ES2724129A1 - NUCLEIC ACID PROBES AND ITS USE AS SUBSTRATE IN A METHOD FOR THE DETECTION OF SALMONELA (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Nucleic acid probes and their use as a substrate in a method for the detection of salmonella. The present invention relates to nucleic acid probes designed to be specifically degraded by the nuclease activity of Salmonella bacteria. Thus, the invention further provides a method for the detection and identification of Salmonella, preferably in samples, which comprises the use of these probes as substrates, in addition to kits comprising these probes. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

NUCLEIC ACID PROBES AND ITS USE AS SUBSTRATE IN A METHOD

FOR THE DETECTION OF SALMONELLA

The invention belongs to the field of food safety and health. Specifically, the invention relates both to nucleic acid probes and methods used and to kits in which said probes are used for the rapid and specific identification and detection of Salmonella bacteria in samples such as food or biological samples isolated from animals. . These probes, methods and kits are therefore useful to identify the offensive food that contains Salmonella and that can cause infections in consumers.

STATE OF THE TECHNIQUE

Salmonella is a common cause of foodborne bacterial disease in humans. Poultry and pork products are considered the main carriers of pathogenic Salmonella for humans, although the pathogen can be found in most foods, such as meats, eggs, milk, cheese, vegetables and ready-made products Eat, including nuts and seeds. Although these animals frequently have asymptomatic infections, salmonellosis in humans runs with abundant gastroenteritis and high risk of systemic infections that could cause death. According to the European Food Safety Authority (EFSA), more than 100,000 cases of human salmonellosis occur each year in Europe. This value increases to 1.2 million infections in the US, causing approximately 19,000 hospitalizations and 450 deaths a year, according to the Food and Drug Administration (FDA). Thus, the reduction of salmonellosis is a declared objective worldwide.

To prevent this relevant zoonosis, key changes were made to food safety regulations to improve controls in the food production chain. Despite these measures, large outbreaks of salmonellosis have been detected in the world in the last decade. Therefore, new strategies for the detection of Salmonella are an obvious priority for food security and public health .

Salmonella detection is generally performed by microbiological culture techniques (Odumeru, JA, León-Velarde, CG, 2012, In Mahmoud, BSM (ed.), InTech, University of Guelph, Guelph, Ontario, Canada, pp. 373 - 392; Zadernowska, A., Chajecka, W., 2012, In Mahmoud, BSM (ed.), InTech, University of Warmia and Mazury in Olsztyn, Faculty of Food Sciences Chair of Industrial and Food Microbiology, Poland, pp. 393 - 412; Almeida, C., et al., 2013, Int J Food Microbiol, 161, 16-22; Ahmed, OB, et al., 2014, Journal of Bacteriology & Parasitology, 5, 187-189) that proved to be irrefutable but of low sensitivity, expensive, laborious, require qualified personnel and time, lasting from days to weeks the confirmation of the presence or absence of Salmonella. Alternative methods based on PCR and immunoassays have been developed (Law, JW, et al., 2014, Front Microbiol, 5, 770; Mainar-Jaime, RC, et al., 2013, J Clin Microbiol, 51, 89-94) However, these methods could detect DNA from dead bacteria (for example, after pasteurization) giving false positive results and / or require an early culture phase that delays confirmation results up to several days within the stipulated sensitivity criteria. in the regulations (for example, EN / ISO 6579: 2002 Amd. 2007). These times are clearly incompatible with the rapid speed at which food moves through the production and distribution chain "from the farm to the table", and to which human salmonellosis outbreaks transmitted by food normally appear. Therefore, reliable alternative methods are still a clear priority for the food industry and clinically. The speed of the diagnostic method is important not only to prevent clinical complications, but also to identify the origin of the outbreak and additionally quickly eliminate contaminated products from the market.

The detection of pathogenic bacteria in food products is a critical parameter to determine quality control and food safety. Current Salmonella detection methods are laborious and time-consuming, with procedures primarily based on bacterial cultures. Although several techniques such as immunoassays or PCR have been described, enrichment of samples with 24-hour cultures is still inconvenient. More specifically, the detection of Salmonella in food samples requires days to weeks until confirmation. On the contrary, the global demand for food control is in continuous growth to meet global requirements. Thus, more specific, more sensitive and faster methodologies are needed to identify Salmonella in samples. These improved methods should have detection times that reach the speed of the food supply chain, "from the farm to the table" and should also allow the detection of live bacteria in order to avoid false positive results.

In summary, compared to the current diagnostic tools, the most desired characteristics for an innovative Salmonella detection system are the reduction of the diagnostic time (from days / weeks to hours) and the detection of the live pathogen.

DESCRIPTION OF THE INVENTION

The present invention describes artificial nucleic acid probes (oligonucleotide probes) designed to be specifically degraded by the nuclease activity of Salmonella bacteria . Thus, the invention provides a method for the detection and identification of Salmonella, preferably in samples, which comprises the use of these probes, in addition to kits comprising these probes.

The inventors of the present invention have designed nucleic acid probes for the specific and sensitive detection of live Salmonella in a rapid manner, preferably in less than 8 hours. The present invention takes advantage of the nuclease activity derived from Salmonella as an infection biomarker. Salmonella nucleases , but not the nucleases of other bacteria genera, have a preference to degrade nucleic acid probes designed in the present invention, so that these probes are proposed herein to be used as substrates for nuclease activity of Salmonella and thus for the specific detection and identification of bacteria belonging to the genus Salmonella. The method described herein is thus based on the identification of Salmonella nuclease activity using the aforementioned probes as substrates.

The probes described in the present invention therefore have the ability to differentiate Salmonella from other bacteria that are also commonly found as food contaminants.

In addition, the probes described in the present invention have the ability to recognize at least 50 different Salmonella serotypes and the most frequent serotypes in porcine mesenteric lymph nodes. In particular, the results shown in the examples below demonstrate 100% correlation with the standard methods of ISO 6579: 2002, such as PCR, microbiology and serotyping tests, underlining the great potential of this approach to be implemented as a reliable method. and fast for food safety tests.

