Proteins for detecting streptococcus uberis and detection devices

Abstract

The present invention relates to proteins having 70% identity with the protein of sequence SEQ ID NO: 1 or with the protein of sequence SEQ ID NO: 2, in particular relates to the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, and to the use thereof in the capture and detection of the Streptococcus uberis bacterium. The invention also relates to a lateral flow chromatography (LFA) strip comprising at least one of these proteins.

Description

PROTEINS FOR THE DETECTION OF STREPTOCOCCUS UBERIS AND DETECTION DEVICES TECHNICAL FIELD

[0001] The present invention relates to novel proteins and their use in the capture and detection of the bacterium Streptococcus uberis. It also relates to a lateral flow chromatography (LFA) strip comprising at least one of these proteins. PRIOR TECHNIQUE

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

[0003] When bovine mastitis is caused by a bacterial infection, this disease is mainly 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 euros in France and 6 billion euros in Europe (Neculai-Valeanu et al., 2021). These economic losses are not only linked to a loss of milk yield and quality but are also linked to animal losses due to the sending of contaminated animals to the slaughterhouse (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 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 causative agent of bovine mastitis, but contagious (from cow to cow) (Wente N et al., 2019). Although different risk mitigation strategies, such as improved milking practices, post-milking teat disinfection, and antimicrobial administration, 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. The result of genetic heterogeneity within S. uberis populations is that vaccines designed against one particular strain show low efficacy against others (Collado R et al., 2018).

[0006] Detecting S. uberis is important for herd management and for directing 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 active against this bacterium. While resistance to penicillin and beta-lactams in general pose 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 on the market are mainly based on screening somatic cells found in milk. This technique can indicate that the cow is infected but it does not identify the responsible bacteria. The detection of the responsible bacteria is often carried out by the culture method or by the molecular biology approach (PCR). For the culture method, this requires incubation times of 24 to 48 hours, which does not correspond to the challenges of the sector. The molecular biology approach (PCR and / or quantitative PCR) allows for faster and more specific detection but the PCR technique only detects the presence of bacterial DNA and not the bacteria themselves, which can lead to the detection of false positives (dead bacteria already treated by the cow's immune system or by a previous treatment).In any case, both methods require sending samples to the laboratory. The number of samples must be large to be significant for 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 detect S. uberis.

[0009] It is therefore important to develop a rapid detection method for S. uberis, usable in the field, without requiring large and expensive equipment or sending samples to the laboratory. SUMMARY

[0010] According to one aspect, the present application relates to a protein comprising a sequence having a percentage 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 one aspect, the present application relates to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular it relates to the protein of sequence SEQ ID NO: 1.

[0012] According to another aspect, the present application relates to a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular a protein of sequence SEQ ID NO: 1, and / or - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular a protein of sequence SEQ ID NO: 2.

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

[0014] According to another aspect, the present application relates to the use of an RBP protein comprising a sequence having a percentage 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.

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

[0016] According to another aspect, the present application relates to the use of a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular a protein of sequence SEQ ID NO: 1, and / or - a protein comprising a sequence having a percentage 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.

[0017] According to another aspect, the present application relates to the use of an RBP protein comprising a sequence having a percentage 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.

[0018] According to another aspect, the present application relates to the use of an RBP protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the use of the RBP protein of sequence SEQ ID NO: 1 for the detection of Streptococcus uberis in a sample.

[0019] According to another aspect, the present application relates to the use of a composition comprising: - an RBP protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular a protein of sequence SEQ ID NO: 1, and / or - an RBP protein comprising a sequence having a percentage 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.

[0020] In particular, detection is performed by ELISA, lateral flow chromatography (LFA) or lateral flow chromatography with bacterial growth.

[0021] 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 portion which comprises: - a protein comprising a sequence having a percentage 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, and / or - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, said at least one part comprising in particular the protein of sequence SEQ ID NO: 1.

[0022] 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 comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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.

[0023] 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 comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0024] 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 marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 ligand specific for S. uberis chosen from at least one antibody directed against S. uberis, at least one aptamer specific for S. uberis, at least one bacteriophage specific for S. uberis, at least one protein of at least one bacteriophage specific for S. uberis, at least one bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, at least one protein of a bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein of at least one bacteriophage specific for S. uberis is an RBP protein, more particularly, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% 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.

[0025] 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 marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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 ligand specific for S. uberis chosen from at least one antibody directed against S. uberis, at least one aptamer specific for S. uberis, at least one bacteriophage specific for S. uberis, at least one protein of at least one bacteriophage specific for S. uberis, at least one bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, at least one protein of a bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein of at least one bacteriophage specific for S.uberis is an RBP protein, more particularly, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% 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.

[0026] 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 first marking part which comprises a marking 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 identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, and / or a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a second marking part which comprises a marking area of ​​S. aureus comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage 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 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone of S. aureus comprises the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0027] 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 first marking part which comprises a marking 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 identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said marking 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 identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a second marking part which comprises a marking area of ​​S. aureus comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage 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 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 S. uberis detection zone which comprises a protein comprising a sequence having a percentage 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 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 S. aureus detection zone which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or which comprises a protein comprising a sequence having a percentage 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 / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0028] According to one aspect, the present application relates to a method of detecting S. uberis in a sample comprising: - a step of bringing the sample into contact with the contact zone of a strip as described above; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis in the sample.

[0029] According to one aspect, the present application relates to a method for detecting S. uberis comprising: 1) Addition of a sample to be tested in a nutrient medium optionally comprising 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 in the closed tube at 37°C for 6 to 48 hours; 5) Appearance of colonies, or appearance of coloration in the case where the nutrient medium includes a chromogenic element, along the S. uberis test line in the event of the presence of S. uberis in the sample

[0030] According to one aspect, the present application relates to a method of detecting S. uberis in a sample comprising: - a step of bringing the sample into contact with the contact zone of a strip as described above; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis in the sample

[0031] 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 zone of a strip as described above; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis and the presence or absence of S. aureus in the sample.

[0032] According to one aspect, the present application relates to a kit for detecting S. uberis comprising: 1) A lateral flow chromatography strip to detect S. uberis as described above; 2) A container intended to receive the sample.

[0033] According to one aspect, the present application relates to a kit for detecting S. uberis and S. aureus comprising: 1) A lateral flow chromatography strip to detect S. uberis and S. aureus as described above; 2) A container intended to receive the sample.

[0034] According to one aspect, the present application relates to a kit for detecting S. uberis comprising: 1) A lateral flow chromatography strip to detect S. uberis as described above; 2) A tube capable of closing hermetically; 3) A flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) optionally, a container intended to receive the sample.

[0035] According to one aspect, the present application relates to a kit for detecting S. uberis and S. aureus comprising: 1) A lateral flow chromatography strip to detect S. uberis and S. aureus as described above; 2) A tube capable of closing hermetically; 3) A flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) optionally, a container intended to receive the sample. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1

[0036] [Fig. 1] shows the detection results with an LFA device or strip 1 comprising on the labeling zone 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: Solution without bacteria. Strip 2: solution of S. aureus strain C24B at 1 .10 9 cfu / ml. Strip 3: solution of E. coli strain 4ka at 1.10 9 cfu / ml. Strip 4: solution of S. uberis strain Duchesse at 1.10 7 cfu / ml. Strip 5: solution of S. uberis strain S49 at 1.10 7 cfu / ml. Arrows indicate the test line on the strips. (cfu=colony forming unit) Fig. 2

[0037] [Fig. 2] shows the detection results with an LFA 1 device comprising on the labeling zone 8 the RBP SUB4 protein coupled to a gold nanoparticle (diameter 40 nm) and on the test line 5 of the nitrocellulose membrane, the immobilized RBP SUB4 protein. Strip 1: Solution without bacteria. Strip 2: solution of S. aureus strain C24B at 1.10 9 cfu / ml. Strip 3: solution of E. co / / strain 4ka at 1.10 9 cfu / ml. Strip 4: solution of S. uberis strain Duchesse at 1.10 7 cfu / ml. Strip 5: solution of S. uberis strain S49 at 1.10 7 cfu / ml. Arrows indicate the test line on the strips. Fig. 3

[0038] [Fig. 3] shows the detection results with an LFA 1 device comprising on the labeling zone 8 the RBP 28.7 protein coupled to a gold nanoparticle (diameter 40 nm) and on the test line 5 of the nitrocellulose membrane, the immobilized RBP 28.7 protein. Strip 1: Solution without bacteria. Strip 2: solution of S. aureus strain C24B at 10 9 cfu / ml. Strip 3: solution of E. coli strain 4ka to 10 9 cfu / ml. Strip 4: solution of S. uberis strain Duchesse at 10 7 cfu / ml. Strip 5: solution of S. uberis strain S49 at 10 7 cfu / ml. Arrows indicate the test line on the strips. Fig. 4

[0039] [Fig. 4] shows the detection results with an LFA 1 device comprising on the marking area 8 the mixture of two proteins RBP P3 and SUB4 (OD=5 at 600 nm) coupled to gold nanoparticles (diameter 40 nm). For the control line 9, the protein on the membrane is VHS / G57 (UniProt: Q08487 / GenBank: CAA52080) at 2.22 mg / ml and on the conjugate the antibody used for the detection of this protein is a mouse monoclonal lgG2b anti-VHS / G57, this antibody is coupled to gold at OD=2.5. On the test line 5 are immobilized the mixture of two proteins RBP P3 (0.625 mg / ml) and SUB4 (0.625 g / ml). Strip 1: Solution without bacteria. Strip 2: solution of S. aureus strain C24B at 1.10 9 cfu / ml. Strip 3: solution of S. uberis strain Duchesse at 1.10 7 cfu / ml. Strip 4: solution of S. uberis strain Duchesse at 1.10 6 cfu / ml. Arrow A indicates the control line. Arrow B indicates the test line. Fig. 5

[0040] [Fig. 5] shows the detection results with an LFA 1 device. A. Labeling area 8 including RBP SUB4 protein coupled to gold nanoparticles (diameter 40 nm) and RBP P3 protein immobilized on test line 5 of the nitrocellulose membrane. B. Labeling area 8 including RBP SUB4 protein coupled to gold nanoparticles (diameter 40 nm) and RBP SUB4 protein immobilized on test line 5 of the nitrocellulose membrane. C. Labeling area 8 including RBP SUB4 protein coupled to gold nanoparticles (diameter 40 nm) and RBP P3 and SUB4 proteins immobilized on test line 5 of the nitrocellulose membrane. Strip 1: Solution without bacteria. Strip 2: Solution of S. aureus strain C24B. 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 1.10 9 cfu / ml for S. aureus strains and 1 .10 7 cfu / ml for S. uberis strains. Fig. 6

