Patent Publication Number: US-2005130155-A1

Title: Primers for the detection and identification of bacterial indicator groups and virulene factors

Description:
The present invention relates to new primers for the detection of any of three important bacterial indicator groups used in food microbiology and two virulence factors, which are associated with the aetiology of several types of watery and bloody human diarrhoea. Furthermore, the present invention also relates to use of the primers in a method which enables this detection, as well as the detection of new emerging pathogenic bacteria.  
      There is a great need today for methods to secure safe sanitary (i.e. the absence of harmful bacteria) evaluation of goods for human consumption, in particular water, as well as methods for the detection of pathogen bacteria useful for e.g. clinical diagnostic. Three important target bacterial indicator groups (made of various species) currently used in food microbiology are the Enterobacteriaceae (family),  Escherichia coli  ( E. coli;  a species belonging to the Enterobacteriaceae family) and the fecal enterococci (most species of the  Enterococcus  genus).  
      The Enterobacteriaceae is a coherent well-defined taxonomic unit, which is relevant both to clinical diagnostic and to food and water routine microbiological analysis, as it includes important human pathogens and the total coliform group. Traditional microbiological methods used for the identification of this family rely on biochemical properties of isolated re-grown bacterial colonies. Only few faster alternative methods have been developed so far, and they are based on the identification of a trait or marker specific to the taxon. Sequences of 16S rRNA genes have been widely used for phylogenetic and taxonomic analysis as well as for diagnostic applications, i.e. for the detection of Enterobacteriaceae members (Mittelman et al. 1997).  
      The Enterobacterial Common Antigen (ECA) was first described in 1963 by Kunin et al. (Kunin 1963) and defined as a cross-reactive antigen that is detectable in all genera of Enterobacteriaceae by indirect hemagglutination and by other methods using antiserum to  E. coli.  It was later found to be strictly family specific with diagnostic and prophylactic potential. The only known noticeable reported exceptions are the Enterobacteriacea ECA-negative  Erwinia chrysanthemi  and the non-Enterobacteriacea ECA-positive  Plesiomonas shigelloides,  both of which have disputed taxonomic positions (see review (Kuhn et al. 1988)). The ECA is a glycophospholipid built up by an aminosugar heteropolymer linked to an L-glycerophosphatidyl residue. This surface antigen remained undetected for a long time due to its non-immunogenicity in most Enterobacteriaceae despite its general ability to act as an epitope (hapten). The genes implicated in the synthesis of ECA, rfe and rff, are clustered around 85 min on the  E. coli  genome (Ohta et al. 1991).  
      Immunology-based diagnostic tests have been developed to detect the presence of ECA for clinical applications (Malkamaki 1981) and later to monitor drinking water microbiological quality by detecting bacteria belonging to the Enterobacteriaceae family (Hubner et al. 1992). Such tests rely on the expression of the character being screened, which might be absent or poorly expressed in mutants, although most of the coding material may still remain intact. In this connection DNA-based techniques, i.e. PCR, have been successfully used to decrease the amount of false negatives in diagnostic applications, i.e. beta-glucuronidase enzyme and its coding sequence used for the detection and identification of  E. coli  (Feng et al. 1991). However, in order to be efficient and practical, it is important that the PCR methods that are used are robust, i.e. that they provide a strong and easily reproducible amplification, with no generation of additional product. Furthermore, the use of multiple primer sets in the same PCR reaction (i.e. multiplex PCR; two primer pairs means a duplex PCR, three primer pairs a triplex PCR etc.) is also preferable to the use of a separate PCR protocol for each of the primer sets (i.e. simplex PCR) when multiple targets are searched. This allows saving time and reagents, and thus lowering the cost of the analysis.  
      When applying PCR it is possible to use so called universal primers. Universal primers have the purpose of working for all variants of a given gene or DNA target. Typically the primers will be chosen in the most conserved areas of the gene, ideally identical in all variants. When no conserved identical portion can be used, two strategies can be used to accommodate the ambiguous nucleotide positions: silent mismatch and degenerate primers. In the first case, the primers are designed so that the variable nucleotide positions are placed in the primers to allow amplification to proceed although there is one or more mismatch. Typically, these ambiguous positions will be placed at the 5′ end of the primers. In the case of degenerate primers, all the ambiguous positions are accounted for, and a mix of all possible combination of the variable positions is used. This has the inconvenience of diluting the one full match primer set. However, the advantage is that it will be more efficient for highly variable genes and have more chances of functioning on new unknown variants of the target.  
       E. coli  is a member of the Enterobacteriaceae and the main species of the thermotrophic coliform group, also called the faecal coliform group. In the UK, the Drinking Water Inspectorate advised the committee responsible for revising Report 71 (Public Health Laboratory Services 1994) that for regulatory purposes, confirmed  E. coli  can be regarded as faecal coliforms. Furthermore, as  E. coli  is viewed as the only true faecal coliform and constitutes up to 99% of all faecal coliform isolates, its detection has been recommended for the evaluation of water microbiological quality. Traditional microbiological methods have relied on the expression of specific biochemical properties such as fermenting lactose or manitol at 44° C. with the production of acid and usually gas within 24 hours, and the production of indole from tryptophane. The expression by most  E. coli  strains of the β-glucuronidase has also been exploited. As previously mentioned, such tests rely on the expression of the specific characteristic which may lack or be delayed among certain strains although the genes might still be present. Hence, DNA-based methods, i.e. PCR, not only will reduce the analysis time, but also reduce the amount of false negatives when a reliable specific target gene or signature sequence within the chosen gene is used. Various PCR methods have been developed for detecting  E. coli  based on the detection of uidA (β-glucuronidase) and lamB (maltose high-affinity uptake system) (Bej et al. 1991b), or gadAB (glutamate decarboxylase) (McDaniels et al. 1996) which was also successfully used for pathogenic  E. coli  (Grant et al. 2001).  
       Enterococcus  is catalase-negative Gram-positive facultative anaerobic bacteria, and the two major species of interest to humans are  Enterococcus faecalis  ( E. faecalis ) and  Enterococcus faecium  ( E. faecium ). They are very common intestine commensal but are also responsible for nosocomial bacteremia, surgical wound infection, endocarditis and urinary tract infection. Most infections are caused by  E. faecalis,  which has virulence genes, whereas  E. faecium  has not but is more often resistant to glycopeptides (vancomycin and teicoplanin). Because of the lack of biochemical diversity between enterococcal species, reliable identification using traditional microbiological tests has proven to be difficult. This has become a problem for infection control purposes, as accurate species identification is required for the appropriate antibiotic treatment of enterococci infections. For example,  E. faecalis  usually are susceptible to ampicillin whereas vancomycili-resistant  E. faecium  also express high levels of resistance to β-lactams.  
      In addition of being clinically relevant species,  E. faecalis  and  E. faecium  are the two main species of the faecal enterococci indicator group used for the microbiological assessment of food and water. PCR methods have been developed to detect other enterococcal species using for example the vanC-1, vanC-2 and vanC-3 genes of  E. gallinarum, E. casseliflavus  and  E. flavescens  respectively, coding for their specific intrinsic low resistance to glycopeptides (Dutka-Malen et al. 1995). Other nucleotide based methods have been using housekeeping genes such as 23S rRNA gene (Betzl et al. 1990), super oxide dismutase gene (Bergeron et al. 1999) or randomly selected specific DNA sections (Cheng et al. 1997) for the specific detection of  E. faecalis  and  E. faecium.  Finally, tuf coding for the elongation factor EF-Tu has been used for the identification of the  Enterococcus  genus (Ke et al. 1999). However, none of these methods managed to simultaneously detect  E. faecalis  and  E. faecium  nor did they use a specific gene for the detection. House keeping genes were used which reduces the chances of developing a robust PCR or to further develop a multiplex method.  
      In addition to the detection of bacterial indicator groups, in which different species or sub-species (may) share similar genes for the coding of specific virulence factors, methods to detect the virulence factor themselves are important. Two virulence factors found in e.g. enteropathogenic  E. coli  (EPEC) and enterohemorrhagic  E. coli  (EHEC) are Shiga toxin (Stx) encoded by the gene stx and Intimin encoded by the gene eae. These two virulence factors are known as the two main virulence factors associated with the onset of human diarrhoea symptoms by these bacterial pathogens (i.e. EPEC and EHEC).  
      The Shiga toxin class, as indicated by its name, was first discovered in  Shigella dysenteriae  type 1 bacteria. A similar toxin was later discovered in  E. coli,  characterized as cytotoxic to vero cells and named Vero toxin (VT). The group of  E. coli  producing VT was accordingly named VTEC. The VT was later shown to be related to Shiga toxin, which prompted some authors to rename it Shiga like toxin (SLT), and the term SLTEC was used to describe the bacterial group (i.e.  E. coli ) producing it. As it became more evident that all Shiga toxins are related, a new genetic nomenclature was proposed and widely accepted (and is the one we use in the present study), and consequently this group of  E. coli  is now referred to as Shiga toxin producing  E. coli  (STEC).  
      Many variants of the stx gene have been described and new ones are still characterized. They have been classified in 2 main groups according to their sequence similarity. The first, stx 1 , is found in STEC and are almost identical to the shiga toxin genes of  S. dysenteriae  type 1, stx. The second group, stx 2  and variants, is the most divergent and comprises sub-groups which appear to be found in host-adapted strains and other species than  E. coli,  and also encode for the most potent shiga toxin for humans. Both stx 2  and stx 2c  are mainly hosted by STEC associated with the aetiology of severe human diarrhoea, whereas stx 2d  has been isolated in STEC of both human and cattle origin. Finally, stx 2e  are found in porcine  E. coli  while stx 2f  are found in  E. coli  hosted by birds. Although Stx2e and Stx2f toxins seem to be adapted to their respective hosts, they both have been associated with human disease. Combination of different stx variants can be found in a same bacteria as illustrated by the case of a patient with three different STEC serotypes, each of which was hosting Stx1, Stx2 and Stx2c. Cattle are considered to be the main reservoir of STEC with 50 to 95% of the animals found to be host, although many other domestic animals were also found to host STECs. It was also shown that bacteria carrying stx genes were isolated from marine waters and are commonly found in rivers. Although not all Stx-producing bacteria can have phage induced, all stx genes are considered to be phage borne, including for  S. dysenteriae  serotype 1. In this connection, Shiga toxin-converting bacteriophages are commonly isolated. in sewage and were shown to play an important role in the emergence of new STEC variants. These findings illustrates how ubiquitous Shiga toxins are in our environment spanning from land to sea and air, with the intrinsic potential of horizontally spreading to new bacterial hosts.  
      The Shiga toxin is an A-B toxin type formed of the association of 5 B subunits structured in a ring-alike shape, and one A subunit on top of the ring. The ring is responsible for the recognition and attachment to the eukaryotic Gb3 globotriaosylceramide cell receptor of the toxin whereas the A subunit is the active toxic component that inhibits protein synthesis by removing an adenine from the 28 S rRNA. The two subunits are encoded by two genes organized in an operon in which the B subunit is more transcribed than A, enabling the final molecular ratio of 1/5 for the whole toxin. The stxA gene varies in length from 948 bp for stxA 1  to around 960 bp for stxA 2 , and the “theoretical” maximum length after alignment of all variants is 967 bp and is used as the reference template for numbering the primers as shown in FIG. 9. The B subunit is 267 bp in length. As more stxA sequences were described than stxB, and as stxA is longer, we chose the latter (i.e. stxA) for the development of universal primers to detect the presence of stx.  
      Although the first and main STEC serotype associated with the onset of human disease is O157:H7, over a 100 serotypes have been recognized and thus, the importance of developing methods for detecting them has been emphasized (World Health Organization 1998). The STEC serotype associated with the development of human haemolytic uraemic syndrome (HUS) might vary from a country to another as shown in a recent Australian survey in which non of the 98 HUS cases identified over 4 years were associated to O157:H7. Similarly, in another Australian study, no O157:H7 were isolated among the 23 STEC isolated from bovine faecal samples. Non-O157 STEC were possibly previously underestimated because of the use of diagnostic methods targeting typical phenotypic characteristics of the O157:H7 serotype such as delayed sorbitol fermentation and lack of glucuronidase activity rather than toxin detection. These tests were developed to enable mass screening by routine laboratory but will obviously miss many STEC including atypical O157 isolates. The same critic can be made for serological diagnostic tests, which are specific to the serotype, i.e. O157 detection methods. Other immunological diagnostic methods targeting Shiga toxins have been developed but rely on toxin expression and lack the analytical flexibility DNA-based methods have. To circumvent the unreliability of phenotypic expression, it is clear that a DNA-based method able to detect all variants of the gene encoding Shiga toxin is needed when evaluating human health risk of environmental samples or when identifying aetiological agents of human gastro-enteritis. Although various universal primer pairs for the detection of stx have been described in the literature (see FIG. 1 and Table 4), few are able to detect all variants or have been used in a multiplex assay.  
