WO2010133257A1

WO2010133257A1 - Method for detection and identification of bacterial strains belonging to the classes escherichia coli, salmonella, campylobacter and listeria

Info

Publication number: WO2010133257A1
Application number: PCT/EP2009/056219
Authority: WO
Inventors: Andrea Lauri; Paola Ornella Mariani
Original assignee: Fondazione Parco Tecnologico Padano
Priority date: 2009-05-22
Filing date: 2009-05-22
Publication date: 2010-11-25

Abstract

The present invention relates to new methods and tools for detecting and identifying specific stereotypes (or species for Listeria and Campylobacter) of pathogenic micro-organisms such as Salmonella, Campylobacter, and Listeria and Escherichia Coli in a Sample.

Description

METHOD FOR THE DETECTION AND IDENTIFICATION OF BACTERIAL STRAINS BELONGING TO THE CLASSES ESCHERICHIA COLI, SALMONELLA, CAMPYLOBACTER AND LISTERIA

DESCRIPTION The present invention was granted a foreign filing license issued by the

Ministero delle Attivita Produttive, D.G.S.P.C. UIBM, on the 4^th of February 2009, Protocol number 10558.

TECHNICAL FIELD

The present invention relates to new methods and tools for detecting and identifying specific serotypes (or species for Listeria and Campylobacter) of pathogenic micro-organisms such as Salmonella, Campylobacter, Listeria and Escherichia coli in a sample.

STATE OF THE ART

The detection and identification of pathogenic micro-organisms in a potentially infected sample, such as a food sample, or a biological sample, is an important goal in analytical microbiology. Bacteria identification it is crucial in order to identify the most suitable treatment for an ill patient, in order to identify the food conditions, in order to identify the most suitable treatment for an infected object or zone or area. The number of bacterial species for which an exact identification, defining also the specific serotype or serorvar or, in the case of Listeria and

Campylobacter, the species, is desirable or even necessary, is extremely high.

Amongst those, micro-organisms that can elicit human illness are of particular interest, an example of said micro-organisms is represented by some species of Salmonella with an estimated 1.3 billion cases worldwide of acute gastroenteritis.

Micro-organisms from the genus Salmonella are responsible for the majority of cases of bacterial diarrhoea occurring in humans. The genus has been divided into two species, Salmonella bongori and Salmonella enterica where enterica can be further subdivided into seven subspecies, designated I, II, Ma, HIb, IV, VI, and VII (Reeves et al, 1989). Salmonella enterica subspecies I is preferentially associated with warm blooded animals. Over 99% of all clinical Salmonella isolates are strains belonging to this subspecies, including serotypes typhimurium and enteritidis, which are the major causes of Salmonella induced gastroenteritis in humans, and typhi, the human specific causative organism of typhoid fever, the most severe form of human salmonellosis (Popoff et al, 1992). Salmonella enterica subspecies I consists of over 1300 different serotypes and is preferentially associated with warm-blooded animals (Baumler, 1997). Over 99% of all clinical Salmonella isolates are strains belonging to this subspecies.

Typically, screening and identification of Salmonella serotypes is accomplished by microbial culturing techniques followed by biochemical testing. This takes from one to three days, sometimes ending in equivocal and not specific results. Up to date alternative strategies aimed at replacing or complementing this method have been developed. These include PCR, Real-Time PCR, multiplex PCR, DNA sequencing and DNA microarrays. Molecular methods nowadays allow the identification of bacteria species and serotypes with the direct investigation of the genomic DNA and are generally less expensive and faster than traditional methods. Until now, molecular methods have been extensively used for the detection and identification of bacteria genera or species. Few tools are available for the detection and identification of Salmonella serotypes that belong to Salmonella enterica subspecies I.

Duran et al. (Duran 1996) have laid the foundation for an assay, based on the amplification and hybridisation of the sefA gene, to identify only the presence of S. enteritidis, S. berta, S. dublin, S. gallinarum, and S. pullorum serotypes in a food sample.

The commercial kit Premi®Test Salmonella (manufactured by DSM) is a multiplexed DNA typing test aimed at identifying up to 78 common serotypes of Salmonella enterica. The Premi®Test uses a DNA-based methodology called multiplex Ligation Detection Reaction (LDR) to generate a collection of ligated probes from the template genome that are further PCR amplified by means of a single pair of amplimers. The amplified molecules are subsequently hybridized to a low-density DNA Universal Array (UA) spotted with probe-specific complementary oligonucleotides (WO/2004/106547 fast method for detecting micro-organisms in food samples).

At the present time some Salmonella serotypes are not identifiable with the commercial kit Premi®Test. These include S. decatur, S. emek, S. haifa, S. miami, S. naestved, S. rubislaw, S. sendai, S. setubal, S. Stanleyville, S. tilene, S. typhisuis, S. wien S. parathyphi A. The Premi®Test Salmonella assigns a certain serotype to the strain under investigation based on the hybridisation pattern that follows an LDR investigation via a padlock probe based LDR-UA. In synthesis, the protocol annexed to the above mentioned kit indicates that the first step of the test consists in picking single bacterial colonies grown on non selective agar, enriching each colony by growing it in Modified Semi-Solid Rappaport-Vassiliadis Agar (MSRV), that is a selective medium historically used for the isolation of Salmonella in chocolate and other food products. The enriched aliquot is hence submitted to lysis, and then analysed with a series of recognition steps wherein and LDR and a PCR are carried out. This means that the simultaneous identification of more than one serotype from a bacteria mix is not possible, as the hybridisation pattern would be the overlapping of two or more serotype specific hybridisation patterns, and would correspond to a third hybridisation pattern with the consequent misinterpretation and erroneous identification.

Other important pathogenic micro-organisms that can elicit human illness are represented by various species of Campylobacter, which is the most common cause of foodborne diarrhea in many industrialized countries, species of Listeria such as Listeria monocytogenes, a micro-organism also capable of crossing the placental barrier, that causes listeriosis, a severe disease associated with high mortality rate, and certain strains of Escherichia coli.

Various methods based on PCR, Real-Time PCR, and multiplex PCR have been developed to detect the presence of these micro-organisms (Persson et al. 2005, Perelle et al 2004, Sanchez et al. 2008, Volokhov et al. 2002).

The GeneGen Major Food Pathogens [MFP] Detection Kit (Sabine Vollenhofer schrumpf et al. 2005) is a commercial assay that detects the presence or absence of the four pathogenic micro-organisms Salmonella, Escherichia coli 0157, Listeria monocytogenes and Campylobacter in food, by using multiplex Polymerase Chain Reaction (PCR) combined with a shortened, simplified hybridisation protocol on test strips. This method, although allowing the simultaneous detection of the four micro-organisms in a sample does not allow the simultaneous identification of the presence or absence of specific serotypes of each micro-organism.

There are still several bacterial serotypes that are not directly identifiable and, moreover, the major constraint of the methods known in the art, is that they do not allow the simultaneous detection of the presence/absence of some specific serotypes in a heterogeneous bacteria mix. At present, the simultaneous identification of more than one serotype from a bacteria mix comprising for example a predominant Salmonella serotype plus more serotypes at a lower concentration is not possible with the disclosed methods for a large number of Salmonella serotypes. This means that all the methods and kits available at present, do require the separation of single colonies by plating, and the carrying out of the analysis for each of the picked colonies. Furthermore, still direct identification for several serotypes is not possible and the data obtained must be combined and overlapped in order to identify specific bacterial serotypes.

Hence, there are no methods known so far that enable to detect simultaneously the presence or the absence of pathogenic micro-organisms belonging to the Salmonella, Listeria and Campylobacter genera and Escherichia coli species and to directly identify specific serotypes or serotype groups (or species, for Listeria and Campylobacter) in a heterogeneous bacterial mix.

SUMMARY OF THE INVENTION The applicants are well aware that, to provide means allowing a fast and large identification of serotype/s or species of contaminating micro -organism/s is highly desirable. The inventors have focalised on means that could allow a fast reply, trying to reduce the time for a response by providing means that enable for the identification of several bacterial serotypes and or species directly from an heterogeneous bacterial pool without previous plating of the bacteria in the sample and colony separation. The present specification hence discloses a method and a kit for the direct identification of the presence or absence of several bacterial serotypes from a heterogeneous bacterial sample, without previous culturing of said sample in order to allow separation of bacterial colonies. The method and kit of the invention, hence, allows the analysis of a heterogeneous bacterial sample without separation of single bacterial colonies for at least one or more of the following Salmonella serotypes (that were never identifiable before) selected in the group comprising Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien by genetic analysis of nucleic acids extracted from said pools for the detection of the presence or absence of serotype specific polymorphisms identified and described for the first time in the present application. The identified polymorphisms are selective with respect to all the known serotypes summarised in table 2 below. The detection of serotype specific Sequence Polymorphisms (SPs) as indicated below and in the tables of the present description, indicates the presence, in the pool, of the serotype for which said polymorphism is specific, the non-detection of a serotype specific polymorphism indicates the absence of said serotype.

The present invention discloses a method and a kit for carrying out said method.