The oligonucleotide probes designed in the present invention therefore have a great ability to recognize Salmonella in a sensitive and specific manner. In addition, these oligonucleotide probes have great flexibility to be incorporated in cheap portable kits that could provide on-site detection of Salmonella quickly (preferably in less than 8 hours).

The examples shown below demonstrate that the nucleic acid probes and the method described herein achieve a significant reduction in detection time, while retaining good sensitivity and specificity, making them competitive for a wide range of applications. The nucleic acid probes of the present invention could help adapt / synchronize food safety tests with the food supply chain.

In addition, in the present invention the optimal conditions for the described method have been identified (time and presence of chelating agents and divalent cations) that provide detection speed and specificity to target Salmonella, but not other bacteria.

Another advantage of the present invention is that it can be applied to the detection of live Salmonella bacteria, which allows in vitro detection in isolated food or biological samples and also the identification of Salmonella in the environment or in animals ( Salmonella infection ) . The detection of live Salmonella also avoids obtaining false positive test results. In addition, the method described herein is sensitive, specific and rapid, allowing the detection of Salmonella in preferably less than 8 hours, more preferably in 15 min, and discriminating between Salmonella and other bacteria that do not belong to this genus.

Thus, a first aspect of the present invention relates to an isolated nucleic acid probe consisting of any of the nucleotide sequences. indicated in Table 1 of the present description, that is, any of the nucleotide sequences SEQ ID NO: 1 to SEQ ID NO: 24.

Another aspect of the invention relates to an isolated nucleic acid probe consisting of the nucleotide sequence of SEQ ID NO: 25, hereinafter "the probe of the invention".

SEQ ID NO: 25 corresponds to the nucleotide sequence UCUN ⁴ N5UACN ⁹ UN11C, where the nucleotide in position N4 can be C, A or G, the nucleotide in position N5 can be G or C, the nucleotide in the position N9 can be G or U and the nucleotide in position N11 can be U or A.

Preferably, the probe of the invention is chemically modified. More preferably, at least 9 nucleotides of SEQ ID NO: 25 comprise a ribose ring whose 2 'position comprises an O-methyl group, also called 2'-O-methyl or 2'-OMe group.

In a more preferred embodiment, the nucleotides at positions 1 to 3, 6, 8, 10 and 12 of SEQ ID NO: 25 comprise a ribose ring whose 2 'position comprises an O-methyl group.

In another preferred embodiment, the probe of the invention consists of a nucleotide sequence selected from the list consisting of: SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. More preferably , the probe of the invention consists of the nucleotide sequence SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19.

SEQ ID NO: 10 (UCUCGUACGUUC) is the probe also referred to in the present invention as "Pir 2'-OMe RNA" or "Parental probe", and consists of the nucleotide sequence of SEQ ID NO: 25 where the nucleotide in the position N4 is C, the nucleotide at position N5 is G, the nucleotide at position N9 is G and the nucleotide at position N11 is U. In a more preferred embodiment, nucleotides at positions 1 to 3, 4, 6 , 8, 10, 11 and 12 of SEQ ID NO: 10 comprise a ribose ring whose 2 'position comprises an O-methyl group.

SEQ ID NO: 15 (UCUACUACUUUC) is the probe also named in the present invention "Salt 3_Pir 2'-OMe RNA" or "Salt-3", and consists of the nucleotide sequence of SEQ ID NO: 25 where the nucleotide at position N4 is A, the nucleotide at position N5 is C, the nucleotide at position N9 is U and the nucleotide at position N11 is U. In a more preferred embodiment, nucleotides at positions 1 to 3, 5, 6, 8, 9, 10, 11 and 12 of SEQ ID NO: 15 comprise a ribose ring whose 2 'position comprises an O-methyl group.

SEQ ID NO: 17 (UCUACUACUUAC) is the probe also referred to in the present invention as "Salt 5_Pir 2'-OMe RNA" or "Salt-5", and consists of the nucleotide sequence of SEQ ID NO: 25 where the nucleotide at position N4 is A, the nucleotide at position N5 is C, the nucleotide at position N9 is U and the nucleotide at position N11 is A. In a more preferred embodiment, nucleotides at positions 1 to 3, 5 , 6, 8, 9, 10 and 12 of SEQ ID NO: 17 comprise a ribose ring whose 2 'position comprises an O-methyl group.

SEQ ID NO: 19 (UCUGCUACUUAC) is the probe also referred to in the present invention as "Salt 7_Pir 2'-OMe RNA" or "Salt-7", and consists of the nucleotide sequence of SEQ ID NO: 25 where the nucleotide at position N4 is G, the nucleotide at position N5 is C, the nucleotide at position N9 is U and the nucleotide at position N11 is A. In a more preferred embodiment, nucleotides at positions 1 to 3, 5 , 6, 8, 9, 10 and 12 of SEQ ID NO: 19 comprise a ribose ring whose 2 'position comprises an O-methyl group.

The results shown in the examples below demonstrate the design, screening and identification of two preferred oligonucleotide probes as the most promising probes for detecting Salmonella. As a result, two optimal nucleic acid probes of the invention have been identified consisting of SEQ ID NO: 15 and SEQ ID NO: 17, which were first tested with strains of S. Typhimurium and S. Enteritidis and subsequently with strains of Salmonella of 50 different serotypes plus a set of 12 different strains of Salmonella. Both probes allowed the specific detection of all Salmonella strains tested in vitro. Finally, a double-blind study was performed using swine lymph node samples to determine the sensitivity and specificity of these probes selected to detect Salmonella.

Thus, in a more preferred embodiment, the probe of the invention consists of the nucleotide sequence SEQ ID NO: 15 or SEQ ID NO: 17, even more preferably SEQ ID NO: 17.

In another preferred embodiment, the probe of the invention further comprises at least one fluorophore linked to at least one of its 5 'or 3' ends. More preferably, it comprises a fluorophore linked to its 5 'end. Even more preferably, the probe of the invention further comprises a fluorophore linked to its 5 'end and / or a suppressor linked to its 3' end. In the most preferred embodiment, the probe of the invention further comprises a fluorophore linked to its 5 'end and a suppressor linked to its 3' end. Preferably, the fluorophore is FAM and the suppressor is TQ2.