[0041] [Fig. 6] montre les résultats de détecteur avec un dispositif LFA 1 companding 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: S. uberis (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 (273). 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). Strains were tested at absorbance at OD 600 nm = 1, which is equivalent to 1.10. 9 cfu / ml for S. aureus strains and 1.10 7 cfu / ml for S. uberis strains. Fig. 7

[0042] [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 stuck, nitrocellulose side down, onto 1X BHI agar + 75 pM tellurite + 1.5% agar. Incubation at 37°C for 15 hours is then carried out before reading. Different contaminations of milk by S. uberis are tested: 1= S. uberis Duchess strain (1.10 4 cfu / ml); 2: Strain S. uberis Duchess (1.10 3 cfu / ml); 3 = S. uberis Duchess strain (1.10 2cfu / ml); 4 = NC milk (without bacteria). The arrows indicate the test line. B: The strips are placed in a cassette and a 1X BHI agar + 75 pM tellurite + 0.4% agar is placed on the test zone 5. The agar is then sealed with an 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 10 4 cfu / ml and S. uberis Mona Lisa at 1 10 4 cfu / ml. Fig. 8

[0043] [Fig. 8] shows the results of a LFA duplex for the simultaneous detection of S. aureus and S. uberis. The S. uberis-specific RBP proteins P3 and SUB4, respectively of sequence SEQ ID NO: 1 and SEQ ID NO: 2, were deposited on a test line 5. These same proteins were also conjugated with gold nanoparticles and sprayed on the labeling part at OD=5 (6 μl). The S. aureus-specific RBP proteins PHIL 2 and PHIL 5, respectively of sequence SEQ ID NO: 4 and SEQ ID NO: 5, were deposited on another test line 5. These same proteins were also conjugated with gold nanoparticles and sprayed on the labeling part at OD=5 (6 μl). Four milk samples were tested: 1) Without bacteria. 2) milk contaminated with S. uberis alone, Duchesse strain at OD=1 (1 .10 7 cfu / ml). 3. milk contaminated with S. aureus alone, strain C24B at OD=1 (1 .10 9cfu / nnl). 4. Milk contaminated with both bacteria. During migration, the contaminated milk was diluted 10 times in 1X PBS buffer with 1% Tween. Fig. 9

[0044] [Fig. 9] shows the results of a duplex LFA for the simultaneous detection of S. aureus and S. uberis. The S. uberis-specific RBP proteins P3 and SUB4, respectively of sequence SEQ ID NO: 1 and SEQ ID NO: 2, were deposited on a test line 5. The SUB4 protein was conjugated with red latex beads and sprayed on the marking area 8 at 0.03% dry bead extract. The RBP proteins PHIL 2 and PHIL 5 specific for S. aureus, respectively of sequence SEQ ID NO: 4 and SEQ ID NO: 5, were deposited on another test line 5. These same proteins were conjugated with blue latex beads and sprayed on the marking zone 8 at 0.5% dry extract of beads for PHIL 2 and 0.5% dry extract of beads for PHI 5 (mixture of PHIL 2 and PHIL 5 proteins deposited on a strip). Four milk samples were tested: 1) Without bacteria. 2) Milk contaminated with S. uberis alone, Duchesse strain at DQ=10 (10.10 7cfu / ml). 3) Milk contaminated with S. aureus alone, strain S41 at DQ=10 (10.10 9 cfu / ml). 4) Milk contaminated by the mixture of the two bacteria. During migration, the milk used was diluted 10 times in 1X PBS buffer with 1% Tween20. Fig. 10

[0045] [Fig. 10] shows the results of a duplex LFA for the simultaneous detection of S. aureus and S. uberis. The S. uberis-specific RBP proteins P3 and SUB4, respectively of sequence SEQ ID NO: 1 and SEQ ID NO: 2, were deposited on a test line 5. The SUB4 protein was conjugated with red latex beads and sprayed on the marking area 8 at 0.03% dry bead extract. The S. aureus-specific RBP proteins PHIL 2 and PHIL 5, respectively of sequence SEQ ID NO: 4 and SEQ ID NO: 5, were deposited on another test line 5. An anti-S. aureus antibody was conjugated with blue latex beads and sprayed on the marking area 8 at 0.3% dry bead extract. Four milk samples were tested: 1) Without bacteria. 2) Milk contaminated with S. uberis alone, Duchesse strain at DQ=10 (10.10 7 cfu / ml). 3) Milk contaminated with S. aureus alone, strain S41 at DQ=10 (10.10 9cfu / ml). 4) Milk contaminated by the mixture of the two bacteria. During migration, the milk used was diluted 10 times in 1X PBS buffer with 1% Tween20. Fig. 11

[0046] [Fig. 11] shows the results of an LFA device placed in 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 part 7 and contain in the detection part 4, the RBP proteins P3 and SUB4 specific to S. uberis, respectively of sequence SEQ ID NO: 1 and SEQ ID NO: 2, 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 1.10 5 cfu / ml, 3) Raw milk contaminated with S. uberis at 1.10 4 cfu / ml, 4) Raw milk contaminated with S. uberis at 1.10 3cfu / ml, 5) Raw milk contaminated with S. uberis at 1.10 2 cfu / ml, 6) control line, 7) S. uberis test line. Fig. 12

[0047] [Fig. 12] shows the results of a duplex LFA device placed 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 part 7. In the detection part 4, the RBP proteins P3 and SUB4 specific to S. uberis, of sequence SEQ ID NO: 1 and SEQ ID NO: 2 respectively, were deposited on a test line 5. The RBP proteins PHIL 2 and PHIL 5 specific to S. aureus, of sequence SEQ ID NO: 4 and SEQ ID NO: 5 respectively, were deposited on another 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 (Ctrl), 2) Raw milk contaminated with S. aureus, strain 49 at 1.105 cfu / ml, 3) Raw milk contaminated with S. uberis, Duchesse strain at 1.10 5 cfu / ml, 4) Raw milk contaminated with S. uberis at 1.10 5 cfu / ml and S. aureus at 1.10 5 cfu / ml. Arrow A indicates the test line. Arrow B indicates the S. uberis (SU) detection line. Arrow C indicates the S. aureus (SA) detection line. Fig. 13

[0048] [Fig. 13] shows a schematic of a sandwich-type ELISA test: 1. Label 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

[0049] [Fig. 14] shows the diagram of an LFA strip 1 comprising a contact part 2 which comprises a contact area 3, a marking part 7 which comprises a marking area 8, a detection part 4 which comprises a detection area 5 and a control area 9, and an absorbent part 6, immersed in a sample to be analyzed. The black lines delimit the different parts of the strip. Fig. 15

[0050] [Fig. 15] shows in A the diagram of an LFA strip 1 comprising a contact part 2 which comprises a contact area 3, a detection part 4 which comprises a detection area 5 and a control area 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 growth of bacteria is observed on the test line in the case where the sample is contaminated by said bacteria. Fig. 16

[0051] [Fig. 16] shows in A the diagram of an LFA strip 1 comprising a contact part 2 which comprises a contact zone 3, a detection part 4 which comprises a detection zone 5 and a control zone 9, and an absorbent part 6. In B, the method for detecting a bacterium of interest by the LFA method with nutrient medium. The strip 1 is immersed in the sample to be tested previously mixed with nutrient medium (1). After migration and incubation, the development of bacteria is observed on the test line (2) in the case where the sample is contaminated by said bacterium. DEFINITIONS

[0052] The following are definitions that apply throughout this disclosure.

[0053] For the purposes of the present application, "lateral flow chromatography" (LFA) means a chromatography device in which a liquid sample migrates by capillary action in a flow directed through a strip comprising several parts. LFA is known from the prior art and has in particular 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).

[0054] For the purposes of this application, “at least one” means one, two, three, four, five, six and the following.

[0055] For the purposes of the present application, a “bacteriophage specific for a bacterium of interest” is a bacteriophage capable of binding specifically to this bacterium. This specificity can be determined by the spot method described in application WO2021 / 234321. A bacteriophage is specific for a bacterium if a lysis plaque is observed around a spot comprising said bacteriophage, deposited on a Petri dish covered with a culture of said bacterium in exponential phase, after 18 hours of incubation at a temperature suitable for the bacterium.

[0056] Phage receptor binding proteins (RBPs) determine the specificity of a phage for a host bacterium. In phages, these proteins are found in the tail and are involved in the first step 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 attachment 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).

[0057] RBP proteins are different from cell-binding domains (CBDs). RBPs are involved in the first step of the phage infection cycle, whereas CBDs are involved late in the infection cycle. CBDs are protein domains found in lytic enzymes produced by phages, which allow 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 progeny virion. Endolysins targeting Gram-positive bacteria generally 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 subsequently hydrolyze the cell wall into fragments, leading to osmotic lysis of the cells.

[0058] The specificity of recognition of a phage protein for a bacterium can be established by placing the bacterium and magnetic beads coated with a phage protein in the presence of the bacterium, followed by recovery of the magnetic beads using a magnetic support, then after resuspension in a buffer, spreading on a culture medium (in particular a specific agar). After 24 to 48 hours at a temperature appropriate for the bacterium, a significant difference between the count of bacterial colonies on the culture medium where the magnetic beads coated with the protein have been spread and the count of bacterial colonies on a culture medium where magnetic beads incubated with the bacterium but not coated with the protein, means that the phage protein is capable of binding to the bacterium.

[0059] Specificity can also be assessed by the ELISA test as described in the application. Bacteria are attached to the bottom of the wells of a plate. RBP proteins coupled to a GTS tag during their production are deposited in each well. After incubation, a washing step is carried out to remove unbound proteins. An incubation step with an anti-GTS antibody coupled to HRP or another substrate is then carried out to reveal the presence of the bacteria.

[0060] In this application, the terms “bacteriophage” and “phage” designate the same entity, they are used interchangeably and are interchangeable.

[0061] In this application, the expressions “antibody directed against X” and “anti-X antibodies” are equivalent and interchangeable.

[0062] In this application, the expressions “X conjugated to Y” and “Y conjugated to X” are equivalent. The term “marked” means “conjugated to a marker”. STATEMENT OF THE INVENTION

[0063] A first aspect of the invention relates to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2.

[0064] In particular, the present invention relates to a protein of sequence SEQ ID NO: 2. This protein, also called SUB4 in the present application, is an RBP protein of a bacteriophage specific to S. uberis.

[0065] A second aspect of the invention relates to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1.

[0066] In particular, the present invention relates to a protein of sequence SEQ ID NO: 1. This protein, also called P3 in the present application, is an RBP protein of a bacteriophage specific to S. uberis.

[0067] A third aspect of the invention relates to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3.

[0068] In particular, the present invention relates to a protein of sequence SEQ ID NO: 3. This protein, also called 28.7 in the present application, is an RBP protein of a bacteriophage specific to S. uberis.