The method herein described may be summarised as follows Method for the simultaneous identification of the presence or absence of one or more of: Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien in a sample comprising an heterogeneous bacterial pool, comprising the step of a. lysing the bacteria in the pool without previous separation of single colonies, b. identifying one or more of said bacteria by analysing the bacterial DNA for the presence or absence of one or more serotype specific SPs for each serotype:

- the serotype specific SPs for Salmonella decatur are represented by one or more of T in position 33 of SEQ ID No. 201 , T in position 33 of SEQ ID No 202, T in position 35 of SEQ ID No 114;

- the serotype specific SP for Salmonella emek is represented by T in position 30 of SEQ ID No. 115;

- the serotype specific SPs for Salmonella miami are represented by one or more of T in position 30 of SEQ ID No. 116, G in position 38 of SEQ ID No 117, G in position 29 of SEQ ID No 118, T in position 33 of SEQ ID No 119, A in position 33 of SEQ ID No 204, G in position 39 of SEQ ID No 223;

- the serotype specific SP for Salmonella rubislaw is represented by T in position 32 of SEQ ID No. 224; - the serotype specific SPs for Salmonella setubal are represented by one or more of T in position 27 of SEQ ID No. 155, T in position 26 of SEQ ID No 156, T in position 34 of SEQ ID No 205, T in position 37 of SEQ ID No 225, A in position 41 of SEQ ID No 226, T in position 31 of SEQ ID No 227, A in position 44 of SEQ ID No 228; - the serotype specific SPs for Salmonella Stanleyville are represented by one or more of A in position 33 of SEQ ID No. 123, C in position 33 of SEQ ID No 169, A in position 31 of SEQ ID No 229;

- the serotype specific SPs for Salmonella tilene are represented by one or more of A in position 35 of SEQ ID No. 124, T in position 35 of SEQ ID No 157, T in position 34 of SEQ ID No 158, A in position 36 of SEQ ID No 206, A in position

35 of SEQ ID No 230;

- the serotype specific SP for Salmonella typhisuis is represented by A in position 31 of SEQ ID No. 184;

- the serotype specific SPs for Salmonella wien are represented by one or more of A in position 33 of SEQ ID No. 159, T in position 36 of SEQ ID No 231; wherein the presence of one or more serotype specific SPs of each group indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Salmonella serotype. Object of the invention is also a kit for the simultaneous identification of the presence or absence of one or more of Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien in a sample comprising an heterogeneous bacterial pool, comprising reagents and tools for the detection of one or more of the serotype specific SPs listed above.

GLOSSARY Sequence Polymorphism/s, SP or SPs as herein defined are Sequence

Polymorphisms (SPs) that are described as differences among two or more sequences that can be represented by one or more nucleotides. In the SPs reported in Table 2 of the present description, the underlined and bold nucleotide (also defined as Discriminating Nucleotide, DN) is the nucleotide specific to the serotype(s) reported in the "positive strains" column and, optionally, to some of the strains belonging to the serotype(s) reported in the "possible positive strains" column.

All the other serotypes have a different nucleotide in the same position. The nucleotide sequence flanking the DN are not said to be absolutely conserved among the serotypes defined as "positive strains". In fact, such sequences have the mere function to indicate the DN and to localize it unambiguously in the analysed genomes. Therefore, a given serotype cannot be annotated as "positive strain" if the homology in the flanking regions is below a certain threshold (about 70%) even if the DN is identical.

When a SP is herein described as serotype specific (or species specific for Listeria and Campylobacter) it means that it unambiguously identifies a single bacterial serotype (or species for Listeria and Campylobacter).

As herein used, a positive control SP is a SP wherein the DN is shared by all the serotypes analysed of a given species, or, by all the Listeria and Campylobacter species analysed. Table 2 discloses also control SPs. Heterogeneous Bacterial Pool as herein defined, is a bacterial pool wherein a plurality of different bacterial species and/or serotypes are together, undivided unless a culturing of said pool on plates allowing the separation of the bacteria into distinct colonies is carried out. The pool might be obtained by culturing a sample infected by bacteria in a suitable growth medium (any suitable commercial medium for liquid bacterial growth) or directly the sample itself wherein said sample can be a food product, or a biological sample. Of course what applies to the detection and identification of single bacterial species or serotypes from a bacterial pool, applies to the detection of single bacterial colonies without use of inventive skill, whereas the opposite is not at all true. Identification techniques so far applicable to single bacterial colonies are not functional when applied to an undivided (bacterial colonies are not separated) bacterial pool.

Discriminating Base or Discriminating Nucleotide (DB or DN) in the present description defines the base, in each given SP described, whose presence in a defined position, indicates the presence of one or more serotype or species (when referred to Listeria and Campylobacter) in the analysed pool. The base position, and the specific base with respect to the given SEQ ID (as indicated in Table 2 and explained also above in the SP definition) are identifiers (i.e. in the methods and kits herein described) of a certain number of bacterial serotypes or species, this number varying from 1 to many (e.g. when a base common to all the serotypes analysed to be used by way of example, as a control sequence, in order to verify the functionality of the reagents) and the position and the base indicated in Table 2 are crucial for the identification of the serotype/s or species for which the base is discriminating. In a particular embodiment, i.e. positive control SPs, the DN is the nucleotide shared by several serotypes (or species) as indicated in Table 2.

Abbreviations as normally used in biology, in the present description S. followed by a word in small letters (e.g. such as in Table 2 of the present description) stands for the species Salmonella, L. for Listeria, C. for Campylobacter an E. for Escherichia.

DETAILED DESCRIPTION OF THE TABLES AND SEQUENCES: Table 1 describes the probes (oligonucleotides) useful for carrying out the amplification of a gene or the region of a gene of interest carrying one or more PN of the invention useful when amplification (pre or post amplification) is needed or desirable in the detection method of the invention. One or more primer pairs listed in Table 1 can also be provided in the new kit described below.

The first column indicates the SEQ ID No of the primer, the second column indicates the bacterial species and gene or region of gene amplified by the primer pair, the third column indicates literature reference if any, the fourth column indicates the oligonucleotide probe sequence.

The primer pairs are distinguishable in the table as they are not separated by empty horizontal lines, have a sequential ID number and the infos on second and third column are shared by the two oligonucleotides. Table 2 describes the SNs of the invention, in each lane, the first column gives the SEQ ID No, the second column indicates the gene comprising the SN, the third column the precise nucleotide consisting in the discriminating base, the fourth column the position of the discriminating base in each SEQ ID, the fifth column the only serotype or serotypes (or serotype group or species when referring to Listeria and species for Campylobacter), amongst all the strains indicated in Table 2, for which that discriminating base in that given position in that SEQ ID is present, and the sixth column indicates, when present, strains that are indicated only in part by the presence of said discriminating base (e.g. strains in which that SP is present in allelic forms).

Table 3 shows a comparison between the possibility of Salmonella serotypes identification with the method and the kit herein described and the kit Premi®Test Salmonella (manufactured by DSM) and method of use of the same. The table shows that: a. the commercial kit (column 5) NEVER identifies directly with a unique identifier (without combination of data) specific Salmonella serotypes; b. 9 of the serotypes identified by a unique identifier with the SPs herein described are identified in the commercial kit (lanes in gray and columns 1, 2, 3 and

6); c. further Salmonella serotypes may be identified by combination of the SPs herein described that are not identifiable with the commercial kit (black cells in column 4) Table 4 shows all the possible combination of 9 Salmonella serotypes as claimed in claim 1 as filed for the carrying out of the method and the kit of the invention wherein \=S. decatur, 2=S. emek, 3=S. miami, 4=S. rubislaw, 5=S. setubal, 6=S. Stanleyville, I=S. tilene, 8=5^*. typhisuis, 9=S. wien, each of the combinations there disclosed is separated by a - sign.

DETAILED DESCRIPTION OF THE INVENTION

Salmonella serotypes have been traditionally determined trough cultivation and immunochemical techniques that implement expensive reagents and are time consuming. Molecular methods nowadays allow the identification of bacteria species and serotypes with the direct investigation of the genomic DNA and are generally less expensive and faster than traditional methods.

Until now, molecular methods have been extensively used for the detection and identification of bacteria genera or species. A higher resolution, i.e. the identification of subspecies or serotypes still remains a challenge for molecular methods making traditional techniques preferable for these tasks.

In fact, few tools are available for the detection and identification of Salmonella serotypes that belong to Salmonella enterica subsp I.

To allow the precise determination of serotypes eight genomic sequences relative to seven genes (safA, sefC, sefA, bigA, phsB, invA, fimA) have been sequenced from more than 150 serotypes of Salmonella. A database has been created ah hoc and integrated with publicly available data.

Serotype-specific polymorphisms, such as serotype specific SPs for S. decatur, S. emek, S. miami, S. rubislaw, S. sendai, S. setubal, S. Stanleyville, S. tilene, S. typhisuis, S. wien and other Salmonella serotypes have been individuated, and specific probes have been designed for identifying said serotypes. For some serotypes, more than one specific sequence polymorphism has been identified as listed in Table 2.

A substantial difference between the prior art Premi®Test and the methods and kits herein described, lies in the method used for the identification of the serotype. The Premi®Test assigns a certain serotype to the strain under investigation based on the hybridisation patter that follows an LDR investigation. This means that the simultaneous identification of more than one serotype is not possible, as the hybridisation pattern would be the overlapping of two or more serotype specific hybridisation patterns and would correspond to a third hybridisation pattern. The reconstruction of the first and second hybridisation patterns, hence the determination of the two corresponding serotypes, from the third hybridisation pattern, resulting from their superimposition, might be hard and in most cases impossible. In some cases the third pattern might be identical to that of another serotype, whose presence would be wrongly diagnosed in the sample. The methods and kits of the invention lie on the new identification of serotype-specific sequence polymorphisms (or species specific polymorphisms when referred to Listeria and Campylobacter). The identification of the serotype specific sequence polymorphisms herein disclosed, allows, for the first time, a simultaneous identification of the serotypes identifiable by the SPs above, from a heterogeneous bacterial mix, without previous separating the bacterial strains, because their hybridisation patterns are not overlapping and are independent.