"FAM" is the fluorescein amidite fluorophore and "TQ2" is Tide Quencher 2 acid.

When chemically modified probes of the invention are used flanked at the 5 'end with a fluorophore (FAM) and at the 3' end with a suppressor (TQ2), the reporter molecule (FAM) is inactivated by the close proximity to the suppressor, and only after degradation of the oligonucleotide probe by a specific nuclease the FAM and suppressor residues are diffused, increasing the initial distance. This allows the rest of FAM to recover its functionality as a reporter molecule, and the nuclease activity event can be recorded by fluorescence measurements.

Another aspect of the invention relates to a kit comprising the nucleic acid probe of the invention, hereinafter referred to as the "kit of the invention". In a preferred embodiment, this kit of the invention comprises the nucleic acid probes of the invention consisting of the nucleotide sequences SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. More preferably, this kit of The invention comprises the nucleic acid probes of the invention consisting of the nucleotide sequences SEQ ID NO: 15 and SEQ ID NO: 17, even more preferably SEQ ID NO: 17.

In another preferred embodiment of the kit of the invention, this further comprises EGTA and / or NTA, preferably EGTA, as chelating agents. More preferably, these chelating agents are in a concentration of 50 mM in the kit of the invention.

In a more preferred embodiment of the kit of the invention, it further comprises divalent cations selected from the list consisting of: Mg2 +, Ca2 + or Mn2 + or Any combination thereof. Even more preferably, the kit of the invention comprises the divalent cations Mg2 +, Ca2 + and Mn2 +. Preferably, these divalent cations are in a concentration of 50 mM in the kit of the invention.

In the most preferred embodiment of the kit of the invention, it comprises the probes of the invention consisting of SEQ ID NO: 15 and SEQ ID NO: 17, EGTA and the divalent cations Mg2 +, Ca2 + and Mn2 +.

Another aspect of the invention relates to the use of the probe of the invention or the kit of the invention for the detection of Salmonella, preferably for the in vitro detection of Salmonella in isolated samples.

In a preferred embodiment, Salmonella is Salmonella Typhimurium or Salmonella Enteritidis.

In another preferred embodiment, the "samples" referred to in the present invention are food samples, that is, samples derived from the food industry that include food and machinery used during food processing, or biological samples isolated from an animal .

The sample referred to in the present invention can also be derived from a microbiological culture or from the environment.

The "animal" referred to in the present invention is any animal that is capable of being a carrier of salmonellosis. Animals of this type may be, for example but not limited to, birds or mammals, preferably mammals including humans and non-human mammals More preferably, the animal is a pig or poultry.Any sample or product derived or obtained from these animals is encompassed within the definition of "sample" in the present invention.

In another preferred embodiment, the food sample referred to in the present invention is selected from the list consisting of: meat, eggs, milk, cheese, vegetables, nuts or seeds. Ready-to-eat products are also included in the present invention.

Another aspect of the invention relates to an in vitro method for the detection or identification of Salmonella in samples or an in vitro method for the diagnosis of an infection caused by Salmonella (salmonellosis) in an animal including humans, which comprises:

to. Contacting the isolated sample to be analyzed with the nucleic acid probe of the invention or the kit of the invention,

b. Incubate the mixture from step (a) under conditions that allow degradation of the nucleic acid probe by Salmonella nuclease activity ,

C. detect degradation of the nucleic acid probe, and

d. assign the sample to the group of samples comprising Salmonella when a degradation of the nucleic acid probe has been detected in step (c).

This method will also be referred to herein as the "method of the invention."

In a preferred embodiment of the method of the invention, the detection of the degradation of the nucleic acid probe in step (c) is performed by means of measuring the fluorescence intensity.

In this method, the nucleic acid probes of the invention are used as oligonucleotide substrates for Salmonella nuclease activity .

In another preferred embodiment of the method of the invention, the conditions that allow degradation of the nucleic acid probe by Salmonella nuclease activity in step (b) are:

- incubation for 15 min to 2 h, preferably 2 h, more preferably 1 h, even more preferably 15 min, and

- the presence of EGTA and / or NTA, preferably EGTA, and of divalent cations selected from the list consisting of: Mg2 +, Ca2 + or Mn2 + or any combination thereof.

More preferably, the incubation of step (b) takes place at 37 ° C.

In another preferred embodiment of the method of the invention, Salmonella is Salmonella Typhimurium or Salmonella Enteritidis.

In another preferred embodiment of the method of the invention, the samples are food samples or biological samples isolated from an animal. Preferably, the animal is a pig, a human being, or poultry.

In another preferred embodiment of the method of the invention, the food samples are meat, eggs, milk, cheese, vegetables, nuts or seeds.

This method of the invention may also be useful for monitoring the response to a treatment against salmonellosis in an animal, including humans.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as that commonly understood by a person skilled in the art to which the present invention belongs. Methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. Throughout the description and claims the word "comprises" and its variations are not intended to exclude other technical characteristics, additives, components, or steps. Other objects, advantages and additional features of the invention will be apparent to those skilled in the art upon examination of the description or may be learned by the practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Screening of the nuclease activity derived from Salmonella. The global nuclease activity derived from Salmonella is described as an increase in average fluorescence intensity as a result of degradation by each probe. The bars represent the average of triplicate fluorescence measurements (± of). The data are representative of at least 3 individual experiments.

FIG. 2. Nuclease activity profile of 11 Salmonella Typhimurium cultures using the Parental probe and 12 probes derived from Salmonella. The bars represent the average of triplicate fluorescence measurements (± of). The data are representative of at least 3 individual experiments.

FIG. 3. Nuclease activity profile of 11 Salmonella Enteritidis cultures using the Parental probe and 12 probes derived from Salmonella . The bars represent the average of triplicate fluorescence measurements (± of). The data are representative of at least 3 individual experiments.

FIG. 4. Growth curves . Salmonella Typhimurium (STM1) and Salmonella Enteritidis (SE1) were cultured and CFU / ml determination was carried out at different time points by plate count.