[0069] For the purposes of the present application, a percentage identity of at least 70% may be in particular 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 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 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.

[0070] Thus, within the meaning of the invention, proteins having a percentage 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, are proteins having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or even at least 95% identity with the sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively and which retain the function of specific binding to S. uberis for its detection.

[0071] For example, the specific binding of a protein to S. uberis can be demonstrated by using the protein in an LFA device as described in the present application, with a sample comprising the bacterium S. uberis. In parallel, it is used in the same LFA device with a sample comprising S. aureus. The observation of a signal on the detection line with the sample containing S. uberis and the absence of a signal with the sample containing S. aureus, testifies that the protein allows the specific binding to S. Uberis.

[0072] A fourth aspect of the invention relates to a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and / or - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2.

[0073] In particular, said composition comprises: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2.

[0074] In particular, said composition comprises the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2.

[0075] In particular, said composition comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2.

[0076] Said compositions may further comprise a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3.

[0077] In particular, said composition comprises the protein of sequence SEQ ID NO:1, the protein of sequence SEQ ID NO:2 and the protein of sequence SEQ ID NO:3.

[0078] The compositions may further comprise excipients.

[0079] A fifth aspect of the invention relates to the use of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, for the capture of the bacterium S. uberis in a sample.

[0080] For the purposes of this application, a sample means any solution likely to contain the bacteria of interest. The sample may be diluted in a buffer solution prior to implementing the detection method.

[0081] In particular, the invention relates to the use of 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 for the capture of the bacterium S. uberis.

[0082] In particular, the invention relates to the use of a protein comprising a sequence having a percentage 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 to the use of the protein of sequence SEQ ID NO: 2 for the capture of the bacterium S. uberis in a sample.

[0083] In particular, the invention relates to the use of a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and / or - a protein comprising a sequence having a percentage 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 a composition comprising the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2 for the capture of the bacterium S. uberis in a sample.

[0084] In particular, the invention relates to the use of a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and - a protein comprising a sequence having a percentage 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 a composition comprising the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2 for the capture of the bacterium S. uberis in a sample.

[0085] Said composition may further comprise a receptor binding protein (RBP) of a bacteriophage specific for S. uberis, comprising a sequence having a percentage 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.

[0086] A sixth aspect of the invention relates to the use of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, for the detection of the bacterium S. uberis in a sample.

[0087] In particular, the invention relates to the use of 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 for the detection of the bacterium S. uberis.

[0088] In particular, the invention relates to the use of a protein comprising a sequence having a percentage 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 to the use of the protein of sequence SEQ ID NO: 2 for the detection of the bacterium S. uberis in a sample.

[0089] In particular, the invention relates to the use of a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and / or - a protein comprising a sequence having a percentage 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 a composition comprising the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2 for the detection of the bacterium S. uberis in a sample.

[0090] In particular, the invention relates to the use of a composition comprising: - a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, and - a protein comprising a sequence having a percentage 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 a composition comprising the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2 for the detection of the bacterium S. uberis in a sample.

[0091] Said composition may further comprise a protein comprising a sequence having a percentage 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.

[0092] Said detection can be carried out by ELISA, lateral flow chromatography or lateral flow chromatography with bacterial growth.

[0093] In a first embodiment, the detection is carried out by an ELISA test.

[0094] ELISA (enzyme-linked immunosorbent assay) is an enzyme-linked immunosorbent assay on solid support, based on the specific antigen-antibody reaction. The classic principle of an ELISA, known as a sandwich type, is to use a support, such as a microplate, on which a ligand specific to the bacterium of interest is immobilized. After placing the support in contact 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 label, thus allowing the detection of the analyte in the event of a signal. Figure 13 illustrates detection by the ELISA method.

[0095] A protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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.

[0096] 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 is added, which is coupled to a marker.

[0097] When the marker is an enzyme, the addition of the enzyme substrate makes it possible to obtain a colored reaction that can be measured by absorbance.

[0098] In an alternative embodiment, the antibody may be replaced by another protein having an affinity for the bacterium of interest. This may for example be an RBP protein. Thus, in another embodiment, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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, may be used in an ELISA for the detection of S. uberis, as a replacement for the capture antibody and / or the primary antibody coupled to the marker.

[0099] In a second embodiment, the detection is carried out by lateral flow chromatography (LFA). The principle of LFA detection is described in Example 2 and illustrated in Figure 14.

[0100] Thus, the present invention relates to the use of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in a lateral flow chromatography strip. In particular, it concerns the use of 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 in a lateral flow chromatography strip.

[0101] The lateral flow chromatography strips are as described further in this application.

[0102] Said protein is used in at least a part of the LFA strip. It can be used at the marking area of ​​the marking part and / or at the detection area (test line) of the detection part.

[0103] In particular, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, is used on the marking area.

[0104] In particular, the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2 is used on the marking area.

[0105] In particular, the protein of sequence SEQ ID NO: 2 is used on the marking area. In particular, the proteins of sequences SEQ ID NO: 1 and SEQ ID NO: 2 are used on the marking area.

[0106] In particular, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, is used on the detection zone.

[0107] In particular, the protein of sequence SEQ ID NO: 1 and / or the protein of sequence SEQ ID NO: 2 is used on the detection zone.

[0108] In particular, the protein of sequence SEQ ID NO: 2 is used on the detection area (test line). In particular, the proteins of sequences SEQ ID NO: 1 and SEQ ID NO: 2 are used on the detection area (test line).

[0109] In a third embodiment, the detection is carried out by lateral flow chromatography (LFA) with bacterial growth on the detection part, in particular at the detection zone (test line) which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3.

[0110] In particular, the detection zone comprises 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. More particularly, it comprises the protein of sequence SEQ ID NO: 2, and even more particularly the proteins of sequence SEQ ID NO: 1 and SEQ ID NO: 2.

[0111] This mode of detection with bacterial growth encompasses two concepts detailed later: - after migration, addition of agar to the detection zone, 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] A seventh aspect of the invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample.

[0114] 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.

[0115] Thus, the present invention relates to a 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 identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3. In particular, said at least one part comprises 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.

[0116] 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 comprises a protein comprising a sequence having a percentage 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.

[0117] 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 comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular said at least one part comprises the protein of sequence SEQ ID NO: 1.

[0118] Said part comprising said protein may be a labeling part and / or a detection part.

[0119] In one embodiment, the portion comprising the protein is a detection portion.

[0120] Thus, in a particular embodiment, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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.

[0121] In particular, the lateral flow chromatography strip for detecting S. uberis in a sample, includes in order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0122] In particular, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbent part.

[0123] The contact part (sample pad) includes a contact area intended to be placed in contact with the sample. Its function is to distribute the sample evenly so that the migration of the sample by capillarity to the following parts is smooth, continuous and homogeneous. The contact part and the contact area are a capillary matrix. For example, the contact part and the contact area are made of cellulose or glass fibers.

[0124] The detection pad includes a detection area where the bacterium of interest S. uberis, if present in the analyzed sample, is captured on the strip thanks to a specific RBP protein immobilized there. The detection pad 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 adsorptions. For example, the detection pad and the detection pad are a nitrocellulose membrane.

[0125] The absorbent pad acts as an absorber. It helps maintain the flow rate through the strip and collects excess sample and reagents, preventing sample backflow. For example, the absorbent pad is made of cotton, cotton mixed with glass fibers, cellulose, or nitrocellulose.

[0126] For the purposes of this application, "in order" means the order in which the different parts which make up the strip will be crossed by the flow comprising the sample, but this term does not mean that these parts are necessarily contiguous.

[0127] In a particular embodiment, one of the ends of the contact portion is superimposed with one of the ends of the portion downstream thereof to form a first superposition zone, one of the ends of the detection portion is superimposed with one of the ends of the portion upstream thereof to form a second superposition zone and the other end of the detection portion is superimposed with one of the ends of the portion downstream thereof to form a third superposition zone, and one of the ends of the absorbing portion is superimposed with one of the ends of the portion downstream thereof to form a fourth superposition zone. In particular, the first, second and third superposition zones are distinct from each other. In particular, the contact zone is distinct from the first superposition zone.In particular, the detection zone is distinct from the second and third overlapping zones.

[0128] In a particular embodiment, one of the ends of the detection portion is superimposed with one of the ends of the contact portion and the other end of the detection portion is superimposed with one of the ends of the absorbing portion.

[0129] In a more particular embodiment, one of the ends of the detection portion is superimposed with one of the ends of the contact portion to form a first superposition area, and the other end of the detection portion is superimposed with one of the ends of the absorbing portion to form a second superposition area. In particular, the first, second and third superposition areas are separated from each other. In particular, the contact area is distinct from the first overlapping area. In particular, the detection area is distinct from the first and second overlapping areas.

[0130] In a particular embodiment, the detection zone is a line transverse to the strip, thus forming a detection line or test line.

[0131] Optionally, the strip comprises a system for controlling the migration of the sample from the contact part to the detection part. Thus, optionally, the detection part comprises a control zone, preferably downstream of the detection zone. This control zone is distinct from the detection zone. In particular, it is also distinct from the overlapping zones between the parts of the strip. In particular, the control zone is a line transverse to the strip, thus forming a control line or control line. The control zone comprises a ligand specific for an analyte other than the bacterium of interest S. uberis. The analyte is conjugated to a label. Optionally, the analyte conjugated to a label is deposited on a part of the strip upstream of the detection part and / or is added beforehand to the sample.For example, the labeled analyte is a primary antibody conjugated to a label and the ligand on the control region is a secondary antibody directed against the primary antibody conjugated to a label.

[0132] When the strip comprises three parts, namely a contact part, a detection part and an absorbent part, the detection can be done for example with bacterial growth on the detection part, in particular at the detection zone (test line).

[0133] This mode of detection with bacterial growth encompasses two concepts: - after migration, addition of agar to the detection zone, and - before migration, addition of the sample to a nutrient medium.

[0134] In particular, said agar and said nutrient medium comprise a chromogenic element.

[0135] 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 Figure 15.

[0136] The LFA strip used in this embodiment is a strip as described in the present application which comprises a contact portion, a detection portion and an absorbent portion. It does not comprise a labeling portion (conjugate pad). The sample to be tested migrates directly through the detection portion which comprises a detection zone (test line) where a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3 is immobilized by adsorption. In particular, the detection zone comprises 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.

[0137] Thus, in this embodiment, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular, the detection zone comprises 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.

[0138] In particular, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0139] In particular, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbent part.

[0140] The method for detecting S. uberis by LFA with bacterial growth includes the following steps: 1) Contacting the 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) Removing the strip; 4) Cutting out the detection area (test line); 5) Depositing the cut test line on an agar, or fixing the cut detection zone in a cassette which is then covered with agar; 6) Incubation at 37°C for 6 to 48 hours; 7) Appearance of colonies along the test line if S. uberis is present in the sample.

