only.

This paper will provide an overview of molecular biological detection, identilication and typing techniques and contemplate how the integrated use of such techniques can be implemented in order to assess and improve the microbiological quality of food products. In addition, examples will be given of the ~pplic~ti~~l~ of molecular biolo~i~l techniques for the i~loi~ito~-ing of food spoilage.

2. Classic DNA-based detection and identification methods

Generally, DNA based methods have the advantage over phenotypic identification methods of not being influenced by the environmental conditions of the cells because the nucleotide sequence of the DNA is kept constant during growth (Ness

J.M.B. M. tm der Vossen, H. Hojitrrr 1 Ini. J. Food Microhiolot~~~ .73 (I 996) 3.549 37

et al., 1993). This statement holds only to a certain extent, as it has been shown that

extrachromosomal elements such as plasmids could be highly unstable (McKay, 1983). Chromosomal sequences are also subject to infrequent rearrangements (Polzin et al., 1993). Changes in the genetic material during evolution enable us to differentiate between different microorganisms at different levels. In this section a number of classic DNA based detection, identifi~dtion and typing methods will be presented and discussed with respect to aspects like ease and operation, and the potential of the method for application in the food industry.

Plasmid profiling is a typing method in which extrachromosomal DNA is isolated from a pure culture and subjected to agarose gel electrophoresis. This method enables the discrimination at subspecies level (Hill and Hill, 1986; Davies et al., 1981; Vogel et al., 1991). The plasmid profiling approach seems to be simple but the instability of plasmids interferes negatively with appropriate identifications. More- over, the rigidity of some bacteria hampers plasmid isolation and large plasmids are often degraded, due to rough experimental procedures. All these factors, plus the fact that not all bacteria contain plasmids, causes the method to be of limited value only. Whole genome targeting approaches are more generally applicable. Restric- tion enzyme analysis (REA) proved useful in typing several microorganisms (Stahl et al., 1990). However, the complexity of the patterns, often showing from hundreds to over a thousand bands, hinders proper evaluation and therefore, the suitability of REA for routine identification purposes is very doubtful.

Pulsed field gel electrophoresis (PFGE) in which large DNA molecules are resolved by continuous reorientation of the electric field during gelelectrophoresis (Schwartz and Cantor, 1984). provided new possibilities for typing and subsequently identification of microorganisms. The method is applied after the digestion of the complete genome with rare cutting restriction enzymes such as Apa 1, Stntr 1, &$I, %cIl, Notl, $fil, etc. (Tanskanen et al.. 1991; Le Bourgeois et ai., 1991, 1993; Kelly et al., 1993, 1995). Initiaily, this electrophoresis method was used to investigate the ~hromosomal content of eukaryotic organisms such as yeasts (karyotyping) (Schwartz and Cantor, 1984). Den Dunnen and van Ommen (1993) showed different PFGE systems and provided a clear protocol for using the equipment. Basically, the continuous reorientation of the electric field causes the DNA molecules to stretch in the direction of the field and hook when the field has changed. This results in a migration velocity in the net field direction depending primarily on the size of the DNA molecule. Long molecules need more time to reorientate than smaller molecules.

2.2. Techniques bused on DNA hybridizution

The DNA hybridization technology is the basis for a number of detection, identification and typing techniques. In its basic application, DNA is fixed to a solid phase and a labelled DNA probe is added and allowed to react with its

counterstrand (Southern, 1975; Matthews and Kricka, 1988). In restriction fragment length polymorphism (RFLP) analysis, DNA is cleaved by restriction enzymes and the resulting fragments are separated by gelelectrophoresis, like in REA, described above. Different banding patterns (polymorphism) may be observed after transferring the DNA from the gel to hybridization filters by blotting and hybridization with labelled DNA-probes. After visualization of the label, a typical banding pattern can be observed. DNA probes used in RFLP analysis are often based on highly conserved genes coding for rRNA (in ribotyping) (Grimont and Grimont. 1986: Rodrigues ct al., 1991: Rodtong and Tannock. 1993: Salzano et al., I%@; Johansson et al., 1995). Rodrigues et al. (f991) demol~str~~ted that the ribotyping method enabled dis~rimin~ltion at the species level although some pattern variation was observed at subspecies levels. DNA-probes may also be based on other highly conserved genes as c.g. DNA-gyrase. RNA-polymersse, ATPase. ribosomal protein S12, and elongation factor Ef-TU. These probes proved to be useful and allow identification at II lower level, subspecies to strain. than ribotyping (Hofstra et al., 1994). Looking for the presence and the relative copy number of lactococcal insertion sequences ISSI in lactococci and several other lactic acid bacteria, Polzin et al. (1993) demonstrated that this RFLP analysis discriminates down to the strain level. They also observed that one frozen stock culture consisted of a mixture of cells having different ISSI-hybridizing bands, indicating that stock cultures may contain cells with varying locations of these insertion sequences. RFLP, using probes based on mit~~~hondri~ll DNA is by its nature. only applicable to eukaryotes (yeasts and moulds), as only cukaryotes contain these organclles (Aigle et al.. 3984; Dubourdieu ct al., 1987; Hallet et al., 1988: Vezinhet et al.. 1990).

The integration of PFGE and DNA hybridization which is essentially used for physical and genetic mapping of chromosomes of lactic acid bacteria (Lc Bourgeois et al., 1992) and yeasts (Henriques et al., 1991; Naumova et al., 1993; Tdriik ct al., 1993) certainly provides the opportunity to discriminate at subspecies level or even below. Since chromosome length polymorphism due to rearrangements and aneu- ploidy occurs frequently, the taxonomic interpretation of the karyotypes is difficult.

Though all moIecular typing methods, including RFLP and the other techniques described so far are usually classified as DNA-fingerprinting techniques, we prefer to use this term for the method described by Jeffrey et al. (1985a,b), in order to obtain individual ‘fingerprints” of human DNA. This DNA-fingerprinting method is very similar to RFLP analysis except for the fact that the DNA probe used are small repeated sequences such as microsatalite DNA or minisatalite DNA (Jeffrey et al., 198%; Jeffrey et al., 1985b). This system has shown to be useful for typing of fungi (Meyer et al., 1991; Lieckfeldt et al.. 1992; Meyer et al., 1992; Meyer et al., I993), but is of minor importance for typing bacteria. Only Miteva et al. (1992) showed that the M 13 core sequence was useful for DNA ~ngerprintin~ in lactic acid bacteria.

