WO2003076641A2 - Method of detecting and quickly identifying pathogenic bacteria that can be transmitted through food using the real-time polymerase chain reaction - Google Patents

Abstract

The invention relates to a method of detecting and quickly identifying pathogenic bacteria that can be transmitted through food using the real-time polymerase chain reaction. The inventive method consists in: (i) preparing an assembly of tubes, each tube containing a specific reaction mixture for each bacterium to be detected; (ii) programming an apparatus for the simultaneous monitoring of numerous nucleic acid amplifications, comprising a thermocycler, a light source and a sensor in order to obtain a set of signals; (iii) interpreting the signals obtained; and (iv) determining simultaneously the presence or absence of one or more of said bacteria in the samples tested, using specific oligonucleotides (marked probes and initiators) for each bacterium. The invention is particularly suitable for use in the agri-food and food distribution industry for the detection of pathogenic bacteria, such as Salmonella spp., Shigella spp. and/or Staphylococcus aureus.

Description

METHOD FOR DETECTION E D3ENT - RAPDDA FICATION OF FOOD-TRANSMISSABLE PATHOGRAPHIC BACTERIA THROUGH REAL-TIME POLEVLERASA CHAIN REACTION

FIELD OF THE INVENTION

The invention relates, in general, to the detection and identification of foodborne pathogenic bacteria, such as Salmonella spp., Shigella spp., Or Staphylococcus aureus, by the polymerase chain reaction in real time.

BACKGROUND OF THE INVENTION

The detection of foodborne pathogenic bacteria, such as Salmonella spp., Shigella spp., Or Staphylococcus aureus (S. aureus), is a very important task in the field of medicine and public hygiene and is very interested in agrifood industry, both the producer and the distributor of food products (raw materials and / or processed products), so various methods for their detection and identification have been described.

The Spanish Food Code establishes a regulation in which, for each food, the microorganisms whose presence must be detected are specified, as well as the techniques recommended for these analyzes. In general, these techniques use conventional methodologies based on the culture of the microorganism, which require pre-enrichment in broth for 24-48 hours, subculture in selective media for another 24-48 hours, and, finally, perform an identification biochemistry and serotyping, which requires another 24-48 hours; that is to say, in total they must pass between 72 and 96 hours before obtaining a result. On the other hand, this methodology based on the culture of the microorganism is not totally reliable since many microorganisms are difficult to grow and, even more, to quantify, and, for the time it takes, it requires long periods of storage of raw materials and of processed products, with their corresponding impact on costs and possible decrease in competitiveness both nationally and internationally. The same goes for distribution companies, especially large stores, which must store large quantities of products, some of them perishable, as well as hospitality companies that, on numerous occasions, are harmed by the delay in control of the food they serve. For these reasons, there is a need to increase sensitivity and at the same time reliability and speed in the detection of microorganisms that are harmful to health.

In some cases it will be necessary only to increase the sensitivity of the method to reliably and quickly detect the presence or absence of the microorganism (case of the pathogenic bacteria Salmonella spp., Shigella spp.), While in other cases it will be necessary, in addition, quantify the microorganisms that may be present in order to establish the concentration limits from which the presence of the microorganism may represent a problem for the health of the consumer.

Compared to traditional microorganism culture methods, molecular techniques, especially polymerase chain reaction (PCR) [Saiki, RK, et al., 1988. Science 239: 487-491] are being revealed as a method easy, sensitive and advantageous in its cost-benefit ratio to detect the presence of microorganisms. These methods are rapidly shifting to conventional methods in the diagnosis of multiple diseases, although in the food area they are developing their first advances with only qualitative value.

Of the molecular methods, there are some marketed to detect Salmonella spp. in food, one by means of DNA hybridization (Gene-Trak Systems, Unipath), which is as sensitive as the culture but takes 50 hours, and others by nucleic acid amplification (BAX, FOMS Dupont and Probelia, Sanofí D iagnostic Pasteur) that require At least 24 hours. None of these methods provide results on the same day as food production nor do they quantify the contamination. The amplification techniques used in them make use of conventional thermal cyclers, whose time for the completion of each cycle is greater than one minute, and also requires a post-amplification detection of the amplified product, which increases the time required to obtain the final result and makes the technique more complex.

There is, therefore, a need for a rapid method, which provides results in less than 24 hours, preferably between 8 and 10 hours, accurate and reliable, which allows qualitative detection and, if desired, quantitative, of contaminating microorganisms possibly present in a test sample, and that allows one or more pathogenic microorganisms to be detected and identified simultaneously.

This objective can be obtained by using a real-time polymerase chain reaction (real-time PCR), which comprises amplifying the copy number of a fragment of the genome of a microorganism and the detection of the product obtained in each step of the reaction, using a rapid thermocycler apparatus with real-time detection that incorporates a connection to a computer in which the necessary software for the control of all processes step by step. This is a recent technique whose main characteristic is that amplification and analysis occur in the same reaction tube and information is obtained during the entire time the technique takes place without the need to use isotopes or electrophoresis gels. which allows to reduce the detection time of nucleic acids from 5 hours to 20 minutes.

