PROTEINS FOR THE DETECTION OF STREPTOCOCCUS UBERIS AND DETECTION DEVICES

Abstract

The present invention relates to a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular to the protein with sequence SEQ ID NO: 2 and its use in the detection of the bacterium Streptococcus uberis. It also relates to a lateral flow chromatography (LFA) strip comprising said protein. [Fig. **]

Description

Title of the invention: PROTEINS FOR THE DETECTION OF STREPTOCOCCUS UBERIS AND DETECTION DEVICES technical field

[0001] The present invention relates to a new protein and its use in the detection of the bacterium Streptococcus uberis.EWc also relates to a lateral flow chromatography (LFA) strip comprising said protein. Previous technique

[0002] Mastitis is an infection of the udder of cows during the lactation period. The bacteria responsible are various.

[0003] When bovine mastitis is caused by a bacterial infection, this disease is primarily due to three bacteria: Staphylococcus aureus (S. aureus), Streptococcus uberis (S. uberis), and Escherichia coli (E. coli) (Paramanandham et al., 2021). Economic losses are estimated at €805 million in France and €6 billion in Europe (Neculai-Valeanu et al., 2021). These economic losses are not only related to a loss of milk yield and quality but also to animal losses due to the sending of infected animals to slaughter (Rédiger et al., 2022).

[0004] S. uberis is one of the most commonly identified pathogens responsible for clinical and subclinical (asymptomatic) bovine mastitis (Keane OM et al., 2019). A common constituent of the bovine intestinal flora, S. uberis can become an opportunistic pathogen in hosts with weakened immune systems.

[0005] S. uberis was primarily classified as an environmental causal agent of bovine mastitis, but it is contagious (from cow to cow) (Wente N et al., 2019). Although various risk mitigation strategies, such as improved milking practices, post-milking teat disinfection, and the administration of antimicrobials, have significantly reduced the incidence of mastitis derived from contagious pathogens, environmental strains of S. uberis continue to pose a major threat due to their high degree of contamination. As a result of the genetic heterogeneity within S. uberis populations, vaccines designed against a particular strain show low efficacy against others (Collado R et al., 2018).

[0006] The detection of S. uberis is important for herd management and for guiding the most appropriate treatment. When a cow is infected with a Gram-positive bacterium such as S. aureus or S. uberis, antibiotic treatment is necessary. However, the effectiveness of antibiotics varies depending on the bacterium. Antibiotics such as penicillins G and A are consistently effective against this bacterium. While resistance to penicillin and beta-lactams in general poses recurring problems in the treatment of other pathogens such as staphylococci or enterobacteria, it is important to detect Streptococcus uberis in order to choose the appropriate antibiotic.

[0007] Mastitis diagnostic devices currently on the market are primarily based on the detection of somatic cells found in milk. This technique can indicate that the cow is infected, but it does not identify the causative bacteria. Detection of the causative bacteria is often performed using culture methods or molecular biology techniques (PCR). Culture methods require incubation times of 24 to 48 hours, which is not suitable for the sector. Molecular biology techniques (PCR and / or quantitative PCR) allow for faster and more specific detection, but PCR only detects the presence of bacterial DNA and not the bacteria themselves, which can lead to false positives (dead bacteria that have already been eliminated by the cow's immune system or by a previous treatment).In all cases, both methods require sending samples to the laboratory. The number of samples must be significant to accurately reflect the problems encountered on the farm (4 samples per cow x the number of cows suspected of being infected) and represents a significant analysis cost.

[0008] Application WO2021 / 234321 describes a lateral flow chromatography device for the detection of Staphylococcus aureus based on the use of phage receptor-binding proteins, but this device does not allow the detection of S. uberis.

[0009] It is therefore important to develop a rapid detection method for S. uberis, usable in the field, not requiring large and expensive equipment, nor sending samples to the laboratory. Summary

[0010] In one aspect, the present application relates to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it relates to the protein of sequence SEQ ID NO: 2.

[0011] According to another aspect, the present application relates to a composition comprising: - a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular a protein of sequence SEQ ID NO: 1, and - a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular a protein of sequence SEQ ID NO: 2.

[0012] According to another aspect, the present application relates to the reuse of an RBP protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the use of the RBP protein of sequence SEQ ID NO: 2, for the capture of Streptococcus uberis in a sample.

[0013] According to another aspect, the present application relates to the use of a composition comprising: - a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1, in particular a protein of sequence SEQ ID NO: 1, and - a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular a protein of sequence SEQ ID NO: 2, for the capture of Streptococcus uberis in a sample.

[0014] According to another aspect, the present application relates to the use of an RBP protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the use of the RBP protein of sequence SEQ ID NO: 2 for the detection of Streptococcus uberis in a sample.

[0015] According to another aspect, the present application relates to the use of a composition comprising: - an RBP protein comprising a sequence having at least 70% identity with the SEQ ID NO sequence: 1, in particular a protein of sequence SEQ ID NO: 1, and - an RBP protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular a protein of sequence SEQ ID NO: 2, for the detection of Streptococcus uberis in a sample.

[0016] In particular, detection is carried out by ELIS A, lateral flow chromatography (LFA) or lateral flow chromatography with bacterial growth.

[0017] According to another aspect, the present application relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising at least one part which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, said at least one part comprising in particular the protein of sequence SEQ ID NO: 2.

[0018] In particular, the present application relates to a lateral flow chromatography strip comprising, in order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone of S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0019] In particular, the present application relates to a lateral flow chromatography strip, comprising in the following order: - a contact part comprising a contact area with the sample; - a marking part comprising a marking area which includes a marker; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part; said marker being conjugated to a specific ligand of S. uberis selected from at least one antibody directed against S. uberis, at least one aptamer specific to S. uberis, at least one bacteriophage specific to S. uberis, at least one protein from at least one bacteriophage specific to S. uberis, at least one bacteriophage specific to S. uberis coupled to an antibody directed against S. uberis, at least one protein from a bacteriophage specific to S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein from at least one bacteriophage specific to S.uberisc^ an RBP protein, more particularly, a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular this is the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, in particular said marker is a gold nanoparticle or a colored latex bead.

[0020] In particular, the present application relates to a lateral flow chromatography strip, comprising in the following order: . - a contact part comprising a contact area with the sample; - a first labeling part which includes a labeling area of ​​S. uberis comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said labeling area optionally comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a second labeling part which includes an S. aureus labeling area comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4, in particular the protein of sequence SEQ ID NO: 4 and / or comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the protein of sequence SEQ ID NO: 5; - a detection portion comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said detection zone for S. uberis optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and comprising a detection zone for S. aureus which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone for S. aureus includes the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0021] In one aspect, the present application relates to a method for detecting S. uberis in a sample comprising: - a step of bringing the sample into contact with the contact area of ​​a strip; - a marker detection step on the detection zone; - a step of analyzing the presence or absence of S. uberis in the sample.

[0022] According to one aspect, the present application relates to a method for detecting S. uberis comprising: 1) Addition of a sample to be tested into a nutrient medium containing a chromogenic element; 2) Contacting an LFA strip with the mixture obtained in step 1); 3) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 4) Incubate the strip in the closed tube at 37°C for 15 to 24 hours, preferably between 17 and 20 hours; 5) Appearance of a color along the test line if S. uèerA is present in the sample.

[0023] According to one aspect, the present application relates to a method for detecting S. uberis and S. aureus in a sample comprising: - a step of bringing the sample into contact with the contact area of ​​a strip; - a marker detection step on the detection zone; - a step of analyzing the presence or absence of S. uberis and the presence or absence of S. aureus in the sample.

[0024] According to one aspect, the present application relates to a S. uberis detection kit comprising: 1) A lateral flow chromatography strip to detect S. uberis; 2) A tube capable of closing hermetically; 3) A vial containing the nutrient medium with a chromogenic element, in particular potassium tellurite.

[0025] According to one aspect, the present application relates to a S. uberis detection kit or a S. uberis and S. aureus detection kit comprising: 1) A lateral flow chromatography strip to detect S. uberis or a lateral flow chromatography strip to detect S. uberis and S. aureus; 2) A container for receiving the sample. Brief description of the drawings Fig. 1

[0026] [Fig. 1] shows the detection results with an LFA device or strip 1 comprising, on the labeling area 8, the RBP P3 protein coupled to a gold nanoparticle (diameter 40 nm) and, on the test line 5 of the nitrocellulose membrane, the immobilized RBP P3 protein. Strip 1: Bacteria-free solution. Strip 2: S. aureus strain C24B solution at 1 x 10⁹ cfu / ml. Strip 3: E. coli strain 4ka solution at 1 x 10⁹ cfu / ml. Strip 4: S. uberis strain Duchesse solution at 1 x 10⁷ cfu / ml. Strip 5: S. uberis strain S49 solution at 1 x 10⁷ cfu / ml. The arrows indicate the test line on the strips. (cfu = colony-forming unit) Fig. 2

[0027] [Fig.2] shows the detection results with an LFA 1 device comprising on the Labeling zone 8 shows the RBP SUB4 protein coupled to a gold nanoparticle (40 nm diameter), and on test line 5 of the nitrocellulose membrane, the immobilized RBP SUB4 protein. Strip 1: Bacteria-free solution. Strip 2: S. aureus strain C24B solution at 1 x 10⁹ cfu / ml. Strip 3: E. coli strain 4ka solution at 1 x 10⁹ cfu / ml. Strip 4: S. uberis strain Duchesse solution at 1 x 10⁷ cfu / ml. Strip 5: S. uberis strain S49 solution at 1 x 10⁷ cfu / ml. The arrows indicate the test line on the strips. Fig. 3

[0028] [Fig.3] shows the detection results with an LFA 1 device comprising on the Labeling zone 8: RBP 28.7 protein coupled to a gold nanoparticle (40 nm diameter). On test line 5 of the nitrocellulose membrane: immobilized RBP 28.7 protein. Strip 1: Bacteria-free solution. Strip 2: S. aureus strain C24B solution at 10⁹ cfu / ml. Strip 3: E. coli strain 4ka solution at 10⁹ cfu / ml. Strip 4: S. uberis strain Duchesse solution at 10⁷ cfu / ml. Strip 5: S. uberis strain S49 solution at 10⁷ cfu / ml. Arrows indicate the test line on the strips. Fig. 4

[0029] [Fig.4] shows the detection results with an LFA 1 device comprising on the Labeling zone 8 contains a mixture of two proteins, RBP P3 and SUB4 (OD=5 to 600 nm), coupled to gold nanoparticles (diameter 40 nm). For control line 9, the protein on the membrane is VHS / G57 (UniProt: Q08487 / GenBank: CAA52080) at 2.22 mg / ml, and the antibody used to detect this protein on the conjugate is a mouse IgG2b anti-VHS / G57 monoclonal antibody coupled to gold at OD=2.5. Test line 5 immobilizes a mixture of two proteins, RBP P3 (0.625 mg / ml) and SUB4 (0.625 g / ml). Strip 1: Bacteria-free solution. Strip 2: S. aureus strain C24B solution at 1 x 10⁹ CFU / ml. Strip 3: S. uberis Duchesse strain solution at 1 x 10⁷ cfu / ml. Strip 4: S. uberis Duchesse strain solution at 1 x 10⁶ cfu / ml. Arrow A indicates the control line. Arrow B indicates the test line. Fig. 5

[0030] [Fig. 5] shows the detection results with an LFA device 1. A. Zone of Labeling area 8 includes RBP SUB4 protein coupled to gold nanoparticles (40 nm diameter) and RBP P3 protein immobilized on test line 5 of the nitrocellulose membrane. B. Labeling area 8 includes RBP SUB4 protein coupled to gold nanoparticles (40 nm diameter) and RBP SUB4 protein immobilized on test line 5 of the nitrocellulose membrane. C. Labeling zone 8 comprising the RBP SUB4 protein coupled to gold nanoparticles (diameter 40 nm) and the RBP P3 and SUB4 proteins immobilized on test line 5 of the nitrocellulose membrane Strip 1: Bacteria-free solution. Strip 2: S. aureus strain C24B solution. Strip 3: S. uberis strain F17W-PC solution. Strip 4: S. uberis strain F17W-GC solution. Strip 5: S. uberis strain 4475B solution. Strip 6: S. uberis strain 9038 solution. Strip 7: S. uberis strain 2EB1 solution. Strip 8: S. uberis strain 1912c solution. Strip 9: S. uberis strain 53G solution. Strip 10: S. uberis strain S52 solution. The strains are tested at an OD =1 at 600 nm which is equivalent to 1.109 cfu / ml for S. aureus strains and 1.107 cfu / ml for S. uberis strains. Fig. 6

[0031] [Fig.6] montre les résultats de detection avec un dispositif LFA 1 comprehend sur la zone de marquage 8 les protéines RBP SUB4 et P3 coupled à des nanoparticles d'or (diamètre 40 nm) et les protéines RBP P3 et SUB4 immobilisées sur la ligne de test 5 de la membrane de nitrocellulose. 1 : Without bacteria. 2: S. aureus (C24B). 3: E. coli (4Ka). 4: S. uberis (16B). 5: S. uberis (304T). 6: S. uberis (4475B). 7: S. uberis (50). 8: S. uberis (2192). 9: S. uberis (9038). 10: S. uberis (Duchesse). 11: 5. bountiful (Joconde). 12: S. uberis (Galande). 13: S. uberis (1912c). 14: S. uberis (1210). 15: S. uberis (6134). 16: S. uberis (9817a). 17: S. uberis (2EB1). 18: S. uberis (LMG1438b). 19: S. uberis (F17W-CG). 20: S. uberis (F17W-PC). 21: S. uberis (27J). 22: S. uberis (53PG-PC). 23: S. uberis (1912i). 24: S. uberis (S43). 25: S. uberis (S44). 26: S. uberis (S45). 27: S. uberis (S46). 28: S. uberis (S47). 29: S. uberis (S48). 30: S. uberis (S49). 31: S. uberis (S50). 32: S. uberis (S51).33: S. uberis (S52). The strains were tested for absorbance at OD 600 nm = 1, which is equivalent to 1.109 cfu / ml for S. aureus strains and 1.107 cfu / ml for S. uberis strains. Fig. 7