J.M.B.M. tun der Vossm, N. Ffo&tra /ht. J. Food Microbiology 33 (1996) 35-49 39

A major disadvantage of the techniques described so far is that they are rather laborious and time-consuming, because considerable amounts of relatively pure DNA or RNA are required and the development of banding patterns takes at least several hours. Because of this and the fact that special facilities are essential for these methods, they are not appropriate for routine identifications in the food industry.

3. PCR-mediated methods

In contrast to the methods dealt with so far, PCR methods are generally rapid and easy to execute. Only a small amount of DNA is required, which does not necessarily need to be highly purified. The Taq polymerase can amplify DNA from lysed cells without any further purification.

In PCR, a specific DNA-fragment is amplified by a thermo-cyclic process, in which the DNA is denaturated at high tem~~ture. Subsequently, two specific oligon~tcleotides are hybridized to the complementary strands at a temperature just below their melting temperature: the annealing temperature, and finally DNA polymerase will extend the oligonucleotides at a temperature optimal for its activity. This results in a doubling of the DNA between the two primers to two double strands. By repeating the cycle several times, DNA between the two primers is amplified exponentially (Saiki et al., 1988).

One of the techniques, recently developed and based on PCR technology is the random amplified polymorphic DNA (RAPD) analysis. In the RAPD assay patterns are generated by the amplification of random DNA segments with single small e.g. IO-base primers of arbitrary nucleotide sequence, and a subsequent gel electrophoresis of the amplified DNA (Williams et al., 1990) Repeated cycles of heating and cooling, generally 45 cycles per RAPD assay, lead to an exponential synthesis, and thus many copies of the amplified segments. An amplification of IV-fold copies can be expected (Kocher and Wilson, 1992). RAPD was first applied for the identi~cation of a polymorphism linked to pest and disease resistance in plant cultivars and horticultural species (Martin et al., 1991; Wolff and Peters-van Rijn, 1993) and later shown to be suitable to generate fingerprints from several bacteria (Mazurier and Wernars, 1992; Du Plessis and Dicks, 1995; Stephan et al., 1994) and fungi (Baleiras Couto et al., 1994, 1995; Bidochka et al., 1994; Bostock et ai., 1993; Lieckfeldt et al., 1992; Molnar et al., 1995). The RAPD assay allowed discrimination at the species level, but small differences in patterns of different strains belonging to the same species occur, indicating that it is also possible to djs~r~minate at the subspecies level. To our experience, the arbitrary primer of choice is only to a certain extent important for the level of discrimi~dtion. More important is the number of bands generated in the RAPD assay with a particular primer. Certainly not all IO-base primers are useful for the RAPD assay. Unfortu- nately, RAPD patterns are not always reproducible among or even within laboratories. This problem could only be solved by observing very strict rules ~on~erning the overall temperature profiles, especially the time involved in heating the reaction

from annealing temperature to the temperature of primer-extension during the amplification procedures (Penner et al., 1993).

A related approach is the amplification of fragments by PCR with oligonucleotides specific for simple repetitive DNA sequences (PCR fingerprinting). This technique was developed on the basis of previous knowledge achieved with the DNA fingerprinting analysis (Lieckfeldt et al., 1993). PCR ~n~erprinting with the tni~ros~telite oligonu~leotide primers (GTG),, (GA@?),. (GACA),. the phage M 1 I DNA and the M 13 sequence GAGGGTGGCGGTTCT has been used for yeast identification (Kunze et al., 1993: Lavallee et al.. 1994: Lieckfeldt et al., 3993: Baleiras Couto et al., 1996a.b). This identification approach allows discrimination at the species and subspecies level. Repetitive DNA sequences have been described recently in eubacteria. Based on these sequences oligonucleotides have been designed, matching repetitive extragenic palindromic (REP) elements and enterobacte- rial repetitive intergenic consensus (ERIC) sequences, which enabled the generation of tillgerprints with PCR. Since these repetitive sequences are mainly present in enteric bacteria and some related Gram-negative bacteria. as observed by hybridizution experiments with REP and ERIC probes, the applicability of this fingerprinting method is restricted to these organisms (Versalovic et al., 1991). The fingerprint primer applicable for yeast typing (GTG), appeared also a useful primer for PCR-fingerprinting of lactic acid bacteria (van der Vossen, unpublished).

The PCR-fingerprinting method is more robust than the RAPD method since the annealing temperature is higher. 55°C instead of 37°C. which is closer to the optimal temper~tlire (72°C) of the Taq polymerase so the primer is extended before an increase in temperature may denaturdte the primer from the template DNA. This makes it easier to compare these fingerprints among laboratories and to construct databases.

The small subunit ribosomal RNA encoding genes (ss rDNA) are part of a highly conserved region. These sequences evolve relatively slowly and hence appear adequate to study genetic relationships at the species level (Baleiras Couto et al.. 1995; Hopfer et al., 1993; M~iw~~ld et al., 1994; Shen et al.. 1994; White et al., 1990). Small subunit rDNA fragments of several spoilage yeast species have been amplified by PCR using primers defined on the basis of the conserved regions (Baleiras Couto et al., 1995). Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified ss rDNA can be visualized upon digestion with restriction enzymes and subsequent agarose gel electrophoresis. enabling discrimination of yeasts at the species level (Baleiras Couto et al., 1995: Niesters et al., 1993; Vilgalys and Hester, 1990).