      The eae gene ( E. coli  attaching and effacing) encodes Intimin of pathogenic  E. coli  producing the typical A/E (attaching-and-effacing) histopathology in infected patients. Five different types have been described: α, β, γ, δ &amp; ε. The open reading frame varies in length from 2820 bp for intimin α and β to 2847 bp for intimin ε. Intimin is a protein involved in the intimate adherence of the bacterium to the epithelial cell membrane of the host&#39;s gut. In an experiment with human volunteers, intimin was proven to be necessary for the full development of diarrhoea caused by EPEC. The eae gene is found in the so-called locus of enterocyte effacement (LEE) pathogenecity island of both EPEC and EHEC. The location of LEE on the chromosome rather than on a plasmid, which is often the case for several other virulence factors, is beneficial in terms of stability of that DNA segment. Plasmid loss during sub-culturing has been reported and demonstrates that pathogenic plasmid borne molecular markers might be unreliable.  
      Several patents disclose methods to detect harmful bacteria. U.S. Pat. No. 6,207,818, U.S. Pat. No. 6,060,252, U.S. Pat. No. 6,054,269 and U.S. Pat. No. 5,298,392 all describe the amplification and detection of such harmful bacteria, however, the methods either detect different indicator markers/groups (i.e. bacteria) and/or different virulence markers/factors (i.e. gene(s)) compared to what is disclosed in the present application. Furthermore, the methods used in these patents do not combine the Enterobacteriaceae indicator group with virulence markers.  
      U.S. Pat. No. 6,218,110, U.S. Pat. No. 6,165,724 and U.S. Pat. No. 5,795,717 use oligonucleotides in PCR protocols to detect STEC bacteria. However, as opposed to what is disclosed in the present application, they use 2 or more sets of primers for the detection of stx and all variants.  
      U.S. Pat. No. 5,652,102 use oligonucleotides in a multiplex PCR protocol to detect STEC bacteria, however, as opposed to what is disclosed in the present application, the primer pairs are claimed to be specific to the  E. coli  O157 serogroup and no indicator group is associated to the method.  
      U.S. Pat. No. 5,994,066, U.S. Pat. No. 5,786,147 and U.S. Pat. No. 5,693,469 use a “housekeeping gene” like rRNA and/or probe technology (as opposed to PCR) to detect various bacteria, and is thus more limited and/or cumbersome than the method disclosed in the present application.  
      Two articles by Fratamico et al. (Fratamico et al. 1993) disclose the use of multiplex PCR protocols using universal primers for stx; however, these primers are not able to detect the eae to gene and variants. Furthermore, these protocols use mismatched universal primer pairs developed by Karch et al. (Karch and Meyer 1989), which was later reported not to detect all variants originally claimed.  
      Osek et al. (Osek 2001) have reported the development of a multiplex PCR for the detection of ETEC and  E. coli  by targeting the genes elt and est, as well as the  E. coli  specific stress protein gene uspA, whereas Grant et al. (Grant et al. 2001) have reported the use of multiplex PCR for the detection of STEC and  E. coli  by targeting stx1, stx2 and gadAB. However, both Osek and Grant are using multiplex PCR targeting a smaller indicator group compared to what is done according to the present invention.  
      Enterobacteriaceae has been proposed for the replacement of the currently used faecal coliform (FC) indicator group in the microbial quality assessment of water. The definition of the FC indicator group, and which species to include in it, has been the focus of much debate. Coliform isolation methods were often used to define this group albeit the lack of a rational taxonomic basis and none of the coliforms can function as reliable markers for all enteric pathogens (Leclerc et al. 2001). In contrast to FC, that was created to fit the human concept of indicator/index group, the family Enterobacteriaceae is a consistent and well-defined phylogenic entity, which is easier to define than the FC indicator group. Also, the choice of Enterobacteriaceae makes it possible to include the detection of important water-born pathogens such as  Salmonella  or  Shigella , which are not detected by the FC microbiological tests currently in use. Thus, the use of Enterobacteriaceae would constitute a safer indicator marker for assessing the efficiency of food and water treatment. Furthermore, while  E. faecalis  and  E. faecium  both commonly were considered to be harmless commensal of the human digestive tract, they have recently emerged as important aetiological agents for various nosocomial diseases. Aside from virulence factors they have acquired a steadily increasing pool of resistance determinants to antibiotics (ampicillin, aminoglycosides and glycopeptides) which turned them into resilient potentially life threatening pathogenic bacteria. Thus, similar to  E. coli,  they have cumulated relevant traits as being the major members of traditional indicator groups important for water surveillance, and are potential pathogens important to be identified in the clinical world. With regard to virulence factors, the detection of intimin and the shiga encoding genes are considered important, as they are highly associated with the onset of human diarrhoea caused by e.g. the EHEC, EPEC and  Shigella dysenteriaeae  bacteria. Furthermore, as many species or sub-species of bacteria often share similar genes for the coding of specific virulence factors (e.g. stx and eae), and because these virulence factors often are located on mobile elements, there exist a need for new primers/methods to detect these specific virulence genes in various bacteria groups.  
      It is therefore an object of the present invention to provide new primers, as well as use of the primers in a method, in order to detect relevant bacteria indicator groups, as well as virulence factors, useful for both clinical diagnostic and for the sanitary evaluation of goods for human consumption. Furthermore, it is also an object of the present invention to enable the detection of new emerging bacteria with specific virulence genes. These objects have been obtained by the present invention, characterized by the enclosed claims.  
      The present invention relates to several new, oligonucleoticle primers and universal degenerate oligonucleotide primers according to any of the sequences SEQ ID NO 1 to SEQ ID NO 32 (see Table 2) for the detection of any of three important bacterial indicator groups used in food microbiology, as well as two virulence factors which are associated with the aetiology of several types of watery and bloody human diarrhoea. The three target indicator groups are 1) the Enterobacteriaceae, which includes the total coliform group, 2) the species  E. coli,  a member of the Enterobacteriaceae family, and 3) the two species  E. faecalis  and  E. faecium  belonging to the faecal enterococci indicator group. The target virulence factors are Shiga toxin (Stx) encoded by the gene stx and Intimin encoded by the gene eae. These two virulence factors are found in EPEC and EHEC among others, and recognized as the two main virulence factors associated with the onset of the human diarrhoea symptoms by these bacterial pathogens. Shiga toxin is found in  Shigella dysenteriae  and has been identified in emerging pathogens such as  Shigella sonnei  and  Citrobacter rodentium,  whereas Intimin has been identified in emerging pathogens such as  Hafnia alvei.  The universal primers according to the present invention were designed in an effort to enable detection of all gene variants of the virulence factors, independently of the bacteria hosting them. Hence, the primers according to the present invention enable the possible detection of new emerging pathogenic bacteria.  
      The present invention also relates to use of the primers according to the present invention, wherein the primers have the sequences according to any of the sequences as defined in SEQ ID NO 1 to SEQ ID NO 32.  
      The present invention further discloses the association of indicator markers with virulence markers in a robust and reliable multiplex PCR for the targets of interest wherein, compared to currently used technology, indicator markers also useful for clinical applications are used. Thus, according to another aspect, the present invention also relates to use of the primers according to the present invention, in a method preferably based on multiplex PCRs (i.e. triplex PCR or multiplex PCR depending on the objectives of the analysis and the amount of information needed) which enables the detection of any of three important bacterial indicator groups used in food microbiology and the two virulence factors which are associated with the aetiology of several types of watery and bloody human diarrhoea, as well as the possible detection of new emerging pathogenic bacteria. However, and according to a further aspect of the present invention, simplex PCR may also, depending upon the analysis requirements, be used. Furthermore, and according to the present invention, the method comprise preferably a restriction enzyme digestion and a seminested duplex PCR protocol for the accurate analysis of the stx gene.  
      The present invention will now be described in more detail, with reference to figures and examples. However, the examples are not to be interpreted as restrictive to the scope of the enclosed claims.  
      FIG. 1. Comparison of the alignments of previously described primers aiming at the detection of stx genes and their variants; +, forward primer; −, reverse primer; arrows are also indicating the direction of the primers.  
      FIG. 2. Comparison of the alignments of previously described primers for the detection of eae genes and their variants; +, forward primer; −, reverse primer; arrows are also indicating the direction of the primers.  
      FIG. 3. Agarose (1.7%) gel electrophoresis of mixtures of DNA templates showing the specificity of the multiplex reactions and the optimization of MgCl 2  and primer concentrations. Lanes: 1, negative control; 2 &amp; 15, DNA size markers; 3, 5, 7, 9, 11 and 13,  E. coli  O157:H7; 4, 6, 8, 10, 12 and 14,  Shigella dysenteriae;  3 to 8, 0.1 μM eae28UU18/eae748LU21 primers, 0.2 μM UstxU1/UstxL1 primers and 0.02 μM Meca202UU20/Meca633LU21 primers; 9 to 14, 0.1 μM eae28UU18/eae748LU21 primers, 0.5 μM UstxU1/UstxL1 primers and 0.05 μM Meca202UU20/Meca633LU21 primers.  
      FIG. 4. Agarose (1.7%) gel electrophoresis of mixtures of DNA templates showing the specificity of the multiplex reactions and the optimization of MgCl 2  concentrations. Lanes: 2, negative control; 3 &amp; 14, DNA size markers; 4, 7 and 10,  E. coli  O157:H7; 5, 8 and 11,  E. coli  O157:H7 with  Shigella dysenteriae;  6, 9 and 12,  Shigella dysenteriae.    
      FIG. 5. Agarose (3%) gel electrophoresis showing stx simplex PCR and subsequent Bsr I digestion for 14 different STECs strains and 3  Shigelia dysenteriae  serotype 1. The detailed typing results are shown in Table 9. Lanes 1, 16, 17 &amp; 32, DNA size markers; 2 &amp; 3,  E. coli  O128:H?; 4 &amp; 5,  E. coli  O113:H21; 6 &amp; 7,  E. coli  O157:H7; 8 &amp; 9,  E. coli  O157:H7; 10 &amp; 11,  E. coli  O?: H?; 12 &amp; 13,  Shigelia dysenteriae;  14 &amp; 15,  Shigella dysenteriae;  18 &amp; 19,  Shigella dysenteriae;  20 &amp; 21,  E. coli  O157:H7; 22 &amp; 23,  E. coli  O157:H-; 24 &amp; 25,  E. coli  O157:H?; 26 &amp; 27,  E. coli  O157:H7; 28 &amp; 29,  E. coli  O157:H7; 30 &amp; 31,  E. coli  O157:H7;  
      FIG. 6A. Agarose (1.7%) gel electrophoresis showing the specificity of the triplex PCR reactions using 15 different bacterial strains. Lanes: 1, DNA size markers; 2,  E. coli  O128:H?; 3,  E. coli  O113:H21; 4,  E. coli  O157:H7; 5,  E. coli  O157:H7; 6,  E. coli  O?:H?; 7,  Shigella dysenteriae;  8,  Shigella dysenteriae;  9,  Shigelia dysenteriae;  10,  E. coli  O157:H7; 11,  E. coli  O157:H-; 12,  E. coli  O157:H?; 13,  E. coli  O157:H7; 14,  E. coli  O157:H7; 15,  E. coli  O157:H7; 16,  E. coli  O157:H7.  
      FIG. 6B. Agarose (1.7%) gel electrophoresis showing results from a seminested duplex PCR performed on diluted aliquots of product of triplex PCR shown on FIG. 6A.  
      FIG. 6C. Agarose (1.7%) gel electrophoresis showing results from a seminested duplex PCR performed directly on the same strains used for the triplex PCR shown in FIG. 6A  
      FIG. 7. Agarose (1.7%) gel electrophoresis of mixtures of DNA templates showing the specificity of the simplex PCR using the Meca479UU21 and Meca722LU21 primer pair. Lanes: 2, DNA size markers; 3,  E. coli  environmental isolate; 4,  E. coli  environmental isolate; 5,  Pseudomonas aeruginosa;  6,  Pseudomonas fluorescence;  7,  E. coli  O26:K60 (EPEC); 8,  E. coli  O44:K74 (EPEC); 9,  Aeromonas hydrophila;  10,  Aeromonas sobria;  11,  Shigella flexneri  serotype 2B; 12,  Shigella sonnel;  13,  Enterococcus faecalis;  14,  Enterococcus faecium;  15,  Shigella dysenteriae  serotype 1; 16,  Shigella dysenteriaea  serotype 1.  
      FIG. 8. Agarose (1.7%) gel electrophoresis of simplex stx optimization for MgCl 2  and annealing temperature (T a ). Lane 1, DNA size markers; Lanes 2 to 16, EHEC O113:H21; Lanes 2, 5, 8, 11 and 14, 1.5 mM MgCl 2 ; Lanes 3, 6, 9, 12 and 15, 2 mM MgCl 2 ; Lanes 4, 7, 10, 13 and 16, 3 mM MgCl 2 ; Lanes 2, 3 and 4, 47.0° C. T a ; Lanes 5, 6 and 7, 48.5° C. T a ; Lanes 8, 9 and 10, 52.1° C. Ta; Lanes 11, 12 and 13, 54.0° C. T a ; Lanes 14, 15 and 16, 57.8° C. T a ; All PCR reactions have 0.1 μM Ustx primer and 0.01 μM stx2f primers.  
      FIG. 9. Alignment of different stx primers/sequences.  
      FIG. 10. Alignment of different eae primers/sequences. 
    