SUMMARY OF THE INVENTION

The invention faces, in general, the problem of developing a fast, precise and reliable method, which makes it possible to detect and identify, b, foodborne, nontoxic acterias, such as Salmonella spp., Shigella spp., Or S. aureus, based on the use of molecular techniques. The solution provided by this invention is based on the design and development of a series of reagents (starter oligonucleotides and specific probes) that, under certain reaction conditions, allow amplifying a fragment of the specific genome of Salmonella spp., Shigella spp., Or S. aureus, and detect 1 presence or absence of said bacteria in a test sample by real-time PCR. Briefly, the method provided by this invention to detect and identify, individually or simultaneously, Salmonella spp., Shigella spp., Or S. aureus, in a test sample, by real-time PCR, comprises extracting the DNA possibly present in said test sample, the amplification of a defined sequence of that DNA by PCR and the continuous detection of the amplified products. To perform such amplification and detection it is necessary to use a series of specific reagents and oligonucleotides (primers and probes). Therefore, to amplify and detect each microorganism, on the one hand, a pair of primers has been designed to amplify a sequence of target nucleotides present in the bacterial genome that it is desired to reveal, and, on the other hand, a pair of probes that allow to identify with certainty said target sequence, said probes being complementary to a region present in the amplification products and being marked, one of them, with a fluorophore and the other with a dye that captures the energy released by the fluorophore and the emits in turn in another wavelength detectable by the system. The rapid thermal cycler with real-time detection, at each step of the reaction, duplicates the DNA fragment by means of the initiators and a heat-resistant polymerase, and each of the two resulting fragments binds the probes. These probes attached to the DNA fragment will emit energy at a certain wavelength when excited. This process (initiator binding, duplication of the DNA fragment, probe binding, detection of emitted energy) is repeated a certain number of times, typically about 30 times, obtaining a large amount of the amplified product at the end. With all the measurements carried out, a statistical analysis is carried out that determines the type of amplified DNA, for example, by analysis of the Hybridization Temperature (Tm) and, if desired, the concentration thereof.

For the implementation of the method provided by this invention, a fast real-time thermal cycler is required, which, in a particular embodiment, is linked to a fluorescence detection system, which allows real-time monitoring of the process of amplification after each cycle. In addition, the analysis of the final PCR product is performed once the amplification process is finished without the need for any manipulation. Its integration together with a computer allows to design the amplification, detection and analysis programs of the products.

A method such as that provided by this invention has the advantage that it allows rapid detection and identification, typically in less than 24 hours, and usually between 8 and 10 hours, accurately and reliably, simultaneously, Salmonella spp., Shigella spp. ., or S. aureus, in a test sample, as well as the quantification of the microorganisms eventually present in said test sample

An object of this invention is a method for the detection and identification of Salmonella spp., Shigella spp., Or S. aureus, by real-time PCR. The oligonucleotides (primers and labeled probes) used in carrying out said method, as well as the kits containing them are additional objects of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for the rapid and simultaneous detection and identification of a bacterium selected from the group formed by Salmonella spp. , Shigella spp. ,

Staphylococcus aureus and mixtures thereof, in one or more test samples, by real time polymerase chain reaction (PCR), hereinafter method of the invention, which it comprises: a) extracting the DNA present in said sample or test samples; b) preparing a set of tubes containing each tube a specific reaction mixture for each bacterium to be detected and identified, each reaction mixture comprising the reagents necessary for the enzymatic amplification of DNA and identification of the bacterium to be detected and identified; c) programming an apparatus for the simultaneous control of multiple nucleic acid amplifications comprising (i) a thermal cycler that includes an element that contains a plurality of holes in which said tubes containing the reaction mixture for enzymatic amplification of DNA and identification of the bacterium to be detected and identified, (ii) a light source optically coupled to said thermocycler and adapted to distribute light over said plurality of holes, and (iii) a sensor adapted to simultaneously detect the light emitted from said plurality of holes, in order to obtain a set of signals; d) interpret these signals; and e) simultaneously determine the presence or absence of one or more of said bacteria in said sample or samples tested;

characterized because

i) the reaction mixture for the enzymatic amplification of DNA and specific identification of Salmonella spp. It comprises a pair of initiators identified as Invl [SEC. ID. N °: 1] and Inv2 [SEC. ID. N °: 2], a probe identified as InvFlu [SEC. ID. N °: 3] linked to a fluorophore and a probe identified as InvLCR [SEC. ID. N °: 4] linked to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength;

ii) the reaction mixture for the enzymatic amplification of DNA and specific identification of Shigella spp. it comprises a pair of primers identified as IpaHF [SEC. ID. N °: 5] and IpaHR [SEC. ID. N °: 6], a probe identified as IpaHFL [SEC. ID. N °: 7] linked to a fluorophore and a probe identified as IpaHLCR [SEC. ID. N °: 8] linked to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength;

iii) the reaction mixture for the enzymatic amplification of DNA and specific identification of S. aureus comprises a pair of primers identified as NucAF [SEQ. ID. N °: 9] and NucAR [SEC. ID. N °: 10], a probe identified as NucAFL [SEC. ID. N °: 11] linked to a fluorophore and a probe identified as NucALCR [SEC. ID. N °: 12] linked to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength; and

iv) the presence or absence of each of said bacteria in said sample or samples tested is determined by the appearance of a specific fusion curve of each amplified product, said fusion curve being obtained by hybridization of the specific probes for the identification of each bacteria with the amplification product obtained in each tube, and, if desired, determine the melting temperature characteristic of each amplified product.