[0141] The said incubation at 37°C is carried out for 6h to 48h, in particular 15h to 48h, 15 to 24h, 18h to 24h, more particularly for 17h to 20h.

[0142] In particular, agar includes a chromogenic element.

[0143] The process for detecting S. uberis by LFA with bacterial growth then includes 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) Removing the strip; 4) Cutting out the detection area (test line); 5) Depositing the cut test line onto an agar comprising a chromogenic element, or fixing the cut detection zone in a cassette which is then covered with agar comprising a chromogenic element; 6) Incubation at 37°C for 6 to 48 hours; 7) Appearance of a coloration along the test line in case of presence of S. uberis in the sample.

[0144] The said incubation at 37°C is carried out for 6h to 48h, in particular 15h to 48h, 15 to 24h, 18h to 24h, more particularly for 17h to 20h.

[0145] In steps 1), the strip is dipped into the sample so that the contact area of ​​the contact part is immersed in the sample.

[0146] Optionally, the sample can be diluted in a buffer solution. This facilitates migration.

[0147] In particular, the agar mentioned in steps 5) is an agar comprising BHI 1X (Brain heart infusion) and agar between 0.4% and 2% (weight / volume).

[0148] If the agar contains 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 range, i.e. between 380 nm and 780 nm.

[0149] When the chromogenic element is potassium tellurite, the appearance of a black color along the test line indicates the presence of S. uberis in the sample being tested. Indeed, S. uberis is capable of hydrolyzing potassium tellurite into tellurium, which is a black-colored compound. Conversely, the absence of a black color indicates the absence of S. uberis in the sample.

[0150] In particular, the concentration of potassium tellurite in agar is between 1.5 pM and 150 pM, in particular between 50 and 100 pM, in particular it is 75 pM.

[0151] The present invention also relates to a kit for detecting S. uberis by LFA with bacterial growth comprising: 1) A lateral flow chromatography strip as previously described to detect S. uberis in a sample, 2) An agar flask optionally comprising a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally a container to receive the sample.

[0152] In particular, the S. uberis detection kit by LFA with bacterial growth 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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) An agar flask optionally comprising a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally a container to receive the sample.

[0153] In particular, the S. uberis detection kit by LFA with bacterial growth 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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) An agar flask optionally comprising a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally a container to receive the sample.

[0154] In particular, the S. uberis detection kit by LFA with bacterial growth 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbent part; 2) An agar flask optionally comprising a chromogenic element such as potassium tellurite; 3) Optionally a container intended to receive the sample; 4) Optionally a cassette.

[0155] The concept of the LFA detection method with bacterial growth and, before migration, addition of the sample to a nutrient medium is described in Example 8 and illustrated in Figure 16.

[0156] The LFA strip used in this embodiment is a strip as described in the present application which comprises a contact portion, a detection portion and an absorbent portion. It does not comprise a labeling portion (conjugate pad). The sample to be tested migrates directly through the detection portion which comprises a detection zone (test line) where a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3 is immobilized by adsorption. In particular, the detection zone comprises 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.

[0157] Thus, in this embodiment, said lateral flow chromatography strip for detecting S. uberis 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 comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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.

[0158] In particular, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0159] In particular, said lateral flow chromatography strip for detecting S. uberis 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbent part.

[0160] The method for detecting S. uberis by LFA with bacterial growth includes the following steps: 1) Addition of a sample to be tested in 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) Incubation of the strip at 37°C for 6 to 48 hours; 5) Appearance of colonies along the test line if S. uberis is present in the sample.

[0161] The said incubation at 37°C is carried out for 6h to 48h, in particular 15h to 48h, 15 to 24h, 18h to 24h, more particularly for 17h to 20h.

[0162] In particular, the nutrient medium comprises a chromogenic element.

[0163] The process for detecting S. uberis by LFA with bacterial growth then includes the following steps: 1) Addition of a sample to be tested in a nutrient medium comprising 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) Incubation of the strip at 37°C for 6 to 48 hours; 5) Appearance of a coloration along the test line in case of presence of S. uberis in the sample.

[0164] The said incubation at 37°C is carried out for 6h to 48h, in particular 15h to 48h, 15 to 24h, 18h to 24h, more particularly for 17h to 20h.

[0165] Thus, in particular, the method for detecting S. uberis by LFA comprises the following steps: 1) Addition of a sample to be tested in a nutrient medium optionally comprising 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) Incubation of the strip at 37°C for 6 to 48 hours; 5) Appearance of colonies, or appearance of coloration in the case where the nutrient medium includes a chromogenic element, along the S. uberis test line in the event of the presence of S. uberis in the sample.

[0166] When the migration is complete, that is to say when all the elements of the strip, including the absorbent part, are soaked in solution, there is still enough liquid at the contact part, which helps prevent dehydration of the strip.

[0167] In steps 1), the strip is dipped into the sample so that the contact area of ​​the contact part is immersed in the sample.

[0168] Typically, the sample to nutrient medium ratio is 1:10.

[0169] The nutrient medium can be a 1X BHI (Brain Heart Infusion) medium. Optionally, it can include 1% (weight / volume) of Tween 20.

[0170] If the nutrient medium 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 range, i.e. between 380 nm and 780 nm.

[0171] When the chromogenic element is potassium tellurite, the appearance of a black color along the test line indicates the presence of S. uberis in the sample being tested. Indeed, S. uberis is capable of hydrolyzing potassium tellurite into tellurium, which is a black-colored compound. Conversely, the absence of a black color indicates the absence of S. uberis in the sample.

[0172] 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.

[0173] In particular, steps 2), 3) and 4) are carried out in a tube capable of hermetically closing.

[0174] In fact, the sample to be tested is added to the nutrient medium including in particular a chromogenic element. The mixture is homogenized and poured into a tube capable of closing hermetically. The strip is then immersed in the mixture as described in step 2), so that the contact area of ​​the contact part is immersed in the mixture.

[0175] The tube is then closed and held upright with the strip inside for migration as indicated in step 3), then incubated as described in step 4).

[0176] The hermetically sealable tube has suitable dimensions to hold the LFA strip in an upright position while ensuring a hermetic seal. This prevents the strip from drying out during incubation. In particular, it is a transparent tube which allows the detection result to be read through it, without the need to open the tube.

[0177] The present invention thus also relates to a kit for detecting S. uberis by LFA with bacterial growth comprising: 1) A lateral flow chromatography strip as previously described to detect S. uberis in a sample, 2) A tube capable of closing hermetically; 3) A flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0178] 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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 flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) Optionally, a container to receive the sample.

[0179] 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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 flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) Optionally a container to receive the sample.

[0180] In particular, the S. uberis detection kit by LFA with bacterial growth 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, - an absorbent part; 2) A tube capable of closing hermetically; 3) A flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) Optionally a container to receive the sample.

[0181] In a particular embodiment, said lateral flow chromatography strip makes it possible to detect S. uberis and one or more other bacteria of interest in a sample. The detection part then comprises one or more additional detection zones on which the appropriate ligands are immobilized to detect this or these other bacteria of interest.

[0182] This type of strip is called “Duplex LFA”.

[0183] In a particular embodiment, said lateral flow chromatography strip makes it possible to detect S. uberis and S. aureus in a sample.

[0184] 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 3 parts, namely a contact part, a detection part and an absorbent part, and which comprises an additional detection zone in the detection part. On this additional detection zone, or test line, is immobilized a ligand specific for S. aureus.

[0185] This S. aureus-specific ligand may be at least one antibody directed against S. aureus, at least one aptamer specific for S. aureus, at least one bacteriophage specific for S. aureus, at least one protein of at least one bacteriophage specific for S. aureus, at least one bacteriophage specific for S. aureus coupled to an antibody directed against S. aureus, at least one protein of a bacteriophage specific for S. aureus coupled to an antibody directed against S. aureus. In particular, said at least one protein of at least one bacteriophage specific for S. aureus is an RBP protein. More particularly, it may be a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5. Typically, it may be the protein of sequence SEQ ID NO:4 and / or the protein of sequence SEQ ID NO:5.

[0186] Thus, 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 part comprising a S. uberis detection zone which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 comprises an antibody directed against S. aureus; - an absorbent part.

[0187] 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 S. uberis detection zone which comprises a protein comprising a sequence having a percentage 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 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 comprises an antibody directed against S. aureus; - an absorbent part.

[0188] 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 S. uberis detection zone which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular it comprises the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, and comprising a S. aureus detection zone which comprises an antibody directed against S. aureus; - an absorbent part.

[0189] 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 part comprising a S. uberis detection zone which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the S. uberis detection zone comprises 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 comprises a ligand specific for S.aureus, in particular a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone of S. aureus comprises the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5;. - an absorbent part.

[0190] 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 comprises a protein comprising a sequence having a percentage 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 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 S. aureus detection zone which comprises a ligand specific for S. aureus, in particular a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage 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 / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0191] 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 S. uberis detection zone which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular it comprises the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, and comprising a S. aureus detection zone which comprises a ligand specific for S. aureus, in particular a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the protein of sequence SEQ ID NO: 4 and the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0192] In the detection part, the detection area of ​​S. uberis and the detection area of ​​S. aureus are distinct from each other, i.e., they do not overlap.

[0193] 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 making it possible to detect one or more other bacteria of interest, and after migration, addition of agar to the detection zone.

[0194] 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 comprise a marking portion (conjugate pad).

[0195] In particular, it is an LFA strip for detecting S. uberis and S. aureus in a sample as described previously.

[0196] The detection process for S. uberis and S. aureus then includes 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) Removing the strip; 4) Cutting out the detection area (test line); 5) Depositing the cut test line onto an agar optionally comprising a chromogenic element, or fixing the cut detection zone in a cassette which is then covered with agar optionally comprising a chromogenic element; 6) Incubation at 37°C for 6 to 48 hours; 7) Appearance of colonies or appearance of a coloration, in the case where the agar includes a chromogenic element, along the S. uberis test line in the case of the presence of S. uberis in the sample and / or appearance of colonies or appearance of a coloration, in the case where the agar includes a chromogenic element, along the S. aureus test line in the case of the presence of S. aureus in the sample.

[0197] The said incubation at 37°C is carried out for 6h to 48h, in particular 15h to 48h, 15 to 24h, 18h to 24h, more particularly for 17h to 20h.

[0198] In particular, it is an agar comprising BHI 1X (Brain heart infusion) and agar between 0.4% and 2% (weight / volume).

[0199] If the agar contains a chromogenic element, the chromogenic element is an element that S. uberis and S. aureus are capable of hydrolyzing and whose hydrolysis product is colored, i.e. detectable in the visible range, i.e. between 380 nm and 780 nm.