FIG. 5. Sensitivity determination of the nuclease assay for Salmonella cultures . Fluorescence measurements of the nuclease activity derived from Salmonella Typhimurium (STM1) and Salmonella Enteritidis (SE1) were performed using the Parental, Sal-3 and Sal-5 probes at different time points.

FIG. 6. Study of specificity of Salmonella probes under normal conditions. Various bacterial cultures were tested using the Parental, Sal-3 and Sal-5 probes under normal conditions (incubation time of 1 h and PBS + / +) for the nuclease activity assay.

FIG. 7. Specificity study of Salmonella probes under optimized conditions . Various bacterial cultures were tested using the Parental, Sal-3 and Sal-5 probes under optimized conditions of 15 min incubation time and addition of the EGTA chelating agent.

FIG. 8. Double-blind experiment to determine the presence or absence of Salmonella in pig lymph nodes. The bars represent the specific nuclease activity derived from lymph node samples using the parental oligonucleotide probes (white bars), Sal-3 (gray bars) and Sal-5 (black bars). 100 % correlation was observed between conventional methods and Salmonella probes .

FIG. 9. Incubation time optimization for Salmonella specific oligonucleotide probes .

FIG. 10. Chelating effect on Salmonella specific oligonucleotide probes .

FIG. 11. Effect of divalent cations on the specific oligonucleotide probes of Salmonella

FIG. 12. PCR-InvA of the lymph node samples in BPW of pigs to confirm the presence or absence of Salmonella. C +: positive control; C-: negative control and as a molecular weight marker (Nippon marker). The PCR products were electrophoresed in 1% agarose in TAE buffer, stained with Midori Green and visualized in a UV transilluminator.

FIG. 13. Evaluation of 50 Salmonella serovars using nucleic acid probes. The bars represent the specific nuclease activity observed for each Salmonella serotype . 50 serotypes (96%) were successfully identified using a nucleic acid probe.

EXAMPLES

1. Materials

Solutions and culture media

Two types of phosphate buffered saline (PBS, pH 7.4) were purchased with MgCl2 and CaCl2 (PBS / +) and without this supplement (PBS - / -) from Gibco (Life Technologies, Spain). Ethylenediaminetetraacetic acid (EDTA, CAS number: 60-00-4, pH 8.0), Tris-EDTA, 1x solution (TE, CAS number: 38641-82-6, pH 8.0) and nitrotriacetic acid (NTA) were purchased , CAS number: 139-13-9) from ThermoFisher Scientific (Madrid, Spain). Ethylene glycol bis (2-aminoethyl ether) -N (EGTA, CAS number: 67-42-5), magnesium chloride hexahydrate (CAS number: 7791-18-6), calcium chloride (CAS number: 1043-) was purchased 52-4), zinc chloride (CAS number: 7646-85-7), manganese (II) chloride (CAS number: 7773-01-5) and copper (II) acetate (CAS number: 142 71-2 ) by Sigma-Aldrich (Madrid, Spain).

Buffered peptone water (BPW), Luria Bertani (LA) agar, tryptone soy (TSA) agar, Luria Bertani (LB) and tryptic soy broth (TSB) were purchased in Difco (Pronadisa, Spain) ).

Bacterial strains

A total of 94 bacterial strains were selected, ie 12 S. Typhimurium, 12 S. Enteritidis, 50 Salmonella spp. of different serotypes and 20 different strains of Salmonella of 12 different genera, from the set of bacterial cultures of the Institute of Agrobiotechnology (IdAB, Navarra, Spain). These strains were previously isolated from animal or food samples, phenotypically characterized and typed in the National Reference Centers for Animal Salmonellosis (Algete, Madrid, Spain) and / or the Carlos III ISC Health Institute (Madrid, Spain ). All strains were stored at -80 ° C in 10% skim milk until use. The strains were named by the initials of the serotype, followed by a correlative number, for example STM1, STM2, SE1, SE2, etc., according to the order of appearance in this work.

BPW-MLN samples

A total of 30 1 ml aliquots of samples with BPW from mesenteric lymph nodes (MLN) from previously grown pigs (37 ° C, 24 h) and stored at -20 ° C (BPW-MLN) were selected from the collection of IdAB. Of these, 15 samples were classified as positive for Salmonella and 15 as negative for Salmonella, according to previous bacteriology performed following the conventional procedure of ISO 6579: 2002 Amd. 2007. In order to minimize the probability of a false negative result, the 15 negative BPW samples were selected from farms where all the fattening pigs analyzed were found to be negative.

2. Methods

Synthesis and purification of oligonucleotide probes

The DNA probes were synthesized and purified on Biomers.net (Germany) with fluorescein amidite fluorophore (FAM) at the 5 'end and Tide quencher 2 acid (TQ2) at the 3' end. For this, a conventional method of solid phase phosphoramidite chemistry was used, followed by purification by high performance liquid chromatography (HPLC). The identities of the probes were confirmed with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The purity of the probes was evaluated with HPLC analysis, normally It is greater than 95%. The oligonucleotide sequences of the probes used in this study are described in Table 1.

Table 1. Sequences of oligonucleotide probes and names. "M" means the presence in the nucleotide that follows a ribose ring whose 2 'position comprises an O-methyl group, also called 2'-O-methyl or 2'-OMe group. "F" means the presence in the nucleotide that follows a ribose ring whose 2 'position comprises a fluorine group, also called the 2'-fluorine group.

Preparation of bacterial supernatants for the nuclease assay

To prepare pure culture supernatants, bacteria were precultured in LA or TSA plates (24 h, 37 ° C) and then an individual colony was transferred to LB or TSB and incubated overnight at 37 ° C. The cultures obtained were diluted 1: 500 in fresh LB or TSB and grown (24h, 37 ° C) with stirring at 200 rpm. Each culture was then centrifuged (6,000 g, 20 min) and the supernatant was preserved and kept at 4 ° C Until its use. Similarly, aliquots of BPW-MLN were thawed and centrifuged for supernatant collection. To assess the absence of bacteria, each supernatant was filtered on a Millipore® (Sarsted) of 0.2 qm and 100 ql of each filtrate was seeded in TSA and incubated (48 h, 37 ° C). All supernatants were stored at 4 ° C until use.