    
      The present invention relates to oligonucleotide primers for the detection and partial characterisation of relevant bacteria useful for both clinical diagnostic and for the sanitary evaluation of goods for human consumption, in particular water. It has a DNA-based approach to identify some virulence factors associated with the aetiology of human diarrhoea, as well as species and family. Since different species or sub-species share similar genes for the coding of the specific virulence factors, the conserved parts of the sequence of some virulence genes were used to develop PCR primers universal for the targeted pathogenic trait (i.e. universal primers). Sequence variability within the PCR product obtained by use of the primers according to the present invention, can then be exploited for identification of the species or sub-species.  
      The choosing of either simplex PCR or multiplex PCR will depend upon the objectives of the analysis, as well as the amount of information needed. Routine microbiological control of food, and in particular water, will preferably (only) use the multiplex PCR, i.e. the quadruplex PCR protocol according to one embodiment of the present invention (see example 2), for detecting bacterial indicators, although positive samples might further be analysed with the triplex PCR (see example 1) to assess the presence of virulence factors. When analysing clinical samples, the multiplex PCR, i.e. the triplex PCR protocol according to another embodiment of the present invention, would be chosen for samples originating from patients with diarrhoea, although the quadruplex PCR might be required in other situations where  E. faecium  or  E. faecalis  is suspected and needs to be differentiated from Enterobacteriaceae. Thus, when desired, a more refined analysis using another test sample from the same source can be used for a second round of identification after a positive first round in order to obtain more information. Furthermore, the stx PCR product, may be further analysed by for example using a specific set of nested primers (to perform a second multiplex PCR using the product of the first PCR), specific probes or restriction enzymes. According to the present invention, restriction enzymes are used for this sub-typing, preferably after a simplex or triplex PCR amplification comprising the stx gene, (see example 3). According to the present invention, a set of seminested primers are used to differentiate stx 1  from stx 2  either after a simplex PCR amplification of the stx gene or a triplex PCR comprising the amplification of the stx gene or directly from a sample (see example 4).  
      The virulence genes coding for the chosen pathogenic traits are located on, or associated with, mobile elements which favours inter-species horizontal transfer and the emergence of new pathogens. One of the goals according to the present invention is that the “universal” mode of design of the primers, using degenerate rather than mismatched primers, will provide a tool for the monitoring of these mobile virulence elements thus making possible their detection, including new variants, in previously unknown bacterial hosts. The various bacterial groups and specific virulence genes targeted by use of the primers according to the present invention are presented in Table1. All 42 stx sequences and 14 eae sequences used in this work are shown in FIGS. 9 and 10 respectively  
               TABLE 1                          Multiplex gene targets and their known hosts                                         GenBank               Higher taxon   Gene/marker   accession no.   Taxonomic unit   Reference                 Enterobacteriaceae     Shiga toxin sub-unit A   X67514     Citrobacter freundii     (Schmidt et al. 1993)           (stxA)   M19473     Escherichia coli  stx 1     (Jackson et al. 1987)               L04539   (EHEC)   (Paton et al. 1993a)               AF125520     Escherichia coli  stx 2     (Plunkett, III et al. 1999)               AJ249351   (EHEC)   (Muniesa et al. 2000)               AJ010730       (Schmidt et al. 2000)               Z50754     Enterobacter clocae     (Paton and Paton 1996)                     Serratia marcessens     (Paton and Paton 1997)               M19437     Shigella dysenteriaea     (Strockbine et al. 1988; Unkmeir               AJ271153       and Schmidt 2000)               AJ132761     Shigella sonnei     (Beutin et al. 1999)           Intimin (eae)   AF022236     Escherichia coli  (EHEC/   (Elliott et al. 1998)               U60002   EPEC)   (Agin et al. 1996)               Z11541       (Yu and Kaper 1992)               X60439       (Beebakhee et al. 1992)               L11691     Citrobacter rodentium     (Schauer and Falkow 1993)                   (formerly  C. freundii                     biotype 4280)               L29509     Hafnia alvei     (Frankel et al. 1994; Ridell et al.                       1994; Albert et al. 1992; Albert et                       al. 1991)           Glutamate decarboxylase   M84024     Escherichia coli     (Smith et al. 1992)           (gadAB)   M84025           Enterobacterial common   S75640     Enterobacteriaceae     (Kuhn et al. 1988; Ohta et al.           antigen (rfe)   AF233324       1991)         Enterococcus     Chromosomal determinant   AF152237     Enterococcus faecalis     (An et al. 1999)           (eep) involved in the           production of the peptide           sex pheromone cAD1           Aminoglycoside acetyl   L12710     Enterococcus faecium     (Costa et al. 1993)           transferase (aac(6′)-Ii)                  
 