[0200] 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 sample tested. Indeed, S. uberis and S. aureus are capable of hydrolyzing potassium tellurite into tellurium, which is a black-colored compound. Conversely, the absence of a black coloration indicates the absence of S. uberis and / or S. aureus in the sample.

[0201] In particular, the concentration of potassium tellurite in agar is between 1.5 pM and 150 pM, in particular between 50 and 100 pM, in particular it is 75 pM.

[0202] The present invention thus also relates to a kit for detecting S. uberis and S. aureus comprising: 1) A lateral flow chromatography strip for detecting S. uberis and S. aureus as described in this application; 2) An agar flask optionally comprising a chromogenic element such as potassium tellurite; 3) Optionally a cassette; 4) Optionally a container to receive the sample.

[0203] 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 making it possible to detect one or more other bacteria of interest and, before migration, addition of the sample into a nutrient medium.

[0204] 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 comprise a marking portion (conjugate pad).

[0205] In particular, it is an LFA strip for detecting S. uberis and S. aureus in a sample as described previously.

[0206] The detection process for S. uberis and S. aureus then includes the following steps: 1) Addition of a sample to be tested in a nutrient medium optionally comprising a chromogenic element; 2) Contacting an LFA strip as described in the present 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 6 to 48 hours; 5) Appearance of colonies or appearance of a coloration, in the case where the agar includes a chromogenic element, along the S. uberis test line in the case of the presence of S. uberis in the sample and / or appearance of colonies or appearance of a coloration, in the case where the agar includes a chromogenic element, along the S. aureus test line in the case of the presence of S. aureus in the sample.

[0207] The said incubation at 37°C is carried out for 6h to 48h, in particular 15h to 48h, 15 to 24h, 18h to 24h, more particularly for 17h to 20h.

[0208] Typically, the sample to nutrient medium ratio is 1:10.

[0209] The nutrient medium can be a 1X BHI (Brain Heart Infusion) medium. Optionally, it can include 1% (weight / volume) of Tween 20.

[0210] If the nutrient medium includes a chromogenic element, the chromogenic element is an element that S. uberis and S. aureus are capable of hydrolyzing and whose hydrolysis product is colored, i.e. detectable in the visible range, i.e. between 380 nm and 780 nm.

[0211] 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 sample tested. Indeed, S. uberis and S. aureus are capable of hydrolyzing potassium tellurite into tellurium, which is a black-colored compound. Conversely, the absence of a black coloration indicates the absence of S. uberis and / or S. aureus in the sample.

[0212] In particular, the concentration of potassium tellurite in the nutrient medium is between 5 mg / nnL and 50 nng / mL, more particularly between 15 mg / L and 25 mg / L, in particular it is 20 mg / L.

[0213] In particular, steps 2), 3) and 4) are carried out in a tube capable of hermetically closing.

[0214] The present invention thus also relates to a kit for detecting S. uberis and S. aureus 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 flask comprising the nutrient medium optionally comprising a chromogenic element, in particular potassium tellurite; 4) Optionally a container to receive the sample.

[0215] In one embodiment, the portion comprising the protein is a labeling portion and / or a detection portion.

[0216] Thus, in a particular embodiment, said strip further comprises, between the contact part and the detection part, a marking part which comprises a marking area.

[0217] The marking part (conjugate pad) includes a marking area where bacteria are marked with a marker when the sample flow to be analyzed passes through it. Their role is to hold the marker until the strip is used and to release the marker when there is recognition between the marker and a bacterium, as the sample flow passes through. For example, the marking part and the marking area are made of glass fibers, cellulose or polyesters.

[0218] Thus, in a particular embodiment, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of the bacterium of interest which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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.

[0219] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0220] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of the bacterium of interest which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2; - an absorbent part.

[0221] For the purposes of the present application, the marker may be any marker capable of marking a bacterium. Bacterial markers are well known to those skilled in the art.

[0222] The term "marker" encompasses both unconjugated and conjugated markers.

[0223] For the purposes of the present application, an unconjugated marker is a free marker, i.e. it is not bound to a ligand to label the bacteria. These are, for example, 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 as examples.

[0224] A conjugated label 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. Such conjugated labels are known from the prior art (Sajid et al., 2015).

[0225] In particular, the marker is a gold nanoparticle (AuNP). By "gold nanoparticle" we mean that it can be a gold nanosphere or a gold nanorod. More specifically, these are 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 range, which varies depending on the size of the nanoparticles.

[0226] In particular, the marker is a colored latex bead. For example, the diameter of latex beads is 400 nm. The aggregation of colored latex beads results in the appearance of a color detectable in the visible range. Several colors of latex beads are commercially available.

[0227] In particular, the marker is HRP.

[0228] For the purposes of the present application, a conjugated marker means that the marker is bound to at least one ligand specific to the bacterium of interest. By "conjugated to at least one specific ligand" is meant that a marker may be conjugated to one specific ligand or to several different specific ligands.

[0229] To bind a label to a ligand, a biotin / streptavidin bond can for example be used.

[0230] For the purposes of the present 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.

[0231] In particular, said at least one protein of at least one bacteriophage specific to the bacterium of interest is an RBP protein.

[0232] In particular, it is a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3.

[0233] In particular, it may be 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.

[0234] Thus, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker conjugated to at least one ligand specific for S. uberis, in particular a gold nanoparticle or a bead of colored latex conjugated to at least one ligand specific to S. uberis; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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.

[0235] In particular, said strip for detecting S. uberis in a sample comprises in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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 ligand specific for S. uberis chosen from at least one antibody directed against S. uberis, at least one aptamer specific for S. uberis, at least one bacteriophage specific for S. uberis, at least one protein of at least one bacteriophage specific for S. uberis, at least one bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, at least one protein of a bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein of at least one bacteriophage specific for S.uberis is an RBP protein, more particularly, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% 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.

[0236] In particular, said strip for detecting S. uberis in a sample comprises in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, and which comprises a protein comprising a sequence having a percentage 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 ligand specific for S. uberis chosen from at least one antibody directed against S. uberis, at least one aptamer specific for S. uberis, at least one bacteriophage specific for S. uberis, at least one protein of at least one bacteriophage specific for S. uberis, at least one bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, at least one protein of a bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein of at least one bacteriophage specific for S.uberis is an RBP protein, more particularly, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% 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.

[0237] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the detection zone comprises 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.

[0238] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0239] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises 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 of the bacterium of interest which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the detection zone comprises the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1; - an absorbent part.

[0240] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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.

[0241] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said marking zone optionally comprising a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone optionally comprising a protein comprising a sequence having a percentage 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.

[0242] In particular, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage 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 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2 and a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the detection zone comprises the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, - an absorbent part.

[0243] In another particular embodiment, the present invention relates to a lateral flow chromatography strip for detecting S. uberis in a sample, comprising in order at least: - a contact part comprising a contact area with the sample; - a labeling portion comprising a labeling area which comprises a label conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular the labeling area comprises 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 of the bacteria of interest which comprises at least less a specific ligand of S. uberis; - an absorbent part.

[0244] In particular, the marker is a gold nanoparticle or a colored latex bead.

[0245] Said at least one ligand specific for S. uberis in the detection zone may be at least one antibody directed against the bacterium of interest, at least one aptamer specific for the bacterium of interest, at least one bacteriophage specific for the bacterium of interest, at least one protein of at least one bacteriophage specific for the bacterium of interest, at least one bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest, at least one protein of a bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest. In particular, said at least one protein of at least one bacteriophage specific for the bacterium of interest is an RBP protein.More particularly, it may be a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3. In particular, it may be 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.

[0246] This application also relates to a S. uberis detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis comprising a contact portion, a labeling portion, a detection portion and an absorbing portion as described above; 2) A container intended to receive the sample.

[0247] In a particular embodiment, the present application relates to a kit for detecting S. uberis comprising: - a strip as described in this application; - a container intended to receive the sample; - a container containing a marker.

[0248] In this particular embodiment, said strip thus comprises either a contact part, a detection part and an absorbent part, or a contact part, a marking part, a detection part and an absorbent part, as described in the present application.

[0249] The label may be an unconjugated label or a conjugated label as defined above. The conjugated label may be conjugated to at least one specific ligand as defined above.

[0250] In a particular embodiment, the marker may be included in the container intended to receive the sample.

[0251] This embodiment of the kit may comprise 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 the present application.

[0252] Where the lateral flow chromatography strip includes a labelling portion, the kit optionally includes a container comprising a label.

[0253] Thus, when the lateral flow chromatography strip includes a labeling part, the kit for detecting S. uberis in a sample includes: - said strip; - a container intended to receive the sample.

[0254] In another particular embodiment, the kit for detecting S. uberis in a sample comprises: - a strip comprising at least: a contact part comprising a contact area with the sample; a detection part comprising a detection area of ​​the bacteria of interest which comprises at least one antibody directed against the bacteria of interest and / or at least one aptamer specific for S. uberis, an absorbent part; - a container intended to receive the sample; - a container comprising a marker conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or conjugated to a protein comprising a sequence having a percentage 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.

[0255] In another aspect, the present invention provides a method for detecting S. uberis in a sample using the lateral flow chromatography strips described above.

[0256] A sample is any solution that may contain the bacteria of interest. The sample may be diluted in a buffer solution prior to carrying out the detection process.

[0257] In a particular embodiment, the method for detecting S. uberis in a sample comprises: - a step of marking the bacteria or the bacteria of interest in the sample; - a step of bringing the sample into contact with the contact zone of a strip as previously described; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis in the sample.

[0258] Thus, in one embodiment, the marking step is carried out in the sample, i.e. prior to bringing the sample into contact 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 marking portion. Thus, this embodiment of the detection method applies to all the strips described in the present application. When the strip used in this detection mode contains a marking portion, a double marking of the bacteria of interest is then carried out, the marker used in the marking step in the sample and the marker included in the marking area of ​​the strip being able to be identical or different.

[0259] The labeling step is carried out with an unconjugated label or a conjugated label as defined previously in the application. The conjugated label may be conjugated to at least one specific ligand as defined previously. Those skilled in the art know how to carry out this labeling step depending on the chosen label. In particular, when it is a conjugated label, an incubation of the sample with the conjugated label may be carried out.

[0260] 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, the detection is done 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-sulphonic acid) (ABTS), 1,2-diaminobenzene (OPD), 3,3'-Diaminobenzidine (DAB), 3-Amino-9-ethylcarbazole (AEC) or 3,3',5,5'-tetramethylbenzidine (TMB) for colored reactions; homovanillic acid and tyramine for fluorescence spectroscopy; luminol for light production.

[0261] To bind a label to a ligand, a biotin / streptavidin bond can for example be used.

[0262] In particular, the marker used is a gold nanoparticle or a colored latex bead.

[0263] The presence of the bacteria of interest in the analyzed sample is concluded when the marker is detected on 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 fluorescent signal appears on the detection zone. 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.