Classical techniques such as DNA ~ngerprinting of eukaryotic DNA (Kunze et al., 1993; Lavaliee et al., 1994; Lieckfeldt et al.. 1993), electrophoretic k~ryotypil~~ (Casey et al., 1990; Kunze et al., 1993: Petering et al., 1988; Venzinhet et al., 1992) and RFLP analysis (Magee et al., 1987) have been shown to discriminate below the species level. Discrimination on the strain level using PCR-based techniques has been shown to be possible for deuteromycetous fungi species using RAPD (Bidochka et al., 1994). The spacer region between the ss rDNA and the large subunit rDNA (Is rDNA), termed the internal transcribed spacer (ITS) (White et

J.M. B.M. ran der Vossen, H. Hojkwa /// Int. J. Food Microbiology 33 (1996) 35 -49 41

al., 1990) and the spacer between the ribosomal gene cluster, named non-transcribed spacer (NTS) or intergenic spacer (IGS) (Musters et al., 1990; Welsh and McClelland, 1992) seem also appropriate to discriminate below the species level. These regions are highly variable among and within species, because they are under a lower degree of conservation. Length and sequence polymorphism of these regions can be visualized after restriction enzyme digestion of PCR amplified fragments, enabling differentiation at the subspecies or sometimes strain level (Baleiras Couto et al., 1996a; Molina et al., 1993). We have assessed genomic variability among S. cerevisiue strains using RAPD analysis, PCR fingerprinting, and restriction enzyme analysis of the ITS and NTS regions (Baleiras Couto et al., 1996a). Implementation of the described PCR mediated typing system allowed Baleiras Couto et al. (1996b) to identify spoilage causing yeast in a survey of yeasts present in the production chain of mayonnaises to be Zygosacchuromyces hailii strains. The strains with a particular type appeared to be identical to a certain strain present on a specific production line. This example illustrates that PCR mediated typing techniques are useful, firstly, in discriminating yeast species and recognizing the particular species that is involved in spoilage of an end product and secondly, in tracing back to the origin of a spoilage outbreak.

Therefore, these typing approaches may be useful as a rapid identification technique in quality control monitoring systems, if a data base of types, found in earlier spoilage cases, is available.

4. Detection methods using DNA-probes

A tool for the rapid detection of microorganisms, developed recently, is a spin off from phylogenetic studies. The evolutionary chronometers used in these studies are the ribosomal RNA sequences and based on the comparison of these sequences phylogenetic relations were determined which are helpful for tools in taxonomy (Martinez-Murcia and Collins, 1990; Wallbanks et al., 1990). Based on these rRNA sequences many species specific probes have been developed mainly for the detection of lactic acid bacteria (Betzl et al., 1990; Hertel et al., 1991; Hertel et al., 1993; Nissen and Dainty, 1995; Pot et al., 1993; Vogel et al., 1993). These rRNA-targeted oligonucleotide probes have successfully been used for the detection of some meat related lactobacilli isolated from vacuum- or gas-packed meat in colony blot hybridizations (Nissen and Dainty, 1995). Beimfohr et al. (1993) used rRNA- targeted oligonucleotides for the in situ hybridization of particular species from the genus Lactococcus, Enterococcus, and Streptococcus. They applied fluorescent oligonucleotides for in situ hybridization in combination with epifluorescence microscopy. This enabled the specific detection of single cells in milk samples.

Another method for the direct detection of lactic acid bacteria in fermented food is the reverse dot blot hybridization method developed by Ehrmann et al. (1994). In this method the rRNA-targeted oligonucleotides are used as a capture probe for PCR amplified rDNA sequences which were isolated from the fermented food matrix by a fast DNA extraction protocol. Recently, we (Le Jeune et al., 1995) have

shown, that DNA probes are useful for the rapid screening of a large number of

lactic acid bacteria for the presence or absence of an undesirable property such as histidine decarboxylase activity by targeting the gene involved.

5. Rapid detection by PCR methods

Techniques based on PCR are currently being developed for the direct detection of microorganisms. In order to design a specific PCR detection system, specific oligonucleotides should be defined. With respect to the specific detection of micro-organisms it is of eminent importance to choose the target DNA-sequence in

such a way that no false positive signals occur. This prerequisite for the specific amplification of DNA makes the evaluation of the detection result rather simple, an

amplification product of the expected size is sufficient and makes confirmation by a DNA probe superfluous. In case the PCR-amplification does not display the

desired specificity, a new set of primers should be defined or one should take advantage of the nested PCR approach. In nested PCR one set of primers is used

to amplify DNA-fragments from target DNA and the second set of primers, which are complementary to an internal sequence of the correct PCR product of the first round of PCR, is used to score the result and can be seen as a confirmation-step.

The most common way to obtain specific probes and primers is the selection of random DNA fragments from a gene bank (Chryssanthou et al., 1994: Fitts et al.,

1983; Hofstra and Huis in ‘t Veld, 1990; Miyakawa et al., 1992). In this case fragments are selected on the basis of the specificity that is desired for their

application (Hofstra et al., 1994). In order to obtain a specificity above the species level. definition of the primers can be based on specific DNA sequences of highly

conserved genes, such as the rRNA genes (Brooks et al., 1992; Grant et al., 1993; Herman et al., 1995; Hopfer et al., 1993; M~kirn~Ir~ et al., 1994a; Nakagawa et al..

1994). A PCR detection system for ~.s~~~r~~flzis spp. and P~~?~il~~~l~li~~~ spp. was based on the gene coding for the small subunit rRNA of both organisms respectively (M~kirnlIr~ et al., 1994b). Another source of DNA probes are genes coding for

virulence factors that can be used to differentiate between pathogenic and non- pathogenic strains within a single species (Rubin et al., 1985).

Alternatively, probes can be developed based on outer membrane protein-encoding genes (Speirings et al.; 1989). In order to detect bacteria that display a

particular undesirable trait, genes coding for a specific activity such as the production of histamine by lactic acid bacteria can be targeted by the /z&A gene specific primerset (Le Jeune et al., 1995). All histamine forming lactic acid bacteria and Gram-positive bacteria can be detected with this PCR detection system. These probes can be used for the detection of specific microorganisms with the potential to display a partic&r undesirable activity. A different approach to isolate probes has been demonstrated by Dobrowolski and O’Brien (1993) who selected a specific DNA-probe for Phvtophthora cinnamomi from RAPD amplification products upon careful selection.