      As it can be seen from Table 1, the largest taxonomic unit to be detected by the primers according to the present invention is the bacterial family Enterobacteriaceae. Another important indicator group is the faecal enterococci, including the two major species of importance to humans  E. faecalis  and  E. faecium,  which are specifically detected. Furthermore, the primers according to the present invention was also developed to detect all variations of the genes encoding Intimin and Shiga toxin, since these factors are associated with the onset of human diarrhoea caused by EPEC, EHEC,  Shigella dysenteriaeae  and various emerging pathogenic bacteria. In particular, these virulence factors have been recognized as the most important for the onset of clinical symptoms in humans infected by STEC (Law 2000).  
      In the present invention, and as it can be seen from Table 1, the genes rfe (implicated in the synthesis of ECA), eae and stxA were used to develop universal primers for the specific detection of the Enterobacteriaceae group by PCR. According to the knowledge of the present inventors, the use of the gene rfe in order to detect Enterobacteriaceae has not been described previously. When included in the multiplex PCR, the rfe pair of primers will have the advantage of also acting as a positive control for the PCR reaction in some experimental conditions, such as when testing a sample known to contain  E. coli  (i.e. faeces sample). Furthermore, even though the two genes stx and eae have been extensively used to develop PCR protocols, a need to develop universal primers for clinical diagnostic has been reported in the literature. In addition, the universal primers developed for stx (Ustx) (i.e. the A subunit encoding gene of stx) and eae according to the present invention, have the advantage of being more robust than the few ones already described in the literature, and were designed to work in a multiplex PCR. Also, the use of only one primer pair for the detection of all stx variants (which is the case with the universal primers according to the present invention) as opposed to 2 or 3 primer pairs which is the case in the currently used technology, increases the robustness of the multiplex PCR, as well as the possibility of including more targets to the method. Furthermore, the unique association of Enterobacteriaceae as an indicator marker with virulence markers is useful to help bridge the notions of indicator and index markers.  
      According to the present invention the chromosomal determinant eep (An et al. 1999) (involved in the production of the peptide sex pheromone cAD1 which induces a mating response from  E. faecalis  donors carrying the haemolysin/bacteriocin (cytolysin) plasmid pAD1) was used to develop primers for the specific detection of  E. faecalis  (Table 1), and according to the knowledge of the present inventors, the eep determinant has never been used previously in a PCR protocol as a target for identification of  E. faecalis.  Various sex pheromone are produced for the specific induction of mating response by donors carrying the corresponding specific plasmid, but all sex pheromones appear to be regulated by the eep chromosomal determinant. Also, and in accordance with the present invention, another chromosome located antibiotic resistance gene for the specific detection of  E. faecium  has been used, the aminoglycoside acetyl transferase aac(6′)-Ii (Table 1) (Costa et al. 1993), which confers intrinsic low resistance to aminoglycosides (kanamycin, gentamycin).  
      Two authors, Lin Z. and Read S. C. (Lin et al. 1993;Read et al. 1992) have developed PCR methods which use a forward primer overlapping part of the forward Ustx used by the present inventors but use different reverse primers, thus generating amplicons of a different length. Moreover the overlapping forward primers they use have two mismatched positions, whereas the present inventors use degenerate, as well as longer primers. These last two points are important when evaluating robustness of the method and use of these primers in a multiplex PCR. Furthermore, Lin and Read performed only simplex PCR.  
      Karch (Karch and Meyer 1989) developed a simplex PCR which was later included in multiplex PCR protocols (Fratamico et al. 1993;Fratamico and Strobaugh 1998;Nagano et al. 1998) in which the reverse primer is complementary to a part of the Ustx forward primer of the present inventors. Again, two mismatches are observed with the Karch primer as shown in FIG. 1. Paton et al. in the simplex PCR they developed, (Paton et al. 1993b), used an overlapping degenerate perfect match to the reverse primer, although they used a different forward primer. Finally, Yamasaki S. described a simplex PCR (Kobayashi et al. 2001;Yamasaki et al. 1996) in which both forward and reverse primers are overlapping those of the present technology thus generating an amplicon of almost the same size (5 nucleotide less). The aim and the claim of all these protocols are to detect all variants of the stx gene using a single pair of primers. Few studies have compared and tested extensively such protocols, but two publications (Bastian et al. 1998) among which a study of the latest gene variant stx 2f  (Schmidt et al. 2000), agree in finding that only Lin (Lin et al. 1993) achieves detection of all variants. A closer study of the reverse primers used by Paton and Yamasaki (Paton et al. 1993b;Yamasaki et al. 1996) indicates they would probably also fail because of a on nucleotide insert in the Stx2f variant symbolised by the gap in FIG. 1. To compensate for this gap we have, according to the present invention, extended the notion of degenerate primers by adding an overlapping primer pair with a perfect match to the Stx2f variant. Although it looks like an extra pair of primers, it is only one more combination to cover all the possible ambiguous positions for the same primer pair location on the stx sequence.  
      All the primers according to the present invention may also be used in simplex PCR protocols, and in particular the primers designed for detecting Enterobacteriaceae,  E. faecium  and  E. faecalis.  Also, all primers according to the present invention were designed using the open reading frames of the targeted genes which allows the technology to be used with RNA based amplification techniques such as NASBA (Organon Technika).  
      The new primers and universal degenerate primers according to the present invention were designed with the help of Oligo 6 (Molecular Biology Insights, Inc. USA) software for windows and/or designed manually using the alignments results. Primers and degenerate universal primers were designed to enhance unknown variant detection and integrate them in multiplex PCR protocol. All primers designed and used in the method according to the present invention were compared with primers described previously in the literature. Results for stxA and eae gene alignments, along with primers used in the method according to the present invention and primers previously described in the literature, are shown in FIGS. 9 and 10. A summary of FIG. 9 to compare the Ustx (i.e. universal stx) degenerate primer pair with the most relevant primer pairs used in other PCR protocols, also aiming at the detection of all stx variants with a single pair of primers, is shown in FIG. 1. In a similar way, FIG. 2 summarises the most relevant previously described primers for the detection of the eae gene.  
      Optimization of the triplex PCR developed to simultaneously detect Enterobacteriaceae and the presence or absence of any variants of staA and eae genes are presented in FIGS. 3 and 4. Various primer pairs were designed according to their potential compatibility in a multiplex PCR, and special care was given to the design of the degenerate primers. Simplex PCR for all primer pairs were first optimized independently by varying physical and chemical conditions such as annealing temperature and primer concentration and were then tested for specificity. Multiplex PCR was then optimized varying annealing temperature, primer concentration and also by testing various additives or facilitators such as DMSO, glycerol, bovine serum albumin, formamide and MgCl 2  which are reported helpful for multiplex PCR. We found that only an increase of MgCl 2  improved the reaction as shown in FIGS. 3 and 4 although the other PCR facilitators may still be helpful when analysing complex samples (i.e. faeces). A list of all primers designed and used in connection with the method according to the present invention is presented in Table 2. The international nomenclature for ambiguous bases used for the degenerate positions in the primers is shown in Table 3.  
               TABLE 2                          List of primers                                         Gene       Primers (5′-3′)   SEQ ID   Ta ° C.   Location   Amp(bp)                                                     stxA 1  &amp;   UstxU1   TRTTGARCRAAATAATTTATATGTG   1   52.1   279-303*   526-7           stxA 1 ,   UstxL1   MTGATGATGRCAATTCAGTAT   2       784-805*   (stxA 1 )       universal   UstxL2   CMTGATGATGRCAATTCAGTAT   3       783-806*   523-4                               (stxA 2 )               stxA 2t     UstxU3   AATGGAACGGAATAACTTATATGT   4   49.0   279-303*   523           UstxL3   GGTTGAGTGGCAATTAAGGAT   5       784-804*               StxA 1     nestx1U   GTACAACACTKGATGATCTC   31   49.6   327-347*   200           UstxL1   MTGATGATGRCAATTCAGTAT   2       784-805*               StxA 2     nestx2U   TGACRACGGACAGCAGT   32   54.3   114-130*   410           UstxL1   MTGATGATGRCAATTCAGTAT   2       784-805*               eae intimin   eae626UU21   ATTATGGAACGGCAGAGGTTA   6   68   626-647*   207           eae812LU21   TGAAGACGTTATAGCCCAACA   7       812-833*                   eae626UU21   ATTATGGAACGGCAGAGGTTA   6   60   626-647*   351           eae956LU21   GGCGCTCATCATAGTCTTTCT   8       956-977*                   eae28UU18   ACCCGGCACAAGCATAAG   9   53.4   28-45*   741           eae748LU21   CGTAAAGCGRGAGTCAATRTA   10       748-768*                   eae28UU18   ACCCGGCACAAGCATAAG   9   51.8   28-45*   949           eae956LU21   GGCGCTCATCATAGTCTTTCT   11       956-977*           eae28UU18   ACCCGGCACAAGCATAAG   9   54.0   28-45*   805           eac812LU21   TGAAGACGTTATAGCCCAACA   7       812-833*               rfe for ECA   Meca479UU21   TGGATATGGTGGCGATTATGT   12   53.2   479-499    261       specific for   Meca722LU18   TCCAGGCMCGCTTAATGC   13       722-739    264         Enterobacteriaceae     Meca722LU21   CYTTCCAGGMCGCTTAATGC   14       722-742                    Meca582UU18   TTCCCGYCAGGCRTTTGT   15   55   582-599    265           Meca826LU21   CMGGYAWTGGTTGTGTCATCR   16       826-846                    Meca202UU20   GGGTTRTCCWGCGTCTCRTT   17   58.6   202-223    452           Meca633LU21   TATTCTGCCRKYACGCCWAYK   18       633-653                gadA &amp; gadB   gad259U21   AAAGAAGAATATCCGCAATCC   19   55   259-279    160       glutamate   gad402L17   GCCATTTCATCGCCATC   20       402-418        decargoxylase   gad658U19   CCACAACCGCTGCACGATG   21   60   658-676    135       of  E. coli     gad772L21   CAGGCGGAAGTCCCAGACGAT   22       772-792                eep, Chromosomal gene   efam1U   AATGCCGTGGGTAATGTGGTT   23   60   855-875    494       involved in the   efam1L   GGCTTTTCGGGGTTCTTCTG   24       1329-1348        production of the   efam2U   TTGAGTTAAATGCCGTGGGTA   25   53   257-277    284       peptide sex pheromone   efam2L   CATGGGTCCCGCAAAG   26       525-540        cAD1 of  Enterococcus           faecalis                 aac(6′)-Ii,   efuaac1U   GGGGGAAGACGTATGATAATC   27   56   191-211    258       chromosomal   efuaac1L   TCGGGAGCTTTCTACAACTAA   28       428-448        aminoglycoside   efuaac2U   GGCGTATTTAACTTAGTCGT   29   58   1257-1276    212       acetyl   efuaac2L   TTTGCGTCTTCTCGTAATTT   30       1449-1468        transferase of         Enterococcus           faecium                   *Numeration is done using the longest hypothetical gene obtained after alignment of all variants (see FIGS.  6  and  7 )             
 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                   
               