The method provided by this invention allows to detect, identify and, if desired, quantify, foodborne pathogenic bacteria, selected from the group formed by Salmonella spp., Shigella spp., S. aureus and mixtures thereof, in one or more samples of assay, by real time PCR. The test sample may be any sample containing DNA in which it is desired to know the possible contamination by said bacteria. In a particular embodiment, said test sample is a sample of a food product, for example, a sample of a raw material used in food production or a sample of a processed food product.

Before contacting the DNA of the bacteria possibly present in said sample or test samples to be analyzed with the reaction mixture for the enzymatic amplification of DNA, all or part of the DNA present in the test samples is extracted. DNA extraction can be performed by conventional methods, including those that do not require purification of the extracted DNA. In one embodiment In particular, DNA extraction was performed using Chelex 100 resin (see the Examples accompanying this description).

For the implementation of the method of the invention a set of tubes is prepared, in which each contains a specific reaction mixture for each bacterium to be detected and identified, each reaction mixture comprising the reagents necessary for enzymatic DNA amplification. and identification of the bacteria to be detected and identified. In general, said reaction mixture contains the reagents necessary for carrying out the method of the invention, for example, water quality PCR, dNTPs (dATP, dCTP, dGTP and dTTP), a suitable ampule for the enzymatic amplification reaction, a Thermostable DNA polymerase, a magnesium salt, etc., together with a pair of primers and a pair of specific labeled probes for the bacteria to be tested.

The primers used in the method of the invention allow to amplify a sequence of target nucleotides or specific fragment of the bacterial genome that it is desired to reveal. In general, any fragment of the bacterial genome that allows the identification of a battery, at the level of genus or species, can be amplified. The pair of labeled probes allows to identify with certainty said specific fragment of the bacterial genome to be detected and comprise a nucleotide sequence complementary to a region present in the amplification products and a marker. In a particular embodiment, one of said probes is marked with a fluorophore that acts as an energy donor and the other probe with a dye that captures the energy released by the fluorophore and emits it in turn at another wavelength detectable by the system. present in the apparatus for the implementation of the method of the invention.

In a particular embodiment, the reaction mixture for the enzymatic amplification of DNA and specific identification of Salmonella spp. It comprises a pair of initiators identified as Invl [SEC. ID. N °: 1] and Inv2 [SEC. ID. N °: 2], a probe identified as InvFlu [SEC. ID. N °: 3] linked to a fluorophore and a probe identified as hivLCR [SEC. ID. N °: 4] linked to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength. Said pair of Invl initiators [SEC. ID. N °: 1] and Inv2 [SEC. ID. N °: 2] allows the amplification of a 457 base pair (bp) fragment of the invA / Ε gene of Salmonella spp.

In another particular embodiment, the reaction mixture for the enzymatic amplification of DNA and specific identification of Shigella spp. comprises a pair of identified initiators as IpaHF [SEC. ID. N °: 5] and IpaHR [SEC. ID. N °: 6], a probe identified as IpaHFL [SEC. ID. N °: 7] linked to a fluorophore and a probe identified as IpaHLCR [SEC. ID. N °: 8] attached to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength. Said pair of IpaHF initiators [SEC. ID. N °: 5] and IpaHR [SEC. ID. N °: 6] allows the amplification of a 603 bp fragment of the ipaH gene of Shigella spp.

In another particular embodiment, the reaction mixture for the enzymatic amplification of DNA and specific identification of S. aureus comprises a pair of primers identified as NucAF [SEQ. ID. N °: 9] and NucAR [SEC. ID. N °: 10], a probe identified as NucAFL [SEC. ID. N °: 11] linked to a fluorophore and a probe identified as NucALCR [SEC. ID. N °: 12] attached to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength. Said pair of NucAF initiators [SEC. ID. N °: 9] and NucAR [SEC. ID. N °: 10] allows the amplification of a 267 bp fragment of the nucA gene of S. aureus. As a fluorophore, any group or fluorescent substance capable of donating energy after being activated can be used, and as a colorant any compound capable of capturing the energy emitted by the activation of the fluorophore can be used and emitting it at a different wavelength. In a particular embodiment, said fluorophore is fluorescein and said dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength is the Red640 dye.

Additional examples of fluorophores and dyes that can be used for the implementation of the method of the invention are shown in Table 1.

Table 1 Fluorophores and Dyes

The method of the invention is performed in an apparatus for simultaneous control of multiple nucleic acid amplifications comprising (i) a thermocycler that includes an element that contains a plurality of holes in which said tubes containing the reaction mixture for the enzymatic amplification of DNA and identification of the bacterium to be detected and identified are introduced , (ii) a light source optically coupled to said thermocycler and adapted to distribute light over said plurality of holes, such as a laser radiation source; and (iii) a sensor adapted to simultaneously detect the light emitted from said plurality of holes, in order to obtain a set of signals, such as a fluorescence detector. Such devices are known [see, for example, European patent application EP 640 828 A]. The rapid thermal cycler with real-time detection, at each step of the reaction duplicates a DNA fragment (target sequence present in the bacterial genome to be detected) by using the corresponding inidade-.es and a polymerase heat resistant and, to each of the two resulting fragments, the labeled probes are attached. These probes attached to the DNA fragment will emit energy at a certain wavelength when excited. This process (union of the initiators, duplication of the DNA fragment, union of the probes, detection of emitted energy) is repeated a certain number of times, typically about 30 times, obtaining in the end a large quantity of the amplified product. With all the measurements carried out, a statistical analysis is performed that determines the type of amplified DNA, for example, by means of Hybridization Temperature (Tm) analysis and, if desired, the concentration thereof is determined.