[0264] The absence of the bacteria of interest in the sample is concluded when the marker is not detected on the detection zone.

[0265] When the lateral flow chromatography strip used for the detection method includes a labeling portion, the step of labeling the bacteria or the bacteria of interest in the sample is optional.

[0266] Thus, when the lateral flow chromatography strip used for the detection method comprises a labeling portion, the method for detecting S. uberis in a sample may comprise: - a step of bringing the sample into contact with the contact zone of a strip as previously described; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis in the sample.

[0267] 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 marker conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 zone of a strip comprising at least one contact part comprising a contact zone with the sample; a detection part comprising a detection zone of S. uberis which comprises at least one antibody directed against S. uberis and / or at least one aptamer specific for S. uberis, an absorbent part; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis in the sample.

[0268] According to 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 portion, a labeling portion, a detection portion and an absorbing portion 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 identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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.

[0269] In a particular embodiment, said lateral flow chromatography strip makes it possible to detect S. uberis and one or more other bacteria of interest in a sample. The detection part then comprises one or more additional detection zones and one or more additional labeling parts on which the appropriate ligands and labels are immobilized to detect this or these other bacteria of interest.

[0270] This type of strip is called “Duplex LFA”.

[0271] In a particular embodiment, said lateral flow chromatography strip makes it possible to detect S. uberis and S. aureus in a sample.

[0272] 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 absorbent part, and which comprises: - an additional detection zone in the detection part. This additional detection zone, or test line, includes a ligand specific to S. aureus, - a second marking part which comprises a marking area with a marker conjugated to a ligand specific to S. aureus.

[0273] 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 of at least one S. aureus-specific bacteriophage, at least one S. aureus-specific bacteriophage coupled to an antibody directed against S. aureus, at least one protein of a S. aureus-specific bacteriophage coupled to an antibody directed against S. aureus. In particular, said at least one protein of at least one S. aureus-specific bacteriophage is an RBP protein. In particular, it may be a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5. More particularly, it may be the protein of sequence SEQ ID NO:4 and / or the protein of sequence SEQ ID NO:5.

[0274] Several types of Duplex LFA strips can therefore be produced, depending on whether: - the S. uberis marking area comprises a marker coupled to a ligand specific to S. uberis or a marker coupled to a specific protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or the sequence SEQ ID NO: 2 and / or the sequence SEQ ID NO: 3; - the S. aureus marking area comprises a marker coupled to a ligand specific to S. aureus or a marker coupled to a specific protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or the sequence SEQ ID NO:5; - the S. uberis detection zone comprises a marker coupled to a ligand specific to S. uberis or a marker coupled to a specific protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or the sequence SEQ ID NO: 2 and / or the sequence SEQ ID NO: 3, - the S. aureus detection zone comprises a marker coupled to a ligand specific to S. aureus or a marker coupled to a specific protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or the sequence SEQ ID NO: 5.

[0275] Table 1 shows different types of LFA Duplex that can be made, for example, in which: - the ligand specific to 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 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. In particular, said at least one protein of at least one bacteriophage specific to the bacterium of interest is an RBP protein. - the specific protein may be for S. uberis a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 3, in particular it may be 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 for S. aureus the specific protein may be a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage 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.

[0276] [Table 1]

[0277] The detection zone of S. uberis and the detection zone of S. aureus are distinct from each other, i.e., they do not overlap.

[0278] The first marking part and the second marking part can be interchanged with respect to the migration direction.

[0279] Similarly, the detection zones can be in one order or the other depending on the direction of migration.

[0280] Thus, in a particular embodiment, such a strip comprises in order at least: - a contact part comprising a contact area with the sample; - a first marking part which comprises 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 identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 marking part which comprises a marking 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 identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 detection zone of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage 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.

[0281] In particular, said 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 first marking part which comprises a marking 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 identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, said marking 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 identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a second marking part which comprises a marking area of ​​S. aureus comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage 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 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 S. uberis detection zone which comprises a protein comprising a sequence having a percentage 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 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 S. aureus detection zone which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or which comprises a protein comprising a sequence having a percentage 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 / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0282] In particular, said 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 first marking part which comprises a marking 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 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 identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1; - a second marking part which comprises a marking area of ​​S. aureus comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4, 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 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the detection zone of S. uberis comprises the protein of sequence SEQ ID NO: 1 and the protein of sequence SEQ ID NO: 2, and comprising a detection zone of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone of S. aureus comprises the protein of sequence SEQ ID NO: 4 and the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0283] In particular, said 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 first marking part which comprises a marking 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 marking part which comprises a marking area of ​​S. aureus with a marker conjugated to an antibody directed against S. aureus - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, and / or a protein comprising a sequence having a percentage 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 detection zone of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or a protein comprising a sequence having a percentage 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.

[0284] In particular, said 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 first marking part which comprises a marking 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 comprises a labeling area of ​​S. aureus with a label conjugated to an antibody directed against S. aureus; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular it comprises the protein of sequence SEQ ID NO: 2, said detection zone of S. uberis optionally comprising a protein comprising a sequence having a percentage 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 of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone of S. aureus comprises the protein of sequence SEQ ID NO: 4 and / or the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0285] In particular, said 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 first marking part which comprises a marking 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 marking part which comprises a marking area of ​​S. aureus with a marker conjugated to an antibody directed against S. aureus - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2 and which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the detection zone of S. uberis comprises the protein of sequence SEQ ID NO: 2 and the protein of sequence SEQ ID NO: 1, and comprising a detection zone of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone of S. aureus comprises the protein of sequence SEQ ID NO: 4 and the protein of sequence SEQ ID NO: 5; - an absorbent part.

[0286] The present 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 portion, a labeling portion, a detection portion and an absorbing portion as described above; 2) A container intended to receive the sample.

[0287] The present 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 for detecting S. uberis and S. aureus comprising a contact part, a marking part, a detection part and an absorbent part as described previously; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis and the presence or absence of S. aureus in the sample.

[0288] If the marker is a gold nanoparticle or a colored latex bead, the presence of the bacteria will result in the appearance of a color on the detection zone. EXAMPLES

[0289] Production of RBP proteins

[0290] 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) including 100 mg / L of ampicillin (Fisher bioreagent, BPHIL 2760-25). Induction is performed when the bacteria reach an optical density ~ 0.5. After induction with IPTG, the bacteria are incubated at 25°C, with shaking. The cultures are centrifuged then resuspended and concentrated in a lysis buffer and lysed with a sonicator. The lysates are then centrifuged at 14000 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 a sintered glass, washed with 50 mM HEPES buffer pH7 and then stored at -80°C.

[0291] Example 1: Comparison of different RBPs of S. uberis with an ELISA test

[0292] 1.1. Materials and Methods

[0293] ELISA (enzyme-linked immunosorbent assay) is a solid-state enzyme-linked immunosorbent assay based on a 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 a colored reaction to be obtained that can be measured by absorbance.

[0294] To determine the specificity of the RPB protein, the ELISA principle was adapted as follows.

[0295] Bacteria are attached to the bottom of the wells of a microplate (ELISA) at (OD=0.05) for 1h30 at 37°C. A saturation step with a solution comprising 3% BSA and 0.05% Tween is carried out, then the BSA solution is removed. Phage proteins are deposited in each well of 10 pg / mL in a PBS buffer (1% BSA; 0.05% Tween). An incubation for 1 h at 37°C is then carried out. The wells are washed (at least 5 times) with the PBS buffer including 0.05% Tween. An incubation step with the antibody -GST coupled to HRP (Invitrogene, ref, 434323) at 0.25 pg / mL, solution in PBS buffer 1% BSA, 0.05% Tween20 for 1 h at 37°C, then the streptavidin is removed. The wells are washed (at least 5 times) with PBS buffer including 0.05% Tween.

[0296] For detection, 100 μl of TMB solution is added and incubated for 15 to 30 minutes at room temperature. Then, 50 μl of 2 M sulfuric acid is added to stop the reaction. The plate is then read at 450 nm.

[0297] The affinity of the protein to the bacteria is expressed by the amount of protein retained by each bacterial strain. This amount is assessed by the absorbance linked to the anti-GST antibody coupled to HRP.

[0298] 1.2. RBP proteins tested

[0299] 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.

[0300] [Table 2]

[0301] 1.3. Results of RBP proteins alone or in combination

[0302] 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 significant values.

[0303] [Table 3]

[0304] Of the 30 strains of S. uberis 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 allowed the detection of 25 strains (83.3%). Therefore, the mixture of the two proteins allowed a coverage of 83% to be achieved.

[0305] These results were confirmed for detection by the LFA method (presented below).

[0306] Example 2: Detection of S. uberis strains with a Lateral Flow Assay (LFA) device including RBP proteins

[0307] 2.1. Composition of the LFA device

[0308] Lateral flow chromatography (LFA) devices were performed using RBP P3 and SUB4 proteins.

[0309] The LFA device or strip 1 was designed in the following way with 4 parts: - a contact part 2: part in contact with the solution to be analyzed; - a marking part 7: part where the RBP proteins conjugated to gold nanoparticles (diameter 40 nm) are deposited at the level of the marking zone 8; - a detection part 4 comprising a test line 5 where the RBP proteins are immobilized; - an absorption part 6 for establishing the flow through the device from the contact part 2.

[0310] 2.2. Implementation of the LFA device for the detection of S. uberis

[0311] S. uberis bacterial strains are seeded onto Petri dishes and incubated overnight in an incubator at 37°C.

[0312] The next day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0313] Bacterial suspensions at the desired bacteria concentration are prepared in 1X PBS comprising 10% whole UHT milk and 1% Tween 20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0314] For each suspension prepared, 250 μl are taken and then placed in the wells of a 96-well plate.

[0315] Strips as described above are used. The different configurations listed in Table 4 are tested with RBP P3 and SUB4 proteins. When used as a conjugate on labeling part 7, they are coupled with gold nanoparticles and when used on test line 5 of detection part 4, they are in solution in 1X PBS with 20% sucrose.

[0316] Each strip is dipped into a well at contact part 2 and left for 30 minutes while it migrates to test line 5. The bacterial strains were tested for absorbance at OD 600 nm = 1 (equivalent to approximately 1.10 9 cfu / ml for S. aureus strains and 1.10 7 cfu / ml for S. uberis strains). The migration buffer is a 1X PBS solution, including 10% sterile whole UHT milk and 1% Tween 20. After migration, the strips are photographed.

[0317] [Table 4]

[0318] 2.3. Results

[0319] For proof of concept, LFA devices of configuration 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.

[0320] Figures 1, 2, 3, and 4 show that the presence of the SUB4 protein in the conjugate allows for better signal intensity. In addition, the RBP proteins do not detect anything in samples including the blank or control strains. This shows that the RBP proteins deposited on the LFA strip are functional because they bind the target bacteria. In addition, these proteins are able to migrate onto the membrane used on the strip.