This is of particular interest when a product is contaminated, by way of example, with a Salmonella serotype (and or a predominant Listeria serotype group and/or species and/or Campylobacter species) plus more serotypes or other species such as Listeria and or Campylobacter at an equal or lower concentration. In an embodiment the method of the invention is a method for the simultaneous identification of the presence or absence of one or more of: Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or

Salmonella wien in a sample comprising an heterogeneous bacterial pool, comprising the step of: a. lysing the bacteria in the pool without previous separation of single colonies, b. identifying one or more of said bacteria by analysing the bacterial DNA for the presence or absence of one or more serotype specific SPs for each serotype as defined in the summary of the invention, wherein the presence of one or more serotype specific SP of each group indicates the presence of said serotype in the sample, the absence of serotype specific SNPs of one or more group indicates the absence of the corresponding Salmonella serotype. According to the present description, hence, the method (as well as the related kit discussed later) can be set in order to allow the identification, from a heterogeneous bacterial pool, of at least one of the Salmonella selected in the group: Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien by identification, in the DNA of said bacterial pool, of one or more of the SPs listed above specific for said serotypes. In brief, the method and the kit encompass also a method and a kit for the identification of the presence/absence of each of one, two, three, four, five, six, seven, eight or all nine the serotypes listed above, in each possible combination, from a heterogeneous bacterial pool, by the identification of the presence or absence of one or more serotype specific SP amongst the serotypes and their relative discriminating base indicated above as well as in Table 2. By simply indexing each bacterial strain (i.e. I=S. decatur, 2=S. emek, 3=S. miami, 4=S. rubislaw, 5=S. setubal, 6=S. Stanleyville, I=S. tilene, S=S. typhisuis, 9=S. wien) all possible combinations can be obtained by simple computer programs available to the public, the combinations, here disclosed one by one separated by a - sign are indicated in Table 4. The method disclosed above and as claimed in claim 1 as originally filed and as defined in detail in the summary of the invention, can be carried out by using several detection methods commonly used in the art.

It is clear that once the serotype specific sequences and discriminating bases are disclosed as in Table 2 of the present specification, the skilled person can design suitable detection protocols without use of inventive skills, merely following the teachings of the art.

The skilled person is made aware, by the present specification, that the SPs listed in Table 2, enable the researcher to either directly recognise, in a bacterial pool comprising one or more or all of the listed bacteria, all the serotypes that are specifically and individually identified by one or more of the disclosed SPs directly from the heterogeneous bacterial pool without separation of said bacteria into single colonies and picking and testing each single colony, or, with a second step, to identify by colony separation, several other serotypes (or, in the case of Listeria, and Campylobacter, species) with the combinations obtained with the SPs that identify a group of serotypes.

As clear from Table 2, the present specification discloses certain SPs that are specific for a single serotype amongst all the serotypes indicated in Table 2, SPs that are specific for a group of serotypes, the combination of which allows the identification of other serotypes and of other species compared to the ones indicated above, and SPs that, on the other hand, are common to all the serotype listed for each species, and are hence useful as positive control in the detection methods herein disclosed. As the skilled person knows, detection methods in biotechnologies are often carried out with positive or negative controls, that are not a must in said methods but that are, when available, of interest and useful for the researcher or the technician as they validate the correct functioning of the reactions carried out.

The methods herein described may hence further comprise the detection of one or more positive control SPs, each of which is directly identifiable in Table 2 as it identifies all the serotypes of a given species or subspecies of a given bacteria. Each of said SPs singularly or in combination with other of said SPs may be included in the method claimed in the present description as positive control SP.

Said one or more positive control SPs may be selected, for Salmonella, in the group of SEQ ID NOs 150, 194, 195, 196, 214, 215, 216, 217 and the discriminating base will be according to Table 2. Said one or more positive control SPs may be selected, for Listeria in the group of SEQ ID NOs 265, 260, 261, 269 and 279, wherein SEQ ID NO 265 discloses a SP that may serve as a positive control for all the serotypes and subspecies listed in Table 2, whereas SEQ ID NOs 260, 261, 269 and 270 may be used as a positive control for the species L. monocytogens. Also in this embodiment, the discriminating base will be according to Table 2. Said one or more positive control SPs may be selected, for Campylobacter in the group of SEQ ID NOs 23 and 24, the discriminating base will be according to Table 2. Said one or more positive control SPs may be selected, for E. coli in the group of SEQ ID NOs 53-58, 67, the discriminating base will be according to Table 2. The method may be hence carried out by identifying also one or more of the positive control SPs indicated above or any combination thereof.

Tools for the detection and identification of one or more of the positive control SPs listed above may be comprised in any kit according to the present specification. The realisation of said tools is identical as for the tools for the detection and identification of any serotype specific SP as herein described.

According to the present description the bacteria can be lysed with any of the methods currently known in the art and DNA purification can be carried out in order to clean the sample to be analysed. The skilled person is well aware that several reactions (such as PCR) can be carried out also on non purified DNA, but it is obviously simpler to carry out a DNA identification experiment on a "clean" sample wherein DNA has been purified. Bacterial DNA extraction and purification is nowadays common knowledge for the skilled person, detailed protocols for mini, midi and maxi preps are available in laboratory manuals such as "Molecular cloning : a laboratory manual / T. Maniatis, E. F. Fritsch, J. Sambrook", articles such as in Letters in applied microbiology Rapid extraction of bacterial genomic DNA with guanidium thiocyanateD.G. PITCHER et al, and several extraction kits such as from Promega, Quiagen and others, are commercially available so that no further indication on bacterial DNA extraction is needed in order to enable the skilled person to carry out the method or kit of the invention. Several DNA extraction protocols are available also when the starting sample is milk, or a body fluid, a food product (e.g. meat, fish, cheese, yoghourts, raw and cooked vegetables and others), faeces, liquids, environmental samples, etcetera. A pre culturing of the bacterial heterogeneous pool can be carried out in order to increase the bacterial number if desired.

Each of the embodiments herein described for the carrying out of the methods claimed and described can include a pre-amplifϊcation step carried out on the bacterial pool DNA, with one or more of the amplification primer pairs listed in Table 1. The pre-amplifϊcation step is not necessary but may be useful for better detecting serotypes that might be in extremely low concentrations. The selection of the primers to be used in this optional pre-amplifϊcation step of the bacterial pool DNA (or when the step of single colony picking is to be carried out, an amplification step preceding the SPs detection and identification can be carried out on the single colony DNA) is obvious from Tables 1 and 2. Table 1 indicates the primers for amplifying a particular gene or gene region, Table 2 indicates, in column 3, the bacterial gene comprising each SP and the sequence of each SP. It is hence obvious that, if, by way of example, detection/identification of one or more SPs located in the Salmonella SefA gene is envisaged, a pre-amplifϊcation with the primer pair of SEQ ID NOs 244 and 245 can be carried out.

The same applies to all others SPs of Table 2 and primer pairs of Table 1. The detection/identification of each SP can be carried out with any method suitable, known to the skilled person. By way of example, Oligo microarray can detect the SPs described herein implementing two different technologies (i.e. the competing probe method or the APEX (Arrayed Primer Extension) system). In both cases the amplification of target DNA is possible, but not absolutely required, depending on the protocol, on the genome size of the organism under investigation, etc.

Competing Probe Method: two kinds of probes are included in an array for each polymorphism, one is a perfectly matching the target sequence probe (Perfect Match (PM) probe), the other(s) contain mutation in any point of the probe, typically in the middle (MisMatch (MM) probe). The competing probe is designed in the same way as the hybridisation probe except that it differs from the hybridisation probe in a single base at the position of the polymorphism. One of the MM probes can represent one allele of the polymorphism. The bacterial DNA pool is labelled with a fluorophore by techniques normally used to this purpose. For instance, the technique can be based on the incorporation, by Taq polymerase and random primers, of a fluorescently labelled nucleotide or of a biotin marked nucleotide subsequently indirectly labelled with a fluorophore labelled streptavidine. A second DNA pool can be used as control and benchmark and compared to the first DNA pool. The second DNA pool must be labelled with a fluorophore different from that used to label the first DNA pool. A hybridisation with the bacterial pool DNA or a single colony DNA is carried out at conditions not allowing unspecific hybridisation which are standardised in the art in terms of temperature and composition of the hybridisation mixture (i.e. salt concentration, formamide concentration and so on). The DNA can be optionally pre-amplified with one or more primer pairs listed in Table 1. The specific position of each probe on the array will allow the user to determine several serotypes in one single experiment. A simple computer program, carried on a computer storage medium could be used to implement the method herein described and to analyse the data obtained by the present assay in order to determine which serotype specific SPs are present and hence, which bacterial serotypes are present in the heterogeneous pool under examination. The hybridisation signal relative to each probe and their ratio, indicates the allelic composition of the target DNA. The signal of the PM probe must be always stronger than the MM probe, otherwise the HP signal could be due to unspecific hybridisation. An other example is given by the case that PM and MM represent two alleles of the same polymorphic sequence. If for instance PM represents allele A and MM allele B, the PM/MM ratio indicates if the sample is AA, AB or BB.

The PM/MM strategy has been implemented in the diagnosis and discrimination of pathogens and reported in literature (e.g. by Lin B, Wang Z, Vora GJ, Thornton JA, Schnur JM, Thach DC, Blaney KM, Ligler AG, Malanoski AP, Santiago J, Walter EA, Agan BK, Metzgar D, Seto D, Daum LT, Kruzelock R, Rowley RK, Hanson EH, Tibbetts C, Stenger DA (2006) Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays. Genome Res 16:527-535).

In this case, the MM probe, competing with the PM probe has the function of demonstrating the absence of the other alleles and hence to indicate that only the serotype of interest is present. It is obvious that, given the fact that the DB (Discriminating Base) specific for a given serotype that is disclosed in Table 2, the MM probes can have, at the DB position, any other nucleotide excluding the DB.

APEX system: the probes, immobilised on the array, are designed on one or more SPs such to anneal the target DNA (bacterial pool DNA or single bacterial colony DNA) and have the 3' end nucleotide immediately before the polymorphic base as indicated in Table 2. The target DNA (genomic DNA of the bacterial pool or of a single colony, depending on the protocol may be pre-amplified by PCR with one or more primer pairs listed in Table 1) including the polymorphic region is hybridised on the array as described above. A DNA polymerase is used to extend the probe of one single base, using labelled dNTPs, each with a different fluorophore. This sort of "mini-sequencing" determines the polymorphism of the target DNA. Alternatively, the same method can be carried out, not as an oligo array system, using a SNaPshot system, with the difference that in the SNaPshot system probes are not immobilised on a chip surface but free and are than run on a capillary. In the SNaPshot system, the detection is carried out as follows: target DNA containing polymorphisms are preferably pre-amplifϊed. PCR products are denatured and specific primers (or probes) as described at point 2 above are added to the reaction. Said primers anneal the target DNA and one base carrying the fluorophore is extended. It is important to note each primer is preferably designed with a different length, in order to render the separation on the capillary more effective. The reaction is cycled to bring to a linear amplification of the signal (i.e. n amplification cycles to obtain n amplification of the signal). The product is run on a capillary in order to separate the extended primers. Each primer (and the corresponding polymorphic site) is expected at a give position. The fluorescence colour tells the nucleotide/s that is/are present on each polymorphic site.