In a particular embodiment, the apparatus used for the implementation of the method of the invention comprises a real-time rapid thermal cycler attached to a fluorescence detection system, which allows real-time monitoring of the amplification process after each cycle, such as the LightCycler thermal cycler (Roche Biochemiclas) [see the Example that accompanies this description]. In this case, the analysis of the final PCR product is performed once the amplification process is finished without the need for any manipulation. Its integration together with a computer allows to design the amplification, detection and analysis programs of the products.

After interpreting the obtained signals, the presence or absence of Salmonella spp., Shigella spp. Can be determined simultaneously. and / or S. aureus. In a particular embodiment, the presence or absence of each of said bacteria in said sample or samples tested is determined by the appearance of a specific fusion curve of each amplified product, said melting curve being obtained by hybridization of the specific probes for the identification of each bacterium with the amplification product obtained in each tube. Additionally, if desired, the characteristic melting temperature of each amplified product as well as its concentration can be determined. The method of the invention allows to detect, identify and, if desired, quantify, foodborne pathogenic bacteria, such as Salmonella spp. , Shigella spp. or S. aureus, in test samples, by real-time PCR. Therefore, said method can be applied to the microbiological analysis of all foods susceptible to transmit infection to consumers by said bacteria, allowing its detection in a few hours, generally in less than 24 hours, typically between 8 and 10 hours, collaborating with this mode in the prevention of food poisoning caused by said bacteria and shortening the storage time prior to the commercialization of the food product. Additionally, said method could also be used in Clinical Microbiology (human and veterinary) to detect the existence of a possible infection c austed by said bacteria in a subject (person or animal) from a test sample containing DNA from said subject .

In a particular embodiment, the method of the invention is used in the rapid detection (qualitative and quantitative) in foods of the genome of human pathogenic bacteria transmissible by food (Salmonella spp., Shigella spp. And S. aureus). The foods that can be analyzed can be both raw materials, for example, milk, eggs, meats, vegetables, etc., as well as their derivatives, processed or non-processed foods, oils, sauces, etc., both in the producing companies and in the distributors and hospitality.

A method for the detection and identification of pathogenic bacteria such as that provided by this invention has, among others, the following advantages: a) it requires a substantially shorter time than the commonly used methods, typically between 8 and 10 hours compared to the methods conventional that require between 48 and 96 hours; b) once the pre-enrichment and DNA extraction has been carried out, the entire process is carried out in the same tube, avoiding manipulations and external contamination; c) it is a quantitative method, which not only detects and identifies the bacteria that may be present in a test sample but also allows if Desires to determine the concentration of said bacteria present in the test sample; d) its complete automation is possible; and e) allows the detection of several bacteria simultaneously. The invention also provides an oligonucleotide for the detection and identification of foodborne pathogenic bacteria, such as Salmonella spp., Shigella spp. or S. aureus, hereinafter oligonucleotide of the invention, which has a nucleotide sequence selected from the group consisting of the sequences identified as Invl [SEQ. ID. N °: 1], Inv2 [SEC. ID. N °: 2], InvFlu [SEC. ID. N °: 3], InvLCR [SEC. ID. N °: 4], IpaHF [SEC. ID. N °: 5], IpaHR [SEC. ID. N °: 6], IpaHFL [SEC. ID. N °: 7], IpaHLCR [SEC. ID. N °: 8], NucAF [SEC. ID. N °: 9], NucAR [SEC. ID. N °: 10], NucAFL [SEC. ID. N °: 11] and NucALCR [SEC. ID. N °: 12].

Also, the invention provides a labeled probe, hereinafter labeled probe of the invention, comprising (i) an oligonucleotide, selected from the group consisting of the oligonucleotides identified as InvFlu [SEC. ID. N °: 3], InvLCR [SEC. ID. N °: 4], IpaHFL [SEC. ID. N °: 7], IpaHLCR [SEC. ID. N °: 8], NucAFL [SEC. ID. N °: 11] and NucALCR [SEC. ID. N °: 12], and (ii) a marker, such as a fluorophore, for example, fluorescein, or a dye capable of capturing the energy emitted by the activation of the fluorophore and emitting it at a different wavelength, for example, the Red640 dye. In a particular embodiment, said labeled probe of the invention is selected from the group consisting of: the oligonucleotide identified as InvFlu [SEC. ID. N °: 3] attached to fluorescein, the oligonucleotide identified as InvLCR [SEC. DD. N °: 4] attached to the Red640 dye, the oligonucleotide identified as IpaHFL [SEC. ID. N °: 7] attached to fluorescein, the oligonucleotide identified as IpaHLCR [SEC. ID. N °: 8] attached to the dye

Red640, the oligonucleotide identified as NucAFL [SEC. ID. N °: 11] attached to fluorescein, the oligonucleotide identified as NucALCR [SEC. ID. N °: 12] attached to the Red640 dye, and mixtures thereof.