               
                 Ambiguous bases nomenclature 
               
            
           
           
               
               
            
               
                 Symbol 
                 Meaning 
               
               
                   
               
               
                 B 
                 Not A 
               
               
                 D 
                 Not C 
               
               
                 H 
                 Not G 
               
               
                 I 
                 Inosine 
               
               
                 K 
                 G or T 
               
               
                 M 
                 A or C 
               
               
                 N 
                 A or C or G or T 
               
               
                 R 
                 A or G 
               
               
                 S 
                 C or G 
               
               
                 V 
                 Not T 
               
               
                 W 
                 A or T 
               
               
                 Y 
                 C or T 
               
               
                   
               
            
           
         
       
     
      According to Table 2, the following primers correspond to the following sequences: 
          UstxU1 corresponds to SEQ ID NO 1     UstxL1 corresponds to SEQ ID NO 2     UstxL2 corresponds to SEQ ID NO 3     UstxU3 corresponds to SEQ ID NO 4     UstxL3 corresponds to SEQ ID NO 5     eae626UU21 corresponds to SEQ ID NO 6     eae812LU21 corresponds to SEQ ID NO 7     eae956LU21 corresponds to SEQ ID NO 8     eae28UU18 corresponds to SEQ ID NO 9     eae748LU21 corresponds to SEQ ID NO 10     eae956LU21 corresponds to SEQ ID NO 11     Meca479UU21 corresponds to SEQ ID NO 12     Meca722LU18 corresponds to SEQ ID NO 13     Meca722LU21 corresponds to SEQ ID NO 14     Meca582UU18 corresponds to SEQID NO 15     Meca826LU21 corresponds to SEQID NO 16     Meca202UU20 corresponds to SEQ ID NO 17     Meca633LU21 corresponds to SEQ ID NO 18     gad259U21 corresponds to SEQ ID NO 19     gad402L17 corresponds to SEQ ID5 NO 20     gad658U19 corresponds to SEQ ID NO 21     gad772L21 corresponds to SEQ ID NO 22     efam1U corresponds to SEQ ID NO 23     efam1L corresponds to SEQ ID NO 24     efam2U corresponds to SEQ ID NO 25     efam2L corresponds to SEQ ID NO 26     efuaac1U corresponds to SEQ ID NO 27     efuaac1L corresponds to SEQ ID NO 28     efuaac2U corresponds to SEQ ID NO 29     efuaac2L corresponds to SEQ ID NO 30     nestx1U corresponds to SEQ ID NO 31     nestx2U corresponds to SEQ ID NO 32        