[0032] [Fig.7] shows the results of the detection of S. uberis in a milk sample using an LFA system and agar detection. A: The strips are directly adhered, nitrocellulose side down, to BHI IX + 75 pM tellurite + 1.5% agar. Incubation at 37°C for 15 hours is then performed before reading. Different S. uberis milk contaminations are tested: 1 = S. uberis Duchess strain (1 x 10⁴ cfu / ml); 2 = S. uberis Duchess strain (1 x 10³ cfu / ml); 3 = S. uberis Duchess strain (1 x 10² cfu / ml); 4 = NC milk (without bacteria). The arrows indicate the test line. B: The strips are placed in a cassette and a BHI IX + tellurite 75 pM + 0.4% agar is deposited on test area 5. The agar is then sealed with a adhesive. An incubation at 37°C for 15 hours is then carried out before reading. 1 = Milk without bacteria. 2 = Milk contaminated by two strains of S. aureus (C24B) 1.104 cfu / ml and S. uberis Joconde at 1.104 cfu / ml. Fig. 8

[0033] [Fig-8] shows the results of a duplex LFA for the simultaneous detection of S. S. aureus and S. uberis. The S. uberis-specific RBP P3 and SUB4 proteins, with SEQ ID NO: 1 and SEQ ID NO: 2 respectively, were loaded onto test line 5. These same proteins were also conjugated with gold nanoparticles and sprayed onto the labeling area at OD=5 (6 pl). The S. aureus-specific RBP PHIL 2 and PHIL 5 proteins, with SEQ ID NO: 4 and SEQ ID NO: 5 respectively, were loaded onto another test line 5. These same proteins were also conjugated with gold nanoparticles and sprayed onto the labeling area at OD=5 (6 pl). Four milk samples were tested: 1) Bacterial-free. 2) Milk contaminated with S. uberis alone, Duchesse strain at OD=1 (1 x 10⁷ cfu / ml). 3. Milk contaminated with S. aureus alone, strain C24B at OD=1 (1 x 10⁹ cfu / ml). 4. Milk contaminated with both bacteria. During migration, the contaminated milk was diluted 10-fold in 1% tween PBS IX buffer. Fig. 9

[0034] [Fig.9] shows the results of a duplex LFA for the simultaneous detection of S. S. aureus and S. uberis. The S. uberis-specific RBP P3 and SUB4 proteins, with SEQ ID NO: 1 and SEQ ID NO: 2 respectively, were loaded onto test line 5. The SUB4 protein was conjugated with red latex beads and sprayed onto the labeling area 8 with 0.03% dry bead extract. The S. aureus-specific RBP PHIL 2 and PHIL 5 proteins, with SEQ ID NO: 4 and SEQ ID NO: 5 respectively, were loaded onto another test line 5. These same proteins were conjugated with blue latex beads and sprayed onto the labeling area 8 with 0.5% dry bead extract for PHIL 2 and 0.5% dry bead extract for PHIL 5 (a mixture of PHIL 2 and PHIL 5 proteins loaded onto a strip). Four milk samples were tested: 1) No bacteria. 2) Milk contaminated with S. uberis alone, Duchesse strain at OD=10 (10 x 10⁷ cfu / ml). 3) Milk contaminated with S. aureus alone, S41 strain at OD=10 (10 x 10⁹ cfu / ml).4) Milk contaminated by a mixture of the two bacteria. During the migration, the milk used was diluted 10 times in PBS IX buffer with 1% tween20. Fig. 10

[0035] [Fig. 10] shows the results of a duplex LFA for the simultaneous detection of S. aureus and S. uberis. The S. uberis-specific RBP P3 and SUB4 proteins, with SEQ ID NO: 1 and SEQ ID NO: 2 sequences respectively, were deposited on a Test line 5. The SUB4 protein was conjugated to red latex beads and sprayed onto the labeling area 8 with 0.03% dry bead extract. The S. aureus-specific RBP proteins PHIL 2 and PHIL 5, with SEQ ID NO: 4 and SEQ ID NO: 5 respectively, were loaded onto another test line 5. An anti-S. aureus antibody was conjugated to blue latex beads and sprayed onto the labeling area 8 with 0.3% dry bead extract. Four milk samples were tested: 1) Bacterial-free. 2) Milk contaminated with S. uberis alone, Duchesse strain at OD=10 (10 x 10⁷ cfu / ml). 3) Milk contaminated with S. aureus alone, S41 strain at OD=10 (10 x 10⁹ cfu / ml). 4) Milk contaminated by a mixture of the two bacteria. During the migration, the milk used was diluted 10 times in PBS IX buffer with 1% tween20. Fig. 11

[0036] [Fig. 11] shows the results of an LFA device brought into contact with a sample supplemented with nutrient medium and a chromogenic compound (tellurite), for the detection of S. uberis. The strips do not have a labeling portion 7 and have in the detection portion 4 the S. uberis-specific RBP P3 and SUB4 proteins, with SEQ ID NO: 1 and SEQ ID NO: 2 respectively, immobilized on the test line 5. The photos of the strips are taken after 15 hours of incubation at 37°C. Different samples were tested: 1) uncontaminated milk, 2) Raw milk contaminated with S. uberis at 1.105 cfu / ml, 3) Raw milk contaminated with S. uberis at 1.104 cfu / ml, 4) Raw milk contaminated with S. uberis at 1.103 cfu / ml, 5) Raw milk contaminated with S. uberis at 1.102 cfu / ml, 6) control line, 7) S. uberis test line. Fig. 12

[0037] [Fig. 12] shows the results of a duplex LFA device in contact with a sample supplemented with nutrient medium and a chromogenic compound (tellurite), for the detection of S. uberis and S. aureus. The strips do not have a labeling section 7. In the detection section 4, the S. uberis-specific RBP P3 and SUB4 proteins, with SEQ ID NO: 1 and SEQ ID NO: 2 sequences respectively, were deposited on one test line 5. The S. aureus-specific RBP PHIL 2 and PHIL 5 proteins, with SEQ ID NO: 4 and SEQ ID NO: 5 sequences respectively, were deposited on another test line 5. The strips were photographed after 15 hours of incubation at 37°C. Different samples were tested: 1) uncontaminated milk (Ctrl), 2) Raw milk contaminated with S. aureus, strain 49 at 1.105 cfu / ml, 3) Raw milk contaminated with S. uberis, strain Duchesse at 1.105 cfu / ml, 4) Raw milk contaminated with S. uberis at 1.105 cfu / ml and S. aureus at 1.105 cfu / ml.Arrow A. indicates the test line. Arrow B indicates the S. uberis detection line (SU). Arrow C indicates the S. aureus detection line (SA). Fig. 13

[0038] [Fig. 13] shows a diagram of a sandwich-type ELISA test: 1. Marker coupled to an antibody. 2. Antibody directed against the analyte. 3. Analyte (bacteria). 4. Capture antibody directed against the analyte and immobilized on the support. Fig. 14

[0039] [Fig. 14] shows the diagram of an LFA strip 1 comprising a contact portion 2 which includes a contact zone 3, a labeling portion 7 which includes a labeling zone 8, a detection portion 4 which includes a detection zone 5 and a control zone 9, and an absorbing portion 6, immersed in a sample to be analyzed. The black lines delimit the different parts of the strip. Fig. 15

[0040] [Fig. 15] shows in A the diagram of an LFA strip 1 comprising a contact part 2 which includes a contact zone 3, a detection part 4 which includes a detection zone 5 and a control zone 9, and an absorbent part 6. In B, the method for detecting S. uberis and S. aureus by the LFA method with detection on agar with a strip as shown in A comprising two detection lines (test line) 5, one for S. uberis 5.1 and the other for S. aureus 5.2. After migration of the sample through the strip (I), nutrient medium containing agar is deposited (II), then after closing the system and incubating at 37°C (III), the development of bacteria is observed on the test line in the case where the sample is contaminated by said bacterium. Fig. 16

[0041] [Fig. 16] shows in A the schematic of an LFA strip 1 comprising a contact portion 2 which includes a contact zone 3, a detection portion 4 which includes a detection zone 5 and a control zone 9, and an absorbent portion 6. In B, the method for detecting a bacterium of interest by the LFA method with nutrient medium is shown. The strip 1 is immersed in the sample to be tested, which has been previously mixed with nutrient medium (1). After migration and incubation, bacterial growth is observed on the test line (2) if the sample is contaminated by said bacterium. DEFINITIONS

[0042] The following definitions are given which apply to the whole of this disclosure.

[0043] For the purposes of this application, "lateral flow assay" (LFA) means a chromatography device in which a The liquid sample migrates by capillary action in a directed flow through a strip comprising several parts. LFA is known from the prior art and has notably been described for the immunological detection of an analyte of interest in a liquid sample, using a primary antibody and a secondary antibody (EP1086372B1; US ​​8 399 261 B2; WO 2017 / 072078; Nanerjee and Jaiswal, 2018).

[0044] For the purposes of this application, "at least one" means one, two, three, four, five, six and so on.

[0045] For the purposes of this application, a "bacteriophage specific to a bacterium of interest" is a bacteriophage capable of binding specifically to that bacterium. This specificity can be determined by the spot method described in application WO2021 / 234321. A bacteriophage is specific to a bacterium if a plaque of lysis is observed around a spot containing said bacteriophage, deposited on a Petri dish covered with a culture of said bacterium in the exponential phase, after 18 hours of incubation at a temperature suitable for the bacterium.

[0046] Phage receptor-binding proteins (RBPs) determine the specificity of a phage for a host bacterium. In phages, these proteins are located in the tail and are involved in the first stage of the phage infection cycle (lytic or lysogenic). They ensure initial contact with the receptor on the host cell envelope. Indeed, RBPs recognize target motifs on the surface of bacteria and thus allow the specific binding of a phage to a host bacterium. The RBPs of each phage strain are different and thus determine the spectrum of activity of each phage (ability to recognize several bacterial strains).

[0047] RBP proteins are distinct from cell wall-binding domains (CBDs). RBPs are involved in the early stages of the phage infection cycle, whereas CBDs are involved in the later stages. CBDs are protein domains found in lytic enzymes produced by phages that enable the recognition of peptidoglycans in cell walls. For example, endolysin is an enzyme produced in the final stage of the lytic cycle to cleave the bacterial cell wall and release the descending virion. Endolysins targeting Gram-positive bacteria typically have a modular structure comprising an enzymatically active domain (EAD) for hydrolysis and a cell wall-binding domain (CBD) for peptidoglycan recognition.Endolysins specifically recognize the peptidoglycan (PG) structure of the host bacterium and then hydrolyze the cell wall into fragments, leading to osmotic cell lysis.

[0048] The recognition specificity of a phage protein for a bacterium can be established by placing the bacterium in contact with coated magnetic beads of a phage protein, followed by the retrieval of magnetic beads using a magnetic support, and then, after resuspension in buffer, spreading onto a culture medium (in particular, a specific agar). After 24 to 48 hours at a temperature suitable for the bacteria, a significant difference between the bacterial colony count on the culture medium where the protein-coated magnetic beads were spread and the bacterial colony count on a culture medium where magnetic beads incubated with the bacteria but not coated with the protein indicates that the phage protein is capable of binding to the bacteria.

[0049] Specificity can also be evaluated by the ELISA test as described in the application. Bacteria are fixed to the bottom of the wells of a plate. RBP proteins, coupled to a GTS tag during their production, are deposited into each well. After incubation, a washing step is performed to remove unbound proteins. An incubation step with an anti-GTS antibody coupled to HRP or another substrate is then performed to reveal the presence of the bacteria.

[0050] In the present application, the terms “bacteriophage” and “phage” refer to the same entity, are used interchangeably and are interchangeable.

[0051] In the present application, the expressions "antibody directed against X" and "anti-X antibody" are equivalent and interchangeable.

[0052] In the present application, the expressions "X conjugate to Y" and "Y conjugate to X" are equivalent. The term "marked" means "conjugated to a marker". Description of the invention

[0053] A first aspect of the invention relates to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2.

[0054] In particular, the present invention relates to a protein of sequence SEQ ID NO: 2. This protein, also referred to as SUB4 in the present application, is an RBP protein of a specific bacteriophage of S. uberis.

[0055] A second aspect of the invention relates to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1.

[0056] In particular, the present invention relates to a protein of sequence SEQ ID NO: 1. This protein, also referred to as P3 in the present application, is an RBP protein of a specific bacteriophage of S. uberis.

[0057] A third aspect of the invention relates to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3.

[0058] In particular, the present invention relates to a protein of sequence SEQ ID NO: 3. This protein, also referred to as 28.7 in the present application, is an RBP protein of a specific bacteriophage of S. uberis.

[0059] For the purposes of this application, a percentage of identity of at least 70% may, in particular, be at least 75%, at least 80%, at least 85%, more particularly at least 90% or even at least 95%. This definition applies in particular to proteins having a percentage of identity of at least 70% with one of the proteins of sequence SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, as described above, as well as to proteins having a percentage of identity of at least 70% with one of the proteins of sequence SEQ ID NO:4 or of sequence SEQ ID NO:5 as described below.

[0060] A fourth aspect of the invention relates to a composition comprising: - a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, and - a protein comprising a sequence having a percentage of identity of at least 70% with the SEQ ID NO sequence: 2.

[0061] In particular, said composition comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2.

[0062] Said composition may further comprise a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3.

[0063] In particular, said composition comprises the SEQ ID NO:1 sequence protein, the SEQ ID NO:2 sequence protein and the SEQ ID NO:3 sequence protein.

[0064] A fifth aspect of the invention relates to the use of a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, for the capture of the bacterium S. uberis in a sample.

[0065] For the purposes of this application, a sample means any solution likely to contain the bacterium of interest. The sample may be diluted in a buffer solution prior to carrying out the detection method.

[0066] In particular, the invention relates to the use of the SEQ ID NO:1 sequence protein and / or the SEQ ID NO:2 sequence protein, and / or the SEQ ID NO:3 sequence protein for the capture of the bacterium S. uberis.

[0067] In particular, the invention relates to the use of a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2 for the capture of the bacterium S. uberis in a sample, more particularly on the use of the protein of sequence SEQ ID NO: 2 for the capture of the bacterium S. uberis in a sample.

[0068] In particular, the invention relates to the use of a composition comprising: - a protein comprising a sequence having at least 70% identity with the SEQ ID NO sequence: 1, and - a protein comprising a sequence having at least 70% identity with the SEQ ID NO sequence: 2 for the capture of S. uberis bacteria in a sample, more specifically on the use of a composition comprising the SEQ ID NO:1 sequence protein and the SEQ ID NO:2 sequence protein for the capture of S. uberis bacteria in a sample.

[0069] Said composition may further comprise a receptor-binding protein (RBP) of a specific S. uberis bacteriophage, comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular it may further comprise the protein of sequence SEQ ID NO: 3.

[0070] A sixth aspect of the invention relates to the use of a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, for the detection of the bacterium S. uberis in a sample.

[0071] In particular, the invention relates to the use of the SEQ ID NO:1 sequence protein and / or the SEQ ID NO:2 sequence protein, and / or the SEQ ID NO:3 sequence protein for the detection of the bacterium S. uberis.