Nuclease Activity Assays

Nuclease activity assays were performed as described previously (Hernandez, LI, et al., 2016, Chemical Communications, 52, 12346-12349) with slight modifications. Specifically, for each reaction, 9 ql of sample (i.e., Salmonella culture supernatants , other bacteria or mesenteric lymph nodes, control medium used for cultures, or control buffer) were combined with 1 ql (50 pmoles, substrate) nuclease) of the oligonucleotide probe to be tested and this mixture was incubated at 37 ° C, for different times: 7.5 min, 15 min, 30 min, 60 min and 120 min.

Once it was determined that 1 h was the optimal incubation time, the study of the chemical conditions was carried out by mixing 8 ql of sample plus 1 ql of any of the chelating agents EDTA, EGTA or NTA 50 mM or 50 mM of divalent cations Mg2 +, Ca2 + , Zn2 +, Mn2 + or Cu2 +, and more than 1 ql (50 pmoles) of the corresponding probe. Each mixture was incubated at 37 ° C, for 1 h.

In all cases, the reaction was stopped by adding 295 ql of PBS - / - supplemented with 10 mM EDTA. Next, 95 ql of each sample was loaded in triplicate into 96-well black plates (96F untreated black well plate, Thermo Scientific). The fluorescence intensity was measured with a fluorescence microplate reader (Synergy HT, BioTek) using the appropriate filter parameters for FAM (excitation / emission 494/521 nm). Three independent experiments were performed with each sample. The results were expressed as the mean ± SD (n = 3) of the fluorescence intensity.

DNA probe screening in Salmonella and other bacterial cultures

Initial screening was performed with 12 probes selected (Salt 1-Salt 12, Table 1) using culture supernatants from two strains of Salmonella (i.e., STM 14028 or STM1 and SE 408 or SE1) and three other common pathogens ( Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes). As a result, the Pir 2'-OMe RNA probe was identified as the "Parental probe" (Figure 1) that was additionally used to design another 12 "derivative probes", called Exit to Sal12 (Table 1).

To assess the sensitivity of these 12 "derived probes" to detect Salmonella nucleases , supernatants from 20 additional strains (n = 10) of S. Typhimurium (STM2-STM11) and S. Enteritidis (SE2-SE11) were selected. the Parental Pir 2'-OMe RNA probe as a control of nuclease activity in the supernatants As a result, the Sal3 and Sal5 probes were selected as "final probes".

The two final Sal3 and Sal5 probes selected were tested with a total of 50 Salmonella strains , selected from each different serotype of the IdAB Salmonella culture collection.

Preparation of supernatants from Salmonella for growth curves and detection limit test

The in vitro detection limit of the technique based on nuclease activity in pure culture supernatants of STM1 and SE1 was determined. For this, each Salmonella reserve culture was grown in LA plates (at 37 ° C, 24 h) and collected in BPW, to prepare bacterial suspensions adjusted by spectrophotometry (optical density at 600 nm = 0.160 absorbance) to contain «2x 108 CFU / ml. The exact number of CFU was retrospectively evaluated by serial dilutions in BPW and sowing in LA (at 37 ° C, 24 h). Growth curves were determined from a suspension containing "102 CFU / ml in BPW (prepared by mixing 6 ml of" 103 CFU / ml and 54 ml of fresh BPW) and subsequent culture at 37 ° C for 24 h. Samples were collected at different times 0, 2, 4, 6, 8, 10 and 24 h after incubation, centrifuged (6,000 g, 20 min), and supernatants diluted tenfold in PBS and used for the assay nuclease, as explained above, with the final probes Sal3 and Sal5, in addition to the Parental Pir 2'-OMe RNA probe as a control. Fluorescence intensity was measured.

In addition, the number of CFU was quantified at each time selected by sowing in triplicate of the appropriate dilutions in LA and incubation (37 ° C, 24 h). Each sample was analyzed in triplicate. Bacterial counts were transformed in logarithms and its mean ± SD (n = 3) was represented.

Specificity of the probes selected in different conditions

The specificity of the 2 "final probes" was determined with 20 additional supernatants prepared from 12 different bacterial species different from Salmonella that are frequently found in food, animals and / or environment. STM1 and SE1 supernatants and probe were used. Parental Pir 2'-OMe RNA as controls These supernatants were first analyzed under conventional conditions (incubation time of 1 h and PBS + / +) for the nuclease activity test To avoid the observed nonspecificity, the same preparations were analyzed and probes under optimized conditions (15 min incubation and addition of the chelating agent EGTA).

Sensitivity of nuclease activity against PCR and bacteriology in BPW-MLN samples

A double-blind experiment was performed in order to determine the presence or absence of Salmonella in pig MLN by nuclease activity, compared to previous bacteriology results obtained by ISO 6579: 2002 Amd. 2007 and with a PCR- / nvA previously described (Mainar-Jaime, RC, et al., 2013, J Clin Microbiol, 51, 89-94). For this, supernatants from the 30 samples of BPW-MLN described above were used.

Nuclease activity experiments were carried out using the selected probes (Sal3 and Sal5) and the conditions selected before (ie, 15 min incubation and addition of the EGTA chelator). The PCR reactions were performed with the DNA extracted from 0.5 ml of each thawed aliquot of BPW-MLN and the amplification of the 229 bp DNA fragments expected by conventional electrophoresis in 1% (weight / volume) of gel was analyzed. agarose in TAE buffer, stained with Midori Green and visualized on a UV transilluminator. STM1 (positive control) and E. coli and sterile ultra-pure water (MilliQ) (negative controls) were used as controls.

3. Results

Nuclease activity screening in cultures of Salmonella

It was intended to identify the specific nuclease activity derived from Salmonella by designing oligonucleotide substrates containing different sequences and chemical modifications. Screening began with 12 different libraries that were incubated with the target bacteria ( Salmonella), along with three non-specific cultures of E. coli, S. aureus and S. pyogenes, as controls. The oligonucleotide probes used in this screening were divided into three categories: i) natural probes; DNA and RNA, ii) 2'-fluorine probes and their derivatives, and iii) 2'-O-methyl probes and their derivatives (Table 1). Figure 1 shows the preference of the nuclease activity detected in Salmonella and the control cultures for each probe tested. Natural probes (DNA and RNA) have demonstrated nuclease activity for both Salmonella and control bacteria, suggesting the presence of multiple nucleases in each of the bacterial cultures. DNA probes are easily degraded by S. aureus cultures . On the other hand, the RNA probe was slightly better degraded by Salmonella cultures compared to control bacteria. These results suggest that Salmonella cultures have a preference for RNA over the DNA oligonucleotide probes analyzed in this study, and this could indicate the presence of active RNases in the cultures.

