WO2014006244A1 - Method and device for the rapid diagnosis of diseases caused by yersinia, in faecal samples - Google Patents

Method and device for the rapid diagnosis of diseases caused by yersinia, in faecal samples Download PDF

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Publication number
WO2014006244A1
WO2014006244A1 PCT/ES2013/070402 ES2013070402W WO2014006244A1 WO 2014006244 A1 WO2014006244 A1 WO 2014006244A1 ES 2013070402 W ES2013070402 W ES 2013070402W WO 2014006244 A1 WO2014006244 A1 WO 2014006244A1
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Prior art keywords
yersinia
sample
immunochromatographic
microparticles
immunoreactive
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PCT/ES2013/070402
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Spanish (es)
French (fr)
Inventor
Carlos GUSTAVO ASÍN
Oscar Landeta Elorz
Beatriz Velasco Michelena
Yolanda GARCÍA MIGUEL
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Certest Biotec, S.L.
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Publication of WO2014006244A1 publication Critical patent/WO2014006244A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia

Definitions

  • the present invention falls within the area of microbiology and biotechnology and specifically in the diagnosis of diseases caused by Yersinia.
  • the object of the invention is a quick and simple method of detecting serotypes 0: 3 and 0: 9 of the bacterium Yersinia enterocolitica (Y. enterocolitica).
  • Yersinia enterocolitica is an facultative anaerobic, gram-negative and negative oxidase species.
  • Y. enterocolitica is highly heterogeneous and can be classified into several serotypes, of which only a few have been associated with disease in humans.
  • Six different biotypes biotype 1A, 1 B, 2-5) and numerous serotypes of Y. enterocolytic have been described. Eleven of these serotypes have been most frequently associated with infections in humans.
  • Most strains of Y. enterocolitica that are associated with human yersiniosis belong to serotypes 0: 9 and 0: 3.
  • Enterolocolytic yersinia is a relatively common pathogen in both humans and animals.
  • the infection caused by Y. enterocolitica can cause, depending on the age of the infected person, a great variety of symptoms.
  • the predominant clinical symptom is abdominal pain and diarrhea with or without fever.
  • There are other complications such as arthritis and erythema nodosum.
  • This Y. enterocolitic infection occurs in young children.
  • the most common symptoms are fever, abdominal pain and diarrhea, often bloody. These symptoms develop between 4 and 7 days after exposure and can last up to 1 or 3 weeks or even longer.
  • foods, particularly pork products are an important source of infection in humans.
  • the most common way to diagnose diseases caused by Yersinia is an immunoassay to detect the presence of antibodies (IgG or IgA) against Yersinia in the serum.
  • the immunoassay can be an agglutination, an immunoblot (such as recomLine Yersinia from Mikrogen GmbH, Neuried, Germany) or an ELISA (such as Serion Elisa Yersinia from Serion Immundiagnostica GmbH, Würzburg, Germany).
  • an immunoblot such as recomLine Yersinia from Mikrogen GmbH, Neuried, Germany
  • an ELISA such as Serion Elisa Yersinia from Serion Immundiagnostica GmbH, Würzburg, Germany.
  • cross reactions with Salmonella, Brucella, Escherichia coli and Vibrio may occur.
  • the use of other more specific Yersinia antigens is proposed, such as those described in US 201 1/0306515 A1.
  • the object of the present invention is a diagnostic device of the immunochromatographic type and its method of use, for the rapid and simultaneous determination of the 0: 3 and 0: 9 antigens of Yersinia enterocolitica in faecal samples.
  • the strains of Yersinia enterocolitica with 0: 3 and 0: 9 antigens are by far the most frequent of all those that cause disease and between the two they make up the great majority of the cases found.
  • the method has the following advantages: It is fast (just resuspend the sample in the diluent, add a few drops in the device and wait 10 minutes), simple (no complicated laboratory instruments are required), the result is easy to interpret (a line appears indicating if the test is positive or not), and cheap (especially because it does not require initial investment and because it can be carried out by non-specialized personnel).
  • the device has the following advantages: a. A specific vial is used that allows the sample to be taken easily and hygienically. b. Reliable It incorporates a control line whose appearance verifies the correct functioning of the test. C. It detects the presence of the bacteria and not the antibodies that the infected organism produces. The 0: 3 and 0: 9 antigens of Yersinia enterocolitica are identified simultaneously and separately.
  • the diagnostic method of the present invention consists in: taking a representative portion of the sample, its dispersion in a specific diluent, the application of this dispersion of the sample in a specific immunochromatographic device, the formation of complexes between the antibodies of the device and sample antigens and visualization of these complexes on the device membrane.