In a further embodiment, the identification and detection of the SPs, can be carried out by the MALDI TOF technique.

MALDI TOF is a mass spectrometry application that bases the identification of molecules on their "time of flight" that measures the molecular mass. Once the molecular mass is determined one can understand which allele the target is because of different molecular composition. It can be used with multiple polymorphisms as different sequences have different masses. Practically, the method can be carried out basically as follows: a primer extension reaction is carried out. In this reaction oligonucleotides are designed to bind directly to the 5 'end of the desired SP sequence. Appropriate dideoxy nucleotides (ddNTPs), which cannot be elongated, are substituted for one of the 4 deoxy nucleotides (dNTPs) in the reaction mix. Therefore, at the DN of the SP of interest, a complementary ddNTP will be incorporated and different allele specific fragments will be created. Supposing that the SP examined has as determining nucleotide an A in position 24, in the case of the serotype specific allele ddATP will be incorporated straight away, which results in a product of 24 nucleotides. In the case of the 2nd allele, the first two nucleotides (dTTP and dGTP) are incorporated as normal and the extension proceeds up to the first T (e.g. in position 26) where a ddA is incorporated resulting in a 26 nucleotide long extension product. The two extension products, a 24-mer and a 26-mer, have different masses. The extension products can hence be spotted on microchips (known in the art, e.g. www.abgene.com) and analysed in the MALDI TOF Mass Spectrometer. A laser fires on the products spotted on the microchip and the DNA is accelerated in vacuum to a detector. Smaller molecules (e.g. a 24-mer) are faster than larger molecules (e.g. a 26-mer) and are detected earlier (figure 4). The mass of every extended product is determined and can be "translated" into one allele of the SNP, in the exemplified case T or A.

Alternatively, an Illumina type Bead Express of GoldenGate method can be carried out: Two competing probes (CPl and CP2) are designed to anneal each of the alleles of the polymorphism (in the present case one probe will have the DB and the other one will be as described above) and carry, each, a different universal primer annealing site. One of the probes (or both in case both alleles are present in the target DNA) anneals the genomic DNA (no amplification needed) and is extended up to the site where a Universal Probe (UP) is annealed (the UP is common to all alleles and carries a ZIP Code, similar to that of the LDR-UA). A DNA ligase binds the UP together with the extended probe. The ligation product is amplified with universal primers contained in the two probes. The universal primer annealing the first probe carries a fluorophore of different colour depending on the allele probe.

After the amplification step, the product is captured by the corresponding bead via the ZIP Code sequence, whose complementary is present on the bead. The bead also carries a bar code that can be read by a laser.

The instrument reads the fluorescence colour and the bar code at the same time and therefore determines the allelic fraction of each investigated site.

The detection/identification can also be carried out by the Ligation Detection Reaction - Universal Array (LDR-UA) technique (Gerry, N. P., N. E. Witowski, J. Day, R. P. Hammer, G. Barany, and F. Barany, Universal DNA microarray method for multiplex detection of low abundance point mutations J. MoI. Biol. (1999) 292:251-262; US patent US06852487) that allows the detection of Single Nucleotide Polymorphisms (SNPs) in DNA molecules. In the present description each SP comprises an equivalent of an SNP identified by the DN indicated. The LDR-UA technique takes advantage of two different probes, called Common Probe (CP) and Discriminating Probe (DP), which are designed to anneal juxtaposed target single strand DNA. DP anneals the DNA at the 5' end of CP. The two probes can be ligated by a thermo-stable ligase such as the Pfu Ligase. If the complementarity between the 3' end of the DP and the target DNA is not perfect, the ligation reaction is compromised. For this reason the last base at the 3' end of the DP is also called Discriminating Base (DB). According to the present invention, the DP will start, at the 3' with the DN of a given SP as indicated in Table 2 and continue with 10 or more immediately upstream adjacent nucleotides (i.e. the nucleotide upstream of the DB and so on) of the SP sequence whereas the corresponding CP will end, at the 5', with the nucleotide immediately following and adjacent to the DB of the same SP sequence and comprises at least 10 immediately downstream adjacent nucleotides (i.e. the nucleotide downstream the DB and so on). The system requires that the probes carry the following modifications:

Each DP that carries a different DB corresponding to a different allele of the SP must be labelled with a distinct fluorophore at its 5' end.

The CP must be phosphorylated at its 5 ' end and must be extended at its 3' end with a "cZIP Code" sequence, that is the complementary and inverse of the "Zip Code" sequence spotted on the Universal Array. Every CP corresponds to a different "ZIP Code".

Alternatively, the system might comprise a Universal Labelling Sequence located at the 5' of a DP for LDR-UA and a complementary Universal Labelling Sequence directly labelled with a fluorophore at least at one or both ends or internally so to allow indirect labelling as described in detail in EP application

08161325.9 on the name of the same applicant.

The LDR-UA platform allows performing all detection reactions in one tube rendering therefore possible the simultaneous identification of all serotype in one experiment and with one fluorophore only (if one DP per SP is used).

The protocols therein described for indirect labelling of the LDR-UA may be used by the skilled person for indirect labelling in the present LDR-UA method.

The ligation product is hybridised to the UA. Each UA spot is composed of different "Zip Code" DNA sequences that capture the corresponding "cZIP Code" sequences at the CP 3' end. The CP-DP ligation event positions the fluorophore at the 5' end of the DP on the corresponding UA spot, which is visualised by the subsequent scanning of the UA. The signal from a spot therefore indicates the perfect match between the DP and the target DNA.

The method above can be also modified in order to carry out a LDR and optionally afterwards, a PCR. As herein described, PCR is an option so far described before the LDR. In order to swap the order of the protocol described above and, when a PCR is desired, the same is to be carried out after the LDR and the primers should be designed differently from the ones shown in Table 1 , design of new probe and primers would be however evident to the skilled person and must be done such that they amplify the ligated probes. Primers are normally universal and are directed against universal sites included in the DP and CP and that span the ZIP Code. All the arrays herein described may comprise one or several copies/spot of each oligonucleotide/probe herein described. A high number of copies render the signal stronger and easily detectable. A dedicated software can be used in order to analyse the data obtained from each of the embodiment indicated above, wherein the said software is apt to carry out step b of the method described above and can be provided, on a computer readable storage support. By computer readable storage support it is intended any support suitable for the storage of software that allows the installation of said software on a computer, e.g. CD, DVD, TAPES, USBPen, EPROM, disks, hard disks, etc. and/or the software may be downloadable from a network. It is to be understood that the analysis of the obtained results and the identification of the serotypes (species for Listeria and Campylobacter) detected by the methods of the invention can be provided as a service through a web-service via an online connection and/or the software can be downloadable from a network.

The software will comprise a database with all the SPs of Table 2 and the procedures and steps for recognising the known polymorphism detected by the method herein described. Within such database, providing a report with the results of the polymorphisms identified and, if an input for the association of a given polymorphisms or polymorphic pattern with a given result (i.e. polymorphism X equals serotype/s, or, for Listeria and Campylobacter species, Y) has been provided to the software, more elaborated results can be provided by the software (e.g. direct identification of one or more given serotype in a sample, etc.).

Furthermore, the method according to the present description, may enable for the direct identification from a heterogeneous bacterial pool without previous separation of single colonies, also for the presence or absence of one or more serotype specific SPs for each of one or more Salmonella serotype selected in the group of: Salmonella agona, Salmonella anatum, Salmonella derby, Salmonella duisburg; Salmonella enteritidis, Salmonella gallinarum, Salmonella hadar, Salmonella heidelberg, Salmonella indiana, Salmonella infantis, Salmonella livingstone; Salmonella mbandaka, Salmonella panama, Salmonella paratyphi A, Salmonella pullorum, Salmonella reading, Salmonella Stanley, Salmonella thompson, Salmonella typhi, Salmonella typhimurium by analysing the bacterial DNA obtained after lysing the bacteria in the pool without previous separation of single colonies, also for the presence or absence of one or more serotype specific SPs for each serotype: - the serotype specific SP for Salmonella agona is represented by C in position 27 of SEQ ID No. 186;

- the serotype specific SP for Salmonella anatum is represented by A in position 30 of SEQ ID No. 113;

- the serotype specific SPs for Salmonella derby are represented by one or more of A in position 33 of SEQ ID No. 160, T in position 38 of SEQ ID No 234;

- the serotype specific SP for Salmonella duisburg is represented by A in position 38 of SEQ ID No. 141; - the serotype specific SPs for Salmonella enteritidis are represented by one or more of T in position 35 of SEQ ID No. 104, C in position 28 of SEQ ID No 105, T in position 31 of SEQ ID No 147;

- the serotype specific SP for Salmonella gallinarum is represented by T in position 35 of SEQ ID No. 174;

- the serotype specific SNPs for Salmonella hadar are represented by one or more of T in position 38 of SEQ ID No. 107, T in position 35 of SEQ ID No 108, T in position 31 of SEQ ID No 175, G in position 27 of SEQ ID No 176, T in position 45 of SEQ ID No 177; - the serotype specific SP for Salmonella heidelberg is represented by T in position 31 of SEQ ID No. 161;

- the serotype specific SPs for Salmonella indiana are represented by one or more of T in position 30 of SEQ ID No. 135, G in position 47 of SEQ ID No. 171;

- the serotype specific SPs for Salmonella infantis are represented by one or more of T in position 33 of SEQ ID No. 110, G in position 25 of SEQ ID No. 193;