Subsequently, both the RNA and DNA probe sequences were chemically modified (completely or partially), incorporating two well-established chemical modifications (2'-fluorine and 2'-O-methyl) of the nucleosides during oligonucleotide synthesis (Table one). Thus, the resulting chimeras are expected to confer additional resistance and / or specificity characteristics for a target nuclease activity associated with Salmonella cultures . In fact, it was observed that 2'-fluorine modified probes were better digested by non-specific control bacteria than by Salmonella cultures , suggesting that 2'-fluoride chemistry is not the most appropriate modification for nuclease identification Salmonella More specifically, Salmonella cultures have shown a lower signal for the completely modified probe with 2'-fluorine (All 2'-F), and for 2'-fluorine-DNA chimeras (Pir-2'-F-DNA and Pur-2'-F-DNA). Interestingly, the 2'-fluorine-RNA chimeras (Pir-2'-F-RNA and Pur-2'-F-RNA) have shown only slightly lower fluorescent values compared to the controls, indicating that the RNA-containing probes are more susceptible to degradation by Salmonella nucleases .

Next, the probes modified with 2'-O-methyl were tested. It was found that the completely 2'-O-methyl sequence (All 2'-OMe) is the most resistant probe for all bacterial cultures analyzed, including Salmonella cultures . On the other hand, the 2'-O-methyl-DNA chimeras (Pir-2'-OMe-DNA and Pur-2'-OMe-DNA) generated the highest fluorescence intensity values, and thus the highest nuclease activity for S. aureus cultures , and limited nuclease activity for Salmonella cultures . In contrast, very promising results were observed for 2'-O-methyl-RNA chimeras, where the Pir-2'-OMe-RNA probe has shown the best ability to discriminate Salmonella from control bacterial cultures (Figure 1, dashed line rectangle). A surprising difference was also observed between the Pir-2'-OMe-DNA and Pir-2'-OMe-RNA probes, probably attributed to the presence of the DNA and RNA purines, respectively. This observation suggests that the three RNA purines in the Pir-2'-OMe-RNA sequence (see Table 1) are essential for directing Salmonella nucleases . Thus, the present inventors further optimized the probe by modifying the number and sequence of these three RNA purines, in order to obtain a specific probe for Salmonella.

Design and characterization of an optimal probe for Salmonella

Based on the satisfactory identification of Pir-2'-OMe-RNA ("Parental probe") as a promising probe to identify Salmonella, the present inventors were motivated to design additional oligonucleotide probes by changing the three purine RNA nucleotides present in The parental probe: To do this, the 12-mer sequence of the Parental probe was modified to assess the effect where the incorporation of a single (adenosine or guanosine) or several purines could improve the sensitivity of the probe compared to the Parental. In mind, 12 new probes (called Sal-1 to Sal-12) were designed that were tested under the same conditions used in the initial screening Figures 2 and 3 show the degradation efficiency of all probes (Parental to Sal- 12) using 11 cultures of Salmonella Typhimurium (Figure 2) and 11 cultures of Salmonella Enteritidis (Figure 3) Interestingly, a variety of probe degradation profiles were observed s The Parental probe was used as a baseline value to assess the degradation efficiency of the new derived probes. The first two probes, Sal-1 and Sal-2, were designed by reducing the number of RNA purines in the sequence, from three purines in the parental sequence to a single adenosine for Sal-1, and a single guanosine, for Sal- 2. For both derived sequences, a clear reduction in probe degradation was observed. In addition, the most drastically reduced fluorescence signal was in Sal-2, indicating that the presence of guanosine increases the resistance of the probe towards Salmonella nucleases . Next, the Sal-3 and Sal-4 derived probes, which contained two adenosines and two guanosines, respectively, were tested. While the Sal-4 probe showed a slight improvement compared to Sal-2, suggesting an additive effect, it was even less efficiently degraded when compared to the parental. In contrast, Sal-3 proved to be a better probe, indicating that adenosine nucleotides are a better substrate for Salmonella nucleases . The incorporation of three adenosines for Sal-5 and three guanosines for Sal-6 were then tested, thus confirming once again the preference of both cultures; S. Typhimurium and S. Enteritidis for adenosine as a substrate, with a more consistent signal. Finally, the combination of three purines was analyzed by placing adenosines and guanosines in different positions (Salt-7 to Salt-12), however, no further improvement was observed. These results suggest that Salmonella nucleases have an amazing ability to degrade adenosines, and this ability is reduced in the presence of guanosines. Thus, these data show that the Sal-3 and Sal-5 probes (Figs. 2 and 3 asterisks) that were modified with two and three adenosines, respectively, are the most promising candidates to select as Salmonella target . Subsequent sensitivity and specificity studies were performed using these probes.

Sensitivity of the probes of Salmonella candidates

To assess the sensitivity of the candidate probes, a growth curve was first performed for Salmonella Typhimurium (STM 14028) and Salmonella Enteritidis (SE 408). For this, several samples of both Salmonella cultures were collected at different times of time and microbiology and nuclease activity assays were performed. Figure 4 shows the number of log10 Colony forming units (log10 CFU) per ml that was determined by microbiology analysis and plotted against the collection time (Figure 4). As a result, the logarithmic, exponential and stationary phases of both Salmonella cultures were represented . Bacterial growth was observed in both cultures at 2 hours. Next, it was investigated to determine the sensitivity that could be achieved by the nuclease activity assay. Therefore, the test was performed at different growth times using the Parental, Sal-3 and Sal-5 probes, as indicated in Figure 5. In particular, since 8-hour Salmonella cultures were able to promote the degradation of probes, indicating that nuclease activity offers a rapid response that could be used for the detection of these bacteria. In addition, the Sal-3 and Sal-5 probes were more efficiently degraded than the Parental probe, with the Sal-5 probe showing the highest sensitivity for Salmonella at 8 hours, according to the nuclease activity derived from a bacterial load of 5x106 CFU / ml. In particular, the time required to perform the nuclease activity test in these experiments was 1 hour, which represents a great improvement over the conventional microbiological plate count method that requires 24 hours. Together, these results demonstrate that this system is accompanied by a significant reduction in detection time, while retaining good sensitivity, which makes it competitive for a wide range of applications.