  • the immunochromatographic device used is for single use. No instrumentation is required to carry out the method or interpret the result. As a sample, feces or enrichment cultures are used.
  • the sample preparation time is about two minutes and the test development time is 5 to 10 minutes, which means between 10 and 20 times less than the EIA (ELISA) tests, much more complex to carry out and they need laboratory instrumentation. Because of its simplicity, the test can be carried out in the doctor's office, and even, with the appropriate instructions, by the patient himself when the sample is faecal.
  • the device of the invention can also be used for the identification of Yersinia enterocolitica from cultures or coprocultures in enrichment media, whether selective or not, and are made in liquid or semi-solid medium (agar).
  • agar liquid or semi-solid medium
  • the use of the CIN (Cefsulodine-Irgasan-Novobiocin) medium grown between 25 ° C and 37 ° C for 24-48h is appropriate.
  • the culture is carried out on agar plates, as in this case, one or more colonies are dispersed in the diluent before adding it to the immunochromatographic device of the invention. In this case, the procedure should be performed in a laboratory with the appropriate safety measures and experience.
  • Figure 1 Scheme of the stages of the diagnostic method.
  • the specific device or vial for the collection of faecal samples (Figure 1 A) is a plastic vial of about 3 ml_ capacity, in which 1 ml of diluent has been previously dispensed, with a screw cap that has a stem whose end, designed for this purpose, allows the collection of a stool sample (Figure 1 B) solid or semi-solid and its introduction into the vial. After this, it is closed and stirred vigorously (Figure 1 C) until complete dispersion of the sample. Finally, the opposite part of the device is broken ( Figure 1 D) and a few drops are added on the place of application of the device sample ( Figure 1 E).
  • FIG. 1 Views of the immunochromatographic device.
  • FIG. 2A View of the immunochromatographic device in strip format.
  • Said strip includes the following elements: an absorbent material (1), a porous membrane (2) with a control line (3), a line for the detection of Yersinia enterocolitica 0: 3 (4) and another line for the detection of Yersinia enterocolitica 0: 9 (5), a place of application of the sample (6) and a place for the antibody-marker conjugate (7).
  • Figure 2B Top view of the open diagnostic device in which the two immunochromatographic strips are observed, one for 0: 3 and one for 0: 9, in parallel placement on the bottom of the device.
  • Figure 2C Top view of the diagnostic device, in which the result windows (A, B) and the sample application points (S) are observed on the upper face of the device.
  • Immunochromatographic (IC) devices for the detection of both antigens and antibodies are described in the state of the art (for example, in EP 12481 12).
  • the device of the present invention can be performed in a single immunochromatographic (IC) strip or in two immunochromatographic strips.
  • one strip is used for the detection of Yersinia enterocolitica 0: 3 and another strip for the detection of Yersinia enterocolitica 0: 9 as shown in Figure 2B.
  • monoclonal antibodies For the detection of 0: 3 and 0: 9 antigens, specific monoclonal antibodies are used for each of them and are commercially available. With these monoclonal antibodies, conjugates with colored microparticles are prepared separately. These immunoreactive microparticles are deposited in one of the materials of the immunochromatographic device and act as a mobile phase. The same antibodies are immobilized separately on the porous membrane that acts as a fixed phase and in which the immunological capture occurs separately from the 0: 3 and 0: 9 antigens of Yersinia enterocolitica. The membrane and the material with the conjugate are mounted with the help of other materials on a plastic support and this is cut in strip form.
  • the stool sample is resuspended in the dispersion diluent of the sample in an approximate proportion 1/10. If the sample is liquid, 100 ⁇ of the sample is mixed with 1 ml of the diluent. If the sample is solid, 100 mg of sample are separated with the help of a spatula and dispersed in 1 ml of diluent. It is very appropriate to use the sampling vial in which the sample is introduced with the help of a stem after which it is closed with the screw cap and vigorously shaken to subsequently break the top of the cap and add 4-5 drops on the point or points of application of the immunochromatographic device sample. In this way the manipulation of the sample is a quick and hygienic simple procedure.
  • the test is considered negative if only one line appears, the control line (for example, green) in the results window; positive for Yersinia enterocolitica 0: 3 if in addition to the control line a line appears in the position of Yersinia enterocolitica 0: 3 (for example, blue); positive for Yersinia enterocolitica 0: 9 if in addition to the control line a line appears in the position of Yersinia enterocolitica 0: 9 (for example, red); and positive for both (0: 3 and 0: 9) if three lines appear (for example, one green, one red and one blue).