J. M.B.M. L’CM cler Vossen. H. Ho/.itra / Inr. J. Food Microhiolog_v 33 (1996) 35-49 43

Although PCR detection systems for a wide range of medically important

microorganisms were developed (Olsen et al., 1995), only a few systems have been

developed for the rapid detection of spoilage organisms in foods so far. A multiprimer PCR for discriminating between S. crreuisiue, 2. bailii and Z. rouxii

was developed by Pearson and McKee (1992). Additionally, non-radioactive PCR- coupled ligase detection was developed to discriminate between the food spoilage yeasts Z. bailii and Z. bisporus (Stubbs et al., 1994). A few PCR detection systems

have been developed for some important spoilage bacteria such as Brochothrix in meat (Grant et al., 1993), Crrrnobacterium in meat (Brooks et al., 1992) alcohol

tolerant lactic acid bacteria in wine and beer (Nakagawa et al., 1994; Tsuchiya et al., 1992) and Clostridium tyrobutyricum spores in raw cheese milk (Herman et al.,

1995). The implementation of PCR detection systems in food is hampered by the fact

that the DNA polymerase in the PCR reaction is often inhibited by substances present in the food (Rossen et al., 1992). Additionally, the sample size imposed by low contamination levels to be detected cannot be accommodated by the small PCR

volumes (50- 100 111). Another hurdle for PCR detection in foods is the presence of non-viable target microorganisms, which may induce false-positive results if DNA from these cells has remained intact. In order to overcome such problems, creative combinations of purification, cell concentration and culturing methods should be adapted to pre-treat each specific sample (Huis in ‘t Veld, 1991). A selective

enrichment of microorganisms prior to amplification can also be applied in order to increase the number of microorganisms and to dilute the food components (Hofstra

et al., 1994; Swaminathan and Feng, 1994). In addition, the use of immuno magnetic beads in order to separate specific bacteria from its environment add to

the sensitivity of the PCR detection system (Hofstra et al., 1994) and also the separation of Gram-positive organisms by magnetic beads on which lectin is immobilized enabled the direct detection of low numbers of Brochothrix in meat samples in PCR (Grant et al., 1993). For the direct and specific detection of clostridial spores in raw milk, the milk samples were chemically extracted and the

spores were opened by intensive microwave treatment to release their DNA for

PCR detection (Herman et al., 1995). In contrast to food pathogens, food spoilage organisms are sometimes allowed to

be present in the food product. For these occasions, it may be essential to quantify the initial number of a particular spoilage organism in order to assess the risk of its presence and to predict the shelf-live. The initial count of a spoilage microorganism

is an important parameter which could not be easily determined by traditional microbiological methods. PCR enables us to detect low numbers of specific

microorganisms. Although PCR is not suited for quantification purposes, most- probable-numbers can be assessed by taking precautions prior to the sample pretreatment. At present, genus specific primers and a protocol have been designed to assess the initial contamination-level of Pseudomonas, which is an important genus involved in the spoilage of refrigerated food products, by PCR (van der Vossen, unpublished).

Alternatively, information on the initial load of spoilage organisms may be important to adapt the production process in such a way that spoilage incidents are eliminated. For example, in order to prevent late blowing of cheese, milk with a high level of spores will be subjected to bactofugdtion and/or nitrate will be added in the process of cheese production. Spoilage organisms such as Z. ~~~~il~~ and Z. rousii are highly detrimental to high acid and high sugar containing products in which they could proliferate. The presence of only one in the product will cause a problem sooner or later. In this case. a detection system is needed that indicates the presence or absence of the spoilage organism.

6. Conclusions

Rapid PCR based detection techniques can be used in the food industry as an integral part of quality-control and -assurance programmes. Essential for the implementation of these novel PCR based techniques in routine laboratories of the food industry is, that they should be adapted in such a way that they become robust so that they are easy to handle. In addition, intervention systems should be designed in order to act adequately on the data provided by the monitoring system.

The typing methods enable the rapid identification of spoilage microorganisms and facilitate the design of appropriate intervention measures. Moreover. some of these typing methods provide a solid basis for the assessment of the taxonomicul position of microbial isolates. This is essential when designing species-specific PCR detection systems for specific microorganisms. Species-specific probes and primers must recognize all strains belonging to the species and never react with other organisms. Only if these conditions are assured, the species-specific detection system could form a basis for an unambiguous quality control programme.

The ~v~~il~lbility of species specific primers for the specific PCR detection of undesired mi~roorg~nisIl1 is no longer a problem. Sample pretre~tmellt needs, however, more attention, especially when the pretre~~tmellt protocol should be in~plemel~ted in a routine I~bo~tol-~/.

References

Aigle. M., Erbs. D. and Mall. M. (1984) Some molecular structure in the genome of lager brewing yeast.

Am. Sec. Brew. Chem. 42, I-7. Baleiras Couto, M.M., Eijsma, 8.. Hofstra, II., lluis in ‘I Vcld, J.II.J. and van dcr Vossen. J.M.B.M.

(IBYha) Evaluation of molecular typing techniques to assign genetic diversity among strains of Suc,c,l~rrrotl~~r.r~ c~erccisiur. App. Envrion. Microbial. 62. 4 I --46

Baleiras Couto, M.M., Ha-tog, B.J.. Huis in ‘t Veld, J.H.J.. Hofstra. H. and van der Vossen, J.M.B.M. (1996b) Identification of spoilage yeasts in a food production chain by microsatellite PCR finper-

printing. Food Microbial. 13, 59-67. Baleira Couto, M.M., van der Vossen, J.M.B.M., Hofstra, H. and Huis in ‘t Veld. J.H.J. (1994) RAPD

analysis: a rapid technique for differentiation of spoilage yeasts. Int. J. Food Microbial. 24, 249 260.

Baleiras Couto, M.M., Vogels, J.T.W.E., Hofstra, H.. Huis in ‘t Veld, J.H.J. and van der Vossen, J.M.B.M. (1995) Random amplified polymorphic DNA and restriction enzyme analysis of PCR

amplified rDNA in taxonomy: two identification techniques for food-borne yeasts. J. Appl. Bacterial.

79, 525-535.

Beimfohr, C., Krause, A., Amann, R.. Ludwig, W. and Schleifer, K.-H. (1993) hz siru identification of

lactococci, enterococci, and streptococci. System. Appl. Microbial. 16, 450-456.

Betzl. D., Ludwig, W. and Schleifer, K.-H. (1990) Identification of lactococci and enterococci by colony

hybridization with 23s rRNA-targeted ohgonucleotide probes. Appl. Environ. Microbial. 56, 2927-

2929.

Bidochka, M.J.. McDonald, M.A., Leger, R.J.S. and Roberts, D.W. (1994) Differentiation of species

and strains of entomopathogenic fungi by random amplification of polymorphic DNA (RAPD).

C’urr. Genet. 25, 107-l 13.

Bostock, A., Khattak, M.N.. Matthews, R. and Burnie, J. (1993) Comparison of PCR ~ngerprinting, by

random amplificatiun of polymorpi~ic DNA, with other molecular typing methods for C~~~~~~~~

trfbictr,ts. J. Gen. Microbial. 139. 2170-2 184. Brooks, J.L.. Moore, A.S., Patchett. R.A., Collins, M.D. and R.G. Kroll. (1992) Use of the poiymerase

chain reaction and oligonucleotide probes for the rapid detection and identification of Cnmohac-

tcrirr,?? species from meat. J. App. Bacterial. 72, 2944301.