- the serotype specific SPs for Salmonella livingstone are represented by one or more of C in position 35 of SEQ ID No. 145, A in position 34 of SEQ ID No. 213;

- the serotype specific SP for Salmonella mbandaka is represented by G in position 24 of SEQ ID No. 212; - the serotype specific SP for Salmonella panama is represented by A in position 39 of SEQ ID No. 172;

- the serotype specific SPs for Salmonella paratyphi A are represented by one or more of T in position 46 of SEQ ID No. 97, G in position 30 of SEQ ID No 98, A in position 35 of SEQ ID No 112, T in position 38 of SEQ ID No 120, A in position 38 of SEQ ID No 200;

- the serotype specific SPs for Salmonella pullorum are represented by one or more of A in position 35 of SEQ ID No. 152, T in position 39 of SEQ ID No. 164;

- the serotype specific SP for Salmonella reading is represented by T in position 35 of SEQ ID No. 129; - the serotype specific SP for Salmonella Stanley is represented by A in position 37 of SEQ ID No. 130;

- the serotype specific SP for Salmonella thompson is represented by T in position 35 of SEQ ID No. 144;

- the serotype specific SPs for Salmonella typhi are represented by one or more of A in position 35 of SEQ ID No. 94, G in position 29 of SEQ ID No 99, C in position 23 of SEQ ID No 100, T in position 40 of SEQ ID No 192;

- the serotype specific SP for Salmonella typhimurium is represented by A in position 32 of SEQ ID No. 161; wherein the presence of one or more serotype specific SP of each group indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Salmonella serotype.

All embodiments herein described and defined so far for methods and kits for detection and identification of the presence in a heterogeneous bacterial sample of one or more of Salmonella serotypes selected in the group of Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien (first group) apply also to the embodiment above comprising the further detection and identification of the presence in a heterogeneous bacterial sample of one or more of Salmonella serotypes selected in the group of: Salmonella agona, Salmonella anatum, Salmonella derby, Salmonella duisburg; Salmonella enteritidis, Salmonella gallinarum, Salmonella hadar, Salmonella heidelberg, Salmonella Indiana, Salmonella infantis, Salmonella livingstone; Salmonella mbandaka, Salmonella panama, Salmonella paratyphi A, Salmonella pullorum, Salmonella reading, Salmonella Stanley, Salmonella thompson, Salmonella typhi, Salmonella typhimurium (second group) as defined above. Moreover, the method of the invention, may further enable for the direct identification from a heterogeneous bacterial pool without previous separation of single colonies, also for the presence or absence of one or more serotype specific SPs for each of one or more of Listeria seeligeri, Listeria ivanovii, Listeria monocytogens I Listeria monocytogens II, Listeria monocytogens III, Listeria grayii in said sample by analysing the bacterial DNA obtained after lysing the bacteria in the pool without previous separation of single colonies, also for the presence or absence of one or more serotype specific SPs for each serotype:

- the serotype specific SPs for Listeria seeligeri are represented by one or more of G in position 43 of SEQ ID No. 258, C in position 45 of SEQ ID No. 268; - the serotype specific SPs for Listeria ivanovii are represented by one or more of C in position 44 of SEQ ID No. 259, C in position 40 of SEQ ID No. 267;

- the serotype specific SPs for Listeria monocytogens I are represented by one or more of C in position 52 of SEQ ID No. 262, A in position 43 of SEQ ID No. 271; - the serotype specific SPs for Listeria monocytogens Il are represented by one or more of C in position 40 of SEQ ID No. 263, T in position 51 of SEQ ID No. 272; - the serotype specific SPs for Listeria monocytogens III are represented by one or more of C in position 39 of SEQ ID No. 264, A in position 47 of SEQ ID No.

273;

- the serotype specific SP for Listeria grayii is represented by T in position 35 of SEQ ID No. 266; wherein the presence of one or more serotype specific SP of each group indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Listeria species or serotypes (L. monocytogenes, L. grayii, L. innocua, etc are species whereas L. monocytogenes I, II e /// are serotype groups).

All embodiments herein described and defined so far for methods and kits for detection and identification of the presence in a heterogeneous bacterial sample of one or more of Salmonella serotypes selected in the group of Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien and, optionally, of one or more of Salmonella serotypes selected in the group of: Salmonella agona, Salmonella anatum, Salmonella derby, Salmonella duisburg; Salmonella enteritidis, Salmonella gallinarum, Salmonella hadar, Salmonella heidelberg, Salmonella indiana, Salmonella infantis, Salmonella livingstone; Salmonella mbandaka, Salmonella panama, Salmonella paratyphi A, Salmonella pullorum, Salmonella reading, Salmonella Stanley, Salmonella thompson, Salmonella typhi, Salmonella typhimurium as defined above, apply also to the embodiment above comprising the further detection and identification of the presence in a heterogeneous bacterial sample of one or more of Listeria species and/or serotypes selected in the group of Listeria seeligeri, Listeria ivanovii, Listeria monocytogens I, Listeria monocytogens II, Listeria monocytogens III, Listeria grayii.

Hence, the method of the invention, comprises the identification of one or more of the Salmonella serotype selected in the group of Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien, and may comprise the identification of further Salmonella serotype/s as indicated above, and/or Listeria species and/or serotypes as indicated above, and/or Campylobacter species as indicated below.

Indeed, the method of the invention, may further enable for the direct identification from a heterogeneous bacterial pool without previous separation of single colonies, also for the presence or absence of one or more species specific SPs for each of one or more of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis in said sample by analysing the bacterial DNA obtained after lysing the bacteria in the pool without previous separation of single colonies, also for the presence or absence of one or more species specific SPs for each species: - the specie specific SPs for Campylobacter jejuni are represented by one or more of T in position 50 of SEQ ID No. 15, A in position 60 of SEQ ID No 16, A in position 51 of SEQ ID No 7, A in position 36 of SEQ ID No 8;

- the specie specific SPs for Campylobacter coli are represented by one or more of T in position 50 of SEQ ID No. 17, A in position 56 of SEQ ID No 18, C in position 48 of SEQ ID No 11, G in position 61 of SEQ ID No 12;

- the specie specific SPs for Campylobacter lari are represented by one or more of A in position 56 of SEQ ID No. 19, A in position 51 of SEQ ID No 20, G in position 52 of SEQ ID No 9, C in position 55 of SEQ ID No 10;

- the specie specific SPs for Campylobacter upsaliensis are represented by one or more of C in position 47 of SEQ ID No. 21, T in position 44 of SEQ ID No

22, G in position 39 of SEQ ID No 13, G in position 37 of SEQ ID No 14; wherein the presence of one or more species specific SP of each group indicates the presence of said serotype in the sample, the absence of species specific SPs of one or more group indicates the absence of the corresponding Campylobacter serotype.

The skilled person is well aware that Campylobacter and Listeria are divided in species and not in serotypes in the current literature.

All embodiments herein described and defined so far for methods and kits for detection and identification of the presence in a heterogeneous bacterial sample of one or more of Salmonella and or Listeria species and/or serotype groups apply also to the embodiment above comprising the further detection and identification of the presence in a heterogeneous bacterial sample of one or more of Campylobacter species.

As already indicated when discussing the positive control SPs, in the method above said bacterial DNA may be analysed also for one or more SP common to all the Salmonella serotypes or to all the Listeria species and/or serotype groups or to all the Campylobacter species to be identified.

As disclosed in detail, in the method above, whether it detects and identifies only the presence or absence of one or more of Salmonella serotypes selected in the first group, whether it also detects and identifies the presence or absence of one or more of the Salmonella serotypes selected in the second group, and/or whether it also detects and identifies the presence or absence of one or more of the Listeria serotype group or species selected in the Listeria species and/or serotype groups disclosed, and/or whether it also detects and identifies the presence or absence of one or more of the Campylobacter species selected in Campylobacter species group disclosed, said one or more polymorphisms may be detected by means of: PCR, TaqMan, LDR- UA, PCR-LDR-UA, SNaPshot, Oligo microarray, SNParray, DNA sequencing, or LDR.

The same method may further comprises, in step a., as already discussed, a DNA extraction after bacterial lysis, may also or further comprise a step in which said bacterial DNA is amplified by PCR with primers specific for a region comprising the SPs under analysis.

Herein is also disclosed as an embodiment of the invention, a kit for the simultaneous identification of the presence or absence of one or more of: Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien in a sample comprising an heterogeneous bacterial pool, comprising reagents and tools for the detection of one or more of the serotype specific SPs listed above as serotype specific for the Salmonella serotypes of group 1, also disclosed in Table 2 of the present specification.

The kit of the invention may comprise the suitable reagents depending on the detection method selected for the realization of the said kit.

The detection methods are fully described above and the SPs to be identified, their specificity and their relevant nucleotide (DN) are fully described in Table 2.

Depending on the detection method chosen, the skilled person would know how to construct the suitable probes and reagents for the detection and identification of the SPs above described.

By way of example, for a competing probe method, the kit will comprise one or more microarray carrying a PM and a MM probe as described above for each SP to be detected and identified, and optionally one or more aliquots of primer pairs for amplification as listed in Table 1. The PM and MM will be designed as oligonucleotides of about 20-45 bp, perfectly matching the sequence disclosed in Table 2 (PM) or with one or more nucleotide exchange in correspondence of the DN for the selected SP.

The probes will be blocked on a suitable microarray and the kit might further comprise a carrier for a computer program or a password and or id for an online service in order to readily analyse the results obtained on the microarray providing an elaborated response indicating the bacterial serotypes and, optionally, species detected in the sample analysed. The kit may further comprise one or more vial of reagent solutions (i.e. one or more of PCR buffer, dNTPs, enzyme, salt, hybridisation mixture, reagents for indirect labelling) suitable for the reactions to be carried out for the detection of the results. In case of a kit for carrying out a detection for APEX system kit will comprise one or more microarray carrying one or more probe designed on the SPs to be detected such to anneal the target DNA and have a 3' nucleotide immediately preceding the DN (or DB) and optionally one or more vial of DNA polymerase and/or of labelled terminator nucleotides and reagents suitable for carrying out the detection according to the method.