The invention also provides a kit for the rapid and simultaneous detection and identification of a bacterium selected from the group formed by Salmonella spp. , Shigella spp. , Staphylococcus aureus, and mixtures thereof, in one or more test samples, by real-time PCR, comprising at least one oligonucleotide of the invention or a labeled probe of the invention. In a particular embodiment, said kit comprises 1 oligonucleotides identified as Invl [SEQ. ID. N °: 1], Inv2 [SEC. ID. N °: 2], IpaHF [SEC. ID. N °: 5], IpaHR [SEC. ID. N °: 6], NucAF [SEC. ID. N °: 9] and NucAR [SEC. ID. N °: 10], and the labeled probes composed of the oligonucleotide InvFlu [SEC. ID. N °: 3] attached to fluorescein, the oligonucleotide InvLCR [SEC. ED. N °: 4] attached to the Red640 dye, the oligonucleotide IpaHFL [SEC. ID. N °: 7] attached to fluorescein, the oligonucleotide IpaHLCR [SEC. ID. N °: 8] attached to the Red640 dye, the NucAFL oligonucleotide [SEC. ED. N °: 11] bound to fluorescein and the oligonucleotide NucALCR [SEC. ID. N °: 12] attached to the Red640 dye.

In general, the kits of the invention contain the pair of initiators corresponding to each bacterium to be detected in an individualized tube, as well as the pair of probes labeled for each bacterium, in order to be able to choose between performing the detection of a single bacterium (using only the tube of its initiators and that of its marked probes), several or all depending on the needs.

The kits provided by this invention may be presented in the form of a kit containing, in addition to containers with one or more of said oligonucleotides and / or labeled probes mentioned above, containers with all or part of the rest of reagents necessary for performing the method in question, for example, ultrapure water, dNTPs (dATP, dCTP, dGTP and dTTP), a buffer suitable for the enzymatic amplification reaction, a thermostable DNA polymerase (for example, Taq DNA polymerase), a magnesium salt (for example, magnesium chloride), etc. Additionally and optionally, the kits provided by this invention may include vessels with Salmonella spp., Shigella spp., Staphylococcus aureus DNA, for use as positive controls. The following example illustrates the invention and should not be considered as limiting its scope.

EXAMPLE 1 Detection and identification of Salmonella spp., Shigella spp., And S. aureus in a food product by real-time PCR

1. Materials and Methods The diagnostic equipment designed for the detection of Salmonella spp. Bacteria, Shigella spp., And S. aureus in food products consists of several tubes each containing the initiators, labeled probes, thermostable DNA polymerase, MgCl ₂ solution and sterile double-distilled water, necessary to carry out the amplification method designed to said team. In this specific case, the equipment used includes all the basic components for the amplification of up to 100 samples and controls. The control DNA provided is composed of a mixture of DNA extracted from laboratory strains of all bacteria to be detected.

The pair of primers corresponding to each bacterium to be detected is presented in an individualized tube, as is the pair of labeled probes for each bacterium.

To carry out the method, a microcentrifuge, a LightCycler thermocycler (Roche Biochemicals), micropipettes and filter micropipette tips have been used.

The method planning is shown in Table 2. Although the handling time has been calculated for a single sample, several samples can be manipulated simultaneously without just increasing the times.

Table 2 Method planning

The method can be performed following the standard protocol described below:

1. Sampling (25 g of the product) and preparation by pre-enrichment in buffered peptone water for 8-10 hours at 37 ° C.

2. DNA extraction by extraction with a reliable method, for example, using Chelex 100 resin. 3. Amplification and detection: A capillary containing the necessary components for the reaction, thermostable DNA polymerase, the four dNTPs, MgCl ₂ is prepared. , Extracted DNA of the sample, primers and probes labeled specific for the bacteria to be detected. If 3 different bacteria are to be detected, it will be necessary to prepare 3 tubes that will be differentiated from each other in the different primers and labeled probes specific to each bacterium placed in each tube. Additionally, a tube is prepared with the control DNA and the primers and probes of the bacteria (s) to be detected.

4. Place the prepared capillaries in the thermocycler in real time and schedule the completion of the amplification and detection process: temperature, time, number of cycles and reading time according to the scheme.

5. Start the program (see Table 3).

Table 3 Program

6. At the end of the program, the results are analyzed starting with the positive control, continuing the analysis if it is correct. Otherwise, the test is discarded.

7. The presence or absence of a bacterium is determined by the appearance of a specific fusion curve between the probes and the amplicon of each bacteria sought. In the capillary corresponding to each bacterium, the specific labeled probes will be joined only with the amplicon derived from the bacteria sought if it is present and the fusion curve that occurs at the end of the protocol will have a specific and unique Tm for that same microorganism. Under the conditions tested, using Invl initiator pairs [SEC. ED. N °: 1] and Inv2 [SEC. ID. N °: 2] for the detection of Salmonella spp .; IpaHF [SEC. ED. N °: 5] and IpaHR [SEC. ED. N °: 6] for the detection of Shigella spp .; and NucAF [SEC. ED. N °: 9] and NucAR [SEC. ED. N °: 10] for the detection of S. aureus; and the pairs of labeled probes composed of the oligonucleotide InvFlu [SEC. ED. N °: 3] bound to fluorescein and the oligonucleotide InvLCR [SEC. ED. N °: 4] attached to the Red640 dye, for the detection of Salmonella spp. ; by the oligonucleotide IpaHFL [SEC. ED. N °: 7] bound to fluorescein and oligonucleotide IpaHLCR [SEC. ED. N °: 8] attached to the Red640 dye, for the detection of Shigella spp .; and by the oligonucleotide NucAFL [SEC. ED. N °: 11] bound to fluorescein and the oligonucleotide NucALCR [SEC. ID. N °: 12] together with the Red640 dye, for the detection of S. aureus, the corresponding Tm are shown in Table 4.