Casey, G.P., Pringlc, A.T. and Erdman, P.A. (1990) Evaluation of recent techniques used to identify

idividual strains of Succhurom?;ces yeasts. Am. Sot. Brew. Chem. 48, 100-106

Chryssanthou, E.. Andersson. B., Petrini, B., Lofdahl, S. and Tollemar, J. (1994) Detection of Can&~z

cr/l>ic,cril.s DNA in serum by polymerase chain reaction. Stand. J. Infect. Dis. 26, 479-485.

Davies, F.L.. Underwood, H.M. and Gasson, M.J. (1981) The value of plasmid profiles for strain

identilication in lactic streptococci and the relationship between Srre/>rococcus luctis 712, M 13, and

(2. J. Appl. Bacterial. 51, 325-337.

Desk. T. (1991) Food borne yeasts. Adv. Appl. Microbial. 39, 179 278.

Den Dunnen. J.T. and van Ommen, G.J.B. (1993) Methods for pulsed-field gel electrophoresis. Appl.

Biochem. Biotechnol. 38. 161-177.

Dobrowolski. M.P. and O’Brien. P.A. (1993) Use of RAPD-PCR to isolate a species specific DNA probe

for ~/z~f~~~~~~~~~~~~ c%mumomi. FEMS Microbial. Lett. 1 13, 43348.

Du Plessis. E.M. and Dicks, L.M.T. (1995) Evaluation of random amplified polymorphic DNA

(RAPD)-PCR as a method to differentiate Lwtohacillus rrcidophilus, Loc~to/~ot~i//u.s c~riqmootus, Lucto-

htrc,ilht.s am~~olocor-us, Lucroha~il1u.s ~ollinurun~, Luc~tohuci1lu.s ~mscri, and Lactohuc~il1u.s ,johnsmii.

C‘urr. Microbial. 31. 114 118.

Dubourdieu. D.. Sokol. A., Zucca, J., Thalouarn, P., Datte, A. and Aigle. M. (lY87) Identification des

souches dc levures isolees de vins par I’analyse de leur ADN mitochondrial. Connaiss. Vigne Vin 2 1, 261. 278.

Ehrmann. M., Ludwig, W. and Schleifer, K.-H. (1994) Reverse dot blot hybridization: a useful method

for the direct identification of lactic acid bacteria in fermented food. FEMS Microbiol. Lett. 117.

143- 149.

Fitts. R., Diamond, M., Hamilton, C. and Neri, M. (1983) DNA-DNA h~bridi~atio?l assay for the

detection of ~ff~~~~~ll~ spp. in foods. Appl. Environ. Microbial. 43, t 146- I 151.

Grant, K.A., Dickinson, J.H., Payne. M.J.. Campbell, S.. Collins, M.D. and Kroll, R.G. (1993) Use of

the polymerase chain reaction and 16s rRNA sequences for the rapid detection of Brochothrix spp.

in foods, J. Appl. Bacterial. 74, 260-267.

Crimont, F. and Grimont, P.A.D. (1986) ribosomal rebonucleic acid gene restriction as potential

taxonomic tools. Ann. L’inst. Pdsteur/Microbiol. 137B, 165 175.

Hallet, J.-N., Craneguy, B., Zucca, J. and Poulard, A. (1988) Caracterisation de differentes souches

industrielles de levures oenologiques par les profils de restriction de leur ADN mitochondrial. Prog.

Agric. Vitic. 105, 328-333.

Henriques, M., Sa-Nogueira, I., Gimenez-Juradao,G. and van Uden, N. (1991) Ribosomal DNA spacer

probes for yeast identification: studies in the genus Metscimiknwia. Yeast 7, 167.--172.

Herman, L.M.F.. de Block, J.H.G.E. and Waes, G.M.A.V.J. (1995) A direct PCR detection method for

Closir~~~u~z f~ro~~f~~~~~~ spores in up to 100 milliliters of raw milk. Appl. Environ. Microbial. 61,

4141.--4146.

Hcrtei. C.. Ludwig. W.. Pot. B.. Kersters. K. and Schleifer. K.H. (1993) L3ifferenti;ition of lactobacilli

occurring in fermented milk products by using oligonucleotide probes and slcctrophoretic protein

profiles. Syst. Appl. Microbial. 16. 463 -467.

Herte1.C.. Ludwig, W.. Obst. M., Vogel. R.I-.. Hamnw~. W.P. anti Schleifer. K.-H. ( 1991) 7.X rRNA-targeted oligonttcleotide probes for the rapid idcntitication of tnwt lactobacilli. Syzt. i\ppl.

Microbial. 13. 17.1. 177.

Hill. H.n. and frill. J.E. ( I OR6) The value of plasmmid proliiing in monitoring tc/c,f,,hirc.i//ia ~~/~/~~~~/~r//~~

in silage fermentations. Cut-r. Microbial. l3:OI 94.

Hofstra, H.. van dcr Vossen. J.M.B.M. and van der Plas, J. (1994) Microbes in food processing

technology. I-‘LIMS ~~icrobiol. Rev. 15. I75 I X.3. Hofstra. H. and I lttis in ‘t Veld. J.H.J. (1990) Bi~)-ttilly~i~~l methods: The application of monoslonai

antibodies and DNA hybridisation in food mtcrobiology. In: P Zettthen et al. (editors). I’roccssing

and Quality of Foods. Elscvicr, London. pp XI 287.

Ilopfcr, R.L.. Wuldcn. P., Setterquist, S. and Highsmith. W.1:. ( 1993) Detection and differentiation 01

fungi in clinical speciments using polymerwe chain reaction (PCR) amplification and restricition

enzyme analysis. J. Med. Vet. Mycol. .il, 65 75.

Huis in ‘t V&i. J.H.J. and Hofstra. H. (1991 I ~i(~te~~llr~~~lo~~ and the quality assurance of foods, Food

Biotechnol. 5, 313 372.

Jeffrey. A.J.. Wilson. V. and Thein. S.I.. (1985;t) H~l~~r\~lri~tble ~~~~i~iis~ttellile’ regions in human DNA.

