The invention relates to a set of primers for amplifying the nucleotide sequence of the recA gene of Salmonella enterica sp. bacteria, a method for detecting Salmonella enterica sp. bacteria, a method for detecting an infection caused by Salmonella enterica sp. bacteria, and a kit for detecting an infection caused by Salmonella enterica sp. bacteria. The invention is applicable in medical diagnostics, clinical and veterinary diagnostics, as well as in the food industry.

Salmonella spp . bacteria belong to the Enterobacteriaceae family, grouping Gram-negative or anaerobic (glucose-fermenting) bacilli. Salmonellosis is one of the most common infectious diseases - both in humans and in animals. Animals and animal products are considered the main source of Salmonella. Infection in humans can occur as acute enteritis, intestinal fever (typhoid or paratyphoid fever), and bacteraemia with or without clinical symptoms. The vast majority of cases of human salmonellosis are caused by a limited number of Salmonella serovars, and the most common cause of the disease is Salmonella enterica. Strains of the Salmonella genus spread rapidly by horizontal transmission between susceptible organisms, and frequent and long-term carriage promotes the spread of infections. The source of infection is not only the carriers of microorganisms, but also the environment into which Salmonella bacilli enter. Microbiological diagnostics is the primary tool to detect and identify the etiological factor of infection. The basic methods of bacteriological diagnostics include: culture and isolation of bacilli, determination of biochemical profiles, drug resistance assays and serological testing. Sometimes determination of the exact antigenic structure of Salmonella bacilli is insufficient for definitive identification and additional biochemical differentiation is required. Both direct identification of Salmonella bacteria present in the tested material and their indirect identification, based on bacterial culture, as well as demonstration of characteristic types of drug resistance (R- type) can also be performed using molecular methods. The choice of molecular method should be tailored to the problem that is posed to be solved: detection, identification, differentiation, or taxonomic studies. Technical aspects are also important, i.e., the degree of application difficulty, research facilities, ease of interpretation of the results, time required for the analysis, the total cost of the method used and the cost of a single test. Laboratories and food industry companies can use alternative analytical methods, in particular the so-called rapid methods, as long as their use provides equivalent results proven by a validation procedure. Genetic tests are among the quick and relatively inexpensive methods of identifying Salmonella bacteria. Detecting highly conservative regions in the genome allows for classification into the Salmonella species. Genes responsible for basic metabolism are examples of such constant regions, including the recA gene coding for a DNA recombinase, the coefficient of variation of which is (according to phylogenetic studies) negligible in different subspecies of Salmonella enterica. Until now, the most common methods for diagnosis of infections caused by Salmonella enterica sp. bacteria, as well as for the species identification, are assays based on culturing; however, despite their high sensitivity and specificity, they are labour-intensive and time-consuming. The methods characterized by the greatest specificity and sensitivity are those involving the detection of Salmonella nucleic acid in biological material (the so-called NAAT methods - Nucleic Acid Amplification Tests). The most commonly used tests in NAAT technology are assays based on Real-Time PCR method. Many different assays using the Real-Time PCR technique are available on the market, but despite the fierce competition, these methods are still relatively expensive. Moreover, they require highly specialized personnel, expensive devices, as well as mandatory extraction of genetic material from the sample. Furthermore, since cyclic heating and cooling of the reaction mixture is necessary, this method is time-consuming, and the devices used consume relatively large amounts of energy to perform this process. Isothermal methods, including the LAMP (Loop-mediated isothermal amplification) technology, are methods that allow to accelerate the diagnostic process and reduce the cost of energy needed to perform the analysis. Moreover, according to the literature data, these methods are characterized by higher sensitivity and specificity than the aforementioned Real-Time PCR technique, they are also much faster. Their isothermal course does not require specialized equipment. Due to the low equipment requirements, isothermal methods are an ideal diagnostic solution both for primary care units (POCT - point-of-care testing), where the test can be performed in the practice of a general practitioner, as well as for rapid veterinary diagnostics and in the food industry. This solution allows for a quick diagnostic test. The described method is more efficient than traditional PCR amplification, and the gene amplification and detection can be performed at the same time. Its advantages are simplicity, speed, high sensitivity and specificity. It does not require the use of large or expensive instruments and is suitable for rapid laboratory testing and detection. Furthermore, it can be promoted and utilised in laboratories with basic equipment. There is a need to provide a diagnostic method with the use of appropriately designed set of primers used for the diagnosis of Salmonella enterica sp. with the LAMP method, that might enable point-of-care testing also in veterinary diagnostics or utilised by laboratories performing quality control for the food industry. Unexpectedly, the above problems have been solved by the present invention.