[0072] In particular, the invention relates to the use of a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2 for the detection of the bacterium S. uberis in a sample, more particularly on the use of the protein of sequence SEQ ID NO: 2 for the detection of the bacterium S. uberis in a sample.

[0073] In particular, the invention relates to the use of a composition comprising: - a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, and - a protein comprising a sequence having at least 70% identity with the SEQ ID NO sequence: 2 for the detection of S. uberis bacteria in a sample, more specifically on the use of a composition comprising the SEQ ID NO:1 sequence protein and the SEQ ID NO:2 sequence protein for the detection of S. uberis bacteria in a sample.

[0074] Said composition may further comprise a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular it may further comprise the protein of sequence SEQ ID NO: 3.

[0075] Said detection can be carried out by ELIS A, lateral flow chromatography or lateral flow chromatography with bacterial growth.

[0076] In a first embodiment, the detection is carried out using an ELIS A test.

[0077] ELISA (enzyme-linked immunosorbent assay) is a solid-supported enzyme immunoassay based on the specific antigen-antibody reaction. The classic principle of a sandwich-type ELISA is to use a support, such as a microplate, on which a ligand specific to the bacterium of interest is immobilized. After mixing the support with the sample likely to contain the bacterium of interest and rinsing, a primary antibody specific to the bacterium of interest is added. This primary antibody is coupled to a marker, thus enabling the detection of the analyte in the event of a signal. Figure 13 illustrates detection by the ELISA method.

[0078] A protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular, the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, can be used in an ELISA as a specific ligand on the support.

[0079] There is also indirect ELISA, in which the primary antibody is not coupled to a marker, and a secondary antibody directed against the primary antibody, which is coupled to a marker, is added.

[0080] When the marker is an enzyme, the addition of the enzyme substrate allows a color reaction to be obtained that can be measured by absorbance.

[0081] In an alternative embodiment, the antibody can be replaced by another protein having an affinity for the bacterium of interest. This could, for example, be an RBP protein. Thus, in another embodiment, a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 1 sequence and / or a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 2 sequence, and / or a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 3 sequence, in particular, the protein with sequence SEQ ID NO: 1 and / or the protein with sequence SEQ ID NO: 2 and / or the protein with sequence SEQ ID NO: 3, can be used in an ELISA for the detection of S. uberis, replacing the capture antibody and / or the primary antibody coupled to the marker.

[0082] In a second embodiment, detection is carried out by lateral flow chromatography (LFA). The principle of detection by LFA is described in Example 2 and illustrated in [Fig. 14].

[0083] Thus, the present invention relates to the use of a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 2, and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 3, in a lateral flow chromatography strip. In particular, it relates to the use of the protein with sequence SEQ ID NO: 1 and / or the protein with sequence SEQ ID NO: 2, and / or the protein with sequence SEQ ID NO: 3 in a lateral flow chromatography strip.

[0084] The lateral flow chromatography strips are as described later in this application.

[0085] Said protein is used in at least a part of the LFA strip. It can be used in the labeling area of ​​the labeling part and / or in the detection area (test line) of the detection part.

[0086] In particular, the protein with sequence SEQ ID NO: 2 is used in the labeling area. In particular, the proteins with sequence SEQ ID NO: 1 and SEQ ID NO: 2 are used in the labeling area.

[0087] In particular, the protein with sequence SEQ ID NO: 2 is used in the detection zone (test line). In particular, the proteins with sequence SEQ ID NO: 1 and SEQ ID NO: 2 are used in the detection zone (test line).

[0088] In a third embodiment, detection is carried out by lateral flow chromatography (LFA) with bacterial growth on the detection area, in particular at the detection zone (test line) which includes a protein comprising a sequence having a percentage of identity of at least 70% with the SEQ ID NO: 1 sequence and / or a protein comprising a sequence having a percentage of identity of at least 70% with the SEQ ID NO: 2 sequence, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the SEQ ID NO: 3 sequence.

[0089] In particular, the detection zone includes the protein with sequence SEQ ID NO: 1 and / or the protein with sequence SEQ ID NO: 2, and / or the protein with sequence SEQ ID NO: 3. More particularly, it includes the protein with sequence SEQ ID NO: 2, and even more particularly the proteins with sequence SEQ ID NO: 1 and SEQ ID NO: 2.

[0090] This detection method with bacterial growth encompasses two concepts detailed further below: - after migration, add agar to the detection area, and - before migration, add the sample to a nutrient medium.

[0091] In particular, said agar and said nutrient medium comprise a chromogenic element.

[0092] A seventh aspect of the invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample.

[0093] The embodiments of the lateral flow chromatography strip described below are described before use of said strip, i.e. before contact with the sample to be analyzed.

[0094] Thus, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising at least one part which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3. In particular, said at least one part includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3.

[0095] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising at least one part which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular said at least one part comprises the protein of sequence SEQ ID NO: 2.

[0096] Said part comprising said protein may be a labeling part and / or a detection part.

[0097] In one embodiment, the part comprising the protein is a detection part.

[0098] Thus, in a particular embodiment, said lateral flow chromatography strip for detecting S. uberis in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a detection area comprising a detection zone for S. uberis which includes a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 2, and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 3, in particular the detection zone includes the SEQ sequence protein ID NO: 1 and / or SEQ sequence protein ID NO: 2, and / or SEQ sequence protein ID NO: 3, - an absorbing part.

[0099] In particular, the lateral flow chromatography strip for detecting S. uberis in a sample comprises, in order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0100] In particular, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbing part.

[0101] The contact part (sample pad) comprises a contact area intended to be brought into contact with the sample. Its function is to distribute the sample evenly so that the migration of the sample by capillary action to the subsequent parts is smooth, continuous, and homogeneous. The contact part and the contact area are made of a capillary matrix. For example, the contact part and the contact area are made of cellulose or glass fibers.

[0102] The detection pad comprises a detection zone where the bacterium of interest, S. uberis, if present in the sample analyzed, is captured on the strip by means of a specific RBP protein immobilized therein. The detection zone is also called the detection line or test line. The role of the detection pad is therefore to provide good support and binding for the immobilization of specific proteins, while limiting non-specific adsorption. For example, the detection pad and the detection zone are made of a nitrocellulose membrane.

[0103] The absorbent pad serves an absorption role. It helps maintain the flow rate through the test strip and allows for the recovery of excess sample and reagents, thus preventing sample reflux. For example, the absorbent pad is made of cotton, cotton blended with glass fibers, cellulose, or nitrocellulose.

[0104] For the purposes of this application, "in order" means the order in which the different parts that make up the strip will be traversed by the flow comprising the sample, but this term does not mean that these parts are necessarily contiguous.

[0105] In a particular embodiment, one end of the contact portion is superimposed with one end of the portion downstream of the contact portion to form a first superposition zone; one end of the detection portion is superimposed with one end of the portion upstream of the contact portion to form a second superposition zone; the other end of the detection portion is superimposed with one end of the portion downstream of the contact portion to form a third superposition zone; and one end of the absorbing portion is superimposed with one end of the portion downstream of the absorbing portion to form a fourth superposition zone. In particular, the first, second, and third superposition zones are distinct from one another. In particular, the contact zone is distinct from the first superposition zone.In particular, the detection zone is distinct from the second and third overlap zones.

[0106] In a particular embodiment, one end of the sensing part is superimposed with one end of the contact part and the other end of the sensing part is superimposed with one end of the absorbing part.

[0107] In a more particular embodiment, one end of the sensing part is superimposed with one end of the contact part to form a first superposition zone, and the other end of the sensing part is superimposed with one end of the absorbing part to form a second superposition zone. In particular, the first, second, and third superposition zones are separated from each other. In particular, the contact zone is distinct from the first superposition zone. In particular, the sensing zone is distinct from the first and second superposition zones.

[0108] In a particular embodiment, the detection zone is a line transverse to the strip, thus forming a detection line or test line.

[0109] Optionally, the strip includes a system for controlling the migration of the sample from the contact portion to the detection portion. Thus, optionally, the detection portion includes a control zone, preferably downstream of the detection zone. This control zone is separate from the detection zone. detection. In particular, it is also distinct from the overlapping areas between the strip sections. Specifically, the control area is a line transverse to the strip, thus forming a control line or control line. The control area contains a ligand specific to an analyte other than the bacterium of interest, S. uberis. The analyte is conjugated to a marker. Optionally, the marker-conjugated analyte is deposited on a portion of the strip upstream of the detection area and / or is added to the sample beforehand. For example, the labeled analyte is a primary antibody conjugated to a marker, and the ligand on the control area is a secondary antibody directed against the primary antibody conjugated to a marker.

[0110] When the strip comprises three parts, namely a contact part, a detection part and an absorbent part, detection can be carried out for example with bacterial growth on the detection part, in particular at the level of the detection area (test line).

[0111] This detection method with bacterial growth encompasses two concepts: - after migration, addition of agar to the detection area, and - before migration, addition of the sample to a nutrient medium.

[0112] In particular, said agar and said nutrient medium comprise a chromogenic element.

[0113] The concept of the LFA detection method with bacterial growth and, after migration, addition of agar to the detection area is described in Example 3 and illustrated in [Fig. 15].

[0114] The LFA strip used in this embodiment is a strip as described in this application, comprising a contact portion, a detection portion, and an absorbent portion. It does not include a labeling portion (conjugate pad). The sample to be tested migrates directly through the detection portion, which includes a detection zone (test line) where a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 2, and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 3 is immobilized by adsorption. In particular, the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2 and / or the protein of sequence SEQ ID NO: 3.

[0115] Thus, in this embodiment, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact zone with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular, the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, - an absorbing part.

[0116] In particular, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0117] In particular, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbing part.

[0118] The method for detecting S. uberis by LFA with bacterial growth comprises the following steps: 1) Bringing the LFA strip, as described above, into contact with the sample to be tested; 2) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 3) Removal of the strip; 4) Delineation of the detection zone (test line); 5) Deposition of the cut test line onto an agar plate, or fixation of the cut detection area in a cassette which is then covered with agar; 6) Incubation at 37°C for 15 to 48 hours, particularly between 18 and 24 hours; 7) Appearance of colonies along the test line if S. uberis is present in the sample.

[0119] In particular, the agar comprises a chromogenic element.

[0120] The method for detecting S. uberis by LFA with bacterial growth comprises Then the following steps: 1) Contacting an LFA strip as described above with the sample to be tested; 2) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 3) Removal of the strip; 4) Delineation of the detection zone (test line); 5) Deposition of the cut test line onto an agar containing a chromogenic element, or fixation of the cut detection area in a cassette which is then covered with agar containing a chromogenic element; 6) Incubation at 37°C for 15 to 48 hours, particularly between 18 and 24 hours; 7) Appearance of a color along the test line if S. uberis is present in the sample.

[0121] In steps 1), the strip is immersed in the sample so that the contact area of ​​the contact part is immersed in the sample.

[0122] Optionally, the sample can be diluted in a buffer solution. This facilitates migration.

[0123] In particular, the agar mentioned in steps 5) is an agar comprising BHI IX (Brain heart infusion) and agar between 0.4% and 2% (weight / volume).

[0124] If the agar includes a chromogenic element, the chromogenic element is an element that S. uberis is capable of hydrolyzing and whose hydrolysis product is colored, i.e. detectable in the visible, i.e. between 380 nm and 780 nm.

[0125] When the chromogenic element is potassium tellurite, the appearance of a black coloration along the test line indicates the presence of S. uberis in the tested sample. This is because S. uberis is capable of hydrolyzing potassium tellurite into tellurium, which is a black compound. Conversely, the absence of a black coloration indicates the absence of S. uberis in the sample.

[0126] In particular, the concentration of potassium tellurite in the agar is between 1.5 pM and 150 pM, in particular between 50 and 100 pM, in particular it is 75 pM.

[0127] The present invention relates to a kit for the detection of S. uberis by LFA with bacterial growth comprising: 1) A lateral flow chromatography strip as previously described for detecting S. uberis in a sample, 2) A vial of agar optionally containing a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally, a container to receive the sample.

[0128] In particular, the S. uberis detection kit by LFA with bacterial growth comprises: 1) A lateral flow chromatography strip for detecting S. uberis in a sample includes, in order at least: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, - an absorbing part; 2) A vial of agar optionally containing a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally, a container to receive the sample.

[0129] In particular, the S. uberis detection kit by LFA with bacterial growth comprises: 1) A lateral flow chromatography strip for detecting S. uberis in a sample includes, in order at least: - a contact part comprising a contact area with the sample; - a detection portion comprising a detection zone for S. uberis which includes a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1, in particular the protein with sequence SEQ ID NO: 1; - an absorbent part. 2) A vial of agar optionally containing a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally, a container to receive the sample.

[0130] In particular, the S. uberis detection kit by LFA with bacterial growth comprises: 1) A lateral flow chromatography strip for detecting S. uberis in a sample includes, in order at least: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbing part; 2) A vial of agar optionally containing a chromogenic element such as potassium tellurite; 3) Optionally, a container for receiving the sample; 4) Optionally, a cassette.

[0131] The concept of the LFA detection method with bacterial growth and, prior to migration, addition of the sample to a nutrient medium is described in Example 8 and illustrated in [Fig. 16].

[0132] The LFA strip used in this embodiment is a strip as described in this application, comprising a contact portion, a detection portion, and an absorbent portion. It does not include a labeling portion (conjugate pad). The sample to be tested migrates directly through the detection portion, which includes a detection zone (test line) where a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 2, and / or a protein comprising a sequence having at least 70% identity with sequence SEQ ID NO: 3 is immobilized by adsorption. In particular, the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2 and / or the protein of sequence SEQ ID NO: 3.

[0133] Thus, in this embodiment, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact zone with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, - an absorbing part.

[0134] In particular, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0135] In particular, said lateral flow chromatography strip for detecting S. uberis in a sample, comprises in at least the following order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbing part.

[0136] The method for detecting S. uberis by LFA with bacterial growth comprises the following steps: 1) Addition of a sample to be tested into a nutrient medium; 2) Contacting an LFA strip as described above with the mixture obtained in step 1); 3) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 4) Incubate the strip at 37°C for 15 to 24 hours, preferably between 18 and 20 hours; 5) Appearance of colonies along the test line if S. uberis is present in the sample.

[0137] In particular, the nutrient medium comprises a chromogenic element.

[0138] The method for detecting S. uberis by LFA with bacterial growth then comprises the following steps: 1) Addition of a sample to be tested into a nutrient medium containing a chromogenic element; 2) Contacting an LFA strip as described above with the mixture obtained in step 1); 3) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 4) Incubate the strip at 37°C for 15 to 24 hours, preferably between 18 and 20 hours; 5) Appearance of a color along the test line if S. uberis is present in the sample.