Table 4 Tm of the amplicon and probes

2. Application of the method to the detection of Salmonella spp. in mayonnaise sauce

This test was performed to illustrate the effectiveness of the method of the invention in the analysis of a food to reveal possible contamination. Specifically, the test of a sample of mayonnaise sauce is described in which it is intended to detect a possible contamination with Salmonella spp. 25 g of the mayonnaise sauce sample are collected and homogenized in 225 ml of buffered peptone water (pH 7.2) and incubated at 37 ° C with stirring for 8 hours. After 8 hours of incubation, to extract the DNA possibly present in the test sample, a 300 μl aliquot is extracted with a Chelex 100 resin, for which 300 μl of a 10% suspension in Chelex 100 water is added Stir vigorously, incubate for 45 minutes at 45 ° C with periodic stirring of the tubes to prevent precipitation of the resin, heat at 95 ° C for 5 minutes and centrifuge for 5 minutes at 13,000 xg, removing the supernatant which is used in the next step (Amplification and detection). The extracted DNA as previously indicated will be used directly for amplification and detection, which is done in a closed capillary tube with a total volume of 20 μl. To this end, in each capillary the appropriate components of the amplification mixture are added in the amounts indicated in Table 5.

Table 5 Mixing amplification and detection

(a): Invl [SEC. ED. N °: 1] and Inv2 [SEC. ED. N °: 2]

(b): InvFlu [SEC. ED. N °: 3] and InvLCR [SEC. ED. N °: 4]

Once all components were added, they were mixed by a pulse in a microcentrifuge. Next, the capillaries were placed in the carousel of the thermal cycler

LightCycler (Roche Biochemicals) and underwent the amplification and detection process, which required about 30 minutes. The conditions for PCR are the following:

Denaturation for 10 seconds at 98 ° C Amplification Protocol:

Parameters Values Cycles 35

Type Quantification

Segment 1 Segment 2 Segment 3 Temperature (° C) 95 52 72

Incubation time (s) 0 5 10 Change rate T ^at (° C / s) 20 20 20

Reading mode None Individual None

Reading values Fl = l F2 = 10 F3 = 10

After amplification, a program was applied to calculate the Tm, slowly increasing the temperature (0, 1 ° C / s) and measuring the fluorescence continuously. The conditions were as follows:

Parameters Values

Cycles 1

Melting Type Segment 1 Segment 2 Segment 3

Temperature (° C) 95 50 95

Incubation time (s) 10 10 0 Change rate T ^at (° C / s) 20 20 0.1 Reading mode None None Continuous Reading values Fl = l F2 = 10 F3 = 10

It was finally cooled to 40 ° C. To interpret the results, the system software automatically analyzes the data obtained, offering the results in the form of an integration curve, thus allowing to obtain the Melting Temperature (Tm) characteristic of each amplified product. In this case, for the detection of Salmonella spp. Under the conditions tested, the Tm probes is 68.3 ° C.

3. Application of the method to the detection of Shisella spp. on lettuce leaves This test was performed to illustrate the effectiveness of the method of the invention in the analysis of a food to reveal possible contamination. Specifically, the test of a sample of salad lettuce leaves in which it is intended to detect possible contamination with Shigella spp.

25 g of the sample of lettuce leaves are collected and crumbled in a stomach placed in a sterile bag with 225 ml of buffered peptone water (pH 7.2) and the supernatant is incubated at 37 ° C with stirring for 8 hours . After 8 hours of incubation, to extract the DNA possibly present in the test sample, a 300 μl aliquot is extracted with a Chelex 100 resin, for which 300 μl of a suspension is added 10% in Chelex 100 water, vigorously stirred, incubated for 45 minutes at 45 ° C with periodic agitation of the tubes to prevent precipitation of the resin, heated at 95 ° C for 5 minutes and centrifuged for 5 minutes. minutes at 13,000 xg, removing the supernatant used in the next step (Amplification and detection).

The extracted DNA as previously indicated will be used directly for amplification and detection, which is done in a closed capillary tube with a total volume of 20 μl. To this end, in each capillary the appropriate components of the amplification mixture are added in the amounts indicated in Table 6.

Table 6 Mixing amplification and detection

(a): IpaHF [SEC. D. N °: 5] and IpaHR [SEC. ED. N °: 6] (b): IpaHFL [SEC. ID. N °: 7] and IpaHLCR [SEC. ED. N °: 8]

Once all components were added, they were mixed by a pulse in a microcentrifuge. Next, the capillaries were placed in the carousel of the LightCycler thermal cycler (Roche Biochemicals) and underwent the amplification and detection process, which required approximately 30 minutes. The conditions for PCR are the following:

Denaturation for 10 seconds at 98 ° C Amplification Protocol:

Parameters Values

Cycles 45 Type Quantification

Segment 1 Segment 2 Segment 3 Temperature (° C) 95 50 72

Incubation time (s) 0 5 10 Change rate T ^at (° C / s) 20 20 20

Reading mode None Individual None

Reading values Fl = l F2 = 10 F3 = 10

After amplification, a program was applied to calculate the Tm, slowly increasing the temperature (0, 1 ° C / s) and measuring the fluorescence continuously. The conditions were as follows:

Parameters Values

Cycles 1

Melting Type

Segment 1 Segment 2 Segment 3 Temperature (° C) 95 50 95

Incubation time (s) 10 10 0 Change rate T ^at (° C / s) 20 20 0.1 Reading mode None None Continuous

Reading values Fl = l F2 = 10 F3 = 10 Finally cooled to 40 ° C.