The first subject of the invention is a set of primers for amplifying the nucleotide sequence of the recA gene of Salmonella enterica sp., characterized in that it comprises a set of internal primers with the following nucleotide sequences a) and b), as well as a set of external primers containing the following nucleotide sequences c) and d) specific for a selected fragment of the recA gene of Salmonella enterica sp. bacteria: a) (nucleic sequence SEQ ID NO: 3 or its reverse and complementary sequence), linked from the 3' end, preferably by TTTT bridge, to the sequence (nucleic sequence SEQ ID NO: 4 or its reverse and complementary sequence) b) (nucleic sequence SEQ ID NO: 5 or its reverse and complementary sequence), linked at the 3' end, preferably by TTTT bridge, to the sequence (nucleic sequence SEQ ID NO: 6 or its reverse and complementary sequence) c) nucleic sequence SEQ ID NO: 1 or its reverse and complementary sequence, and d) nucleic sequence SEQ ID NO: 2 or its reverse and complementary sequence.

In a preferred embodiment of the invention, the primer set comprises a set of loop primer sequences comprising a nucleotide sequence that is reverse and complementary to the Salmonella enterica sp. recA gene with SEQ ID NO: 7 - or its reverse and complementary sequence, and a second nucleotide sequence identical to the Salmonella enterica sp. recA gene with SEQ ID NO: 8 - or its reverse and complementary sequence.

The second subject of the invention is a method for detecting Salmonella enterica sp. bacteria, characterized in that a selected region of the nucleotide sequence of the Salmonella enterica sp. genome (recA gene fragment) is amplified using a primer set as defined in the first subject of the invention, the amplification method being the LAMP method. In a preferred embodiment of the second subject of the invention, the amplification is carried out with a temperature profile: 68°C, 40 min. In a further preferred embodiment of the invention, the end-point reaction is carried out with additional temperature profile of 80°C, 5 min.

The third subject of the invention is a method for detecting the presence of Salmonella enterica sp. bacteria, characterized in that it comprises the detection method defined in the second subject of the invention.

The fourth subject of the invention is a kit for detecting the presence of Salmonella enterica sp. bacteria, characterized in that it comprises a set of primers as defined in the first subject of the invention. In a preferred embodiment of the invention, the infection detection kit comprises 5.0 pl WarmStart LAMP Master Mix. In a further preferred embodiment of the invention, individual amplification primers as defined in the first subject of the invention, the primers having the following concentrations: 0.13 μM F3, 0.13 μM B3, 1.06 μM FIP, 1.06 μM BIP, 0.26 μM LoopF, 0.26 μM LoopB; D- (+)-Trehalose dihydrate - 6%; mannitol - 1.25%; fluorescent marker interacting with double-stranded DNA - EvaGreen ≤1X (Biotium) or Fluorescent Dye (New England Biolabs) in the amount of ≤1 μl or Syto-13 ≤16 μM (ThermoFisher Scientific) or SYTO-82 ≤16 μM (ThermoFisher Scientific) or another fluorescent dye interacting with double- stranded DNA at a concentration that does not inhibit the amplification reaction.

The advantage of the primer sets of the invention for detecting Salmonella enterica sp., as well as the method of detecting the amplification products is the possibility of using them in medical diagnostics at the point of care (POCT) in the target application with a portable genetic analyser. The set of primers, which is the subject of this application, detects individual subspecies and serovars of bacteria of the Salmonella enterica sp. species, which was demonstrated by a bioinformatic analysis and experimentally by analysing both the recA gene sequence among the subspecies, as well as the sequences of individual primers included in the set. Freeze-drying of the reaction mixtures of the invention allows the diagnostic kits to be stored at room temperature without reducing the diagnostic parameters of the tests. In turn, the use of a fluorescent dye to detect the amplification product increases the sensitivity of the method, allows to lower the detection limit (down to 10 genome copies/reaction), as well as it enables the quantitative measurement of the bacteria in the test sample.