The same kit might alternatively comprise, or optionally further comprise as means for detection in a SNaPshot system instead or in addition of oligo arrays. The same probes are provided as free of substrate and will undergo the same hybridisation and extension procedure as described for APEX. The extended probes will then be separated and visualized on a capillary.

Hence, the kit may provide tools for carrying out a primer extension as described above and for two possible detection systems or two kits can be produced, one for APEX detection and the other for SNaPshot.

By way of example, the array may be designed for detection of Salmonella serotypes of group 1 and optionally, group 2 serotypes and/or Listeria serotypes and/or Campylobacter species, whereas the SNaPshot detection could be used for the detection of the serotypes of group 1.

The kit of the invention may comprise, when a Maldi Tof detection is envisaged, aliquots of one or more oligonucleotide specific for one or more of the SPs disclosed (depending on the serotypes to be identified the specification fully enables for the selection of the suitable SPs) and, optionally, one or more aliquot of reagents suitable for the elongation reaction as detailed above.

Where an illumina GoldenGate method (BeadXpress) is the detection method envisaged, the kit will comprise at least one or more aliquot of each of the primers (CPl and CP2, and UP as described above) and optionally of suitable reagents for carrying out the detection.

The kit can alternatively comprise tools for an LDR-UA detection. In this case the kit will comprise one or more microarray comprising one or more CP as defined below and one or more aliquot of one each corresponding DP. According to the present invention, the DP will start, at the 3' with the DB of a given SP as indicated in Table 2 and continue with 10 or more immediately upstream adjacent nucleotides (i.e. the nucleotide upstream the DB and so on) of the SP sequence whereas the corresponding CP will end, at the 5', with the nucleotide immediately preceding and adjacent to the DB of the same SP sequence and comprise at least 10 immediately downstream adjacent nucleotides (i.e. the nucleotide downstream the DB and so on). Each DP detecting a different DB of a SP will be labelled with a distinct fluorophore at its 5 ' end.

The CP will be phosphorylated at its 5 ' end and will be extended at its 3 ' end with a "cZIP Code" sequence, which is the complementary and inverse of the "Zip Code" sequence spotted on the UA. Every CP corresponds to a different "ZIP Code". Alternatively, the system might comprise a ULS located at the 5' of a DP for

LDR-UA and a complementary ULS directly labelled with a fluorophore at least at one or both ends or internally so to allow indirect labelling as described in detail in EP application 08161325.9 on the name of the same applicant.

As for any kit above, the kit may further comprise a storage medium for a computer program or a login and password for an online service made with a computer program for the analysis and elaboration of the results as explained above.

All the components of the kit as herein described for each detection method, may apply to the kit embodiments described below when further SPs are detected.

Hence the kit of the invention may comprise the tools for the detection and identification of: one or more serotype Group 1 serotypes and, one or more of either, Group 2, Listeria serotypes listed above, Campylobacter species listed above; or all of them; or any combination of one or more of Group 1 with one or more of group 2; or any combination of one or more of Group one with one or more of group 2 and one or more of Listeria serotypes listed above; or any combination of one or more of Group 1 with one or more of group 2 and one or more of Campylobacter species listed above; or any combination of one or more of Group 1 with one or more of Listeria species and/or serotype group listed above and one or more of Campylobacter species listed above; or any combination of one or more of Group 1 with one or more of Listeria species and/or serotype group listed above; or any combination of one or more of Group 1 with one or more of Campylobacter species listed above.

Each of these embodiments for the kit of the invention may be produced for each of the detection systems as described above. The kit may further comprise one or more aliquot of primer pairs for DNA pre-amplifϊcation as disclosed in Table 1 and thoroughly explained above.

The method for the identification of these strains is divided into two steps: first DNA of the bacteria, optionally cultivated according to specific growth conditions (e.g LB media at 37°C for Salmonella, Bolton broth at 30⁰C in microaerophilic conditions for Campylobacter, etc.) is extracted. Secondly the extracted DNA sequences (optionally pre-amplified by PCR with one or more primer pairs of Table 1 or other suitable pairs amplifying the same genes or regions), are used as templates for the identification reaction. The identification of E. coli and some Salmonella serotype indicated below

(group 3) requires the preventive isolation of single strains. This can be easily done by plating dilutions of bacteria suspensions and by picking isolated colonies.

The tool is fully customizable with respect to the number and nature of strains the user intend to detect and characterise. Hence it is herein disclosed also a method, according to any of the embodiments of the method of the invention so far described, wherein the detection and identification of the presence or absence of all the remaining SPs or of further SPs selected from one or more of the SPs subgroups:

SEQ ID NOs 137, 138, 150, 170, 194, 195, 196, 199, 214, 215, 216, 217, 220, 222, 232 and 233, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 95, 150, 194, 195, 196, 197, 214, 215, 216, 217, 219 and 220, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 126, 127, 150, 165, 166, 181, 194, 195, 196, 203, 210, 214, 215, 216, 217 and 218, wherein the discriminating nucleotide is as indicated in Table 2; SEQ ID NOs 150, 162, 194, 195, 196 and 199, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 149, 150, 153, 194, 195, 196, 199 and 215, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 126, 127, 139, 146, 150, 165, 166, 187, 188, 194, 195, 196, 198, 209, 214, 215, 216 and 217, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 95, 96, 150, 194, 195, 196, 197, 214, 215, 216, 217 and 219, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 150, 194, 195, 196, 197, 214, 215, 216, 217, 222 and 235, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 150, 178, 189, 194, 195, 196, 198, 209, 210, 214, 215, 216, 217 and 222, wherein the discriminating nucleotide is as indicated in Table 2; SEQ ID NOs 111, 137, 138, 150, 194, 195, 196, 199, 214, 215, 216, 217, 220, 222, 232 and 233, wherein the discriminating nucleotide is as indicated in Table 2;

SEQ ID NOs 150, 154, 167, 168, 194, 195, 196, 197, 210, 214, 215, 216, 217 and 222, wherein the discriminating nucleotide is as indicated in Table 2; SEQ ID NOs 150, 194,195, 196, 198, 211, 214, 215, 216 and 217 wherein the discriminating nucleotide is as indicated in Table 2; is carried out on the heterogeneous sample and wherein, when one or more of said further SPs subgroups are detected, further step c. is carried out in which said heterogeneous bacterial pool is plated and DNA extracted from single colonies is analysed for the presence or absence of said detected further polymorphisms thus identifying one or more Salmonella serotypes selected in the group consisting of: Salmonella brandenburg (the identification of it requiring all of SEQ ID NOs 137, 138, 150, 170, 194, 195, 196, 199, 214, 215, 216, 217, 220, 222, 232 and 233) , Salmonella dublin (the identification of it requiring all of 95, 150, 194, 195, 196, 197, 214, 215, 216, 217, 219 and 220), Salmonella haifa (the identification of it requiring all of 126, 127, 150, 165, 166, 181, 194, 195, 196, 203, 210, 214, 215, 216, 217 and 218), Salmonella kentucky (the identification of it requiring all of 150, 162,

194, 195, 196 and 199), Salmonella montevideo (the identification of it requiring all of 149, 150, 153, 194, 195, 196, 199 and 215), Salmonella muenchen (the identification of it requiring all of 126, 127, 139, 146, 150, 165, 166, 187, 188, 194,

195, 196, 198, 209, 214, 215, 216 and 217), Salmonella naestved (the identification of it requiring all of 95, 96, 150, 194, 195, 196, 197, 214, 215, 216, 217 and 219), Salmonella newport (the identification of it requiring all of 150, 194, 195, 196, 197, 214, 215, 216, 217, 222 and 235), Salmonella paratyphi B (the identification of it requiring all of 150, 178, 189, 194, 195, 196, 198, 209, 210, 214, 215, 216, 217 and 222), Salmonella schwarzengrund (the identification of it requiring all of 111, 137, 138, 150, 194, 195, 196, 199, 214, 215, 216, 217, 220, 222, 232 and 233), Salmonella sendai (the identification of it requiring all 150, 154, 167, 168, 194, 195,

196, 197, 210, 214, 215, 216, 217 and 222), Salmonella senftenberg (the identification of it requiring all 150, 194,195, 196, 198, 211, 214, 215, 216 and 217),

(herein also defined as group 3 when referring to Salmonella serotypes) wherein the presence of one or more SPs subgroup indicates the presence of said serotype in the sample, the absence of serotype specific SNPs of one or more group indicates the absence of the corresponding Salmonella serotype. Hence, each subgroup of SPs as indicated above is serotype specific when the analysis is carried out on single colonies.

For a completely positive result, in this embodiment, the absence of other SPs that resulted positive in the first part of the method when the analysis was carried out on the heterogeneous bacterial pool. Moreover, Table 2 indicates also certain SPs that are present in part of the population.