To interpret the results, the system software automatically analyzes the data obtained, offering the results in the form of an integration curve, thus allowing to obtain the Melting Temperature (Tm) characteristic of each amplified product.

In this case, for the detection of Shigella spp. Under the conditions tested, the Tm probes are 69.4 ° C.

4. Application of the method to the detection of Staphylococcus aureus in custard This test was carried out to illustrate the efficacy of the method of the invention in the analysis of a food to reveal possible contamination. Specifically, the test of a sample of custard cream is described in which it is intended to detect a possible contamination with Staphylococcus aureus. 25 g of the pastry cream sample are collected and disintegrated in a stomach placed in a sterile bag with 225 ml of buffered peptone water (pH 7.2) and incubated at 37 ° C with stirring for 8 hours. After 8 hours of incubation, to extract the DNA possibly present in the test sample, a 300 μl aliquot is extracted with a Chelex 100 resin, for which 300 μl of a 10% suspension in Chelex 100 water is added Stir vigorously, incubate for 45 minutes at 45 ° C with periodic stirring of the tubes to prevent precipitation of the resin, heat at 95 ° C for 5 minutes and centrifuge for 5 minutes at 13,000 xg, removing the supernatant which is used in the next step (Amplification and detection).

The extracted DNA as previously indicated will be used directly for amplification and detection, which is done in a closed capillary tube with a total volume of 20 μl. For this, the appropriate components of the amplification mixture are added to each capillary in the amounts indicated in Table 7.

Table 7 Mixing amplification and detection

(a): NucAF [SEC. ID. N °: 9] and NucAR [SEC. ED. N °: 10]

(b): NucAFL [SEC. ED. N °: 11] and NucALCR [SEC. ED. N °: 12]

Once all components were added, they were mixed by a pulse in a microcentrifuge. Next, the capillaries were placed in the carousel of the LightCycler thermocycler (Roche Biochemicals) and were subjected to the amplification and detection process, which requires about 30 minutes. The conditions for PCR are the following:

Denaturation for 10 seconds at 98 ° C Amplification Protocol: Parameters Values

Cycles 45

Type Quantification

Segment 1 Segment 2 Segment 3 Temperature (° C) 95 55 72 Incubation time (s) 0 5 10

Rate of change T (° C / s) 20 20 20 Reading mode None Individual None

Reading values Fl = l F2 = 10 F3 = 10

After amplification, a program was applied to calculate the Tm, slowly increasing the temperature (0, 1 ° C / s) and measuring the fluorescence continuously. The conditions were as follows:

Parameters Values

Cycles 1

Melting Type Segment 1 Segment 2 Segment 3

Temperature (° C) 95 50 95

Incubation time (s) 10 10 0 Change rate T ^at (° C / s) 20 20 0.1 Reading mode None None Continuous Reading values Fl = l F2 = 10 F3 = 10

It was finally cooled to 40 ° C. To interpret the results, the system software automatically analyzes the data obtained, offering the results in the form of an integration curve, thus allowing to obtain the Melting Temperature (Tm) characteristic of each amplified product. In this case, for the detection of S. aureus under the conditions tested, the Tm probes are 64.2 and 61, C. 5. Sensitivity and specificity The sensitivity and specificity values obtained in the LightCycler thermocycler using the pairs of primers selected for each bacterium are shown in Table 8.

Table 8 Sensitivity and specificity of initiator couples

Likewise, the sensitivity and specificity values obtained in the LightCycler thermocycler using the pairs of labeled probes selected for each bacterium are shown in Table 9.