Exemplary embodiments of the invention are presented in the drawing, in which Fig. 1 and Fig. 2 show the characteristics of the method, where a specific signal was obtained with templates isolated from environmental samples: Salmonella enterica subsp. enterica and Salmonella enterica subsp. enterica serovar typhimurium, previously characterized according to Guidelines for serotyping of Salmonella spp. ISO/TR 6579-3:2014 where the identification of Salmonella bacilli was carried out according to the Regulation of the European Commission in accordance with ISO PN-EN ISO 6579-1:2017-04 Salmonella enterica sp., wherein the species profiling was performed using slide agglutination assay based on the Kauffmann-White antigenic scheme, polyvalent and monovalent sera were used for somatic antigens determination, while anti-Vi and anti-H sera were used for the detection of the Vi and H antigens, where Fig. 1 illustrates the result of electrophoresis performed in a 2% agarose gel: lane 1: mass marker (Quick-Load® Purple 100 bp DNA Ladder, NewEngland Biolabs); lane 2: NTC; lanes 3 and 4: S. enterica, sample no. 55; lane 5: NTC; lanes 6 and 7: S. enterica, sample no. All; lane 8: NTC; lanes 9 and 10: S. enterica, sample no. 75; and Fig. 2 illustrates the characteristics of the method where a specific signal was obtained with Salmonella enterica subsp. enterica serovar typhimurium obtained from environmental samples and the result of the performed 2% agarose gel electrophoresis: lane 1: mass marker (Quick-Load® Purple 100 bp DNA Ladder, NewEngland Biolabs); lane 2: NTC; lanes 3 and 4: S. enterica, serovar typhimurium, sample no. 124; lane 5: NTC; lanes 6 and 7: 2x S. enterica, serovar typhimurium, sample no. 125; lane 8: NTC; lanes 9 and 10: S. enterica, serovar typhimurium, sample no. 126; and Fig. 3 shows the specificity of the product resulting from the detection of Salmonella enterica and Salmonella enterica subsp. enterica serovar thypimurium measured with the amplification curve of the amplification product using classified environmental samples by measuring the fluorescence in real time for a specific reaction product, where the following microorganisms were included in the specificity test: Enterococcus faecalis, Pseudomonas aeruginosa, Lactobacillus gasseri, Escherichia coli, Campylobacter jejuni, Candida albicans, Proteus sp., Klebsiella sp., Citrobacter sp.

Tables 1 and 2 show the specificity of the method of the invention with standard templates of a number of pathogens potentially present in the tested biological material as natural physiological flora, those which may result from co-infections or those which share similar genomic sequences. A positive result was obtained with the templates of Salmonella enterica sample no. 55, Salmonella enterica sample no. 53, Salmonella enterica sample no. Alli, Salmonella enterica sample no. 75, Salmonella enterica sample no. 665 and S. enterica, serovar typhimurium sample no. 124, S. enterica, serovar typhimurium sample no. 125,

S. enterica, serovar typhimurium sample no. 126. There was no product with the other DNA standards tested (according to Tables 1 and 2).

Example 1. Primer sequences

The sequences of specific oligonucleotides used for the detection of Salmonella enterica sp. genetic material using LAMP technology are presented and characterized below.

1. The Salmonella enterica sp. recAF3 oligonucleotide sequence: is a sequence identical to the Salmonella enterica sp. recA gene (5'-3' strand), which from the 3' end precedes the F2 oligonucleotide by 5 nucleotides.

2. The Salmonella enterica sp. recAB3 oligonucleotide sequence: is a complementary fragment of the Salmonella enterica sp. recA gene (5'-3' strand) 182 nucleotides away from the 3' end of the oligonucleotide 1.

3. The Salmonella enterica sp. recAF2 oligonucleotide sequence: is a sequence identical to the Salmonella enterica sp. recA gene (5'-3' strand) 5 nucleotides away from the 3' end of the oligonucleotide 1.

4. The Salmonella enterica sp. recAB2 oligonucleotide sequence: is a complementary fragment of the Salmonella enterica sp. recA gene (5'-3' strand) 155 nucleotides away from the 3' end of the oligonucleotide 1. 5. The Salmonella enterica sp. recAF1c oligonucleotide sequence: is a complementary fragment of the Salmonella enterica sp. recA gene (5'-3' strand) 47 nucleotides away from the 3' end of the oligonucleotide 1.