All the modes of carrying out the method as herein described may comprise a step c. in which said heterogeneous bacterial pool is plated and DNA extracted from single colonies is analysed for the detection and identification of the presence or absence of Escherichia coli serotypes or strains selected from the group: Escherichia coli 0145, Escherichia coli 0157, Escherichia coli 0111, Escherichia coli 026,

Escherichia coli strains with gene for eaeA-gammal , Escherichia coli strains with gene for eaeA - gamma2/teta, Escherichia coli O103/ strains with gene for eaeA- epsilon, Escherichia coli VTEC, by analysing the single colony bacterial DNA obtained after lysing the bacteria for the presence or absence of one or more species or strain specific SPs for each species or strain:

- the species specific SPs for Escherichia coli 0145 are represented by one or more of T in position 34 of SEQ ID No. 59, T in position 41 of SEQ ID No 60;

- the species specific SPs for Escherichia coli 0157 are represented by one or more of T in position 37 of SEQ ID No. 66, C in position 44 of SEQ ID No 86, T in position 55 of SEQ ID No. 87;

- the species specific SPs for Escherichia coli Olll are represented by one or more of T in position 55 of SEQ ID No. 63, T in position 56 of SEQ ID No 84, T in position 57 of SEQ ID No. 85;

- the species specific SPs for Escherichia coli 026 are represented by one or more of C in position 53 of SEQ ID No. 68, A in position 39 of SEQ ID No. 69;

- the strain specific SPs for Escherichia coli strains with gene for eaeA- gammal are represented by one or more of A in position 43 of SEQ ID No. 70, A in position 51 of SEQ ID No 71;

- the strain specific SPs for Escherichia coli strains with gene for eaeA - gamma2/teta are represented by one or more of G in position 53 of SEQ ID No. 72, C in position 34 of SEQ ID No 73; - the strain specific SPs for Escherichia coli O103/ strains with gene for eaeA-epsilon are represented by one or more of A in position 45 of SEQ ID No. 74, C in position 40 of SEQ ID No 75, T in position 51 of SEQ ID No 80;

- the species specific SPs for Escherichia coli VTEC are represented by one or more of G in position 33 of SEQ ID No. 92, A in position 37 of SEQ ID No 93; wherein the presence of one or more species or strain specific SP of each group indicates the presence of said serotype or strain in the sample, the absence of species or strain specific SNPs of one or more group indicates the absence of the corresponding E. coli.

When one of the two methods comprising the further single colony separation are carried out, any of the detection systems described above can be applied. So no further description of detection systems will be necessary in order to enable the skilled person to carry out the method of the invention comprising the further step c. of separating the bacterial colonies.

The kit of the invention may hence further comprise (in any possible form as described above for any detection method indicated above) suitable primers, oligos, arrays and means for detecting the Salmonella serotypes of group 3 and or the E. coli species or strains indicated above.

The following examples indicate the best mode to carry out the invention when most of the serotypes and species are analysed in the sample, the examples, however, do not intend to imitate the invention to the sole modes described as the difference of modes lies mainly in the detection technique selected that, once the SPs of the invention and their relative DN are disclosed, is to the reach of the ordinarily skilled in the art.

EXAMPLES

DNA was extracted from the heterogeneous bacterial pool with a commercial kit (e.g. Gentra, QIAGEN, etc,). The LDR-UA primers were designed as described in the specification and the

CP and DP were designed as described.

The SPs detection was performed on the DNs thus extracted as well as on DNA wherein the target sequences were amplified by PCR with specific primers (reported in the attached Table 1). PCR conditions were corrected to facilitate the product yield rather than the specificity of the reaction.

When PCR was performed, PCR products were pooled in one vial and purified by NaAC/EtOH precipitation or a suitable PCR purification kit (e.g. Sigma or Qiagen).

10 uL of purified PCR product were added to 10 uL of LDR mix (I U Pfu Ligase (Stratagene), 2 uL 1Ox buffer, DP-CP probe mix at a final concentration of 5OmM each).

Reaction mix was denatured at 94°C for 5', followed by 30 cycles of 30" at 94°C and 4' at 65°C.

The reaction was added to the hybridisation solution (10 M NaCl, 0,11 Sodium Citrate, 7 μg Salmon Sperm), incubated 3' at 95°C and immediately hybridised on the UA.

Hybridisation is performed for 1-2 hours. Afterward the UA slide is washed with pre-warmed 2x SSC 0,3M NaCl, 0,03M Sodium Citrate) and 0,1% SDS for 5'. Subsequent rinsing steps are in room temperature 2x SSC, Ix SSC and 05x SSC, 5' each.

The UA slide is then dried and scanned with a laser Scanner (Perkin Elmer). Fluorescence intensities are converted in values by QuantArray software.

Signals are considered positive if they exceed 2 times benchmark values, both in terms of absolute fluorescence and signal/noise values.

Protocol without pre-amplifϊcation step:

CP and DP are typically included in one probe that goes under the name of padlock probe. Specifically the padlock probe is a 100 bp circa oligo with the "DP" at the 3' extremity and the "CP" at the 5' end. Between these two there are two annealing sites of universal primers that span the zip code sequence.

The padlock probe is circularised upon target recognition and ligation. DNA pool including ligated and non ligated probes is treated with a DNA exonuclease to degrade unligated DNA (exonucleotide treatment is not always necessary).

Circularised padlock probes are amplified with the universal primers, with one of the two primers marked with a fluorophore.

Amplification product is hybridised to the UA. The rest of the protocol follows the procedure as described for the "normal" LDR-UA.

Claims

1. Method for the simultaneous identification of the presence or absence of one or more of: Salmonella decatur, Salmonella emek, Salmonella miami,

Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien in a sample comprising an heterogeneous bacterial pool, comprising the step of a. lysing the bacteria in the pool without previous separation of single colonies, b. identifying one or more of said bacteria by analysing the bacterial DNA for the presence or absence of one or more serotype specific SPs for each serotype:

- the serotype specific SPs for Salmonella decatur are represented by one or more of T in position 33 of SEQ ID No. 201, T in position 33 of SEQ ID No 202, T in position 35 of SEQ ID No 114;

- the serotype specific SPs for Salmonella miami are represented by one or more of T in position 30 of SEQ ID No. 116, G in position 38 of SEQ ID No 117, G in position 29 of SEQ ID No 118, T in position 33 of SEQ ID No 119, A in position 33 of SEQ ID No 204, G in position 39 of SEQ ID No 223; - the serotype specific SP for Salmonella rubislaw is represented by T in position 32 of SEQ ID No. 224;

- the serotype specific SPs for Salmonella setubal are represented by one or more of T in position 27 of SEQ ID No. 155, T in position 26 of SEQ ID No 156, T in position 34 of SEQ ID No 205, T in position 37 of SEQ ID No 225, A in position 41 of SEQ ID No 226, T in position 31 of SEQ ID No 227, A in position 44 of SEQ ID No 228;

- the serotype specific SPs for Salmonella Stanleyville are represented by one or more of A in position 33 of SEQ ID No. 123, C in position 33 of SEQ ID No 169, A in position 31 of SEQ ID No 229; - the serotype specific SPs for Salmonella tilene are represented by one or more of A in position 35 of SEQ ID No. 124, T in position 35 of SEQ ID No 157, T in position 34 of SEQ ID No 158, A in position 36 of SEQ ID No 206, A in position 35 of SEQ ID No 230;

- the serotype specific SPs for Salmonella wien are represented by one or more of A in position 33 of SEQ ID No. 159, T in position 36 of SEQ ID No 231; wherein the presence of one or more serotype specific SP of each group indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Salmonella serotype.

2. Method according to claim 1 wherein said simultaneous identification is carried out for at least one of each possible combination of two, three, four, five, six, seven, eight, nine of said Salmonella serotypes.

3. The method, according to any one of claims 1 or 2, wherein one or more of

Salmonella agona, Salmonella anatum, Salmonella derby, Salmonella duisburg; Salmonella enteritidis, Salmonella gallinarum, Salmonella hadar, Salmonella heidelberg, Salmonella Indiana, Salmonella infantis, Salmonella livingstone;

Salmonella mbandaka, Salmonella panama, Salmonella paratyphi A, Salmonella pullorum, Salmonella reading, Salmonella Stanley, Salmonella thompson, Salmonella typhi, Salmonella typhimurium in said sample are further identified by analysing the bacterial DNA obtained after lysing the bacteria in the pool without previous separation of single colonies, also for the presence or absence of one or more serotype specific SPs for each serotype:

- the serotype specific SP for Salmonella agona is represented by C in position 27 of SEQ ID No. 186; - the serotype specific SP for Salmonella anatum is represented by A in position 30 of SEQ ID No. 113;

- the serotype specific SP for Salmonella duisburg is represented by A in position 38 of SEQ ID No. 141;

- the serotype specific SPs for Salmonella enteritidis are represented by one or more of T in position 35 of SEQ ID No. 104, C in position 28 of SEQ ID No 105, T in position 31 of SEQ ID No 147;

- the serotype specific SP for Salmonella mbandaka is represented by G in position 24 of SEQ ID No. 212;

- the serotype specific SP for Salmonella panama is represented by A in position 39 of SEQ ID No. 172; - the serotype specific SPs for Salmonella paratyphi A are represented by one or more of T in position 46 of SEQ ID No. 97, G in position 30 of SEQ ID No 98, A in position 35 of SEQ ID No 112, T in position 38 of SEQ ID No 120, A in position 38 of SEQ ID No 200;

- the serotype specific SP for Salmonella reading is represented by T in position 35 of SEQ ID No. 129;

- the serotype specific SP for Salmonella Stanley is represented by A in position 37 of SEQ ID No. 130; - the serotype specific SP for Salmonella thompson is represented by T in position 35 of SEQ ID No. 144;

- the serotype specific SPs for Salmonella typhi are represented by one or more of A in position 35 of SEQ ID No. 94, G in position 29 of SEQ ID No 99, C in position 23 of SEQ ID No 100, T in position 40 of SEQ ID No 192; - the serotype specific SP for Salmonella typhimurium is represented by A in position 32 of SEQ ID No. 161; wherein the presence of one or more serotype specific SP of each group indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Salmonella serotype.

4. The method according to anyone of claim 1 to 3 wherein one or more of Listeria seeligeri, Listeria ivanovii, Listeria monocytogens I, Listeria monocytogens II, Listeria monocytogens III, Listeria grayii in said sample are further identified by analysing the bacterial DNA obtained after lysing the bacteria in the pool without previous separation of single colonies, also for the presence or absence of one or more serotype group or species specific SPs for each serotype group or species:

- the species specific SPs for Listeria seeligeri are represented by one or more of G in position 43 of SEQ ID No. 258, C in position 45 of SEQ ID No. 268;

- the species specific SPs for Listeria ivanovii are represented by one or more of C in position 44 of SEQ ID No. 259, C in position 40 of SEQ ID No. 267;

- the serotype group specific SPs for Listeria monocytogens I are represented by one or more of C in position 52 of SEQ ID No. 262, A in position 43 of SEQ ID

No. 271;

- the serotype group specific SPs for Listeria monocytogens II are represented by one or more of C in position 40 of SEQ ID No. 263, T in position 51 of SEQ ID No. 272; - the serotype group specific SPs for Listeria monocytogens III are represented by one or more of C in position 39 of SEQ ID No. 264, A in position 47 of SEQ ID No. 273;

- the species specific SP for Listeria grayii is represented by T in position 35 of SEQ ID No. 266; wherein the presence of one or more serotype specific SP of each group indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Listeria serotype group or species.