[0321] After validating the concept of detecting S. uberis strains on LFA strips using RBP proteins, tests were performed on other S. uberis strains using additional configurations.

[0322] Figure 5 part A shows that when P3 is alone on test line 5, only strain 9038 is very well detected on the 8 strains of S. uberis. Figure 5 part B shows that using the SUB4 protein alone in the conjugate and on the test line allows the detection of 7 strains tested out of 8. Strain 1912c is not recognized but, however, the latter could be weakly detected when test line 5 contains the P3 and SUB4 proteins. Therefore, the mixture of the two RBP proteins allowed the detection of more strains than the use of a single protein on test line 5 (Figure 5 part C).

[0323] 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 S. uberis strains used in the previous experiment. Of the 30 S. uberis strains tested, 28 could be detected using the RBP protein mixture. This system allows the recognition of more than 90% of the strains tested.

[0324] Example 3: LFA system with growth of S. uberis and addition of agar to the detection zone (test line)

[0325] 3.1. Concept of detection with bacterial growth

[0326] The concept of the LFA method with bacteria growth on detection zone 5 (test line) includes the following steps: 1) The LFA device 1 comprises a contact part 2, a detection part 4 and an absorption part 6. It does not comprise a labeling part 7. The sample to be tested migrates directly through the detection part 4 which comprises a detection zone 5 or test line where a ligand specific to the bacterium of interest is immobilized by adsorption; 2) When the bacteria of interest arrives by capillarity 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 bacteria of interest.

[0327] 3.2. Detection of S. uberis with the LFA system with bacterial growth on the detection area

[0328] 250 µl of bacterial suspension in 1X PBS + 10% whole UHT milk + 1% TWEEN 20 at the desired bacterial concentration are placed in a well of a 96-well plate.

[0329] An LFA device comprising at the detection part, a test line 5 with the RBP proteins SUB4 and P3 (1 pl deposited; composition: 0.625 mg / ml of the SUB4 protein, 0.625 mg / ml of the P3 protein, PBS 1X, 10% sucrose).

[0330] The strip is immersed at contact part 2 for 30 minutes in the well containing the sample to be tested.

[0331] After migration, test line 5 is cut out, and is either deposited: - on a Petri dish agar (Figure 7 A) comprising 1X BHI, 75 pM potassium tellurite and 1.5% agar. - in a cassette (Figure 7 B). The strip is fixed with double-sided tape inside the cassette. The cassette is then closed. An agar comprising 1X BHI, 75 pM potassium tellurite and 0.4% agar is deposited on the strip 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.

[0332] Potassium tellurite is used as a chromogenic element to improve the detection of bacterial multiplication.

[0333] The strips are then incubated at 37°C for 15 hours and a photo is then taken.

[0334] The photos in Figure 7 clearly show that in the sample without bacteria, no black line appears, which indicates the absence of bacteria immobilized on the test line. On the other hand, for the samples containing S. uberis tested at 1.10 4cfu / ml and, a black line appeared at the test line corresponding to the multiplication of the bacteria of interest S. uberis and the reduction of tellurite to tellurium, a chromogenic element improving the visibility of the result.

[0335] Example 4: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus

[0336] 4.1. Composition of the LFA device

[0337] 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 marking part 7 n°1: part where the RBP P3 and SUB4 proteins specific to S. uberis are deposited, conjugated to gold nanoparticles (diameter 40 nm) - a marking part 7 n°2: part where the RBP proteins PHIL 2 and PHIL 5 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 for establishing the flow through the device from the contact part 2.

[0338] [Table s]

[0339] 4.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0340] The bacterial strains of S. uberis and S. aureus are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0341] The next day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0342] Bacterial suspensions of S. aureus, S. uberis, or S. aureus + S. uberis at the desired bacterial concentration are prepared in 1X PBS containing 10% whole UHT milk and 1% Tween 20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0343] For each suspension prepared, 250 μl are taken and then placed in the wells of a 96-well plate.

[0344] Strips as described above are used. The S. uberis-specific RBP proteins P3 and SUB4 are immobilized on test line 5 no. 1 (1 μl of a 0.625 mg / ml solution of P3, 0.625 mg / ml of SUB4, 1X PBS including 10% sucrose), and the S. aureus-specific RBP proteins PHIL 2 and PHIL 5 are immobilized on test line 5 no. 2 (1 μl of a 0.75 mg / ml solution of PHIL 2, 0.75 mg / ml of PHIL 5, 1X PBS including 10% sucrose). For their immobilization they are coupled with GST.

[0345] On the labeling part 7 n°1 are deposited 6 μl of a solution of RBP P3 proteins specific to S. uberis, conjugated to gold nanoparticles (diameter 40 nm). The protein solution is at an OD=10 with 20% sucrose, 0.25% BSA and PBS1X.

[0346] On the labeling part 7 n°2 are deposited 6 μl of a solution of RBP proteins PHIL 2 and PHIL 5 specific to S. aureus, conjugated to gold nanoparticles (diameter 40 nm). The protein solution is at an OD=10 with 20% sucrose, 0.5% BSA and PBS1X.

[0347] Both parts of marking 7 are of identical reference (Ahlstrom-Grade 800).

[0348] Each strip 1 is dipped into a well at the contact part 2 and left for 30 minutes while it migrates to the test line 5. The bacterial strains were tested for absorbance at OD 600 nm = 1 (equivalent to approximately 1.10 9cfu / ml for S. aureus strains and 1.10 9 cfu / ml for S. uberis strains). The migration buffer is a 1X PBS solution, including 10% sterile whole UHT milk and 1% Tween 20. After migration, the strips are photographed.

[0349] 4.3. Results

[0350] As shown in Figure 8, with the well without bacteria, only control line 9 is visible. With a sample containing S. uberis, two lines appear: control line 9 and S. uberis test line 5. With a sample containing only S. aureus, two lines appear: control line 9 and S. aureus test line 5. With a sample containing both bacteria, all 3 test lines appear.

[0351] This clearly shows that this device is suitable for the simultaneous detection of S. aureus and S. uberis.

[0352] Example 5: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus

[0353] 5.1. Composition of the LFA device

[0354] 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 marking part 7 n°1: part where the RBP P3 and SUB4 proteins specific to S. uberis are deposited, conjugated to latex beads with a diameter of 400 nm; - a marking part 7 n°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 for establishing the flow through the device from the contact part 2.

[0355] 5.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0356] The bacterial strains of S. uberis and S. aureus are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0357] The next day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0358] Bacterial suspensions of S. aureus, S. uberis, or S. aureus + S. uberis at the desired bacterial concentration are prepared in 1X PBS containing 10% whole UHT milk and 1% Tween 20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0359] For each suspension prepared, 250 μl are taken and then placed in the wells of a 96-well plate.

[0360] Strips as described above are used. The S. uberis-specific RBP proteins P3 and SUB4 are immobilized on test line 5 no. 1 (1 μl of a 0.625 mg / ml solution of P3, 0.625 mg / ml of SUB4, 1X PBS including 10% sucrose), and the S. aureus-specific RBP proteins PHIL 2 and PHIL 5 are immobilized on test line 5 no. 2 (1 μl of a 0.75 mg / ml solution of PHIL 2, 0.75 mg / ml of PHIL 5, 1X PBS including 10% sucrose). For their immobilization they are coupled with GST.

[0361] On the labeling part 7 n°1 are deposited 6 μl of a solution of RBP P3 and SUB4 proteins specific to S. uberis, conjugated to latex beads. The protein solution is at an OD=10 with 20% sucrose, 0;25% BSA and PBS1X.

[0362] On the labeling part 7 n°2 are deposited 6 μl of a solution of RBP PHIL 2 and PHIL 5 proteins specific to S. aureus, conjugated to latex beads. The protein solution is at an OD=10 with 20% sucrose, 0;25% BSA and PBS1X.

[0363] Both marking parts are of identical reference (Ahlstrom-Grade 800).

[0364] Each strip 1 is dipped into a well at the contact part 2 and left for 30 minutes while it migrates to the test line 5. The bacterial strains were tested for absorbance at OD 600 nm = 1 (equivalent to approximately 1.10 9 cfu / ml for S. aureus strains and 1.10 9 cfu / ml for S. uberis strains). The migration buffer is a 1 X PBS solution, comprising 10% sterile whole UHT milk and 1% Tween 20. After 30 min of migration, the strips are photographed.

[0365] 5.3. Results

[0366] As shown in Figure 9, with a sample containing S. uberis, S. uberis test line 5 appears. With a sample containing only S. aureus, S. aureus test line 5 appears. With a sample containing both bacteria, both test lines appear.

[0367] This clearly shows that this device is suitable for the simultaneous detection of S. aureus and S. uberis.

[0368] Example 6: Duplex LFA device for the simultaneous detection of S. uberis and S. aureus

[0369] 6.1. Composition of the LFA device

[0370] The LFA device or strip was designed as follows: - a contact part 2: part in contact with the solution to be analyzed. - a marking part 7 n°1: part where the RBP P3 and SUB4 proteins specific to S. uberis are deposited, conjugated to latex beads with a diameter of 400 nm; ; - a marking part 7 n°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 for establishing the flow through the device from the contact part 2.

[0371] 6.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0372] The bacterial strains of S. uberis and S. aureus are inoculated onto Petri dishes and incubated overnight in an incubator at 37°C.

[0373] The next day, the bacteria are resuspended in sterile PBS buffer and the OD is read at 600nm.

[0374] Bacterial suspensions of S. aureus, S. uberis, or S. aureus + S. uberis at the desired bacterial concentration are prepared in 1X PBS containing 10% whole UHT milk and 1% Tween 20. A bacteria-free solution is also prepared to serve as a negative control (blank).

[0375] For each suspension prepared, 250 μl are taken and then placed in the wells of a 96-well plate.

[0376] Strips as described above are used. The S. uberis-specific RBP proteins P3 and SUB4 are immobilized on test line 5 No. 1 (1 μl of a 0.625 mg / ml solution of P3, 0.625 mg / ml of SUB4, 1X PBS including 10% sucrose), and the S. aureus-specific RBP proteins PHIL 2 and PHIL 5 are immobilized on test line 5 No. 2 (1 μl of a 0.75 mg / ml solution of PHIL 2, 0.75 mg / ml of PHIL 5, 1X PBS including 10% sucrose). For their immobilization they are coupled with GST.

[0377] On the labeling part 7 n°1 are deposited 6 μl of a solution of RBP P3 and SUB4 proteins specific to S. uberis, conjugated to latex beads. The protein solution is at an OD=10 with 20% sucrose, 0.25% BSA and PBS1X.