  • each strip is interpreted separately in a similar way.
  • the culture in CIN medium is suitable for the isolation of Yersinia spp. (aerobic conditions, 24 h, 37 ° C).
  • CIN agar Cefsulodin-Irgasan-Novobiocin agar
  • the CIN plates are examined after incubation and 3-4 small colonies (1-2 mm in diameter) are selected with the sunken red center and with the lighter edge, no color, very defined and intact
  • the vial for sample dilution is opened and the 3 or 4 suspicious Yersinia colonies are chopped with the stem.
  • the vial is closed, shaken and after breaking the part top of the cap, 4-5 drops are added on the point or points of application of the immunochromatographic device sample.
  • the diagnostic device and the vial with the diluent can be grouped, together with the instructions for use, in the form of a kit for performing one or more diagnostic tests.
  • a conjugate of the 0: 3 anti-Yersinia enterocolitica monoclonal antibody is prepared with polystyrene microparticles. Colored particles with carboxyl groups on their surface with a nominal diameter of 300 nm are used (K1 030 of the Estapor brand, Merck, Darmstadt, Germany). 1 ml_ of 10% (w / v) particles are washed by centrifugation and resuspension in 10 mM MES (2- (N-morpholino) ethanesulfonic acid) pH 6 buffer and EDC (1-ethyl-3- ( 3- dimethylaminopropyl) -carbodiimide) to a concentration of 5 mM.
  • MES N-morpholino
  • EDC 1-ethyl-3- ( 3- dimethylaminopropyl) -carbodiimide
  • Conjugates with control line particles are obtained in the same way but using streptavidin (S4762, Sigma-Aldrich) at a final surface concentration of 1 mg / m 2 .
  • Example 2 Test preparation of an IC strip.
  • TRIS buffer tris (hydroxymethyl) aminomethane
  • This solution is deposited at a rate of 14 ⁇ / ⁇ in a winding material of non-interwoven polyester fibers between 20-30 mm wide that is dried in an air stream at 45 ° C after deposition (5 min) and for 24 h in a chamber at 30 degrees and 20% relative humidity.
  • anti-Yersinia enterocolitica 0: 3 anti-Yersinia enterocolitica 0: 9 and anti-streptavidin antibodies (for the control line) are dialyzed in PBS, brought to a concentration between 0.5 and 1 mg / mL and deposited linearly in parallel on a laminated nitrocellulose membrane (Millipore Hi-Flow Plus) 25 mm wide and pore size between 10 and 30 ⁇ at a rate of 1 ⁇ / cm after which it is dried in a stream of air at 45 ° C after deposition (2 min) and immediately afterwards for 24 hours in a chamber at 30 ° C and 20% relative humidity.
  • a laminated nitrocellulose membrane Micropore Hi-Flow Plus
  • the material with the conjugate, the membrane and the absorbent material are assembled as indicated in Figure 2A on a plastic support with an adhesive sheet and the strips are cut transversely to their assembly to a width of 4 mm.
  • the strip for the detection of Yersinia enterocolitica 0: 3 is prepared in the same way as described above but without using the anti-Yersinia enterocolitic antibody 0: 9 on the membrane and without depositing the colloid anti-Yersinia enterocolitica 0: 9 in the absorbent.
  • the strip for the detection of Yersinia enterocolitica 0: 9 is prepared in the same manner but without the anti-Yersinia enterocolitic antibody 0: 3 in the membrane and without the anti-Yersinia enterocolytic colloid 0: 3 in the absorbent.
  • the two IC strips (0: 3 and 0: 9) are arranged inside a plastic housing that has been designed in such a way that it has a housing for each of the strips, two different windows for the addition of the samples and two other windows for viewing the results.
  • the plastic housing can be marked or printed for the correct indication to the user of the position of each strip.
  • a solution is prepared for the dispersion of stool samples consisting of an aqueous solution of 150 mM sodium chloride, 1% Triton X-100, nonspecific mouse IgG antibodies 250 ⁇ g / mL and nonspecific rabbit IgG antibodies at 100 ⁇ g / mL in a 200 mM TRIS buffer at a pH of 9.1.
  • This preparation is dispensed in vials for sampling at a rate of 1 mL / vial. These vials are used to collect and prepare the sample.