      As multiplex PCR methods have been previously developed for the detection of various virulence factors associated with human gastrointestinal diseases, in particular stxAB 1 , stxAB 2  and variants, eae, hlyA, ipaH and eltAB, an extensive literature search have been performed to look into redundancy with prior art and is presented in Tables 4 and 5.  
               TABLE 4                          Relevant PCR protocols for the detection of virulence factors and indicator groups                                 Pathogenic markers       Indicator marker                                                 All       elt       Serotyping       16S                                                                                         Reference   stx 1     stx 2     stx   eae   hlyA   AB   est   invA   eae   rfb   fli   uidA   uspA   UidA/R   Gad AB   lamB   lacZ   rRNA               (Bej et al. 1991b; Bej et al.                                                       +       +   +           1990; Bej et al. 1991a)       (Bellin et al. 2001)   +   +       (Call et al. 2001)   +   +       +   +       (Carroll et al. 2000)                                                                       +       (Cebula et al. 1995)   +   +                                       +       (Chen and Griffiths 1998)                                                   +       (Chen and Griffiths 2001)   +   +                       +       (China et al. 1996)   +   +       +       (Desmarchelier et al. 1998)                                       +       (Fach et al. 2001)           +       (Fagan et al. 1999)   +   +           +               +       (Feng and Monday 2000)   +   +       +   +                           +       (Fratamico and Strobaugh           +       +           +   +       1998)       (Fratamico et al. 2000)   +   +       +   +                       +       (Fratamico et al. 1993)           +       +               +       (Gannon et al. 1997)   +   +                           +       +       (Gannon et al. 1992)   +   +       (Grant et al. 2001)   +   +                                                   +       (Heuvelink et al. 1995)   +   +       +       (Karch et al. 1993)               +       (Karch and Meyer 1989)           +       (Klausegger et al. 1999)                                                                       +       (Lang et al. 1994)   +   +               +       (Lin et al. 1993)           +       (McDaniels et al. 1996)                                                           +       (McDaniels et al. 1996)                                                       +       (Meng et al. 1996; Meng et               +       al. 1997)       (Mittelman et al. 1997)                                                                       +       (Nagano et al. 1998)           +                           +   +       (Olsvik et al. 1991; Olsvik   +   +       and Strockbine 1993)       (Osek 2001)                       +   +                       +       (Pass et al. 2000)   +   +       +       (Paton and Paton 1998)   +   +       +   +       (Paton et al. 1993b)           +       (Paton and Paton 1998)                                       +       (Pollard et al. 1990)   +   +       (Radu et al. 2001)   +   +                                   +       (Read et al. 1992)           +       (Tsen and Jian 1998)   +   +               +   +       (Yamasaki et al. 1996)           +                  
 
                     TABLE 5                          Relevant PCR protocols for detection of  E. faecalis  and  E. faecium                                                               Size of           Target       Reference &amp;           product       strains, Patent   PCR   primer name   Target gene(s)   Sequence (5′-3′)   (bp)                                                   Enterococcus  &amp;   PCR   (Ke et al.   tuf, chromosomal,   AAYATGATIACIGGIGCIGCICARATGGA   803           Universal,   (nested)   1999),   elongation   AYRTTITCICCIGGCATIACCAT               Universal   factor EF-Tu   TACTGACAAACCATTCATGATG   112               degenerate U1       AACTTCGTCACCAACGCGAAC               &amp; U2 Ent1 &amp;               Ent2                 Enterococcus     Multipplex   (Knijff et al.   ddl, chromosomal   TTATGTCCCWGTWTTGAAAAATCAA   186         durans,     (2),   2001), DuHifF,   D-Ala D-Ala   TGAATCATATTGGTATGCAGTCCG   377         Enterococcus         DuR &amp; HiR   ligase for   TTTTGTTAGACCTCTTCCGGA         hirae             peptidoglycan                   final synthesis                   step (sensitive                   to glycopeptides)                 Enterococcus     Probe   (Betzl et al.   23S rRNA   TAGGTGTTGTTAGCATTTCG         faecalis     technology   1990), DB8         Enterococcus         DB6       CACACAATCGTAACATCCTA         faecium                   Enterococcus     Cycle Probe   (Modrusanet   VanA and VanB-B2   TTAATAACCCAAAAGGCGGGAGTAGCT         faecium/     Technology   et al. 1999),   glycopeptide   TACATTCTTACAAAAAATGCGGGCATC       faecalis-   (CPT), 5′   vanA811L-27   resistance genes       glycopeptide   terminal   VanB467-27       resistance   labeled with           [γ- 32 P]-ATP.                 Enterococcus     PCR (1)   (Cheng et al.   Randomly selected   ACGCAACAATGGTGGTGGACA   658         faecium         1997), EM1A &amp; B   species specific   TCTTGATTTGCAGTAGAGGTAATAG                     E. faecium  DNA                   sequence, unknown                   function                 Enterococcus     PCR (1)   (Bergeron et   sod, superoxide   ACGCAACAATGGTGGTGGACA         faecium/         al. 1999)   dismutase   TCTTGATTTGCAGTAGAGGTAATAG       US5,994,066                         Enterococcus     Multiplex   (Clark et   vanC-1, D-Ala D-Ser   GAAAGACAACAGGAAGACCGC   796         gallinarum     (3)   al. 1998)   ligase for peptido-   ATCGCATCACAAGCACCAATC               (Angeletti et   glycan final               al. 2001)   synthesis step               (Satake et   (constitutive low               al. 1997)   resistance to                   glycopeptides)         Enterococcus         vanC2-1 &amp; -2   vanC-2   CGGGGAAGATGGCAGTAT   484         casselifavus                 CGCAGGGACGGTGATTTT         Enterococcus             vanC-3,   GCCTTTACTTATTGTTCC   224         flavescens                 GCTTGTTCTTTGACCTTA                 Enterococcus     Multipplex   (Petrich et   vanA, D-Ala D-Lac   Biotin-GCTGCGATATTCAAAGCTCA   545       glycopeptide   (2)   al. 1999),   ligase for peptido-   CAGTACAATGCGGCCGTTA       resistance       VanA1 &amp; VanA2   glycan final   ATTGCGTAGTCCAATTC-               VanA3   synthesis step   Fluorescein                   (aquired high                   resistance to                   vancomycin &amp;                   teicoplanin)           EIA detection   (Petrich et   vanB, D-Ala D-Lac   Biotin-ATGGGAAGCCGATAGTC   368           in microtiter   al. 1999)   ligase for peptido-   GTTACGCCAAAGGACGAAC           plates with   (Dutka-Malen   glycan final   GACAATTCAAACAGACC-           anti-FITC   et al. 1995),   synthesis step   Flourescein           HRP conjugate   VanB1 &amp;   (aquired high resis-               VanB3   tance to vancomycin)               VanB4                 Enterococcus-         (Dutka-Malen   vanA, D-Ala D-Lac   GGGAAAACGACAATTGC   732       glycopeptide       et al. 1995)/   ligase for peptido-   GTACAATGCGGCCGTTA       resistance       A 1  &amp; A 2 ,   glycan final               (Roger et   synthesis step               al. 1999)   (aquired high               (Angeletti et   resistance to               al. 2001)   vancomycin &amp;               (Satake et   teicoplanin)               al. 1997)               B1 &amp; B2   vanB, D-Ala D-Lac   ATGGGAAGCCGATAGTC   635               (Isenberg   ligase for peptido-   GATTTCGTTCCTCGACC               1998)   glycan final                   synthesis step                   (aquired high                   resistance to                   vancomycin)         Enterococcus     Multiplex   p662, C 1  &amp; C 2     vanC-1, D-Ala D-Ser   GGTATCAAGGAAACCTC   822         gallinarum     (6)   (Dutka-Malen   ligase for peptido-   CTTCCGCCATCATAGCT               el al. 1995)/   glycan final               (Isenberg   synthesis step               1998) p662   (constitutive low                   resistance to                   glycopeptides)         Enterococcus         D 1  &amp; D 2     vanC-2 &amp; van C-3,   CTCCTACGATTCTCTTG   439         casselifavus  &amp;           D-Ala D-Ser ligase   CGAGCAAGACCTTTAAG         flavescens             for peptidoglycan                   final synthesis                   step (constitutive                   low resistance to                   glycopeptides)         Enterococcus         E 1  &amp; E 2     ddl E. faecalis ,   ATCAAGTACAGTTAGTCT   941         faecalis             chromosomal D-Ala   ACGATTCAAAGCTAACTG                   D-Ala ligase for                   peptidoglycan final                   synthesis step                   (sensitive to                   glycopeptides)         Enterococcus         F 1  &amp; F 2     ddl E. faecium ,   TAGAGACATTGAATATGCC   550         faecium             chromosomal D-Ala   TCGAATGTGCTACAATC                   D-Ala ligase for                   peptidoglycan final                   synthesis step                   (sensitive to                   glycopeptides)                 Enterococcus     PCR, tRNA   (Baele et   tRNA genes flanking   AGTCCGGTGCTCTAACCAACTGAG   variable       species   intergenic   al. 2000),   conserved edges   AGGTCGCGGGTTCGAATCC           spacer PCR,   T5A &amp;           capillary   T3B*(TET)           electro-           phoresis of           amplicons                 Enterococcus     PCR (broad   (Poyart et   sod, superoxide   CCITAYICITAYGAYGCIYTIGARCC   480       species   range) with   al. 2000)   dismutase   ARRTARTAIGCRTGYTCCCAIACRTC         Streptococcus     degenerate   (Poyart et   Universal degenerated       species &amp; Gram   primers,   al. 1995),   primers for the       positive bacteria   sequencing   d1 &amp; d2   amplification of a           of the   (Poyart et   480 Nu internal           product and   al. 1998)   fragment sodA int             comparison           to a database                    
 Best Mode 
 