Specificity of the probes of Salmonella candidates

The specificity of Salmonella probes was analyzed by increasing the number of control bacteria used in the initial screening. A set of pathogens (previously characterized by IdAB, Spain) that are normally found as contaminants in food products was tested. Figure 6 shows the degradation profile of the Parental, Sal-3 and Sal-5 probes that were incubated with Salmonella STM1 and SE1 cultures and 20 additional supernatants prepared from 14 different bacterial species as negative controls. Cross reactivity was observed with several of the control cultures tested. Bacterial cultures of E. coli, K. pneumoniae and S. aureus have shown a significant background signal that could limit the applicability of this technology in real samples. To solve the problems of cross-reactivity, the optimization of Salmonella probes was performed by applying various conditions that can modulate nuclease activity, such as: time, chelating agents and divalent cations. This probe optimization process aims to eliminate or reduce unwanted nuclease activity and enhance or maintain the activity for Salmonella target nucleases . Figure 9 shows the optimization of Salmonella Sal-3 and Sal-5 probes, using different incubation times (7.5 to 120 min, Figure 9), where no significant differences in fluorescence signal were observed between 15 and 120 min. Next, the function of several chelators was tested (EDTA, EGTA and NTA, Figure 10), where the activity of Salmonella nucleases was clearly affected by EDTA, but not by the other chelators, suggesting the utility of EGTA and NTA to block the nuclease activity of non-specific bacteria. Then, the effect of a variety of divalent cofactors (Mg2 +, Ca2 +, Zn2 +, Mn2 +, Cu2 +, Figure 11) was evaluated. It was observed that Mg2 +, Ca2 + and Mn2 + were essential cofactors for nuclease activity. As a result of the optimization data, it was concluded that 15 min and the addition of EGTA are the conditions that favor the activity of nucleases associated with Salmonella, but not with other bacteria. Next, the specificity studies were repeated using the newly optimized conditions for Salmonella (Figure 7). In particular, a significant reduction in nuclease activity was observed for all bacteria with cross-reactivity. In conclusion, a set of conditions that can provide detection speed and specificity to target Salmonella was identified .

Double blind study using relevant samples

To demonstrate the usefulness of the two final probes and the optimal conditions of the nuclease activity test to detect Salmonella, a double-blind study was designed to be carried out on samples previously identified as positive or negative, by the standard micro65 method ISO6579: 2002 Amd. 2007. To avoid false negative samples, BPW-MLN samples were selected from a farm where all the pigs analyzed were negative. For this study, supernatant from 30 samples of porcine mesenteric lymph nodes, the standard sample used to determine the presence of Salmonella in pig samples, was used. Lymph nodes were removed and cultured as indicated in the materials and methods section. To confirm the presence or absence of Salmonella in these samples, three independent analyzes were carried out as follows: i) PCR analysis and biochemical identification by IdAB were performed (Figure 12, Table 2, respectively), ii) analysis was performed Microbiological with serotype identification by the Spanish National Reference Laboratory (Table 2), and iii) nuclease activity was tested internally.

Table 2. Samples of mesenteric lymph nodes and respective biochemical tests for the presence of Salmonella and the consequent determination of Salmonella serotypes

Biochemical test

of Salmonella

Name of the (IdAB) Salmonella Serovariety (NRL) sample

BPW 1 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 2 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 3 S. Typhimurium 1.4, [ 5], 12: i: 1.2 BPW 4 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 5 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 6 S. Typhimurium 1.4, [5], 12: i: -BPW 7 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 8 S. Typhimurium 1.4, [5] , 12: i: 1.2 BPW 9 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 10 S. arizonae 48: z4, z23, z32: -BPW 11 S. Derby 4.12 : f, g: -BPW 12 Enteritidis 9,12: g, m: -BPW 13 S. Bard 8: e, h: 1,2

BPW 14 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 15 S. Typhimurium 1.4, [5], 12: i: 1.2 BPW 16-BPW30 - NA

The results were compared without prior knowledge of each part. Figure 8 shows the results obtained by three different techniques. And, more importantly, the method described herein based on the identification of nuclease activity of Salmonella cultures using the aforementioned probes, showed 100% correlation with the standard reference methodologies ( gold standard methodologies), such as PCR, microbiology tests and serotyping. These surprising results underscore the enormous potential of Salmonella probes described in this document and their applicability to the analysis of real samples. In summary, oligonucleotide probes have been developed with the great ability to recognize Salmonella cultures in a sensitive and specific way. Interestingly, the nuclease activity test was carried out in 15 min, in 8-hour cultures. In addition, the oligonucleotide probes described in the This document has great flexibility to be incorporated into cheap portable devices that could provide on-site detection in less than 8 hours.

4. List of strains and samples of BPW-MLN used in this work

Table 3 below shows the list of strains and samples of BPW-MLN used in this work.