Abstract

The invention relates to an immunochromatographic device, and to a method for using said device, which allows the simultaneous and differentiated detection of O:3 and O:9 antigens of the bacteria Yersinia enterocolitica, in a faeces sample or an enrichment culture applied to the device, by means of the formation of conjugates with coloured particles and the capture thereof in a porous membrane.

Description

MÉTODO Y DISPOSITIVO PARA EL DIAGNÓSTICO RÁPIDO DE  METHOD AND DEVICE FOR THE QUICK DIAGNOSIS OF
ENFERMEDADES CAUSADAS POR YERSINIA, EN MUESTRAS FECALES  DISEASES CAUSED BY YERSINIA, IN FECAL SAMPLES
Campo de la invención Field of the Invention
La presente invención se encuadra dentro del área de microbiología y biotecnología y en concreto en el diagnóstico de enfermedades causadas por Yersinia. El objeto de la invención es un procedimiento rápido y sencillo de detección de los serotipos 0:3 y 0:9 de la bacteria Yersinia enterocolítica (Y. enterocolítica). The present invention falls within the area of microbiology and biotechnology and specifically in the diagnosis of diseases caused by Yersinia. The object of the invention is a quick and simple method of detecting serotypes 0: 3 and 0: 9 of the bacterium Yersinia enterocolitica (Y. enterocolitica).
Estado de la técnica State of the art
Yersinia enterocolítica es una especie anaeróbica facultativa, gram-negativa y oxidasa negativa. Y. enterocolítica es altamente heterogénea y puede clasificarse dentro de varios serotipos, de los cuales sólo unos pocos han sido asociados con enfermedad en humanos. Se han descrito seis diferentes biotipos (biotipo 1A, 1 B, 2- 5) y numerosos serotipos de Y. enterocolítica. Once de esos serotipos se han asociado con mayor frecuencia con infecciones en humanos. La mayoría de las cepas de Y. enterocolítica que se asocian con la yersiniosis humana pertenecen a los serotipos 0:9 y 0:3. Yersinia enterocolitica is an facultative anaerobic, gram-negative and negative oxidase species. Y. enterocolitica is highly heterogeneous and can be classified into several serotypes, of which only a few have been associated with disease in humans. Six different biotypes (biotype 1A, 1 B, 2-5) and numerous serotypes of Y. enterocolytic have been described. Eleven of these serotypes have been most frequently associated with infections in humans. Most strains of Y. enterocolitica that are associated with human yersiniosis belong to serotypes 0: 9 and 0: 3.
Yersinia enterolocolítica es un patógeno relativamente común tanto en humanos como en animales. La infección provocada por Y. enterocolítica puede provocar, dependiendo de la edad de la persona infectada, gran variedad de síntomas. El síntoma clínico predominante es dolor abdominal y diarrea con o sin presencia de fiebre. Existen otras complicaciones como artritis y eritema nodoso. A menudo esta infección por Y. enterocolítica ocurre en niños pequeños. Los síntomas más comunes son fiebre, dolor abdominal y diarrea, a menudo sanguinolenta. Estos síntomas se desarrollan entre 4 y 7 días tras exposición y pueden durar hasta 1 o 3 semanas o incluso más. Hay cierta evidencia indirecta de que los alimentos, particularmente productos derivados del cerdo, son una fuente importante de la infección en humanos. Enterolocolytic yersinia is a relatively common pathogen in both humans and animals. The infection caused by Y. enterocolitica can cause, depending on the age of the infected person, a great variety of symptoms. The predominant clinical symptom is abdominal pain and diarrhea with or without fever. There are other complications such as arthritis and erythema nodosum. Often this Y. enterocolitic infection occurs in young children. The most common symptoms are fever, abdominal pain and diarrhea, often bloody. These symptoms develop between 4 and 7 days after exposure and can last up to 1 or 3 weeks or even longer. There is some indirect evidence that foods, particularly pork products, are an important source of infection in humans.
Problema a resolver Tradicionalmente el diagnóstico de las enfermedades causadas por Yersinia, especialmente enterocolítica y pseudotuberculosis ha sido el cultivo y aislamiento a partir del mismo de la bacteria. Se puede cultivar con relativa facilidad a partir de fluidos corporales (fluidos cerebroespinal y peritoneal) y de tejidos. El aislamiento a partir de heces es mucho más complicado por la interferencia de la flora fecal, requiriéndose medios selectivos y cultivo a bajas temperaturas durante largos tiempos. En todo caso el procedimiento es largo y laborioso y requiere personal experimentado. Problem to solve Traditionally the diagnosis of diseases caused by Yersinia, especially enterocolitic and pseudotuberculosis has been the culture and isolation from it of the bacteria. It can be grown relatively easily from body fluids (cerebrospinal and peritoneal fluids) and tissues. Isolation from feces is much more complicated by the interference of faecal flora, requiring selective media and cultivation at low temperatures for long periods. In any case, the procedure is long and laborious and requires experienced personnel.