      Triplex PCR: For sample analysis (i.e. the detection of bacteria), individual colonies (i.e. test samples) from Luria agar culture plates are suspended in sterile distilled water. The preparation is then either used for direct PCR of bacteria or first boiled 10 min before use for the PCR. Samples (10 μl) were amplified in 50 μl final reaction mixtures using a BioTest Biometra PCR machine. The mixtures contained 0.1 mM each dATP, dCTP, dGTP, dUTP, 1× final concentration of the 10× buffer solution and 1 U of DyNazyme II (Finnzymes) DNA polymerase per reaction. MgCl 2  is adjusted from 1.5 mM standard concentration in the 1× buffer to 3 mM final concentration. Primer concentrations are as following, 0.05 μM for Meca202UU20 and Meca633LU21; 0.5 μM for UstxU1; 0.3 μM for UstxL1; 0.02 μM for UstxU3 and UstxL3 (The triplex is not as robust when using UstxU3 and UstxL3 and should therefore not be added if the subtype Stx 2f  is not researched); and 0.15 μM for eae28UU18 and eae748LU21. The PCR conditions consisted of 2 min preheating at 94° C. for one cycle followed by 15 s denaturation at 94° C., 30 s annealing at 57° C. and 60 s at 72° C. for 40 cycles; and 5 min final elongation at 72° C. Reaction products are separated by agarose (1.7%) gel electrophoresis with 0.5× Tris-borate-EDTA buffer and stained with ethidium bromide 0.5 μg/ml. PCR products are separated by applying seventy-five volts and 25 mA across the gel for about 1 h 30 min.  
      Quadruplex PCR: For direct sample analysis (i.e. the detection of bacteria) of water, the sample is centrifuged at 12000 rpm for 5 min and the pellet is re-suspended in sterile distilled water two times before it is re-suspended in a 50 μl final volume when PCR inhibitors are expected to be found. The preparation is then boiled 10 min before use for the PCR. Samples (10 μl) were amplified in 50 μl final reaction mixtures using a BioTest Biometra PCR machine. The mixtures contained 0.1 mM each dATP, dCTP, dGTP, dUTP, 1× final concentration of the 10× buffer solution and 1 U of DyNazyme II (Finnzymes) DNA polymerase per reaction. MgCl 2  is adjusted from 1.5 mM standard concentration in the 1× buffer to 3 mM final concentration. Primer concentrations are as following, 0.5 μM for efam1U and efam1L; 0.05 μM for Meca582UU18 and Meca826LU21; 0.5 μM for efuaac2U and efuaac2L; and 0.5 μM for gad259U21 and gad402L17. The PCR conditions consisted of 2 min preheating at 94° C. for one cycle followed by 15 s denaturation at 94° C., 30 s annealing at 57° C. and 60 s at 72° C. for 40 cycles; and 5 min final elongation at 72° C. Reaction products are separated by agarose (1.7%) gel electrophoresis with 0.5× Tris-borate-EDTA buffer and stained with ethidium bromide 0.5 μg/ml. PCR products are separated by applying seventy-five volts and 25 mA across the gel for about 1 h 30 min.  
      BsrI endonuclease restriction: The restriction endonucleases Bsr I with the recognition site ACTGG(1/−1) was purchased at New England Biolab. Digestion was performed using 16 μl PCR product (from stx simplex PCR or tiplex PCR), 10 U BsrI with the provided NEB3 buffer 1× final in a total volume of 20 μl. PCR tubes were used and digestion was carried out at 65° C. for 2h30min in the thermocycler. Reaction products are separated by agarose (3%) gel electrophoresis with 0.5× Tris-borate-EDTA buffer and stained with ethidium bromide 0.5 μg/ml. PCR products are separated by applying seventy-five volts and 25 mA across the gel for about 1 h.  
      Stx seminested duplex PCR: For typing (stx 1 /stx 2 ) the product of a first positive simplex or triplex PCR (as described in Example1). The PCR product must be diluted to 10 −3  before 5 μl sample is used in a 50 μl final reaction mixtures using a BioTest Biometra PCR machine. The mixtures contained 0.1 mM each dATP, dCTP, dGTP, dUTP, 1× final concentration of the 10× buffer solution and 1 U of DyNazyme II (Finnzymes) DNA polymerase per reaction. MgCl 2  is adjusted from 1.5 mM standard concentration in the 1× buffer to 3 mM final concentration. Primer concentrations are as following 0.1 μM for UstxL1, 0.3 μM for nestx1U and 0.05 μM for nestx2U. The PCR conditions consisted of 2 min preheating at 94° C. for one cycle followed by 15 s denaturation at 94° C., 30 s annealing at 57° C. and 60 s elongation at 72° C. for 30 cycles; and 5 min final elongation at 72° C. Reaction products are separated by agarose (1.7%) gel electrophoresis with 0.5× Tris-borate-EDTA buffer and stained with ethidium bromide 0.5 μg/ml. PCR products are separated by applying seventy-five volts and 25 mA across the gel for about 1 h. The method can also be used directly on DNA samples.  
     EXAMPLES  
     Example 1  
     Simultaneous Detection of stx and eae Virulence Genes and Enterobacteriaceae in a Multiplex (Triplex) PCR  
      For sample analysis (i.e. the detection of bacteria), individual colonies (i.e. test samples) from Luria agar culture plates are suspended in sterile distilled water. The preparation is then either used for direct PCR of bacteria or first boiled 10 min before use for the PCR. Samples (10 μl) are amplified in 50 μl final reaction mixtures using a BioTest Biometra PCR machine. The mixture contains 0.1 mM each dATP, dCTP, dGTP, dUTP, 1× final concentration of the 10× buffer solution and 1 U of DyNazyme II (Finnzymes) DNA polymerase per reaction. Concentrations for MgCl 2  and primers were optimized for each multiplex and are shown in Table 6 and 7. The PCR conditions consists of 2 min preheating at 94° C. for one cycle followed by 15 s denaturation at 94° C., 30 s annealing at 57° C. and 60 s elongation at 72° C. for 40 cycles; and 5 min final elongation at 72° C. Reaction products are separated by agarose (1.7%) gel electrophoresis stained with ethidium bromide (0.5 μg/ml), and the results are shown in FIGS. 3 and 4. 0.5× Tris-borate-EDTA buffer is used for the electrophoresis. Seventy-five volts and 25 mA are applied across the gel for about 1 h 30 min to separate the PCR products.  
               TABLE 6                          Sequences of primers, conditions to perform the triplex PCR and product sizes.                                                                         Product   Primers   MgCl 2                 Gene           Primers (5′-3′)   Location   size (bp)   μM   mM   Ta ° C.                                                             rfe for   Meca202UU20   SEQ ID NO 17   GGGTTRTCCWGCGTCTCRTT   202-223   452   0.05   3   57           ECA   Meca633LU21   SEQ ID NO 18   TATTCTGCCRKYACGCCWAYK   633-653               stxA 1 &amp;   UstxU1   SEQ ID NO 1   TRTTGARCRAAATAATTTATATGT    279-303*   526 (stxA 1 )   0.5       stxA 2 ,   UstxL1   SEQ ID NO 2   MTGATGATGRCAATTCAGTAT    784-805*   523 (stxA 2 )   0.3       universal               STXa 2t     UstxU3   SEQ ID NO 4   AATGGAACGGAATAACTTATATGT   279-303   523 (stxA 2t )   0.02           UstxL3   SEQ ID NO 5   GGTTGAGTGGCAATTAAGGAT   784-804               eae   eae28UU18   SEQ ID NO 9   ACCCGGCACAAGCATAAG    28-45*   741   0.15       intimin   eae748LU21   SEQ ID NO 10   CGTAAAGCGRGAGTCAATRTA   748-768                 *Numeration is done using the longest hypothetical gene obtained after alignment of all variants (see FIGS.  9  and  10 ).             
 
      The results show that only an increase in the concentration of MgCl 2  improved the reaction with an optimum concentration reached at 3 mM. Similarly optimum concentrations for the primers as indicated in Table 6 was demonstrated as shown in FIG. 3 where column 9 to 14 have optimum primer concentration. Furthermore, specificity is demonstrated in FIG. 4, as only the samples containing  E. coli  O157H7 produces the expected band of 741 bp that indicates the presence of the intimin gene eae. Finally, samples containing either  E. coli  O157H7 or  Shigella dysenteriaea  both show amplification products for the stx and rfe gene as expected.  
     Example 2  
     Simultaneous Detection of Enterobacteriaceae,  E. coli, E. faecalis  and  E. faecium  in a Multiplex (Quadruplex) PCR  
      The quadruplex PCR operational characteristics are similar to those of the triplex described in example 1 apart from specific conditions given in Table 7.  
               TABLE 7                          Sequences of primers, conditions to perform the quadruplex PCR and product sizes.                                                                         Product   Primers   MgCL 2     Ta           Gene           Primers (5′-3′)   Location   size (bp)   μM   mM   ° C.                                                             eep,   efam1U   SEQ ID NO 23   AATGCCGTGGGTAATGTGGTT   855-575   494   0.5   3   55           Chromosomal   efam1L   SEQ ID NO 24   GGCTTTTCGGGGTTCTTCTG   1329-1348       gene of  E.           faecalls                 rfe for ECA   Meca582UU18   SEQ ID NO 15   TTCCCGYCAGGCRTTTGT   582-599   265   0.05           Meca826LU21   SEQ ID NO 16   CMGGYAWTGGTTGTGTCATCR   826-846               aac(6′)-Ii,   efuaac2U   SEQ ID NO 29   GGCGTATTTAACTTAGTCGT   1257-1276   212   0.5       chromosomal   efuaac2L   SEQ ID NO 30   TTTGCGTCTTCTCGTAATTT   1449-1468       gene of  E.           faecium                 gadAB of  E.     gad259U21   SEQ ID NO 19   AAAGAAGAATATCCGCAATCC   259-279   160   0.5         coli     gad402L17   SEQ ID NO 20   GCCATTTCATCGCCATC   402-418                  
 