Nature 316. 67 73.

Jeffrey. A.J.. Wilson, V. and Thein. S.L. (198Sh) Individrt~tl-specific ‘fingerprints‘ of human DNA.

Nature 316, 76 79.

Johansson, M.-L.. Molin. G., Pcttcrsson, B.. Uhlcn. M. and Ahrne, S. (1995) Char;tctcrizatioll and

species recognition of Lnc,tohtrc,i//rr., p/tn~/lrrrlm strains by restriction fragment length polymorphtstn

(RFLP) of the IhS I-RNA gene. J. Appl. Bacterial. 79. 536 541.

Kelly. W.J.. Huang. C.M. and Asmundwn. R.V. ( 1993) Comparison of Lc~~?c~.Y~oc IX~OJ b,trains b)

pulsed-field gel clectrophoresis. Appi. Environ. Microbial. 59. 3969 3972.

Kelly. W.J., Asmundson. K.V., Harrison. G.L. and Huang. c’.h$. (lW5) Differetl~i~tiotl of deutran-pro-

ducing ~~,t~~,~/~?~~.s~~~~ strains from ferl~lel~tc[~ rice cake (puto) using l~ttl~ed-meld gel clcclr~~pll~~r~si~. 1111.

.I. Food Microbioi. 26. 345 ~152.

Kocher. T.D. and Wilson. A.C. (1992) DNA amplification by the polymeruse chain reaction. In: A.R.

Hoelzel (editor), Molecular Genetic Analysis of Populalions. A Practical Approach. IRL I’rcsh.

Oxford University Press. Oxford. pp 185 --X)7.

Kunre. G.. Kunzc. I.. Barner. A.. and Schulz. R. ( 190.3) Classilictttion of ,~~~~,(,/~NI.oII~~.[.(,.Y wwr.ritrc~

stratns by genetical and biochemical methods. Momttsschrifi fiir Rr~tttnissellsch;tl‘I~iss~iisch~tf~ 46. 112.. 136. Lavallec. I-., Salva, Y.. Lam). S., Thomas. D.Y.. Dcgrc, R. and Duhtu. I.,. (1994) PC’R and DNA

fingerprinting used as quality control in the production of wine yeast strains. Am. J. Enol. Vitic. 45.

S6- 91.

Le Bourgeois, P., Lautier, M.. Mata. M. and Ritzenthaler. P. (1992) Physical and genetic map of the

chromosome of Lnc~rrwocws kfa.ti.c subsp. iwrir IL 1403. J. Bacterial. 174, 67X--6762.

Le Bourgeois, P., Mata, M. and Ritzenthalcr. P. (1’391) Pulsed-field gel electrophoresis as tt tool for

studying the phylogeny and genetic history of Iactococcal strains. In: G.M. Dunny. P.P. Clwry and

L.L. McKay (editors) Genetics and molecular biology of Streptococci. Lactococci and Enterococci.

ASM, Washington, DC. pp. 14@ 145.

Lc Bourgeois, P., Lautier, M. and Ritzenthaler, P. (1993) Chromosome mapping in lactic acid bacteria.

FEMS Microbioi. Rev. 12, 109 125.

Lc Jeune, C., LoI~v~tu~~-Fuliel, A., ten Brink. I)., HofStra, H. and van drr Vossen. J.M.B.M. (1995)

Developmei~t of a detection system for histidine dec~rb~~xyl~itin~ Incric acid bacteria based on DNA

probes, PCK and activity test. J. Appl. Bacterial. 7X. 316. 326.

Lieckfeldt. E.. Meyer, W., Kuhls. K. and Biirner. T. (1992) C:haracterization of filatnentous fungi and

yeasts by DNA fingerprinting and random amplified polymorphic DNA. Belg. J. Bot. 125, 226 232.

Lieckfeldt. E., Meyer, W. and Borner. T. (1993) Rapid identification and diffcrcntiation of yeasts by

DNA and PCR fingerprinting. J. Basic Microbial. 33, 413 426.

J.M.B.M. van der Vossen, H. H&m j Int. J. Food Microbiology 33 (1996) 35-49 41

Magee, B.B., D’Souza, T.M. and Magee. P.T. (1987) Strain and species identification by restriction

fragment length polymorphisms in the ribosomal DNA repeat of Candida species. J. Bacterial. 169,

1639- 1643.

Maiwald, M.. Kappe, R. and Sonntag, H.G. (1994) Rapid presumptive identification of medically

relevant yeasts to the species level by polymerase chain reaction. J. Med. Vet. Mycol. 32, I IS- 122.

Makimura, K., Murayama, S.Y. and Yamaguchi, H. (1994a) Detection of a wide range of medically

important fungi by the polymerase chain reaction. J. Med. Microbial. 40, 358-364.

Makimura, K., Murayama, S.Y. and Yamaguchi, H. (1994b) Specific detection of Aspergilhrs and

penicilhn species from respiratory specimens by polymerase chain reaction (PCR). Jpn. J. Med. Sci.

Biol. 47, 141-I%.

Martin, G.B., Williams, J.G.K. and Tanksley, S.D. (1991) Rapid identification of markers linked to a

Pseu&~nzorrus resistance gene in tomato by using random primers and near-isogenic lines. Proc. Natl.

Acad. Sci. 88, 23362340.

Martinez-Murcia, A.J. and Collins, M.D. (1990) A phylogenetic analysis of the genus Leuconosfoc based

on reverse transcriptax sequencing of 16s rRNA. FEMS Microbial. Lett. 70. 73-84.

Matthews, J.A. and Kricka, L.J. (1988) .Analytical strategies for the use fo DNA probes. Analytical

strategies for the use of DNA probes. Anal. Biochem. 169,1-25.

Mazurier. S.I. and Wernars, K. (1992) Typing of Listeria strains by random amplified polymorphic

DNA. Rcs. Mierobiol. 14.1. 499-505.

McKay, L.L. (1983) Functional properties of plasmids in lactic streptococci. Antonie van Leeuwenhoek.

J. Microbial. 49, 259-274.

Meyer, W., Lieckfeldt, E., Kuhls, K., Freedman, E.Z., Biirner, T. and Mitchell, T.G. (1993) DNA- and

PCR-fingerprinting in fungi. In: S.D.J. Pena, R. Chakroborty, J.T. Epplen and A.J. Feffreys

(editors), DNA Fingerprinting: State of the Science. Birkhauser Verlag, Basel, Switzerland, pp

3 I l-320.

Meyer, W., Morawetz, R., Borner, T. and Kubicek, C.P. (1992) The use of DNA-fingerprinting analysis

in the classification of some species of the Trichodema aggregate. Curr. Genet. 21. 27-30.