[0139] When the migration ends, i.e. when all the elements of the strip, including the absorbent part, are soaked with solution, there is still enough liquid left at the contact part, which prevents dehydration of the strip.

[0140] In steps 1), the strip is immersed in the sample so that the contact area of ​​the contact part is immersed in the sample.

[0141] Typically, the sample / nutrient medium ratio is 1:10.

[0142] The nutrient medium may be BHI IX (Brain Heart Infusion) medium. Optionally, it may comprise 1% (weight / volume) of Tween 20.

[0143] If the nutrient medium includes a chromogenic element, the chromogenic element is an element that S. uberis is able to hydrolyze and whose hydrolysis product is colored, i.e. detectable in the visible, i.e. between 380 nm and 780 nm.

[0144] When the chromogenic element is potassium tellurite, the appearance of a black coloration along the test line indicates the presence of S. uberis in the tested sample. This is because S. uberis is capable of hydrolyzing potassium tellurite into tellurium, which is a black compound. Conversely, the absence of a black coloration indicates the absence of S. uberis in the sample.

[0145] In particular, the concentration of potassium tellurite is between 5 mg / mL and 50 mg / mL, more particularly between 15 mg / L and 25 mg / L, in particular it is 20 mg / L.

[0146] In particular, steps 2), 3) and 4) are carried out in a tube capable of being hermetically sealed.

[0147] Indeed, the sample to be tested is added to the nutrient medium, which includes, in particular, a chromogenic element. The mixture is homogenized and poured into a hermetically sealed tube. The test strip is then immersed in the mixture as described in step 2), so that the contact area of ​​the test strip is submerged in the mixture.

[0148] The tube is then closed and held vertically with the strip inside for migration as indicated in step 3), then incubated as described in step 4).

[0149] The hermetically sealed tube has dimensions suitable for holding the LFA strip in a vertical position while ensuring an airtight seal. This prevents the strip from drying out during incubation. In particular, it is a transparent tube, allowing the detection result to be read through it without needing to open the tube.

[0150] The present invention also relates to a kit for the detection of S. uberis by LFA with bacterial growth comprising: 1) A lateral flow chromatography strip as previously described for detecting S. uberis in a sample, 2) A tube capable of closing hermetically; 3) A vial containing the nutrient medium with a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0151] In particular, the S. uberis detection kit includes: 1) A lateral flow chromatography strip for detecting S. uberis in a sample includes, in order at least: - a contact part comprising a contact area with the sample; - a detection portion comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, - an absorbent part; 2) A tube capable of closing hermetically; 3) A vial containing the nutrient medium with a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0152] In particular, the S. uberis detection kit includes: 1) A lateral flow chromatography strip for detecting S. uberis in a sample includes, in order at least: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part; 2) A tube capable of closing hermetically; 3) A vial containing the nutrient medium with a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0153] In particular, the S. uberis detection kit by LFA with bacterial growth comprises: 1) A lateral flow chromatography strip for detecting S. uberis in a sample includes, in order at least: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbing part; 2) A tube capable of closing hermetically; 3) A vial containing the nutrient medium with a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0154] In a particular embodiment, said lateral flow chromatography strip enables the detection of S. uberis and one or more other bacteria of interest in a sample. The detection portion then comprises one or more additional detection zones on which the appropriate ligands for detecting this or these other bacteria of interest are immobilized.

[0155] This type of strip is called “Duplex LFA”.

[0156] In a particular embodiment, said lateral flow chromatography strip enables the detection of S. uberis and S. aureus in a sample.

[0157] In a particular embodiment, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample is a strip as described above, comprising three parts, namely a contact part, a detection part, and an absorbing part, and which includes an additional detection zone within the detection part. A specific ligand of S. aureus is immobilized on this additional detection zone, or test line.

[0158] This S. aureus-specific ligand may be at least one antibody directed against S. aureus, at least one S. aureus-specific aptamer, at least one S. aureus-specific bacteriophage, at least one protein from at least one S. aureus-specific bacteriophage, at least one S. aureus-specific bacteriophage coupled to an antibody directed against S. aureus, or at least one protein from an S. aureus-specific bacteriophage coupled to an antibody directed against S. aureus. In particular, said at least one protein from at least one S. aureus-specific bacteriophage is an RBP protein. More specifically, it may be a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 5. Typically, this could be the SEQ ID NO:4 sequence protein and / or the SEQ ID NO:5 sequence protein.

[0159] Thus, for example, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample comprises in the following order at least: - a contact part comprising a contact zone with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, and comprising an S. aureus detection zone which includes an antibody directed against S. aureus; - an absorbent part.

[0160] In particular, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample comprises, in order at least: - a contact part comprising a contact area with the sample; - a detection portion comprising an S. uberis detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said S. uberis detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and comprising an S. aureus detection zone which includes an antibody directed against S. aureus; - an absorbent part.

[0161] In particular, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample comprises, in order at least: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular it includes the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, and comprising a detection zone for S. aureus which includes an antibody directed against S. aureus; - an absorbent part.

[0162] For example, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample comprises, in order at least: - a contact part comprising a contact area with the sample; - a detection portion comprising a S. uberis detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the S. uberis detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, and comprising an S. aureus detection zone which includes an S. aureus specific ligand, in particular a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the S. aureus detection zone includes the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0163] In particular, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample comprises, in order at least: - a contact part comprising a contact area with the sample; - a detection portion comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said detection zone for S. uberis optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and comprising a detection zone for S. aureus which includes a specific ligand for S. aureus, in particular a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone for S. aureus includes the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0164] In particular, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample comprises, in order at least: - a contact part comprising a contact area with the sample; - a detection portion comprising a S. uberis detection zone which includes a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2 and a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1, in particular it includes the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, and comprising an S. aureus detection zone which includes a specific ligand of S. aureus, in particular a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 4 and a protein comprising a sequence having at least 70% identity with the SEQ ID NO sequence: 5, in particular the protein with SEQ ID NO sequence: 4 and the protein with SEQ ID NO sequence: 5; - an absorbent part.

[0165] In the detection part, the detection zone of S. uberis and the detection zone of S. aureus are distinct from each other, i.e. they do not overlap.

[0166] In a particular embodiment, the detection of S. uberis and another bacterium of interest is carried out by the concept of the LFA detection method with bacterial growth with an LFA strip also allowing the detection of one or more other bacteria of interest, and after migration, addition of agar on the detection area.

[0167] The LFA strip used in this embodiment is a so-called "Duplex" LFA strip as described above, which comprises a contact portion, a detection portion, and an absorbent portion. It does not include a marking portion (conjugal pad).

[0168] In particular, this is an LFA strip for detecting S. uberis and S. aureus in a sample as described above.

[0169] The method for detecting S. uberis and S. aureus then comprises the following steps: 1) Contacting an LFA strip as described in this application with the sample to be tested; 2) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 3) Removal of the strip; 4) Delineation of the detection zone (test line); 5) Deposition of the cut test line onto an agar plate optionally comprising a chromogenic element, or fixation of the cut detection area in a cassette which is then covered with agar plate optionally comprising a chromogenic element; 6) Incubation at 37°C for 15 to 48 hours, particularly between 18 and 24 hours; 7) Appearance of colonies or appearance of a color, in the case where the agar includes a chromogenic element, along the test line of S. uberis in the event of the presence of S. uberis in the sample and / or appearance of colonies or appearance of a color, in the case where the agar includes a chromogenic element, along the test line of S. aureus in the event of the presence of S. aureus in the sample.

[0170] In particular, it is an agar comprising BHI IX (Brain heart infusion) and agar between 0.4% and 2% (weight / volume).

[0171] If the agar includes a chromogenic element, the chromogenic element is an element that S. uberis and S. aureus are able to hydrolyze and whose hydrolysis product is colored, i.e. detectable in the visible, i.e. between 380 nm and 780 nm.

[0172] When the chromogenic element is potassium tellurite, the appearance of a black coloration along the test line indicates the presence of S. uberis and / or S. aureus in the tested sample. Indeed, S. uberis and S. aureus are capable of hydrolyzing potassium tellurite into tellurium, which is a black compound. Conversely, the absence of a black coloration indicates the absence of S. uberis and / or S. aureus in the sample.

[0173] In particular, the concentration of potassium tellurite in the agar is between 1.5 pM and 150 pM, in particular between 50 and 100 pM, in particular it is 75 pM.

[0174] The present invention also relates to a S. uberis and S. aureus detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis and S. aureus as described in this application; 2) A vial of agar optionally containing a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally, a container to receive the sample.

[0175] In a particular embodiment, the detection of S. uberis and another bacterium of interest is carried out by the concept of the LFA detection method with bacterial growth with an LFA strip also allowing the detection of one or more other bacteria of interest and, before migration, addition of the sample in a nutrient medium.

[0176] The LFA strip used in this embodiment is a so-called "Duplex" LFA strip as described above, which comprises a contact portion, a detection portion, and an absorbent portion. It does not include a marking portion (conjugal pad).

[0177] In particular, this is an LFA strip for detecting S. uberis and S. aureus in a sample as described above.

[0178] The method for detecting S. uberis and S. aureus then comprises the following steps: 1) Addition of a sample to be tested into a nutrient medium optionally including a chromogenic element; 2) Contacting an LFA strip as described in this application with the mixture obtained in step 1); 3) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 4) Incubation at 37°C for 15 to 24 hours, especially between 6 and 8 pm; 5) Appearance of colonies or appearance of a color, in the case where the agar includes a chromogenic element, along the test line of S. uberis in the event of the presence of S. uberis in the sample and / or appearance of colonies or appearance of a color, in the case where the agar includes a chromogenic element, along the test line of S. aureus in the event of the presence of S. aureus in the sample.

[0179] Typically, the sample / nutrient medium ratio is 1:10.

[0180] The nutrient medium may be BHI IX (Brain Heart Infusion) medium. Optionally, it may comprise 1% (weight / volume) of Tween 20.

[0181] If the nutrient medium includes a chromogenic element, the chromogenic element is an element that S. uberis and S. aureus are able to hydrolyze and whose hydrolysis product is colored, i.e. detectable in the visible, i.e. between 380 nm and 780 nm.

[0182] When the chromogenic element is potassium tellurite, the appearance of a black coloration along the test line indicates the presence of S. uberis and / or S. aureus in the tested sample. Indeed, S. uberis and S. aureus are capable of hydrolyzing potassium tellurite into tellurium, which is a black compound. Conversely, the absence of a black coloration indicates the absence of S. uberis and / or S. aureus in the sample.

[0183] In particular, the concentration of potassium tellurite in the nutrient medium is between 5 mg / mL and 50 mg / mL, more particularly between 15 mg / L and 25 mg / L, in particular it is 20 mg / L.

[0184] In particular, steps 2), 3) and 4) are carried out in a tube capable of being hermetically sealed.

[0185] The present invention also relates to a S. uberis and S. aureus detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis and S. aureus as described in this application; 2) A tube capable of closing hermetically; 3) A vial containing the nutrient medium optionally including a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0186] In one embodiment, the part comprising the protein is a labeling part and / or a detection part.

[0187] Thus, in a particular embodiment, said strip further includes, between the contact part and the detection part, a marking part which includes a marking area.

[0188] The labeling portion (conjugal pad) includes a labeling area where bacteria are labeled with a marker as the sample flow to be analyzed passes through it. Its role is to retain the marker until the strip is used and to release the marker when recognition occurs between the marker and a bacterium as the sample flow passes through. For example, the labeling portion and the labeling area are made of glass fibers, cellulose, or polyesters.

[0189] Thus, in a particular embodiment, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising, in at least the following order: - a contact part comprising a contact area with the sample; - a marking part comprising a marking area which includes a marker; - a detection part comprising a detection zone for the bacterium of interest which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, - an absorbing part.

[0190] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a marking part comprising a marking area which includes a marker; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0191] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a marking part comprising a marking area which includes a marker; - a detection part comprising a detection zone for the bacterium of interest which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2; - an absorbent part.

[0192] For the purposes of this application, the marker may be any marker capable of marking a bacterium. Bacterial markers are well known to those skilled in the art.

[0193] The term “marker” encompasses both non-conjugated and conjugated markers.

[0194] For the purposes of this application, an unconjugated marker is a free marker, that is, one that is not bound to a ligand to label the bacterium. Examples include fluorescent dyes. Fluorescent dyes capable of labeling bacteria are well known to those skilled in the art, such as DAPI (4',6-diamidino-2-phenylindole) or SYTO9, by way of example.

[0195] A conjugated marker can be, for example: a gold nanoparticle (AuNP) or colloidal gold, a colored latex bead, a carbon nanoparticle or colloidal carbon, a magnetic particle, a quantum dot, an upconverting phosphor (UCP), a luminescent molecule including fluorescent molecules (fluorophores) and phosphorescent molecules, a dye, a liposome, or an enzyme such as horseradish peroxidase (HRP) that catalyzes the conversion of chromogenic substrates into colored compounds, or that produces light from chemiluminescent substrates. These conjugated markers are known from the prior art (Sajid et al., 2015).

[0196] In particular, the marker is a gold nanoparticle (AuNP). By "gold nanoparticle" is meant either a gold nanosphere or a gold nanorod. More specifically, it is gold nanospheres with a diameter of 40 nm. The aggregation of the gold nanoparticles results in the appearance of a detectable color in the visible spectrum, which varies according to the size of the nanoparticles.

[0197] In particular, the marker is a colored latex bead. For example, the diameter of the latex beads is 400 nm. The aggregation of the colored latex beads leads to the appearance of a detectable color in the visible spectrum. Several colors of latex beads are commercially available.

[0198] In particular, the marker is the HRP.

[0199] For the purposes of this application, a conjugated marker means that the marker is bound to at least one specific ligand of the bacterium of interest. “Conjugated to at least one specific ligand” means that a marker may be conjugated to one or more different specific ligands.

[0200] To bind a marker to a ligand, a biotin / streptavidin bond can for example be used.

[0201] For the purposes of this application, "said at least one ligand specific to the bacterium of interest" may be at least one antibody directed against the bacterium of interest, at least one aptamer specific to the bacterium of interest, at least one bacteriophage specific to the bacterium of interest, at least one protein of at least one bacteriophage specific to the bacterium of interest, at least one bacteriophage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest, at least one protein of a bacteriophage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest.

[0202] In particular, said at least one protein of at least one bacteriophage specific to the bacterium of interest is an RBP protein.

[0203] In particular, it is a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3.