6. The Salmonella enterica sp. recABlc oligonucleotide sequence: is a sequence identical to the Salmonella enterica sp. recA gene (5'-3' strand) 104 nucleotides away from the 3' end of the oligonucleotide 1.

7. The Salmonella enterica sp. recALoopF oligonucleotide sequence: .

8. The Salmonella enterica sp. recALoopB oligonucleotide sequence: .

The sequences of the Flc and F2 oligonucleotides have preferably been linked by a TTTT bridge and used as FIP. The sequences of the B1c and B2 oligonucleotides have preferably been linked by a TTTT bridge and used as BIP.

Example 2

The method of amplifying the recA gene of Salmonella enterica sp. using the oligonucleotides characterized in Example 1 with LAMP technology and the following composition of the reaction mixture:

5.0 μl WarmStart LAMP 2X Master Mix

0.13 μM F3

0.13 μM B3

1.06 μM FIP

1.06 μM BIP

0.26 μM LoopF 0.26 μM LoopB

D- (+)-Trehalose dihydrate - 6%

Mannitol - 1.25%

Fluorescent marker interacting with double-stranded DNA EvaGreen ≤1X or Fluorescent dye 50X (New England Biolabs) in the amount of 0.5 μl or GreenFluorescent Dye (Lucigen) in the amount of ≤1 μl or Syto-13 ≤16 μM or SYTO-82 ≤16 μM or another fluorescent dye that interacts with double-stranded DNA at a concentration that does not inhibit the amplification reaction.

DNA template extracted from previously characterized environmental samples of Salmonella enterica sp.

Total reaction volume adjusted to 10 μl with DNase and RNase free water.

Example 3

The method of amplifying the recA gene of Salmonella enterica sp. using the oligonucleotides characterized in Example 1,2 with LAMP technology and the composition of the reaction mixture characterized in Example 2 with the following temperature profile :

1) 68°C, 40 min

2) preferably for end-point reactions 80°C, 5 min.

Example 4

The method of amplification and detection of the recA gene of Salmonella enterica sp. using the oligonucleotides characterized in Example 1 with LAMP technology and the composition of the reaction mixture characterized in Example 2 with the temperature profile characterized in Example 3 and the detection method described below.

A fluorescent dye is used, capable of interacting with double- stranded DNA, added to the reaction mixture in an amount of 0.5 μl EvaGreen 20X; 0.5 μl or a concentration of ≤1X; ≤16 μM for GreenFluorescent Dye (Lucigen); SYTO-13 and SYTO-82, respectively, before starting the reaction, real-time and/or end-point measurement. Excitation wavelength in the range similar to the FAM dye - 490-500 nm (optimally 494 nm) for EvaGreen; Fluorescent dye 50X (New England Biolabs), GreenFluorescent Dye (Lucigen); SYTO-13 dyes and 535 nm (optimally 541 nm) for the SYTO-82 dye; emission wavelength in the range of 509-530 nm (optimally 518 nm) for EvaGreen; GreenFluorescent Dye (Lucigen); SYTO-13 dyes and 556 nm (optimally 560 nm) for the SYTO-82 dye, the method of detection, change recording time starting from 7 minutes from the start of the reaction for Salmonella enterica and the negative control.

Example 5. Description of the freeze-drying process

The method of preparation and freeze-drying of reagents for detecting the amplification and detection of the Salmonella enterica sp. recA gene using the oligonucleotides characterized in Example 1 with LAMP technology and the composition of the reaction mixture characterized in Example 2 with the temperature profile characterized in Example 3 and the detection method described in Example 4.

The reaction components were mixed according to the composition described in Example 2, except the template DNA, to a total volume of 10 μl. The mixture was transferred to 0.2 ml tubes and subjected to the freeze-drying process according to the parameters below. The mixture placed in the test tubes was pre-cooled to -80°C for 2 hours. Then the freeze-drying process was carried out at the temperature of -80°C for 3 hours under the pressure of 5 ^-2 mBar.

Tab. 1

Tab. 2

Sequence listing