Table 9 Sensitivity and specificity of the pairs of labeled probes

Claims

1. A method for the rapid and simultaneous detection and identification of a bacterium selected from the group formed by Salmonella spp. , Shigella spp. Staphylococcus aureus, and mixtures thereof, in one or more test samples, by means of the polymerase chain reaction (PCR) in real time, comprising: a) extracting the DNA present in said sample or test samples; b) preparing a set of tubes containing each tube a specific reaction mixture for each bacterium to be detected and identified, each reaction mixture comprising the reagents necessary for the enzymatic amplification of DNA and identification of the bacterium to be detected and identified; c) programming an apparatus for the simultaneous control of multiple nucleic acid amplifications comprising (i) a thermal cycler that includes an element that contains a plurality of holes in which said tubes containing the reaction mixture for enzymatic amplification of DNA and identification of the bacterium to be detected and identified, (ii) a light source optically coupled to said thermocycler and adapted to distribute light over said plurality of holes, and (iii) a sensor adapted to simultaneously detect the light emitted from said plurality of holes, in order to obtain a set of signals; d) interpret these signals; and e) simultaneously determine the presence or absence of one or more of said bacteria in said sample or samples tested; characterized because i) the reaction mixture for the enzymatic amplification of DNA and specific identification of Salmonella spp. It comprises a pair of initiators identified as Invl [SEC. ID. N °: 1] and Inv2 [SEC. ID. N °: 2], a probe identified as InvFlu [SEC. ED. N °: 3] linked to a fluorophore and a probe identified as InvLCR [SEC. D. N °: 4] attached to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a wavelength SUBSTITUTE SHEET (RULE 26) different; ii) the reaction mixture for the enzymatic amplification of DNA and specific identification of Shigella spp. it comprises a pair of primers identified as IpaHF [SEC. ID. N °: 5] and IpaHR [SEC. ED. N °: 6], a probe identified as IpaHFL [SEC. ED. N °: 7] linked to a fluorophore and a probe identified as IpaHLCR [SEC. D. N °: 8] linked to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength; iii) the reaction mixture for the enzymatic amplification of DNA and specific identification of S. aureus comprises a pair of primers identified as NucAF [SEQ. ID. N °: 9] and NucAR [SEC. ID. N °: 10], a probe identified as NucAFL [SEC. ID. N °: 11] linked to a fluorophore and a probe identified as NucALCR [SEC. ED. N °: 12] linked to a dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength; and iv) the presence or absence of each of said bacteria in said sample or samples tested is determined by the appearance of a specific fusion curve of each amplified product, said fusion curve being obtained by hybridization of the specific probes for the identification of each bacteria with the amplification product obtained in each tube, and, if desired, determine the melting temperature characteristic of each amplified product. 2. A method according to claim 1, wherein said sample or test samples is a sample of a raw material used in the production of food or a processed food product. 3. The method of claim 1, wherein said fluorophore is fluorescein and said dye that captures the energy emitted by the activation of the fluorophore and emits it at a different wavelength is the Red640 dye. SUBSTITUTE SHEET (ΛΞGLA 26) 4. The method according to claim 1, wherein said light source optically coupled to said thermal cycler and adapted to distribute light over said plurality of holes is a source of laser radiation. 5. A method according to claim 1, wherein said sensor adapted to simultaneously detect the light emitted from said plurality of holes is a fluorescence detector. 6. An oligonucleotide having a nucleotide sequence selected from the group consisting of the sequences identified as Invl [SEC. ID. N °: 1], Inv2 [SEC. ID. N °: 2], InvFlu [SEC. ED. N °: 3], InvLCR [SEC. ID. N °: 4], IpaHF [SEC. OR). N °: 5], IpaHR [SEC. ED. N °: 6], IpaHFL [SEC. ID. N °: 7], IpaHLCR [SEC. ED. N °: 8], NucAF [SEC. ED. N °: 9], NucAR [SEC. ID. N °: 10], NucAFL [SEC. ID. N °: 11] and NucALCR [SEC. ED. N °: 12]. 7. A labeled probe comprising (i) an oligonucleotide, selected from the group consisting of the oligonucleotides identified as InvFlu [SEQ. ID. N °: 3], InvLCR [SEC. ID. N °: 4], IpaHFL [SEC. ED. N °: 7], IpaHLCR [SEC. ED. N °: 8], NucAFL [SEC. D. N °: 11] and NucALCR [SEC. ID. N °: 12], and (ii) a marker. 8. Probe according to claim 7, wherein said marker is a fluorophore or a dye capable of capturing the energy emitted by the activation of a fluorophore. 9. Probe according to claim 7, selected from the group consisting of: the oligonucleotide identified as InvFlu [SEC. EO N °: 3] attached to fluorescein, the oligonucleotide identified as InvLCR [SEC. OR). N °: 4] attached to the dye Red640, the oligonucleotide identified as IpaHFL [SEC. ID. N °: 7] attached to fluorescein, the oligonucleotide identified as IpaHLCR [SEC. ID. N °: 8] attached to the dye Red640, the oligonucleotide identified as NucAFL [SEC. ED. N °: 11] attached to fluorescein, the oligonucleotide identified as NucALCR [SEC. ED. No: 12] attached to the dye Red640, and E SUBSTITUTE SHEET (RULE 26) their mixtures 10. A kit for the rapid and simultaneous detection and identification of a bacterium selected from the group consisting of Salmonella spp., Shigella spp., Staphylococcus aureus, and mixtures thereof, in one or more test samples, by chain reaction of the Real-time polymerase (PCR), comprising at least one oligonucleotide according to claim 6, or at least one labeled probe according to any of claims 7 to 9. 11. Kit according to claim 10, comprising the oligonucleotides identified as Invl [SEQ. ID. N °: 1], Inv2 [SEC. ID. N °: 2], IpaHF [SEC. ID. N °: 5], IpaHR [SEC. ID. N °: 6], NucAF [SEC. D. N °: 9] and NucAR [SEC. ED. N °: 10], and the labeled probes composed of the oligonucleotide InvFlu [SEC. ED. N °: 3] attached to fluorescein, the oligonucleotide InvLCR [SEC. ID. N °: 4] attached to the Red640 dye, the oligonucleotide IpaHFL [SEC. ID. N °: 7] attached to fluorescein, the oligonucleotide IpaHLCR [SEC. D. N °: 8] attached to the Red640 dye, the NucAFL oligonucleotide [SEC. ID. N °: 11] bound to fluorescein and the oligonucleotide NucALCR [SEC. ID. N °: 12] attached to the Red640 dye. E SUBSTITUTE SHEET (RULE 26)