[0378] On the marking part 7 n°2 is deposited an anti-S. aureus antibody conjugated to latex beads.

[0379] Both marking parts are of identical reference (Ahlstrom-Grade 800).

[0380] Each strip is dipped into a well at contact part 2 and left for 30 minutes while it migrates to test line 5. The bacterial strains were tested for absorbance at OD 600 nm = 1 (equivalent to approximately 1.10 9 cfu / ml for S. aureus strains and 1.10 9cfu / ml for S. uberis strains). The migration buffer is a 1 X PBS solution, comprising 10% sterile whole UHT milk and 1% Tween 20. After 30 min of migration, the strips are photographed.

[0381] 6.3. Results

[0382] As shown in Figure 10, with a sample containing S. uberis, S. uberis test line 5 appears. With a sample containing only S. aureus, S. aureus test line 5 appears. With a sample containing both bacteria, both test lines appear.

[0383] This clearly shows that this device is suitable for the simultaneous detection of S. aureus and S. uberis.

[0384] 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

[0385] 7.1. Composition of the LFA device

[0386] 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 proteins P3 and SUB4 specific to S. uberis are immobilized, and comprising a test line 5 no. 2 where the RBP proteins PHIL2 and PHIL5 specific to S. aureus are immobilized; - an absorption part 6 for establishing the flow through the device from the contact part 2.

[0387] 7.2. Implementation of the LFA system

[0388] 250 µl of bacterial suspension in 1X PBS + 10% whole UHT milk + 1% TWEEN 20 at the desired bacterial concentration are placed in a well of a 96-well plate.

[0389] An LFA 1 device comprising at the detection part 4, a test line 5 with the RBP proteins SUB4 and P3 specific to S. uberis (1 pl deposited; composition: 0.625 mg / ml of the SUB4 protein, 0.625 mg / ml of the P3 protein, PBS 1X, 20% sucrose), and a second test line 5 with the RBP proteins PHIL 2 and PHIL 5 specific to S. aureus.

[0390] The strip is immersed at contact part 2 for 30 minutes in the well containing the sample to be tested.

[0391] After migration, the test line is cut out and plated on a Petri dish containing 1X BHI, 75 pM potassium tellurite, and 1.5% agar.

[0392] The strips are then incubated at 37°C for 15 hours and a photo is then taken.

[0393] The results in Figure 11 show that with the sample without bacteria, no black line appears, which indicates the absence of bacteria immobilized on test line 5. On the other hand, with the sample containing S. uberis bacteria, the appearance of a black line at the level of the S. uberis test line 5 is visible, which corresponds to the multiplication of the bacteria and the reduction of tellurite to tellurium, a chromogenic element improving the visibility of the result. Similarly, with the sample containing S. aureus bacteria, we observe the appearance of a black line at the level of the S. aureus test line 5, thus indicating the multiplication of the bacteria and the reduction of tellurite to tellurium. Finally, with the sample containing S. uberis and S. aureus, two black lines are observed and therefore testify to the presence of these two bacteria in the sample.

[0394] Example 8: LFA device for the detection of S. uberis with bacterial growth and addition of the sample to a nutrient medium

[0395] 8.1. Concept of the LFA device

[0396] The sample to be tested is supplemented with a nutrient medium comprising potassium tellurite in a ratio of 1 / 20. The contact part 2 of an LFA 1 strip is immersed in the mixture. The bacteria migrate and are captured at the test line 5 where the S. uberis-specific RBP proteins are fixed. Everything is 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 tellurium compound. Note that the LFA 1 device does not have a marking part 7.

[0397] 8.2. Implementation of the LFA system

[0398] 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 RBP P3 and SUB4 proteins specific to S. uberis are immobilized (1 μl deposited; composition: 0.625 mg / ml of the SUB4 protein, 0.625 mg / ml of the P3 protein, PBS 1X, 10% sucrose); - an absorption part 6 for establishing the flow through the device from the contact part.

[0399] In a tube, 25 μl of raw milk contaminated with a known Streptococcus concentration was added to 500 μl of a buffer composed of 1X brain heart infusion, 1% TWEEN 20, and 20 mg / ml potassium telluride.

[0400] After mixing the milk and buffer, strip 1 is dipped into the buffer at contact area 2. The tube is closed and incubated at 37°C for 15 hours. After the 15-hour incubation period, the result is read. The presence of a black band at the test line indicates the presence of S. uberis in the sample tested (Figure 12).

[0401] Example 9: LFA Duplex Device for the simultaneous detection of S. uberis and S. aureus with bacterial growth and addition of the sample to a nutrient medium

[0402] 9.1. Composition of the LFA device

[0403] 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 μl deposited; composition: 0.625 mg / ml of the SUB4 protein, 0.625 mg / ml of the P3 protein, PBS 1X, 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 μl deposited; composition: 0.75 mg / ml of the PHIL 5 protein and 0.25 mg / ml of the PHIL 2 protein, PBS 1X, 10% sucrose); - an absorption part 6 for establishing the flow through the device from the contact part 2.

[0404] An anti-rabbit antibody is deposited on control line 9.

[0405] 9.2. Implementation of the LFA device for the detection of S. uberis and S. aureus

[0406] In a tube, 25 μl of raw milk contaminated with a known Streptococcus concentration was added to 500 μl of a buffer composed of 1X brain heart infusion, 1% TWEEN 20, and 20 mg / ml potassium telluride.

[0407] After mixing the milk and the buffer, strip 1 is immersed in the buffer at contact part 2. The tube is closed and incubated at 37°C for 15 hours. After the 15-hour incubation period, the result is read.

[0408] The results in Figure 13 show that with the sample without bacteria, no black line appears, which indicates the absence of bacteria immobilized on test line 5. On the other hand, with the sample containing S. uberis bacteria, the appearance of a black line at the level of the S. uberis test line 5 is visible, which corresponds to the multiplication of the bacteria and the reduction of tellurite to tellurium, a chromogenic element improving the visibility of the result. Similarly, with the sample containing S. aureus bacteria, we observe the appearance of a black line at the level of the S. aureus test line 5, thus indicating the multiplication of the bacteria and the reduction of tellurite to tellurium. Finally, with the sample containing S. uberis and S. aureus, two black lines are observed and therefore testify to the presence of these two bacteria in the sample. List of cited documents

[0409] À toute fin utile, les publications suivantes sont citées dans la demande : 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. Animals, vol. 11 , numéro 6, mai 2021 , p. 1625. Rédiger, et al. Bayesian latent class models to determine diagnostic sensitivities and specificities of two point of care rapid tests (Selma plus, Dipslide) for the detection 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 Lateral 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 lateral 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. 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 October 24, 2016. International application WO 2021 / 234321, Device for detecting a bacterium of interest. Filed on May 20, 2021.

Claims

Claims

1. Use of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and / or of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, for the capture of Streptococcus uberis in a sample.

2. Use of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and / or of a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2, for the detection of Streptococcus uberis in a sample.

3. Use according to claim 2, wherein the detection is carried out by ELISA, lateral flow chromatography or lateral flow chromatography with bacterial growth.

4. A lateral flow chromatography strip for detecting S. uberis in a sample, comprising at least one portion which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular said at least one portion comprises the protein of sequence SEQ ID NO: 1, and / or a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular said at least one portion comprises the protein of sequence SEQ ID NO:

2.

5. A lateral flow chromatography strip according to claim 4, comprising in order: - a contact part comprising a contact area with the sample; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular it comprises the protein of sequence SEQ ID NO: 1, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2; - an absorbent part.

6. A lateral flow chromatography strip according to claim 4, comprising in order: - a contact part comprising a contact area with the sample; - a marking portion comprising a marking area which comprises a marker; - a detection part comprising a detection zone of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular it comprises the protein of sequence SEQ ID NO: 1, and / or a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2; - an absorbent part; said marker being conjugated to a ligand specific for S. uberis chosen from at least one antibody directed against S. uberis, at least one aptamer specific for S. uberis, at least one bacteriophage specific for S. uberis, at least one protein of at least one bacteriophage specific for S. uberis, at least one bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, at least one protein of a bacteriophage specific for S. uberis coupled to an antibody directed against S. uberis, in particular said at least one protein of at least one bacteriophage specific for S.uberis is a phage receptor binding protein (RBP), more particularly, a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1 and / or a protein comprising a sequence having a percentage identity of at least 70% 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.

7. A lateral flow chromatography strip according to claim 4, comprising in order: - a contact part comprising a contact area with the sample; - a first marking part which comprises a marking 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 identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and / or a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 2, in particular the protein of sequence SEQ ID NO: 2; - a second marking part which comprises a marking area of ​​S. aureus comprising a marker, in particular a gold nanoparticle or a colored latex bead, conjugated to a protein comprising a sequence having a percentage 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 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 of S. uberis which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 1, in particular the protein of sequence SEQ ID NO: 1, and / or a protein comprising a sequence having a percentage 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 detection zone of S. aureus which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 4 and / or which comprises a protein comprising a sequence having a percentage identity of at least 70% with the sequence SEQ ID NO: 5, in particular the detection zone of S. aureus comprises the protein of sequence SEQ ID NO:4 and / or protein of sequence SEQ ID NO:5; - an absorbent part.

8. A method of detecting S. uberis in a sample comprising: - a step of bringing the sample into contact with the contact zone of a strip according to claim 6; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis in the sample.

9. A method of detecting S. uberis comprising: 1) Addition of a sample to be tested in a nutrient medium optionally comprising a chromogenic element; 2) Contacting an LFA strip according to claim 4 or 5 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 6 to 48 hours; 5) Appearance of colonies, or appearance of coloration in the case where the nutrient medium includes a chromogenic element, along the S. uberis test line in the event of the presence of S. uberis in the sample.

10. A method of detecting S. uberis and S. aureus in a sample comprising: - a step of bringing the sample into contact with the contact zone of a strip according to claim 7; - a step of detecting the marker on the detection zone; - a step of analysis of the presence or absence of S. uberis and the presence or absence of S. aureus in the sample.

11. S. uberis detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis according to claim 6; 2) A container intended to receive the sample.

12. A kit for detecting S. uberis and S. aureus comprising: 1) A lateral flow chromatography strip for detecting S. uberis and S. aureus according to claim 7; 2) A container intended to receive the sample.

13. S. uberis detection kit comprising: 1) A lateral flow chromatography strip for detecting S. uberis according to claim 5; 2) A tube capable of closing hermetically; 3) A flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) optionally, a container intended to receive the sample.

14. A kit for detecting S. uberis and S. aureus comprising: 1) A lateral flow chromatography strip for detecting S. uberis and S. aureus according to claim 7; 2) A tube capable of closing hermetically; 3) A flask comprising a nutrient medium, which optionally comprises a chromogenic element, in particular potassium tellurite; 4) optionally, a container intended to receive the sample.

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