[0204] In particular, it may be the protein with sequence SEQ ID NO: 1 and / or the protein with sequence SEQ ID NO: 2, and / or the protein with sequence SEQ ID NO: 3.

[0205] Thus, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact zone with the sample; - a labeling part comprising a labeling area which includes a marker conjugated to at least one specific ligand of S. uberis, in particular a gold nanoparticle or a colored latex bead conjugated to at least one specific ligand of S. uberis; - a detection portion comprising a S. uberis detection zone which includes a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 1 sequence and / or a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 2 sequence, and / or a protein comprising a sequence having at least 70% with the SEQ ID NO:3 sequence, in particular the detection area includes the protein of sequence SEQ ID NO:1 and / or the protein of sequence SEQ ID NO:2, and / or the protein of sequence SEQ ID NO:3, - an absorbing part.

[0206] In particular, said strip for detecting S. uberis in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a marking part comprising a marking area which includes a marker; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part; said marker being conjugated to a specific ligand of S. uberis selected from at least one antibody directed against S. uberis, at least one aptamer specific to S. uberis, at least one bacteriophage specific to S. uberis, at least one protein from at least one bacteriophage specific to S. uberis, at least one bacteriophage specific to S. uberis coupled to an antibody directed against S. uberis, at least one protein from a bacteriophage specific to S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein from at least one bacteriophage specific to S.Uberis is an RBP protein, more particularly, a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular it is the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, in particular, said marker is a gold nanoparticle or a colored latex bead.

[0207] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in the order at least: . - a contact part comprising a contact area with the sample; - a labeling part comprising a labeling area which includes an antibody directed against S. uberis conjugated to a marker, in particular an antibody directed against S. uberis conjugated to a gold nanoparticle or a colored latex bead; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3; - an absorbent part.

[0208] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a labeling portion comprising a labeling zone which includes an antibody directed against S. uberis conjugated to a marker, in particular an antibody directed against S. uberis conjugated to a gold nanoparticle or a colored latex bead; - a detection portion comprising an S. uberis detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0209] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a labeling part comprising a labeling area which includes an antibody directed against S. uberis conjugated to a marker, in particular an antibody directed against S. uberis conjugated to a gold nanoparticle or a colored latex bead; - a detection part comprising a detection area for the bacterium of interest which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the detection area includes the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0210] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a labeling part comprising a labeling area which includes a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, - an absorbent part.

[0211] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a labeling part comprising a labeling area which includes a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said labeling area optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a detection portion comprising a detection zone for S. uberisqvù includes a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0212] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a labeling part comprising a labeling area which includes a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular conjugated to the protein of sequence SEQ ID NO: 2, and a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular conjugated to the protein of sequence SEQ ID NO: 1; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the detection zone includes the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, - an absorbing part.

[0213] In another particular embodiment, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in at least the following order: - a contact part comprising a contact area with the sample; - a labeling portion comprising a labeling area which includes a protein-conjugated marker comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 3, in particular the labeling area includes the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3; - a detection portion comprising a detection area for the bacterium of interest which includes at least one ligand specific to S. uberis; - an absorbent part.

[0214] In particular, the marker is a gold nanoparticle or a colored latex bead.

[0215] Said at least one specific ligand of S. uberis in the detection zone may must be at least one antibody directed against the bacterium of interest, at least one aptamer specific to the bacterium of interest, at least one bacteriophage specific to the bacterium of interest, at least one protein from at least one bacteriophage specific to the bacterium of interest, at least one bacteriophage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest, or at least one protein from a bacteriophage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest. In particular, said at least one protein from at least one bacteriophage specific to the bacterium of interest is an RBP protein.More specifically, it may be a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 3. In particular, it may be the protein with sequence SEQ ID NO: 1 and / or the protein with sequence SEQ ID NO: 2, and / or the protein with sequence SEQ ID NO: 3.

[0216] This application also relates to a S. uberis detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis comprising a contact part, a labeling part, a detection part and an absorbing part as described above; 2) A container intended to receive the sample.

[0217] In a particular embodiment, the present application relates to a S. uèerâ detection kit comprising: - a strip as described in this application; - a container intended to receive the sample; - a container containing a marker.

[0218] In this particular embodiment, said strip thus comprises either a contact part, a detection part and an absorbing part, or a contact part, a marking part, a detection part and an absorbing part, as described in this application.

[0219] The marker may be an unconjugated marker or a conjugated marker as defined above. The conjugated marker may be conjugated to at least one specific ligand as defined above.

[0220] In a particular embodiment, the marker may be included in the container intended to receive the sample.

[0221] This embodiment of the kit may include a lateral flow chromatography strip with or without a labeling portion. Thus, this embodiment of the detection kit applies to all the strips described in this application.

[0222] When the lateral flow chromatography strip includes a labeling portion, the kit optionally includes a container containing a marker.

[0223] Thus, when the lateral flow chromatography strip includes a labeling part, the S. uberis detection kit, in a sample, includes: - said strip; - a container intended to receive the sample.

[0224] In another particular embodiment, the S. uberis detection kit, in a sample, comprises: - a strip containing at least: a contact part comprising a contact area with the sample; a detection part comprising a detection zone for the bacterium of interest which includes at least one antibody directed against the bacterium of interest and / or at least one aptamer specific to S. uberis, an absorbing part; - a container intended to receive the sample; - a container comprising a marker conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular conjugated to the protein of sequence SEQ ID NO: 1 and / or conjugated to the protein of sequence SEQ ID NO: 2, and / or conjugated to the protein of sequence SEQ ID NO: 3.

[0225] According to another aspect, the present invention relates to a method for detecting S. uberis in a sample using the lateral flow chromatography strips described above.

[0226] A sample means any solution likely to contain the bacterium of interest. The sample may be diluted in a buffer solution prior to carrying out the detection process.

[0227] In a particular embodiment, the method for detecting S. uberis in a sample comprises: - a step of labeling the bacteria or the bacterium of interest in the sample; - a step of bringing the sample into contact with the contact area of ​​a strip as previously described; - a marker detection step on the detection zone; - a step of analyzing the presence or absence of S. uberis in the sample.

[0228] Thus, in one embodiment, the labeling step is performed in the sample, that is, prior to contacting the sample with the contact area of ​​the strip. This embodiment of the detection method is applicable whether or not the lateral flow chromatography strip used includes a labeling area. Therefore, this embodiment of the detection method applies to all the strips described in this application. When the strip used in this detection method contains a labeling area, double labeling of the bacterium of interest is then performed, the marker used in the labeling step in the sample and the marker included in the labeling area of ​​the strip being either identical or different.

[0229] The labeling step is carried out with an unconjugated marker or a conjugated marker as defined earlier in the application. The conjugated marker may be conjugated to at least one specific ligand as defined earlier. A person skilled in the art knows how to carry out this labeling step depending on the marker chosen. In particular, when a conjugated marker is used, the sample may be incubated with the conjugated marker.

[0230] A person skilled in the art knows how to adapt the detection step according to the marker used. In particular, when the marker is a gold nanoparticle or a colored latex bead, detection is carried out in the visible range. In particular, when the marker used is a fluorescent marker, a fluorescence reader is used. In particular, when the marker used is an enzyme, the substrate of the reaction it catalyzes is added.More specifically, when it comes to horseradish peroxidase (HRP) which catalyzes the conversion of chromogenic substrates into colored compounds, or which produces light from chemiluminescent substrates, the added substrate is for example 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonyl) acid (ABTS), 1,2-diaminobenzene (OPD), 3,3'-Diaminobenzidine (DAB), 3-Amino-9-ethylcarbazole (AEC) or even 3,3',5,5'-tetramethylbenzidine (TMB) for colored reactions; homovanillic acid and tyramine for fluorescence spectroscopy; luminol for light production.

[0231] To bind a marker to a ligand, a biotin / streptavidin link can for example be used.

[0232] In particular, the marker used is a gold nanoparticle or a colored latex bead.

[0233] The presence of the bacterium of interest in the analyzed sample is concluded when the marker is detected in the detection zone. In particular, when the marker is a gold nanoparticle, a colored signal visible to the naked eye appears. In particular, when the marker used is a fluorescent marker (conjugated or not), a signal A fluorescent signal appears in the detection area. When the marker used is an enzyme such as horseradish peroxidase, a substrate such as those listed above is added, and a colored signal visible to the naked eye, a fluorescent signal, or a luminescent signal is observed, depending on the substrate.

[0234] The absence of the bacterium of interest in the sample is concluded when the marker is not detected on the detection area.

[0235] When the lateral flow chromatography strip used for the detection method includes a labeling portion, the step of labeling the bacteria or the bacterium of interest in the sample is optional.

[0236] Thus, when the lateral flow chromatography strip used for the detection method includes a labeling portion, the method for detecting S. uberis in a sample may include: - a step of bringing the sample into contact with the contact area of ​​a strip as previously described; - a marker detection step on the detection zone; - a step of analyzing the presence or absence of S. uberis in the sample.

[0237] In another particular embodiment, the method for detecting S. uberis in a sample comprises: - a step of labeling the bacterium of interest in the sample with a protein-conjugated marker comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3; - a step of bringing the sample into contact with the contact area of ​​a strip comprising at least a contact part comprising a contact area with the sample; a detection portion comprising a S. uberis detection zone which includes at least one antibody directed against S. uberis and / or at least one S. uberis-specific aptamer, an absorbent part; - a marker detection step on the detection zone; - an analysis step to determine the presence or absence of S. uberis in the sample.

[0238] In one aspect, the present application relates to a detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis as described above comprising a contact part, a labeling part, a detection part and an absorbing part as described above; 2) A container intended to receive the sample; 3) A container comprising a marker conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3.

[0239] In a particular embodiment, said lateral flow chromatography strip enables the detection of S. uberis and one or more other bacteria of interest in a sample. The detection portion then comprises one or more additional detection zones and one or more additional labeling portions on which the appropriate ligands and markers for detecting this or these other bacteria of interest are immobilized.

[0240] This type of strip is called "Duplex LFA".

[0241] In a particular embodiment, said lateral flow chromatography strip enables the detection of S. uberis and S. aureus in a sample.

[0242] In a particular embodiment, said lateral flow chromatography strip for detecting S. uberis and S. aureus in a sample, is a strip as described above, comprising 4 parts, namely a contact part, a labeling part, a detection part and an absorbing part, and which includes: - an additional detection zone in the detection part. This additional detection zone, or test line, comprises a specific ligand of S. aureus; - a second labeling part which includes a labeling area with a marker conjugated to a specific S. aureus ligand.

[0243] Said S. aureus-specific ligand may be at least one antibody directed against S. aureus, at least one S. aureus-specific aptamer, at least one S. aureus-specific bacteriophage, at least one protein from at least one S. aureus-specific bacteriophage, at least one S. aureus-specific bacteriophage coupled to an antibody directed against S. aureus, or at least one protein from an S. aureus-specific bacteriophage coupled to an antibody directed against S. aureus. In particular, said at least one protein from at least one S. aureus-specific bacteriophage is an RBP protein. In particular, it may be a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 4 sequence and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO:5. More specifically, it may be the protein of sequence SEQ ID NO:4 and / or the protein of sequence SEQ ID NO:5.

[0244] Several types of Duplex LFA strips can therefore be produced, depending on whether: - the S. uberis labeling area includes a marker coupled to a specific S. uberis ligand or a marker coupled to a specific protein comprising a sequence having a percentage of identity of at least 70% with SEQ ID NO:1 and / or SEQ ID NO:2 and / or SEQ ID NO:3; - the S. aureus labeling area includes a marker coupled to a specific S. aureus ligand or a marker coupled to a specific protein comprising a sequence having a percentage of identity of at least 70% with SEQ ID NO: 4 and / or SEQ ID NO: 5; - the S. uberis detection zone includes a marker coupled to a specific S. uberis ligand or a marker coupled to a specific protein comprising a sequence having a percentage of identity of at least 70% with SEQ ID NO:1 and / or SEQ ID NO:2 and / or SEQ ID NO:3, - the S. aureus detection zone includes a marker coupled to a specific S. aureus ligand or a marker coupled to a specific protein comprising a sequence having a percentage of identity of at least 70% with SEQ ID NO: 4 and / or SEQ ID NO: 5.

[0245] Table 1 presents different types of Duplex LFA which can, for example, be produced, in which: - The specific ligand for the bacterium of interest, S. uberis or S. aureus, may be at least one antibody directed against the bacterium of interest, at least one aptamer specific to the bacterium of interest, at least one bacteriophage specific to the bacterium of interest, at least one protein from at least one bacteriophage specific to the bacterium of interest, at least one bacteriophage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest, or at least one protein from a bacteriophage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest. In particular, said at least one protein from at least one bacteriophage specific to the bacterium of interest is an RBP protein. - the specific protein for S. uberis may be a protein comprising a sequence with at least 70% identity with sequence SEQ ID NO: 1 and / or a protein comprising a sequence with at least 70% identity with sequence SEQ ID NO: 2, and / or a protein comprising a sequence with at least 70% identity with sequence SEQ ID NO: 3, in particular it may be the protein with sequence SEQ ID NO: 1 and / or the protein with sequence SEQ ID NO: 2, and / or the protein with sequence SEQ ID NO: 3; and For S. aureus, the specific protein may be a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular it may be the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5.

[0246] [Tables 1] S. uberis tagging zone S. aureus tagging zone S. uberis detection zone S. aureus detection zone Specific ligand or specific protein Specific protein Specific ligand or specific protein Specific ligand or specific protein Specific ligand or specific protein Specific ligand or specific protein Specific protein 1 XXXX 2 XXXX 3 XXXX 4 XXXX 5 XXXX 6 XXXX 7 XXXX 8 XXXX 9 XXXX 10 XXXX 11 XXXX 12 XXXX 13 XXXX 14 XXXX 15 XXXX 16 XXXX 17 XXXX 18 XXXX

[0247] The detection zone of S. uberis and the detection zone of S. aureus are distinct from each other, that is to say they do not overlap.

[0248] The first marking part and the second marking part can be reversed with respect to the direction of migration.

[0249] Similarly, the detection zones can be in one order or the other depending on the direction of migration.

[0250] Thus, in a particular embodiment, such a strip comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a first marking section which includes a marking area of ​​S. uberis with a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3; - a second labeling part which includes a labeling area of ​​S. aureus with a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, and comprising a S. aureus detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbing part.