     Example 3  
     Sub-Typing of stx by Endonuclease Restriction of stx PCR Amplification Product  
      The restriction endonucleases HaeII, HindIII and BsrI, purchased at New England Biolab, were chosen to obtain the appropriate restriction patterns as shown in Table 8. Endonuclease restriction is performed on the product of simplex PCR using Ustx primers (see example 6).  
               TABLE 8                          Endonuclease restriction results of the PCR amplification of stx gene       variants.                                     stx gene   BsrI   HaeII   Hind III                       stx 1c     396, 130   526*   291, 235           stx 1     334, 130, 62   526*   291, 235           Stx 2 , Stx 2c , Stx 2d     200, 193, 91, 39   369, 154   253*           Stx 2e     200, 193, 130   369, 154   523*           Stx 2f     200, 193, 130   331, 193   523*                         *No restriction             
 
      Results are shown in FIG. 5 and Table 9 for BsrI endonuclease restriction of the stx PCR amplicon. 0.5× Tris-borate-EDTA buffer is used for the electrophoresis. Seventy-five volts and 25 mA are applied across the gel for about 1 h 30 min to separate the digestion products. When using PCR products from the triplex amplification the eae amplicon (741 bp) is discriminated in two groups. Amplicons from eae α, β, δ and ε will give two fragments of 88 and 654 bp while eae γ will give three fragments of 88, 178 and 476 bp.  
               TABLE 9                          Summary of results shown in FIG.  5  (BsrI restriction of stx amplification)                                                 Lane on       stxA   stxA 1c     stxA 1     All stxA 2     All stx − stxA 2     stxA 2,2c,2d             FIG.  5     Species   523-526   396   334   200-193   130   91   Results               2 &amp; 3     E. coli  EHEC O128:H?   +   +   −   +   +   +   stxA 1c  + stxA 2,2c,2d         4 &amp; 5     E. coli  EHEC O113:H21   +   −   +   +   +   +   stxA 1  + stxA 2,2c,2d         6 &amp; 7   EHEC  E. coli  O157:H7   +   −   +   +   +   +       8 &amp; 9     E. coli  EHEC O157:H7   +   −   +   −   +   −   stxA 1         10 &amp; 11     E. coli  EHEC O?:H?   +   −   +   −   +   −       12 &amp; 13     Shigella dysenteriae     +   −   +   −   +   −           Serotype 1       14 &amp; 15     Shigella dysenteriae     +   −   +   −   +   −           Serotype 1       17 &amp; 18     Shigella dysenteriae     +   −   +   −   +   −           Serotype 1       19 &amp; 20     E. coli  EHEC O157:H7   +   −   −   +   −   +   stxA 2,2c,2d         21 &amp; 22     E. coli  EHEC O157:H-   +   −   −   +   −   +       23 &amp; 24     E. coli  EHEC O157:H?   +   −   −   +   −   +       25 &amp; 26     E. coli  EHEC O157:H7   +   −   −   +   −   +       27 &amp; 28     E. coli  EHEC O157:H7   +   −   −   +   −   +       29 &amp; 30     E. coli  EHEC O157:H7   +   −   −   +   −   +                  
 
     Example 4  
     stx Seminested Duplex PCR to Differentiate stx1 and stx2 after a Simplex PCR, Triplex PCR or Directly  
      The seminested duplex PCR operational characteristics are similar to those of the triplex described in example 1 apart from specific conditions given in Table 10. The method can be used on 10 −3  diluted aliquots from a triplex stx PCR as shown in FIGS. 6A and 6B or directly from individual colonies prepared as indicated in example 1 (see results in FIG. 6C). This method is very sensitive and 25 cycles are sufficient when using aliquots from a triplex PCR. The results shown in FIGS. 6B and 6C corroborate those obtained using the BsrI endonuclease restriction method shown in FIG. 5 and Table 9.  
                   TABLE 10                          Sequences of primers, conditions to perform the seminested duplex PCR and           product sizes                                                                 Primer       Product   Primers   MgCl 2                 Gene           sequence (5′-3′)   Location   size (bp)   μM   mM   Ta ° C.                                                             stxA 1  &amp; stxA 2     UstxL1   SEQ ID NO 2   MTGATGATGRCAATTCAGTAT   784-805        0.1   3   57                   stxA 1     Nestx1   SEQ ID NO 31   GTACAACACTKGATGATCTC   327-347*   200   0.3               StxA 2     Nestx2   SEQ ID NO 32   TGACRACGGACAGCAGT   114-130*   410   0.05                 *Numeration is done using the longest hypothetical gene obtained after alignment of all variants (see FIGS.  9  and  10 ).             
 
     Example 5  
     Simplex PCR Example for the Detection of Enterobacteriaceae  
      For sample analysis (i.e. the detection of bacteria), individual colonies (i.e. test samples) from Luria agar culture plates are suspended in sterile distilled water. The preparation is then either used for direct PCR of bacteria or first boiled 10 min before use for the PCR. Samples (10 μl) to are amplified in 50 μl final reaction mixtures using a BioTest Biometra PCR machine. The mixtures contains 0.1 mM each dATP, dCTP, dGTP, dUTP, 1× final concentration of the 10× buffer solution and 1 U of DyNazyme II (Finnzymes) DNA polymerase per reaction and 0.1 μM of each primer Meca479UU21 and Meca722LU21. The PCR conditions consists of 2 min preheating at 94° C. for one cycle followed by 15 s denaturation at 94° C., 30 s annealing at 57° C. and 60 s elongation at 72° C. for 40 cycles; and 5 min final elongation at 72° C. Reaction products are separated by agarose (1.7%) gel electrophoresis stained with ethidium bromide (0.5 μg/ml), and the results are shown in FIG. 6. 0.5× Tris-borate-EDTA buffer is used for the electrophoresis. One hundred volts and 40 mA are applied across the gel for about 50 min to separate the PCR products.  
      Simplex PCR using Meca479UU21 and Meca722LU21 is performed on various Enterobacteriaceae and non-Enterobacteriaceae species to illustrate specificity of the method. As shown in FIG. 7, no non-Enterobacteriaceae was amplified by the simplex PCR.  
     Example 6  
     Simplex PCR Example for the Detection of stx  
      For sample analysis (i.e. the detection of bacteria), individual colonies (i.e. test samples) from Luria agar culture plates are suspended in sterile distilled water. The preparation is then either used for direct PCR of bacteria or first boiled 10 min before use for the PCR. Samples (10 μl) are amplified in 50 μl final reaction mixtures using a BioTest Biometra T gradient PCR machine. The mixtures contains 0.1 mM each dATP, dCTP, dGTP, dUTP, 1× final concentration of the 10× buffer solution and 1 U of DyNazyme II (Finnzymes) DNA polymerase per reaction and 0.1 μM of each primer UstxU1 and UstxL1, and 0.01 μM of each primer UstxU3 and UstxL3. The PCR conditions consists of 2 min preheating at 94° C. for one cycle followed by 15 s denaturation at 94° C., 30 s annealing at 57° C. and 60 s elongation at 72° C. for 40 cycles; and 5 min final elongation at 72° C. Reaction products are separated by agarose (1.7%) gel electrophoresis stained with ethidium bromide (0.5 μg/ml), and the results are shown in FIG. 8. 0.5× Tris-borate-EDTA buffer is used for the electrophoresis. One hundred volts and 40 mA are applied across the gel for about 1 h.  
                   TABLE 11                          Sequences of primers and optimum conditions to perform the simplex stx PCR           and product sizes.                                                                         Product   Primers   MgCl 2                 Gene           Primers (5′-3′)   Location   size (bp)   μM   mM   Ta ° C.                                                             stxA 1  &amp;   UstxU1   SEQ ID NO 1   TRTTGARCRAAATAATTTATATGT   279-303*   526 (stxA 1 )   0.1   3   57           stxA 2 ,   UstxL1   SEQ ID NO 2   MTGATGATGRCAATTCAGTAT   784-805*   523 (stxA 2 )   0.1       universal               stxA 2t     UstxU3   SEQ ID NO 4   AATGGAACGGAATAACTTATATGT   279-303*   523 (stxA 2t )   0.01           UstxL3   SEQ ID NO 5   GGTTGAGTGGCAATTAAGGAT   784-804*                  
 
      Results shown in FIG. 8 demonstrate how important MgCl 2  concentration is to develop a robust PCR amplification.  
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