Meyer, W., Koch A.. Neimann, C., Beyermann, B., Epplen, J.T. and Biirner, T. (1991) Differentiation

of species and strains among filamentous fungi by DNA fingerprinting. Curr. Genet. 19, 239.-242.

Miteva, V.I.. Abadjieva, A.N. and Stefanova, T.T. (1992) Ml3 DNA fingerprinting, a new tool for

classification and identification of Lactohucillus spp. J. Appl. Bacterial. 73, 3499354.

Miyakawa. Y., Mabuchi, T., Kagaya, K., and Fukazawa, Y. (1992) Isolation and characterization of a

species-specific DNA fragment for detection of Candida n1bican.s by polymerase chain reaction. J.

Clin. Microbial. 30. 8944900.

Molina, F.I., Jong, S.-C. and Huffman, J.L. (1993) PCR ampliticaiton of the 3’ external transcribed and

intergenic spacer of the ribosomal DNA repeat unit in three species of Succhuromyces. FEMS

Microbial. Lett. 108, 259264.

Molnar. O., Messner, R., Prillinger, H., Stahl, U. and Slavikova, E. (1995) Genotypic identification of

.Scrcclrrrrom,ce.~ species using random amplified polymorphic DNA analysis. Syst. Appl. Microbial.

18, I36 145.

Mosscl, D.A.A. (1982) Microbial detoriation of foods. Microbiology of foods. The Ecological essentials

of assurance and assessment of safety and quality (3rd ed.). The University of Utrecht, Utrecht, pp.

29-49.

Musters, W., Planta. R.J., van Heerikhuizen, H. and Raue, H.A. (1990) Functional analysis of the

transcribed spacer of ~~~~~~z~~(/~~~[,~~ cereuisiae ribosomal DNA: it takes a precusor to form a

ribosome.In: W.E. Hill. A. Ddhlberg, R.A. Garret, P.B. Moore, D. Schlessinger and J.R. Warner

(eds). The ribosome: Structure, function and evolution. American Society for Microbiology. Wdsh-

ington, DC, pp. 435 --44X

Nakagawd, T., Shimada, M., Mukai, H.. Asada, K., Kato. I., Fujino K. and Sato T. (1994) Detection

of Alcohol-tolerant Hiochi bacteria by PCR. Appl. Environ. Microbial. 60, 637-640.

Naumova. E., Naumov, Cr., Fournier, P., Nguyen, H.-V. and Gaillardin, C. (1993) Chromosomal

polymorphism of the yeast Yarronsia iipolytica and related species: electrophoretic karyotyping and

hybridization with cloned genes. Curr. Genet. 23, 450-454.

Ness. F., Lavallee, F.. Dubourdieu. D.. Aiglc. M. and D&u. L. lYY3. Identification of yeast strains

using the polymerase chain reaction. J. Sci. Food Agric. 62:XY Y4. Niesters. H.G.M.. Goessens. W.H.F.. Meih, J.F.M.G. and Quint, W.G.V. (1993) Rapid. polymerasc

chain reaction-based identification assays for C‘cn~&/r species. J. Clin. Microbial. 31. 904 YIO. Nissen, H. and Dainty, R. (1995) Comparison of the use of rRNA probes and conventional methods 111

identifying strains of Luctohuc~ilhn .wkc and Loc~tohac~i//x\ wrwni.\ isolated from meat. Int. J. Food Microbial. 25. 31 I 315.

Olsen. J.E. Aabo. S.. Hill. W., Notermans. S.. Wernars. K.. Granum. P.E.. Popovic, T.. Rasmussen.

H.N. and Olsvic. 0. (1995) Probes and polymerase chain reaction for detection of food-borne bacterial pathogens. Int. J. Food Microbial. 2X. I 7X.

Pearson, B. M., and McKee. R.A. ( 1992) Rapid identification of Srrcc Irtrwrrr~~~ <‘.\ wwri\itrc,. Z~n~~.wwhtr- wrn~ws hi/ii, and Z~~c~,vuc~c~/~aror~~~~~~~,s rorr.\ii. J. Food Microbial. 16. 6.3 67.

Penner. G.A., Bush. A.. Wise. R.. Kim, W.. Domier. L., Kasha, K.. Laroche, A., Stoles. G., Molnar.

S.J. and Fcda. G. (1993) Reproducibilit) of random amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Protocols and Applications 2. 341 345.

Petering, J., Langridge, P. and Henshke. P. (1988) Fingreprinting wine yeasts. The application of

chromosome electrophoresis. Aust. NZ. Wine Ind. J. 3. 4X 52. Polzin. D.M.. Romer0.D.. Shimizu-Kadota, M.. Klaenhammer. T.R. and McKay. L.L. (19Y3) Copy

number and location of insertion sequence? lSS/ and IS%3/ in lactococci and several other lactic acid bacteria. J. Dairy Sci. 76, 1243 1252.

Pot. B.. Hertel, C.. Ludwig. W.. Descheemaeker. P.. Kersters, K. and Schleifer, K.-H. (1993) Identitica-

tion and classification of Ltr~tohuc~illrr.c trcit/opl~ilus, I_. gmccr~. anti I,. jol~~w~ii strains by SDS-PAGE and rRNA-targeted oligonucleotidc probe hybrization. J. Gen. Microbial. 139. 513 5 17.

Rodrigues, U.M., Aguirre. M., Facklam. R.R. and Collins, M.D. (19Yl ) Specific and intraspecific

molecular typing of lactococci based on polymorphism of DNA encoding rRNA. J. Appl. Bactcriol. 71, so9 516.

Rodtong. S. and Tdnnock,G.W. ( 1993) Diffcrcntiation of Lrrc,/ohtrc,i//rr strains by ribotyping. Appl. Environ. Microbial. 59, 3480-3484.

Rossen, L., Norskov, P.. Holmstrom, K. and Rasmussen. O.F. (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays. and DNA-extraction solutions. Int. J. Food Microbial. 17, 37 45.