[0251] In particular, said strip for detecting S. uberis and S. aureus in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a first labeling portion comprising a labeling zone for S. uberis including a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular the protein with sequence SEQ ID NO: 2, said labeling zone optionally comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% with the SEQ ID NO sequence: 1, in particular the protein with SEQ ID NO sequence: 1; - a second labeling part which includes an S. aureus labeling area comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4, in particular the protein of sequence SEQ ID NO: 4 and / or comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the protein of sequence SEQ ID NO: 5; - a detection portion comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said detection zone for S. uberis optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and comprising a detection zone for S. aureus which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone for S. aureus includes the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0252] In particular, said strip for detecting S. uberis and S. aureus in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a first labeling part which includes a labeling area of ​​S. uberis comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2 and comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a second labeling portion comprising an S. aureus labeling zone including a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 4 sequence, in particular the protein of sequence SEQ ID NO:4 and comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO:5, in particular the protein of sequence SEQ ID NO:5; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone for S. uberis includes the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, and comprising a detection zone for S. aureus which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone for S. aureus includes the protein of sequence SEQ ID NO: 4 and the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0253] In particular, said strip for detecting S. uberis and S. aureus in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a first labeling part which includes a labeling area of ​​S. uberis with a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to an antibody directed against S. uberis; - a second labeling part which includes a labeling area of ​​S. aureus with a marker conjugated to an antibody directed against S. aureus; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 3, in particular the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, and / or the protein of sequence SEQ ID NO: 3, and comprising an S. aureus detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0254] In particular, said strip for detecting S. uberis and S. aureus in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a first labeling part which includes a labeling area of ​​S. uberis with a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to an antibody directed against S. uberis; - a second labeling part which includes a labeling area of ​​S. aureus with a marker conjugated to an antibody directed against S. aureus; - a detection portion comprising a S. uberis detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular it includes the protein of sequence SEQ ID NO: 2, said S. uberis detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and comprising an S. aureus detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and / or which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the S. aureus detection zone includes the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0255] In particular, said strip for detecting S. uberis and S. aureus in a sample comprises, in at least the following order: - a contact part comprising a contact area with the sample; - a first labeling part which includes a labeling area of ​​S. uberis with a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to an antibody directed against S. uberis; - a second labeling part which includes a labeling area of ​​S. aureus with a marker conjugated to an antibody directed against S. aureus; - a detection part comprising a detection zone for S. uberis which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2 and which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the detection zone for S. uberis includes the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, and comprising an S. aureus detection zone which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 4 and which includes a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 5, in particular the S. aureus detection zone comprises the protein of sequence SEQ ID NO: 4 and the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0256] This application also relates to a kit for detecting S. uberis and S. aureus in a sample comprising: 1) A lateral flow chromatography strip for detecting S. uberis and S. aureus as described above comprising a contact part, a labeling part, a detection part and an absorbing part as described above; 2) A container for receiving the sample.

[0257] This application also relates to a method for detecting S. uberis and S. aureus in a sample comprising: - a step of bringing the sample into contact with the contact area of ​​a strip to detect S. uberis and S. aureus comprising a contact part, a labeling part, a detection part and an absorbing part as described previously; - a marker detection step on the detection zone; - a step of analyzing the presence or absence of S. uberis and the presence or absence of S. aureus in the sample.

[0258] If the marker is a gold nanoparticle or a colored latex bead, the presence of the bacterium will result in the appearance of a color on the detection area. Examples

[0259] Production of RBP proteins

[0260] GST-PHIL 2 and GST-P2 proteins are expressed in a bacterial plasmid with an Escherichia coli BL21 star strain in LB medium (BD, 240230) containing 100 mg / L of ampicillin (Fisher bioreagent, BPHIL 2760-25). Induction is performed when the bacteria reach an optical density of ~0.5. After induction with IPTG, the bacteria are incubated at 25°C with shaking. The cultures are centrifuged, then resuspended and concentrated in lysis buffer and lysed by sonication. The lysates are then centrifuged at 14,000 g for 30 minutes. The supernatant is collected and incubated overnight with Glutathione Sepharose 4B resin (Cytiva, 17-0756-01). The resin is then recovered using a column with sintered glass, washed with HEPES 50 mM pH7 buffer and then stored at -80°C.

[0261] Example 1: Comparison of different RBPs of S. uberis with an ELISA test

[0262] 1.1. Materials and Methods

[0263] The ELISA (enzyme-linked immunosorbent assay) is an enzyme-linked immunosorbent assay on a solid support, based on the specific antigen-antibody reaction. The principle of an indirect ELISA is to place an antigen on a microplate and add a primary antibody directed against this antigen. A secondary antibody recognizing the heavy chains of the first antibody, coupled to an enzyme, is then added. The addition of the enzyme substrate allows for a colorimetric reaction that can be measured by absorbance.

[0264] To determine the specificity of the RPB protein, the ELIS A principle was adapted as follows.

[0265] Bacteria are fixed to the bottom of the wells of a microplate (ELISA) at (OD=0.05) for 1.5 hours at 37°C. A saturation step is performed with a solution containing 3% BSA and 0.05% Tween, and then the BSA solution is discarded. Phage proteins are loaded into each well at a concentration of 10 µg / mL in PBS buffer (1% BSA; 0.05% Tween). Incubation is then performed for 1 hour at 37°C. The wells are washed (at least 5 times) with PBS buffer containing 0.05% Tween. An incubation step with the HRP-coupled -GST antibody (Invitrogene, ref, 434323) at 0.25 pg / mL, solution in 1% BSA, 0.05% Tween20 PBS buffer for 1 h at 37°C, then the streptavidin is removed. The wells are washed (at least 5 times) with PBS buffer containing 0.05% Tween20.

[0266] For detection, 100 ql of a TMB solution are added and incubated for 15 to 30 minutes at room temperature. Then, 50 ql of 2 M sulfuric acid are added to stop the reaction. The plate is then read at 450 nm.

[0267] The affinity of the protein for the bacterium is expressed by the amount of protein retained by each bacterial strain. This amount is evaluated by the absorbance associated with the anti-GST antibody coupled to HRP.

[0268] 1.2. RBP proteins tested

[0269] Several RBPs from S. uberis-specific phages (see Table 2) were produced. To characterize the ability of these proteins to detect bacterial strains, ELISA tests were performed.

[0270] [Tables2] RBP Protein Sequence Number Sequence P3 SEQ ID NO:1 MSSLIEIHDIKRDVGNNRVVTTNTSPFLGSVIQRYKIDSKKITVSFSIWTRDRNTVKH QLASIFNQSEPKRLLFSDEPDKYYLALWDEIQMQEASIKRSYGTITFLITDGVAHST SYKKITDFTESDGKVIFNITNNGNVEALPIITAKMNSENGYFGLVNETGVMEIGDREL IDSETLAYSERPFDYSDTGTKIADGFAKGAKNVAILNDSSATLDKNLSILNWLGRDH VVLDGTSVAGTHAGSLTFDLPTEGSLYDYIWWRQIFWAGNINQLGFIKVAVSDTDG KFLFGVETIKRKNGLETEYNVMVADGKGGYKFIDFRKKFSASHKDEENPFNANRG WSDIKRIDDKLSIFWFGSRYERTVPELKGKKSAKLHVALGAIQGKPLVTRMYLDGIK YRKDNVADGYNIPNPYGTGSTVVINSENKTLLVDNIPKLNHWDYSKWLKIPVGTST IEISTSSWNEIKPTFSLAFEERWL SUB 4 SEQ ID NO :2 MGDVYVSGKTDTKSLTVDGFAISKLTILNMIYPVGSIFISTSSANPATTMGGTWTRY GQGRVLVGVNESDTDFSTAGKTGGEKTHLQTVDEMPSHTHGFRGGENNYVRV EPSSTYGYSGNSDKTTNATGGNKPFNIMQPYITTYMWLRTA 28-7 SEQ ID NO :3 LSSLIEIHDIKRDVGNNRWTTTNSPFLFGSVIQRYKIDSKKITVSFSIWTRDRNTVKH QLASIFNQSEPKRLLFSDEPDKYYLALVVDEIQMQEASIKRSYGTITFLITDGVAHST SYKKITDFTESDGKVIFNITNNGNVEALPIITAKMNSENGYFGLVNETGVMEIGDREII DSETRQFSERPFDYTGSTSVNKNLLTNTKDFSGYTNLANWKNVGTFNGNWLENLDTNPVYQQTTITAGKTYTFSAYVKSVTGTPSTIAIGLTGLGTATINITNPTVAITTTMQ RVSVTFTCTATGTIHPRLSSTYTATNKVQISSFKLEENTVATPWTNSTSGIYPISQAL IDGAKNVAISNDSTTILDKNLSIINWLNRDHIVVDGTVATSGNHAGSLTFDLPSSGSL FDRIWWRQIFHIGDPSQFGYIKILVSDTDGKFLYGFETMKRKQGYEAECNFLITDEQ GGYKTTDFQKKFIANEKDTDNPFNDDRGFSDLIRQDDQVQIYWFGSHIKRTFSELK GKKSAKLHVIIGSMAGKPLVTRMYLDGIKYQKDNVAFDYNIPNPYGIGSNWINGEN KTFLVDNIAKLNHWDYSKWLKIPVGTSTIEITTSSWNDIKPTISIDFEERWL

[0271] 1.3. Results of RBP proteins alone or in combination

[0272] The RBP P3 and SUB4 proteins were then tested alone or in combination on about thirty strains of S. uberis. The absorbance data obtained are reported in Table 3. All absorbance values ​​greater than or equal to 0.5 are considered to be significant values.

[0273] [Tables3] Recognition by P3 SUB4 Mixture of P3 and SUB4 S. non aureus L7a1 0.068 0.005 0.023 E. coii 4ka 0.17 0.02 0.08 L. lactis 66A1a 0.12 0.03 0.09 Enrococcijs F3F 0.28 0.05 0.16 S. uberis 50 0.09 0.01 0.05 S. uberis F17WGC 0.197 0.045 0.187 S. uberis JOCONDE 2.66 2.9 2.718 S. uberis 304T 0.002 0.001 0.05 S. uberis 16B 1.215 1.267 1.475 S uberis 2192 0.626 0.348 0.66 S uberis 27J 0.659 0.321 0.549 S. uberis DUCHESSE 1.12 2.012 1.849 S. uberis 4475b 0.348 0.08 0.242 S. uberis 19121 0.268 0.16 0.241 S. uberis 2EB1 0.875 0.402 0.769 S. uberis 9038 0.991 0.665 1.236 S. uberis GALANTE 0.511 0.299 0.558 S uberis 1912c 0.63 0.06 0.55 S. uberis 1210 2.789 3.248 2.77 S. uberis 6134 0.693 0.879 0.931 S. uberis 9817a 0.799 0.899 0.619 S. uberis LMG14389 3.414 3.64 3.406 S. uberis F17WPC 0.4 0.615 0.592 S uberis 53G 0.528 0.439 0.541 S. uberis S43 0.838 1.985 1.139 S. uberis S44 0.569 0.645 0.896 S. uberis S45 1.134 1.45 1.575 S. uberis S46 1.01 0.794 1.606 S. uberis S47 1,359 1,63 1,529 S.uberis S48 0.859 1.14 0.921 S uberis S49 2.654 3.07 3.129 S uberis S50 0.516 0.975 0.836 S. uberis S51 2.835 2.923 2.583 S. uberis S52 0.63 0.706 0.52.

[0274] Of 30 S. uberis strains tested, 24 strains were recognized by the P3 protein (80%), 20 strains by the SUB4 protein (66.7%), and the mixture of P3 and SUB4 proteins detected 25 strains (83.3%). Therefore, the mixture of the two proteins achieved 83% coverage.

[0275] These results were confirmed for detection by the LFA method (presented below).

[0276] Example 2: Detection of S. uberis strains with a Lateral Flow Assay (LFA) device comprising RBP proteins

[0277] 2.1. Composition of the LFA device

[0278] Lateral Flow Assay (LFA) devices were performed using RBP P3 and SUB4 proteins.

[0279] The LFA device or strip 1 was designed as follows with 4 parts: - a contact part 2: part in contact with the solution to be analyzed; - a labeling area 7: area where RBP proteins conjugated to gold nanoparticles (diameter 40 nm) are deposited at the level of the labeling area 8; - a detection part 4 comprising a test line 5 where RBP proteins are immobilized; - an absorption part 6 to establish the flow through the device from the contact part 2.

[0280] 2.2. Implementation of the LFA device for the detection of S. uberis

[0281] S. uberis bacterial strains are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0282] The following day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0283] Bacterial suspensions at the desired bacterial concentration are prepared in PBS IX comprising 10% whole UHT milk and 1% tween20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0284] For each suspension prepared, 250pl are taken and then deposited in the wells of a 96-well plate.

[0285] Strips as described above are used. The different configurations listed in Table 4 are tested with the RBP P3 and SUB4 proteins. When used as a conjugate on the labeling portion 7, they are coupled with gold nanoparticles, and when used on the test line 5 of the detection portion 4, they are in solution in PBS IX with 20% sucrose.

[0286] Each strip is immersed in a well at the contact point 2 and left for 30 minutes to allow migration to the test line 5. The bacterial strains were tested by absorbance at OD 600 nm = 1 (equivalent to approximately 1 x 10⁹ cfu / ml for S. aureus strains and 1 x 10⁷ cfu / ml for S. uberis strains). The migration buffer is a PBS IX solution, comprising 10% sterile whole UHT milk and 1% tween20. After migration, the strips are photographed.

[0287] [Tables4] Conjugate Test Line Tested Strains Recognized Strains Figure Configuration 1 P3 (DO = 10) P3 (1.25 mg / ml) Duchesse / Galande Duchesse and Galande 1 Configuration 2 SUB4 (DO = 10) P3 (1.25 mg / ml) Duchesse / Galande Duchesse and Galande more intense 2 F17W-PC / F17W-GC / 4475b / 9038 / 2EB1 / 1912c / 53G / S52 9038 5A Configuration 3 SUB4 (DO = 10) SUB4 (0.625 mg / mL) F17W-PC / F17W-GC / 4475b / 9038 / 2EB1 / 1912c / 53G / S52 F17W-PC / F17W-GC / 4475b / 9038 / 2EB1 / 53G / S52 5B Configuration 4 SUB4 (OD = 10) P3 (0.625 mg / ml) + SUB4 (0.625 mg / mL) F17W-PC / F17W-GC / 4475b / 9038 / 2EB1 / 1912c / 53G / S52 F17W-PC / F17W-GC / 4475b / 9038 / 2EB1 / 1912C / 53G / S52 5C Configuration 5 P3 (OD = 5) + SUB4 (OD = 5) P3 (0.625 mg / ml) + SUB4 (0.625 mg / ml) Duchesse / Galande Duchesse / Galande 4 30 strains 28 strains 6

[0288] 2.3. Results

[0289] For proof of concept, LFA devices in configurations 1, 2, and 5 (see Table 4) were tested for the detection of S. uberis Duchesse and Galande strains. Tests without bacteria and with non-S. uberis bacteria (S. aureus and E. coli) were also performed.