Code Features

Salmonella strains : Fattening pig field isolates : STM1 S. Typhimurium ATCC 15981

STM2 to STM11 S. Typhimurium of different animals

SE1 to SE11 S. Enteritidis of different animals

S1 S. Single phase Thyphimurium

S2 S.> 4baetetuba

S3 S. Agona

S4 S. Amsterdam

S5 S. Anatum

S6 S. enteric subsp. arizonae

S7 S. Bard

S8 S. Bovismobificans

S9 S. Braenderup

S10 S. Branderburg

S11 S. Bredeney

S12 S. Coeln

S13 S. Derby

S14 S. enteric subsp. diarizonae

S15 S. Gaminara

S16 S. Give

S17 S. Goldcoast

S18 S. Hadar

S19 S. Havana

S20 S. Infantis

S21 S. Indiana

S22 S. Kapemba

S23 S. Kedogou

S24 S. Kottbus

S25 S. Lexington

S26 S. Livingstone

S27 S. London

S28 S. Meleagridis

S29 S. Mikawasirna

S30 S. Mishmarhaek

S31 S. Montevideo

S32 S. Muenchen

S33 S. Newport

S34 S. Nottingham

S35 S. Ohio isolated

S36 S. Oranienburg

S37 S. Poona

S38 S. Reading

S39 S. Rissen

S40 S. enteric subsp. salamae

S41 S. Szentes

S42 S. Tennessee

S43 S. Thompson

S44 S. Toulon

S45 S. Umbilo

S46 S. Urban

S47 S. Wien

S48

S49 9.12: -: - Other strains that are not isolated from the field of fattening pigs:

Salmonella (NS):

NS1 to NS5 Escherichia coli ATCC 25981 and 4 other strains isolated from feed samples and fattening pigs

NS6 Enterococcus faecalis isolated from fattening pigs

NS7 and NS8 Hafnia alvei isolated from the pig environment NS9 Moellerella wisconsensis isolated from food samples

NS10 Oceanobacillus oncorhynchi isolated from food samples

NS11 and NS12 Pseudomonas aeruginosa isolated from fattening pigs

NS13 Proteus mirabilis isolated from fattening pigs NS14 Providencia rustigianii isolated from food samples

NS15 Proteus vulgaris isolated from fattening pigs NS16 and NS17 Staphylococcus aureus isolated from food samples

NS18 Serratia liquefaciens isolated from the environment of the pig NS19 Streptococcus pyogenes isolated from food samples

NS20 Klebsiella pneumoniae isolated from food samples

BPW-MLN samples : 1 ml aliquots of BPW with mesenteric lymph node (MLN) samples from previously grown fattening pigs (37 ° C, 24 h) according to ISO 6579: 2002 Amd. 2007 and kept frozen at -20 ° C.

BPW-MLN1 to BPW-MLN10 Samples positive for S. Typhimurium by ISO 6579: 2002 and PCR-lnvA

BPW-MLN11 S. Single-phase Typhimurium

BPW-MLN12 S. enterica subsp. arizonae

BPW-MLN13 S. Derby

BPW-MLN14 S. Enteritidis

BPW-MLN15 S. Bardo

BPW-MLN16 to BPW-MLN30 Negative samples for Salmonella by ISO 6579: 2002 and PCR-InvA

Claims (25)

1. A nucleic acid probe consisting of the nucleotide sequence of SEQ ID NO: 25. 2. The nucleic acid probe according to claim 1, which consists of a nucleotide sequence selected from the list consisting of: SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 . 3. The nucleic acid probe according to claim 2, consisting of the nucleotide sequence SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19. 4. The nucleic acid probe according to claim 3, consisting of the nucleotide sequence SEQ ID NO: 15 or SEQ ID NO: 17, preferably SEQ ID NO: 17. 5. The nucleic acid probe according to any one of claims 1 to 4, further comprising a fluorophore linked to its 5 'end and / or a suppressor linked to its 3' end. 6. The nucleic acid probe according to claim 5, further comprising a fluorophore linked to its 5 'end and a suppressor linked to its 3' end. 7. The nucleic acid probe according to any of claims 5 or 6, wherein the fluorophore is FAM and the suppressor is TQ2. 8. A kit comprising the nucleic acid probe according to any one of claims 1 to 7. 9. The kit according to claim 8, comprising nucleic acid probes consisting of the nucleotide sequences SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. 10. The kit according to claim 9, comprising the nucleic acid probes consisting of the nucleotide sequences SEQ ID NO: 15 and SEQ ID NO: 17, preferably SEQ ID NO: 17. 11. The kit according to any one of claims 8 to 10, further comprising EGTA and / or NTA, preferably EGTA. 12. The kit according to any one of claims 8 to 11, further comprising divalent cations selected from the list consisting of: Mg2 +, Ca2 + or Mn2 + or any combination thereof. 13. The kit according to claim 12, comprising the divalent cations Mg2 +, Ca2 + and Mn2 +. 14. Use of the nucleic acid probe according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 13 for in vitro detection of Salmonella in samples. 15. Use according to claim 14, wherein Salmonella is Salmonella Typhimurium or Salmonella Enteritidis. 16. Use according to any one of claims 14 to 15, wherein the samples are food samples or biological samples isolated from an animal. 17. Use according to claim 16, wherein the animal is a pig or poultry. 18. Use according to claim 16, wherein the food samples are meat, eggs, milk, cheese, vegetables, nuts or seeds. 19. An in vitro method for the detection of Salmonella in samples comprising: to. Contacting the sample to be analyzed with the nucleic acid probe according to any one of claims 1 to 7 or the kit according to any one of claims 8 to 13, b. incubate the mixture of step (a) under conditions that allow degradation of the nucleic acid probe by Salmonella nuclease activity , c. detect degradation of the nucleic acid probe, and d. assign the sample to the group of samples comprising Salmonella when a degradation of the nucleic acid probe has been detected in step (c). 20. The in vitro method according to claim 19, wherein the detection of the degradation of the nucleic acid probe in step (c) is performed by means of measuring the fluorescence intensity. 21. The in vitro method according to any of claims 19 or 20, wherein the conditions that allow degradation of the nucleic acid probe by Salmonella nuclease activity in step (b) are incubation for 15 min in the presence of EGTA and / or NTA, preferably EGTA, and divalent cations selected from the list consisting of: Mg2 +, Ca2 + or Mn2 + or any combination thereof. 22. The in vitro method according to any one of claims 19 to 21, wherein Salmonella is Salmonella Typhimurium or Salmonella Enteritidis. 23. The in vitro method according to any one of claims 19 to 22, wherein the samples are food samples or biological samples isolated from an animal. 24. The in vitro method according to claim 23, wherein the animal is a pig or poultry. 25. The in vitro method according to claim 23, wherein the food samples are meat, eggs, milk, cheese, vegetables, nuts or seeds.