5. The method according to anyone of claim 1 to 4 wherein one or more of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis in said sample are further identified by analysing the bacterial DNA obtained after lysing the bacteria in the pool without previous separation of single colonies, also for the presence or absence of one or more species specific SNPs for each species: - the specie specific SPs for Campylobacter jejuni are represented by one or more of T in position 50 of SEQ ID No. 15, A in position 60 of SEQ ID No 16, A in position 51 of SEQ ID No 7, A in position 36 of SEQ ID No 8;

6. The method of any one of claims 1 to 5 wherein said bacterial DNA is analysed also for one or more SPs common to all the Salmonella serotypes or to all the Listeria serotype groups or species or to all the Campylobacter species to be identified.

7. The method according to any one of claims 1 to 6 wherein the presence or absence of said one or more polymorphisms is detected by means of: PCR, TaqMan, LDR-UA, PCR-LDR-UA, SNaPshot, Oligo microarray, SNParray, DNA sequencing, or LDR.

8. The method of any one of claims from 1 to 7 wherein step a. further comprises a DNA extraction after bacterial lysis.

9. The method of any one of claim 7, wherein said bacterial DNA is amplified by PCR with primers specific for a region comprising the SPs under analysis.

10. The method of any one of claims 1-9 wherein all the SPs for Salmonella, listed in Table 2 are analysed.

11. The method of any one of claims 1-10, further comprising a step c. wherein said heterogeneous bacterial pool is plated and the DNA of single colonies is analysed for the presence or absence of one or more Salmonella serotypes selected in the group consisting of:

Salmonella brandenburg identified by the presence of all the SPs and DB of SEQ ID NOs 137, 138, 150, 170, 194, 195, 196, 199, 214, 215, 216, 217, 220, 222, 232 and 233 as in table 2; Salmonella dublin identified by the presence of all the SPs and DB of SEQ ID

NOs 95, 150, 194, 195, 196, 197, 214, 215, 216, 217, 219 and 220 as in table 2; Salmonella haifa identified by the presence of all the SPs and DB of SEQ ID NOs 126, 127, 150, 165, 166, 181, 194, 195, 196, 203, 210, 214, 215, 216, 217 and 218 as in table 2; Salmonella kentucky identified by the presence of all the SPs and DB of SEQ

ID NOs 150, 162, 194, 195, 196 and 199 as in table 2;

Salmonella montevideo identified by the presence of all the SPs and DB of SEQ ID NOs 149, 150, 153, 194, 195, 196, 199 and 215 as in table 2;

Salmonella muenchen identified by the presence of all the SPs and DB of SEQ ID NOs 126, 127, 139, 146, 150, 165, 166, 187, 188, 194, 195, 196, 198, 209, 214, 215, 216 and 217 as in table 2;

Salmonella naestved identified by the presence of all the SPs and DB of SEQ ID NOs 95, 96, 150, 194, 195, 196, 197, 214, 215, 216, 217 and 219 as in table 2;

Salmonella newport identified by the presence of all the SPs and DB of SEQ ID NOs 150, 194, 195, 196, 197, 214, 215, 216, 217, 222 and 235 as in table 2;

Salmonella paratyphi B identified by the presence of all the SPs and DB of SEQ ID NOs 178, 189, 194, 195, 196, 198, 209, 210, 214, 215, 216, 217 and 222 as in table 2;

Salmonella schwarzengrund identified by the presence of all the SPs and DB of SEQ ID NOs 111, 137, 138, 150, 194, 195, 196, 199, 214, 215, 216, 217, 220, 222, 232 and 233 as in table 2; Salmonella sendai identified by the presence of all the SPs and DB of SEQ

ID NOs 150, 154, 167, 168, 194, 195, 196, 197, 210, 214, 215, 216, 217 and 222 as in table 2;

Salmonella senftenberg identified by the presence of all the SPs and DB of SEQ ID NOs, 194,195, 196, 198, 211, 214, 215, 216 and 217 as in table 2; wherein the presence of one or more SPs subgroup indicates the presence of said serotype in the sample, the absence of serotype specific SPs of one or more group indicates the absence of the corresponding Salmonella serotype.

12. The method of any one of claims 1-11 , comprising a step c. in which said heterogeneous bacterial pool is plated and DNA from single colonies is analysed for the detection and identification of the presence or absence of Escherichia coli serotypes or strains selected from the group: Escherichia coli 0145, Escherichia coli

0157, Escherichia coli Olll, Escherichia coli 026, Escherichia coli strains with gene for eaeA-gammal , Escherichia coli strains with gene for eaeA - gamma2/teta,

Escherichia coli Ol 03/ strains with gene for eaeA-epsilon, Escherichia coli VTEC, by analysing the single colony bacterial DNA obtained after lysing the bacteria for the presence or absence of one or more species or strain specific SPs for each species or strain:

- the species specific SPs for Escherichia coli 0145 are represented by one or more of T in position 34 of SEQ ID No. 59, T in position 41 of SEQ ID No 60; - the species specific SPs for Escherichia coli 0157 are represented by one or more of T in position 37 of SEQ ID No. 66, C in position 44 of SEQ ID No 86, T in position 55 of SEQ ID No. 87;

- the species specific SPs for Escherichia coli 026 are represented by one or more of C in position 53 of SEQ ID No. 68, A in position 39 of SEQ ID No. 69; - the strain specific SPs for Escherichia coli strains with gene for eaeA- gammal are represented by one or more of A in position 43 of SEQ ID No. 70, A in position 51 of SEQ ID No 71;

- the strain specific SPs for Escherichia coli strains with gene for eaeA - gamma2/teta are represented by one or more of G in position 53 of SEQ ID No. 72,

C in position 34 of SEQ ID No 73;

- the strain specific SPs for Escherichia coli Ol 03/ strains with gene for eaeA-epsilon are represented by one or more of A in position 45 of SEQ ID No. 74, C in position 40 of SEQ ID No 75, T in position 51 of SEQ ID No 80; - the species specific SPs for Escherichia coli VTEC are represented by one or more of G in position 33 of SEQ ID No. 92, A in position 37 of SEQ ID No 93; wherein the presence of one or more species or strain specific SP of each group indicates the presence of said serotype or strain in the sample, the absence of species or strain specific SNPs of one or more group indicates the absence of the corresponding Escherichia coli.

13. A kit for the simultaneous identification of the presence or absence of one or more of: Salmonella decatur, Salmonella emek, Salmonella miami, Salmonella rubislaw, Salmonella setubal, Salmonella Stanleyville, Salmonella tilene, Salmonella typhisuis and/or Salmonella wien in a sample comprising an heterogeneous bacterial pool, comprising reagents and tools for the detection of one or more of the serotype specific SPs listed in claim 1.

14. The kit of claim 13 further comprising reagents and tools for the detection of one or more of the serotype specific SPs listed in claim 3 for the detection of the presence or absence of one or more of Salmonella agona, Salmonella anatum, Salmonella derby, Salmonella duisburg; Salmonella enteritidis, Salmonella gallinarum, Salmonella hadar, Salmonella heidelberg, Salmonella Indiana, Salmonella infantis, Salmonella livingstone; Salmonella mbandaka, Salmonella panama, Salmonella paratyphi A, Salmonella pullorum, Salmonella reading, Salmonella Stanley, Salmonella thompson, Salmonella typhi, Salmonella typhimurium.

15. The kit of claim 13 or 14 further comprising reagents and tools for the detection of one or more of the species or serotype group specific SP listed in claim 4 for the detection of the presence or absence of one or more of Listeria seeligeri, Listeria ivanovii, Listeria monocytogens I, Listeria monocytogens II, Listeria monocytogens III, Listeria grayii.

16. The kit of any one of claims 13 to 15 further comprising reagents and tools for the detection of one or more of the species specific SP listed in claim 5 for the detection of the presence or absence of one or more of Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis.

17. The kit of any one of claims 13-16, further comprising reagents and tools for the presence or absence of one or more of Salmonella brandenburg, Salmonella dublin, Salmonella haifa, Salmonella kentucky, Salmonella montevideo, Salmonella muenchen, Salmonella naestved, Salmonella newport, Salmonella paratyphi B, Salmonella schwarzengrund, Salmonella sendai, Salmonella senftenberg.

18. The kit of any one of claims 13-17, further comprising reagents and tools for the presence or absence of one or more of Escherichia coli 0145, Escherichia coli 0157, Escherichia coli 0111, Escherichia coli 026, Escherichia coli strains with gene for eaeA-gammal , Escherichia coli strains with gene for eaeA - gamma2/teta, Escherichia coli O103 strains with gene for eaeA-epsilon, Escherichia coli VTEC strains.

19. A kit according to any one of claims 10 to 18 wherein said tools are one or more array carrying one or more immobilised common probe for one or more of the SEQ IDs listed in any one of claims 1 to 5, wherein said common probe may comprise all or a part of the downstream adjacent 5' to 3' nucleotides following the SP for each of said SEQ IDs provided that the portion immediately following the 3' of the SP up to the 3' nucleotide adjacent to said SP is part of said common probe, and said reagents are one or more aliquots of labelled discriminating probes corresponding to the common probes immobilised on said array, wherein said discriminating probes consists, at the 5', of the SP nucleotide specific for a given serotype as listed in any one of claims 1 to 5 and 10 or more upstream 5' to 3' nucleotides adjacent to said SP nucleotide.