[0290] Figures 1, 2, 3, and 4 show that the presence of the SUB4 protein in the conjugate results in improved signal intensity. Furthermore, the RBP proteins do not detect anything in samples containing the blank or control strains. This demonstrates that the RBP proteins deposited on the LFA strip are functional, as they bind to the target bacteria. Moreover, these proteins are capable of migrating across the membrane used on the strip.

[0291] After validating the concept of detecting S. uberis strains on LFA strips using RBP proteins, tests were carried out on other S. uberis strains using additional configurations.

[0292] Figure 5, part A, shows that when P3 is alone on test line 5, only the Strain 9038 was readily detected in all eight S. uberis strains. Figure 5, part B, shows that using the SUB4 protein alone in the conjugate and on the test line allowed for the detection of seven out of the eight strains tested. Strain 1912c was not recognized, but it was weakly detected when test line 5 contained both P3 and SUB4 proteins. Therefore, mixing the two RBP proteins allowed for the detection of more strains than using a single protein on test line 5 (Figure 5, part C).

[0293] The mixture of the two proteins was then used as a conjugate and also on test line 5 for the detection of 30 strains of S. uberis. Figure 6 shows that the use of the P3 and SUB4 protein mixture in the conjugate and on test line 5 increases the signal intensity of all the S. uberis strains used in the previous experiment. Of the 30 S. uberis strains tested, 28 were detected using the RBP protein mixture. This system allows for the recognition of more than 90% of the strains tested.

[0294] Example 3: LFA system with S. uberis growth and addition of agar to the detection zone (test line)

[0295] 3.1. Concept of detection with bacterial growth

[0296] The concept of the LFA method with bacterial growth on the detection zone 5 (test line) includes the following steps: 1) The LFA device 1 includes a contact part 2, a detection part 4 and an absorption part 6. It does not include a labeling part 7. The sample to be tested migrates directly through the detection part 4 which includes a detection zone 5 or test line where a specific ligand of the bacterium of interest is immobilized by adsorption; 2) When the bacterium of interest arrives by capillary action on test line 5, it is captured by the ligands on the membrane; 3) At the end of the migration, a nutrient agar is deposited on test line 5; 4) Optionally, chromogenic elements can be added to the agar to improve the detection of the multiplication of the bacterium of interest.

[0297] 3.2. Detection of S. uberis with the LFA system with bacterial growth on the detection zone

[0298] 250 µl of bacterial suspension in PBS IX + 10% whole UHT milk + TWEEN 20 1% at the desired bacterial concentration are placed in one well of a 96-well plate.

[0299] An LFA device comprising, at the detection part, a test line 5 with the RBP SUB4 and P3 proteins (1 pl deposited; composition: 0.625 mg / ml of the SUB4 protein, 0.625 mg / ml of the P3 protein, PBS IX, 10% sucrose).

[0300] The strip is immersed at the contact part 2 for 30 minutes in the well containing the sample to be tested.

[0301] After the migration, test line 5 is cut and is either: - on a Petri dish agar ([Fig.7] A) comprising BHI IX, potassium tellurite at 75 pM and agar at 1.5%. - in a cassette ([Fig. 7] B). The strip is fixed with double-sided tape inside the cassette. The cassette is then closed. An agar containing BHI IX, 75 pM potassium tellurite, and 0.4% agar is deposited on the The strip is trapped in the cassette with a pipette. An adhesive film (ThermoFischer Scientific - 15036) is used to protect the added agar from the environment and evaporation.

[0302] Potassium tellurite is used as a chromogenic element to enhance the detection of bacterial multiplication.

[0303] The strips are then incubated at 37°C for 15 hours and a photo is then taken.

[0304] The photos in [Fig. 7] clearly show that in the sample without bacteria, no black line appears, indicating the absence of bacteria immobilized on the test line. In contrast, for samples containing S. uberis tested at 1.104 cfu / ml, a black line appeared on the test line, corresponding to the multiplication of the bacterium of interest, S. uberis, and the reduction of tellurite to tellurium, a chromogenic element that improves the visibility of the result.

[0305] Example 4: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus

[0306] 4.1. Composition of the LFA device

[0307] The LFA device or strip 1 was designed as follows: - a contact part 2: part in contact 2 with the solution to be analyzed. - a labeling region 7 no. 1: region where the RBP P3 and SUB4 proteins specific to S. uberis are deposited, conjugated to gold nanoparticles (diameter 40 nm) - a labeling region 7 no. 2: region where the RBP PHIL 2 and PHIL 5 proteins specific to S. aureus are deposited, conjugated to gold nanoparticles (diameter 40 nm) - a detection part 4 comprising a test line 5 no. 1 where the RBP P3 and SUB4 proteins specific to S. uberis are immobilized, and comprising a test line 5 no. 2 where the RBP PHIL 2 and PHIL 5 proteins specific to S. aureus are immobilized; - an absorption part 6 to establish the flow through the device from the contact part 2.

[0308] [Tables5] Protein RBP Number of sequence Sequence PHIL 2 SEQ ID NO:4 MALNFTTITENNVIRDLTTQVNNIGEELTKERNIFDITDDLVYNFNKSQKIKLTDDK GLTKSYGNITALRDIKEPGYYYIGARTLATLLDRPDMESLDWLHVVPLDTSSKV VQHLYTLSTNNNQIKMLYRFVSGNSSSEWQFIQGLPSNKNAVISGTNILDIASPG VYFVMMGMTGGMPSGVSSGFLDLSVDANDNRLARLTDAETGKEYTSIKKPTGTY TAWKKEFEPKDMEKYLLSSIRDDGSASFPLLVYTSDSKTFQQAIIDHIDRTGQTT FTFYVQGGVSGSPMSNSCRGLFMSDTPNTSSLHGVYNAIGTDGRNVTGSWG SNWTSPKTSPSHKELWTGAQSFLSTGTTKNLSDDISNYSYVEVYTTHKTTEKTK GNNDNTGTICHKFYLDGSGTYVCSGTFVSGDRTDTKPPITEFYRVGVSFKGSTWT LVDSAVQNSKTQYVTRIIGINMP PHIL 5 SEQID NO :5 MAYGLITSLHSITGEKVVAQHEYNYRLLDNGMSKLEKMFIYHQKEEIYAHTANQI KYLNGSVSDFLAYLNGRFSNMVLGHNGDGINEVKDARVDNTGYAHPTLQDRLY HDYSTLDAFTKKVEKWGEHYKEYQATEYRFEPKEQEPEFITDLSPYTNAVMQS FWVDPRTKIIYMTQARPGNHYMLSRLKPNGQFIDRLLVKNGGHGTHNAYRYIGN ELWIYSAVLDANNNNKFVRFKYRSGEMTYGNEMQDVMPNVFNDRYTSAIYNPI ENLMIFRREYKTSEQQAKNSLNFIEVRSVDDIDKGIDKVLYQMDIPMEYSSLTQP MQGIAYDAGVLYWYTGDSNPANPNYLQGFDIKTKELLFKRRIDIGVNNNFKGD FQEAEGLDMYYDLETGRKALLIGVTIGGPGNNRHHSIYSIGQRGVNQFLKNIAPQVSMTDSGGRVKPLPVQNPAYLSDVTEVGNYYLYSQDTQNALDFPLPKEFRDAG WFFDVLPGHYNGALRQVLTRNSTGRNMLKFERVIDIFNKKNNGSWNFNPQSAG FWEHIPKSITKLSDLKIVGLDFYITTEESKRFTDFPKDFKGIAGWILEVKSNTPGNT TQVLRRNNFPSAHQFLLRNFGTGGVGKWSLFEGKWE

[0309] 4.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0310] Bacterial strains of S. uberis and S. aureus are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0311] The following day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0312] Bacterial suspensions of S. aureus, S. uberis or S. aureus + S. uberis at the desired bacterial concentration are prepared in PBS IX comprising 10% whole UHT milk and 1% tween20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0313] For each suspension prepared, 250pl are taken and then deposited in the wells of a 96-well plate.

[0314] Strips as described above are used. The S. uberis-specific RBP P3 and SUB4 proteins are immobilized on test line 5 No. 1 (1 µl of a solution containing 0.625 mg / ml P3, 0.625 mg / ml SUB4, and PBS IX comprising 10% sucrose), and the S. aureus-specific RBP PHIL 2 and PHIL 5 proteins are immobilized on test line 5 No. 2 (1 µl of a solution containing 0.75 mg / ml PHIL 2, 0.75 mg / ml PHIL 5, and PBS IX comprising 10% sucrose). For immobilization, they are coupled with GST.

[0315] On the labeling area 7 n°l, 6 µl of a solution of S. uberis-specific RBP P3 proteins, conjugated to gold nanoparticles (diameter 40 nm), are deposited. The protein solution has an OD=10 with 20% sucrose, 0.25% BSA and PBS1X.

[0316] On the labeling area 7 no. 2, 6 µl of a solution of RBP PHIL 2 and PHIL 5 proteins specific to S. aureus, conjugated to gold nanoparticles (diameter 40 nm), are deposited. The protein solution has an OD=10 with 20% sucrose, 0.5% BSA and PBS1X.

[0317] Both marking parts 7 are of identical reference (Ahlstrom-Grade 800).

[0318] Each strip 1 is immersed in a well at the contact area 2 and left for 30 minutes to allow migration to the test line 5. The bacterial strains were tested by absorbance at OD 600 nm = 1 (equivalent to approximately 1 x 10⁹ cfu / ml for S. aureus strains and 1 x 10⁹ cfu / ml for S. uberis strains). The migration buffer is a PBS IX solution, comprising 10% sterile whole UHT milk and 1% tween20. After migration, the strips are photographed.

[0319] 4.3. Results

[0320] As shown in [Fig. 8], with the well containing no bacteria, only the control line 9 is visible. With a sample containing S. uberis, two lines appear: the control line 9 and the S. uberis test line 5. With a sample containing only S. aureus, two lines appear: the control line 9 and the S. aureus test line 5. With a sample containing both bacteria, all three test lines appear.

[0321] This clearly shows that this device is capable of the simultaneous detection of S. aureus and S. uberis.

[0322] Example 5: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus

[0323] 5.1. Composition of the LFA device

[0324] The LFA device or strip 1 was designed as follows: - a contact part 2: part in contact 2 with the solution to be analyzed. - a labeling part 7 n°1: part where the RBP P3 and SUB4 proteins specific to S. uberis are deposited, conjugated to latex beads of diameter 400 nm; - a labeling part 7 no. 2: part where the RBP PHIL 2 and PHIL 5 proteins specific to S. aureus are deposited, conjugated to latex beads; - a detection part 4 comprising a test line 5 no. 1 where the RBP P3 and SUB4 proteins specific to S. uberis are immobilized, and comprising a test line 5 no. 2 where the RBP PHIL 2 and PHIL 5 proteins specific to S. aureus are immobilized; - an absorption part 6 to establish the flow through the device from the contact part 2.

[0325] 5.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0326] Bacterial strains of S. uberis and S. aureus are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0327] The following day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0328] Bacterial suspensions of S. aureus, S. uberis or S. aureus + S. uberis at the desired bacterial concentration are prepared in PBS IX comprising 10% whole UHT milk and 1% tween20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0329] For each suspension prepared, 250pl are taken and then deposited in the wells of a 96-well plate.

[0330] Strips as described above are used. The S. uberis-specific RBP P3 and SUB4 proteins are immobilized on test line 5 No. 1 (1 µl of a solution containing 0.625 mg / ml P3, 0.625 mg / ml SUB4, and PBS IX comprising 10% sucrose), and the S. aureus-specific RBP PHIL 2 and PHIL 5 proteins are immobilized on test line 5 No. 2 (1 µl of a solution containing 0.75 mg / ml PHIL 2, 0.75 mg / ml PHIL 5, and PBS IX comprising 10% sucrose). For immobilization, they are coupled with GST.

[0331] On the labeling area 7 n°l, 6 µl of a solution of RBP P3 and SUB4 proteins specific to S. uberis, conjugated to latex beads, are deposited. The protein solution has an OD=10 with 20% sucrose, 0.25% BSA and PBS IX.

[0332] On the labeling area 7 no. 2, 6 µl of a solution of RBP PHIL 2 and PHIL 5 proteins specific to S. aureus, conjugated to latex beads, are deposited. The protein solution has an OD=10 with 20% sucrose, 0.25% BSA and PBS IX.

[0333] Both marking parts are of identical reference (Ahlstrom-Grade 800).

[0334] Each strip 1 is immersed in a well at the contact point 2 and left for 30 minutes to allow migration to the test line 5. The bacterial strains were tested by absorbance at OD 600 nm = 1 (equivalent to approximately 1 x 10⁹ cfu / ml for S. aureus strains and 1 x 10⁹ cfu / ml for S. uberis strains). The migration buffer is a PBS IX solution, comprising 10% sterile whole UHT milk and 1% tween20. After 30 minutes of migration, the strips are photographed.

[0335] 5.3. Results

[0336] As shown in [Fig. 9], with a sample containing S. uberis, the S. uberis test line 5 appears. With a sample containing only S. aureus, the line Test line 5 for S. aureus appears. With a sample containing both bacteria, both test lines appear.

[0337] This clearly shows that this device is capable of the simultaneous detection of S. aureus and S. uberis.

[0338] Example 6: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus

[0339] 6.1. Composition of the LFA device

[0340] The LFA device or strip was designed as follows: - a contact part 2: part in contact with the solution to be analyzed. - a labeling part 7 n°1: part where the RBP P3 and SUB4 proteins specific to S. uberis are deposited, conjugated to latex beads of diameter 400 nm; - a marking part 7 no. 2: part where an anti-S. aureus antibody is deposited, conjugated to latex beads; - a detection part 4 comprising a test line 5 no. 1 where the RBP P3 and SUB4 proteins specific to S. uberis are immobilized, and comprising a test line 5 no. 2 where the RBP PHIL 2 and PHIL 5 proteins specific to S. aureus are immobilized; - an absorption part 6 to establish the flow through the device from the contact part 2.

[0341] 6.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0342] Bacterial strains of S. uberis ct S. aureus are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0343] The following day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0344] Bacterial suspensions of S. aureus, S. uberis or S. aureus + S. uberis at the desired bacterial concentration are prepared in PBS IX comprising 10% whole UHT milk and 1% tween20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0345] For each suspension prepared, 250pl are taken and then deposited in the wells of a 96-well plate.