Rubin. F.A.. Kopecko, D.J.. Noon, K.1:. and Baron. L.S. (1985) Development of a DNA probe to

detect S~lrwnclkr /)plli. J. Clin. Microbial. 22. 600 605. Saiki. R.K.. Gelfand, D.H., Stoffcl, S.. Scharf. S.J.. Higuchj. R.. IIorn, G.T., Mullis. K.B. and Ehrlich,

H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerare. Science 239. 487 49 I

Salrano. G., Moschetti. G., Villani, F., Pepe. 0.. Mauriello, G. and Coppola. S. (1994) Genotypinp of S/w~foc.wc.x, t/rrrr~~o~~hi/u.s evidenced by restriction analysis of ribosomal DNA. Res. Microbial. 145, 65 I 658.

Schwartz, D.C. and Cantor. C.R. (1984) Separation of yeast chromosome-sized DNAr by pulsed-field

gel electrophoresis. Cell 37, 67 75. Shen. P., Jong. S.-C. and Molina, F.I. (1994) Analysis of ribosomal DNA restriciton patterns in the

genus K/zc?,ruro,,l~ces. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbial. 65. 99 IO5

Southern, E.M. (1975) Detection of specific scqucnccs among DNA fragments separated by gel

electrophoresis. J. Mol. Biol. 9X. 503 517. Speirings, G., Hofstra, [I.. Huis in ‘t Veld, J.H.J.. Hoekstra, W. and Tommassen. J. (1989) Development

of enterobacterium-specific oligonucleotide probes based on the surface-exposed regions of outer

membrane proteins. Appl. Environ. Microbial. 55, 3250 3252. Stghl, M., Molin, G., Persson, A., Ahrnk. S. and Stlihl, S. (1990) Restriction endonucleasc patterns and

multivariate analysis as a classification tool for Lac~tohac~i//~.s spp. Int. J. Syst. Bacterial. 40: I89 193. Stephan. R.. Schraft. H. and Untermann, F. (1994) Characterization of BLIC~//US liclzrrz~/orn~i.~ with the

RAPD technique (randomly amplified polymorphic DNA). Lett. Appl. Microbial. 18, 260-263. Stubbs, S., Hutson, R., James, S. and Collins, M.D. (1994) Differentiation of the spoilage yeast

Z1’~~~~ac~/iarom~~,~~ builii from other Z~~o.scrccharom?;~~.s species using IXS rDNA as target for a non-radioactive ligase detection reaction. Lett. Appl. Microbial. 19. 268-272.

Swaminathan. B. and Feng, P. f 1994) Rapid detection of food-borne pathogenic bacteria. Annu. Rev.

Microbial. 48, 401-426.

Tanskanen, E.I., Tulloch, D.L., Hillier, A.J. and Davidson, B.E. (1991) Pulsed-Field gel electrophoresis

of Snza 1 digests of lactococcal genomic DNA, a novel method of strain identification. Appl. Environ. Microbial. 56. 3105-31 I I.

Tarbk. T., Rockhold. D. and King, A.D. Jr. (1993) Use of electrophoretic karyotyping and DNA-DNA hybridization in yeast identification. lnt. J. Food Microbial. 19, 63-80.

Tsuohiya. Y., Kaneda, H., Kano, Y. and Koshino, S. (1992) Detection of beer spoilage organisms by polymerase chain reaction technology. Am. Sot. Brew. Chem. J. 50, 64-67.

Venzinhet. F.. H&et, J.-N.. V&d. M. and Poulard. A. (1992) Ecological survey of wine yeast strdinsby

molecular methods of idcntific~tion. Am. J. Enol. Vitic. 43, 83 86.

Vcrsalovic. J., Koeuth, T. and Lupski. J.R. (1991) Distributioil of repetitive DNA sequences in

eubacteria and application to finge~rintin~ of bacterial genomes. Nucleic Acids Res. 19, 6823~6831.

Vezinhet. F.. Blondin. B. and Hallet. J.-N. (1990) Chromosomal DNA patterns and mitochondrial DNA

polymorphism as tools for identification of enologicai strains of Srrcrlraront_rws rerecisicw. Appl.

Microbial. Biotechnol. 32. 568 571.

Vilgalys. R. and Hester. M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cr~~~oiorws species. J. Bacterial. 172, 4238-4246.

Vogel. R.F.. Lohmann, M., Nguayen, M., Weller. A.N. and Hammes, W.P. (1993) Molecular chardcter- ization of Lwtohuc~ilhrs curcutus and Lac~tohuciNus sake isolated from sauerkraut and their applica-

tion in sausage fermentations. .I. Appl. Bacterial. 74, 295-300.

Vogel. R.F.. Lohmann, M., Weller, A.N., Hugas, M. and Hammes, W.P. 1991. Structural similarity and

distribution of small cryptic plasmids of Lrrctohaciltus cur~~ttu.s and L. s~h-e. FEMS Microbial. Lett.

84: I 8.1 190.

Wallbanks. S.. ~~~lrtincz-Murci~. A.J., Fryer. L.J.. Phillips, B.A. and Collins, M.D. (1990) 1% rRNA

sequence determin~tio~i for members of the genus C~~~(~~U~,~~~~~~?~ and related lactic. acid bacteria

and description of Fu~wwcu.s Suit~?~)~7i~zu~jl{f~ sp. nav. Int. J. Syst. Bacterial. 40. 2744230.

Welsh, J. and McClelland. M. (1992) PCR-amplified length polymorphisms in tRNA intergenic spacers

for categrizing staphylococci. Mol. Microbial. 6, l673- 1680.

White, T.J., Bruns, T., Lee, S. and Taylor. J. (1990) Amplification and direct sequencing of fungal

ribosomal RNA genes for phylogentics. In: M.A. Innis. D.H. Gelfand, J.J. Sninsky and T.J. White

(editors). PCR Protocols: A Giude to Methods and Applications. Academic Press, San Diego. pp 315 322.

Williams. J.G.K., Kubelik, A.E.. Levak, K.J., Rafalski, J.A. and Tingey, S.C. (1990) DN.4 polymor-

phimsms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531 6335.

Wolff. K. and Peters-van Rijn, J. (1993) Rapid detection of genetic variability in chrysanthemum (~~,~?~ru~7//i~~/?~~~ grmdi/&wrr Tzvelev) usin g random primers. Heredity 71, 335-341.

DNA based typing, identification and detection systems...

Documents

Transcript of DNA based typing, identification and detection systems...