[0346] Strips as described above are used. The S. uberis-specific RBP P3 and SUB4 proteins are immobilized on test line 5 No. 1 (1 µl of a solution containing 0.625 mg / ml P3, 0.625 mg / ml SUB4, and PBS IX comprising 10% sucrose), and the S. aureus-specific RBP PHIL 2 and PHIL 5 proteins are immobilized on test line 5 No. 2 (1 µl of a solution containing 0.75 mg / ml PHIL 2, 0.75 mg / ml PHIL 5, and PBS IX comprising 10% sucrose). For immobilization, they are coupled with GST.

[0347] On the labeling area 7 no. 1, 6 µl of a solution of RBP P3 and SUB4 proteins specific to S. uberis, conjugated to latex beads, are deposited. The protein solution has an OD=10 with 20% sucrose, 0.25% BSA and PBS1X.

[0348] On the labeling area 7 no. 2 is deposited an anti-S. aureus antibody conjugated to latex beads.

[0349] Both marking parts are of identical reference (Ahlstrom-Grade 800).

[0350] Each strip is immersed in a well at the contact point 2 and left for 30 minutes to allow migration to the test line 5. The bacterial strains were tested by absorbance at OD 600 nm = 1 (equivalent to approximately 1 x 10⁹ cfu / ml for S. aureus strains and 1 x 10⁹ cfu / ml for S. uberis strains). The migration buffer is a PBS IX solution, comprising 10% sterile whole UHT milk and 1% tween20. After 30 minutes of migration, the strips are photographed.

[0351] 6.3. Results

[0352] As shown in [Fig. 10], with a sample containing S. uberis, the S. uberis test line 5 appears. With a sample containing only S. aureus, the S. aureus test line 5 appears. With a sample containing both bacteria, both test lines appear.

[0353] This clearly shows that this device is capable of the simultaneous detection of S. aureus and S. uberis.

[0354] Example 7: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus with bacterial growth and addition of the sample to a nutrient medium

[0355] 7.1. Composition of the LFA device

[0356] The LFA device or strip 1 was designed as follows: - a contact part 2: part in contact with the solution to be analyzed. - a detection part 4 comprising a test line 5 No. 1 where the RBP P3 and SUB4 proteins specific to S. uberis are immobilized, and comprising a test line 5 No. 2 where the RBP PHIL2 and PHIL5 proteins specific to S. aureus are immobilized; - an absorption part 6 to establish the flow through the device from the contact part 2.

[0357] 7.2. Implementation of the LFA system

[0358] 250 µl of bacterial suspension in PBS IX + 10% whole UHT milk + TWEEN 20 1% at the desired bacterial concentration are placed in one well of a 96-well plate.

[0359] An LFA device 1 comprising, in the detection section 4, a test line 5 with the RBP SUB4 and P3 proteins specific to S. uberis (1 µl deposited; composition: 0.625 mg / ml of SUB4 protein, 0.625 mg / ml of P3 protein, PBS IX, 20% sucrose), and a second line of test 5 with the RBP proteins PHIL 2 and PHIL 5 specific to S. aureus.

[0360] The strip is immersed at the contact part 2 for 30 minutes in the well containing the sample to be tested.

[0361] After migration, the test line is cut out and deposited on an agar in a Petri dish comprising BHI IX, 75 pM potassium tellurite and 1.5% agar.

[0362] The strips are then incubated at 37°C for 15 hours and a photo is then taken.

[0363] The results in [Fig. 1] 1 show that with the sample without bacteria, no black line appears, indicating the absence of bacteria immobilized on test line 5. In contrast, with the sample containing S. uberis bacteria, a black line appears at the S. uberis test line 5, corresponding to bacterial multiplication and the reduction of tellurite to tellurium, a chromogenic element that improves the visibility of the result. Similarly, with the sample containing S. aureus bacteria, a black line appears at the S. aureus test line 5, thus indicating bacterial multiplication and the reduction of tellurite to tellurium. Finally, with the sample containing both S. uberis and S. aureus, two black lines are observed, indicating the presence of these two bacteria in the sample.

[0364] Example 8: LFA device for the detection of S. uberis with bacterial growth and addition of the sample to a nutrient medium

[0365] 8.1. Concept of the LFA device

[0366] The sample to be tested is supplemented with a nutrient medium containing potassium tellurite in a 1 / 20 ratio. The contact portion 2 of an LFA strip 1 is immersed in the mixture. Bacteria migrate and are captured at the test line 5 where the S. uberis-specific RBP proteins are bound. This is all carried out in a tube which is then hermetically sealed to prevent the membrane from drying out. The captured bacteria grow and hydrolyze the black tellurite compound. Note that the LFA 1 device does not have a labeling portion 7.

[0367] 8.2. Implementation of the LFA mechanism

[0368] The LFA device or strip 1 was designed as follows: - a contact part 2: part in contact with the solution to be analyzed; - a detection part 4 comprising a test line 5 where the S. uberis specific RBP P3 and SUB4 proteins are immobilized (1 pl deposited; composition: 0.625 mg / ml of SUB4 protein, 0.625 mg / ml of P3 protein, PBS IX, 10% sucrose); - an absorption part 6 to establish the flow through the device from the contact part.

[0369] In a tube, 25 µl of raw milk contaminated with a known concentration of Streptococcus was added to 500 µl of a buffer composed of heart brain broth IX, TWEEN 20 1% and potassium telluride at 20 mg / ml.

[0370] After mixing the milk and buffer, strip 1 is immersed in the buffer at the contact point 2. The tube is closed and incubated at 37 °C for 15 hours. After 15 hours of incubation, the result is read. The presence of a black band at the test line indicates the presence of S. uberis in the tested sample ([Fig. 1]2).

[0371] Example 9: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus with bacterial growth and addition of the sample to a nutrient medium

[0372] 9.1. Composition of the LFA device

[0373] The LFA device or strip 1 was designed as follows: - a contact part 2: part in contact with the solution to be analyzed. - a detection part 4 comprising a test line 5 no. 1 where the RBP P3 and SUB4 proteins specific to S. uberis are immobilized (1 pl deposited; composition: 0.625 mg / ml of the SUB4 protein, 0.625 mg / ml of the P3 protein, PBS IX, 20% sucrose), and comprising a test line 5 no. 2 where the RBP PHIL 2 and PHIL 5 proteins specific to S. aureus are immobilized (1 pl deposited; composition: 0.75 mg / ml of the PHIL 5 protein and 0.25 mg / ml of the PHIL 2 protein, PBS IX, 10% sucrose); - an absorption part 6 to establish the flow through the device from the contact part 2.

[0374] An anti-rabbit antibody is deposited on control line 9.

[0375] 9.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0376] In a tube, 25 µl of raw milk contaminated with a known concentration of Streptococcus was added to 500 µl of a buffer composed of heart brain broth IX, TWEEN 20 1% and potassium telluride at 20 mg / ml.

[0377] After mixing the milk and buffer, strip 1 is immersed in the buffer at the contact point 2. The tube is closed and incubated at 37 °C for 15 hours. After 15 hours of incubation, the result is read.

[0378] The results in [Fig. 13] show that with the sample without bacteria, no black line appears, indicating the absence of bacteria immobilized on test line 5. In contrast, with the sample containing S. uberis bacteria, a black line appears on test line 5 for S. uberis, which corresponds The results are due to bacterial multiplication and the reduction of tellurite to tellurium, a chromogenic element that improves the visibility of the result. Similarly, with the sample containing S. aureus bacteria, a black line appears at test line 5 for S. aureus, thus indicating bacterial multiplication and the reduction of tellurite to tellurium. Finally, with the sample containing S. uberis and S. aureus, two black lines are observed, confirming the presence of these two bacteria in the sample. List of documents cited

[0379] For the avoidance of doubt, the following publications are cited in the application: Paramanandham et al. An Understanding of the Global Status of Major Bacterial Pathogens of Milk Concerning Bovine Mastitis: A Systematic Review and Meta-Analysis (Scientometrics). Pathogens. 2021 April 30; 10(5):545. Neculai-Valean et al. Nanomaterials and Essential Oils as Candidates for Developing Novel Treatment Options for Bovine Mastitis. Animais, vol. 11, numéro 6, mai 2021, p. 1625. Rédiger,et al. Bayesian latent class models to détermine diagnostic sensitivities and specificities of two point of care rapid tests (Selma plus, Dipslide) for the détection of Streptococcus uberis associated with mastitis in dairy cows. Frontiers in Veterinary Science, vol. 9, décembre 2022, p. 1062056. Keane OM. Symposium review: Intramammary infections—Major pathogens and strain-associated complexity. J Dairy Sci 2019;102:4713-4726. Wente et al. Associations between Streptococcus uberis strains from the animal environment and clinical bovine mastitis cases. J Dairy Sci 2019;102:9360-9369. Collado et al., Study of the efficacy of a Streptococcus uberis mastitis vaccine against an experimental intramammary infection with a heterologous strain in dairy cows. J Dairy Sci 2018 Nov;101 (11): 10290-10302. Nanerjee and Jaiswal., Recent Advances in Nanoparticle-Based Latéral Flow Immunoassay as a Point-Of-Care Diagnostic Tool for Infectious Agents and Diseases (Ruptanu Banerjee et Amit Jaiswal), 2018 143(9):1970-1996. Sajid et al.Designs, formats and applications of latéral flow assay : A literature review. Journal of Saudi Chemical Society, 2015, 19, 689-705. Brevet EP1086372B1. Collection device for single assay or oral fluids. Déposé le 29 Mars 1999 et délivré le 31 Mai 2006. US Patent 8,399,261 B2. Lateral flow assay system and methods for its use. Issued from PCT / US2008 / 0686821 filed on June 27, 2008. International application WO 2017 / 072078, Subtractive immunoassay method and lateral flow immunochromatography assay strip for performing the method. Filed on 24 October 2016. International application WO 2021 / 234321, Device for detecting a bacterium of interest. Filed on 20 May 2021.

Claims

Demands

1. Protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO:

2.

2. Composition comprising: - a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and - an RBP protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO:

2.

3. Use of the protein according to claim 1 for the capture of Streptococcus uberis in a sample.

4. Use of the composition according to claim 2 for the capture of Streptococcus uberis in a sample.

5. Use of the protein according to claim 1 for the detection of Streptococcus uberis in a sample.

6. Use of the composition according to claim 2 for the detection of Streptococcus uberis in a sample.

7. Use according to claim 5 or 6, wherein detection is carried out by ELIS A, lateral flow chromatography or lateral flow chromatography with bacterial growth.

8. Lateral flow chromatography strip for detecting S. uberis in a sample, comprising at least one part which comprises a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 2, in particular said at least one part comprises the protein of sequence SEQ ID NO:

2.

9. A lateral flow chromatography strip according to claim 8, comprising in the following order: - a contact portion comprising a sample contact zone; - a detection portion comprising a S. uberis detection zone comprising a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 2 sequence, in particular comprising the protein with SEQ ID NO: 2 sequence, said detection zone optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - an absorbing part.

10. A lateral flow chromatography strip according to claim 8, comprising in the following order: - a contact portion comprising a sample contact area; - a labeling portion comprising a labeling area comprising a marker; - a detection portion comprising a S. uberis detection area comprising a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 2 sequence, in particular comprising the protein with sequence SEQ ID NO: 2, said detection area optionally comprising a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 1 sequence, in particular the protein with sequence SEQ ID NO: 1; - an absorbing portion; said marker being conjugated to a specific S. uberis ligand selected from at least one antibody directed against S. uberis, at least one S. uberis-specific aptamer.uberis, at least one S. uberis-specific bacteriophage, at least one protein from at least one S. uberis-specific bacteriophage, at least one S. uberis-specific bacteriophage coupled to an antibody directed against S. uberis, at least one protein from an S. uberis-specific bacteriophage coupled to an antibody directed against S. uberis, in particular said at least one protein from at least one S. uberis-specific bacteriophage is an RBP protein, more particularly, a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular this is the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2, in particular said marker is a gold nanoparticle or a colored latex bead.

11. Lateral flow chromatography strip according to claim 8, comprising in the following order: - a contact portion comprising a sample contact area; - a first labeling portion comprising a S. uberis labeling area comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 2 sequence, in particular the protein with sequence SEQ ID NO: 2, said labeling area optionally comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% identity with the SEQ ID NO: 1 sequence, in particular the protein with sequence SEQ ID NO: 1; - a second labeling portion comprising an S.aureus comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 4, in particular the protein with sequence SEQ ID NO: 4 and / or comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 5, in particular the protein with sequence SEQ ID NO: 5; - a detection portion comprising a S. uberis detection zone comprising a protein comprising a sequence having at least 70% identity with the sequence SEQ ID NO: 2, in particular the protein with sequence SEQ ID NO: 2, said S. uberis detection zoneuberis optionally comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO:1, in particular the protein of sequence SEQ ID NO:1, and comprising an S. aureus detection zone comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID NO:4 and / or comprising a protein comprising a sequence having a percentage of identity of at least 70% with the sequence SEQ ID. NO:5, in particular the S. aureus detection zone includes the SEQ ID NO:4 sequence protein and / or the SEQ ID NO:5 sequence protein; - an absorbing part.

12. Method for detecting S. uberis in a sample comprising: - a step of bringing the sample into contact with the contact area of ​​a strip according to claim 10; - a step of detecting the marker on the detection area; - a step of analyzing the presence or absence of S. uberis in the sample.

13. A method for detecting S. uberis comprising: 1) Adding a sample to be tested to a nutrient medium comprising a chromogenic element; 2) Contacting an LFA strip according to claim 9 with the mixture obtained in step 1); 3) Migration between 5 and 45 min, in particular between 15 and 35 min, more particularly between 20 and 30 min, preferably 30 min; 4) Incubating the strip in the closed tube at 37°C for 15h to 24h, preferably between 17h and 20h; 5) Appearance of a color along the test line if S. uberis is present in the sample.

14. Method for detecting S. uberis and S. aureus in a sample comprising: - a step of bringing the sample into contact with the contact area of ​​a strip according to claim 11; - a step of detecting the marker on the detection area; - a step of analyzing the presence or absence of S. uberis and the presence or absence of S. aureus in the sample.

15. S. uberis detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis according to claim 9; 2) A tube capable of being hermetically sealed; 3) A vial comprising the nutrient medium with a chromogenic element, in particular potassium tellurite.

16. A kit for detecting S. uberis or for detecting S. uberis and S. aureus comprising: 1) A lateral flow chromatography strip for detecting S. uberis according to claim 10 or a strip of lateral flow chromatography to detect S. uberis and S. aureus according to claim 11; 2) A container intended to receive the sample.

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