La forma más común de diagnosticar las enfermedades causadas por Yersinia es un inmunoensayo para detectar la presencia de anticuerpos (IgG o IgA) anti Yersinia en el suero. El inmunoensayo puede ser una aglutinación, un inmunoblot (como el recomLine Yersinia de Mikrogen GmbH, Neuried, Alemania) o un ELISA (como el Serion Elisa Yersinia de Serion Immundiagnostica GmbH, Würzburg, Alemania). Dependiendo del antígeno utilizado para realizar el inmunoensayo pueden aparecer reacciones cruzadas con Salmonella, Brucella, Escherichia coli y Vibrio. Para superar estos inconvenientes se propone la utilización de otros antígenos más específicos de Yersinia como los descritos en el documento US 201 1/0306515 A1 . The most common way to diagnose diseases caused by Yersinia is an immunoassay to detect the presence of antibodies (IgG or IgA) against Yersinia in the serum. The immunoassay can be an agglutination, an immunoblot (such as recomLine Yersinia from Mikrogen GmbH, Neuried, Germany) or an ELISA (such as Serion Elisa Yersinia from Serion Immundiagnostica GmbH, Würzburg, Germany). Depending on the antigen used to perform the immunoassay, cross reactions with Salmonella, Brucella, Escherichia coli and Vibrio may occur. To overcome these drawbacks, the use of other more specific Yersinia antigens is proposed, such as those described in US 201 1/0306515 A1.
El diagnóstico de Yersinia por técnicas de cultivo es largo y tedioso y presenta una sensibilidad muy baja. La detección de anticuerpos en el suero, además de tener falsos positivos, solo dice que el sistema inmunitario del individuo ha visto el antígeno, no que haya una enfermedad activa. The diagnosis of Yersinia by culture techniques is long and tedious and has a very low sensitivity. The detection of antibodies in the serum, in addition to having false positives, only says that the individual's immune system has seen the antigen, not that there is an active disease.
Solución del problema Problem solution
El objeto de la presente invención es un dispositivo de diagnóstico del tipo imunocromatográfico y su método de uso, para la determinación rápida y simultánea de los antígenos 0:3 y 0:9 de Yersinia enterocolítica en muestras fecales. Los cepas de Yersinia enterocolítica con antígenos 0:3 y 0:9 son con diferencia las más frecuentes de todas las que causan enfermedad y entre las dos suman la gran mayoría de los casos encontrados. The object of the present invention is a diagnostic device of the immunochromatographic type and its method of use, for the rapid and simultaneous determination of the 0: 3 and 0: 9 antigens of Yersinia enterocolitica in faecal samples. The strains of Yersinia enterocolitica with 0: 3 and 0: 9 antigens are by far the most frequent of all those that cause disease and between the two they make up the great majority of the cases found.
El método presenta las siguientes ventajas: Es rápido (basta resuspender la muestra en el diluyente, añadir unas gotas en el dispositivo y esperar 10 minutos), sencillo (no se requiere ningún complicado instrumental de laboratorio), el resultado es fácil de interpretar (aparece una línea indicando si el test es positivo o no), y barato (especialmente porque no requiere inversión inicial y porque lo puede llevar a cabo personal no especializado). The method has the following advantages: It is fast (just resuspend the sample in the diluent, add a few drops in the device and wait 10 minutes), simple (no complicated laboratory instruments are required), the result is easy to interpret (a line appears indicating if the test is positive or not), and cheap (especially because it does not require initial investment and because it can be carried out by non-specialized personnel).
El dispositivo presenta las siguientes ventajas: a. Se utiliza un vial específico que permite la toma de la muestra de manera fácil e higiénica. b. Fiable. Lleva incorporada una línea de control cuya aparición verifica el correcto funcionamiento del test. c. Detecta la presencia de la bacteria y no los anticuerpos que el organismo infectado produce. Se identifican simultánea y separadamente los antígenos 0:3 y 0:9 de Yersinia enterocolítica. The device has the following advantages: a. A specific vial is used that allows the sample to be taken easily and hygienically. b. Reliable It incorporates a control line whose appearance verifies the correct functioning of the test. C. It detects the presence of the bacteria and not the antibodies that the infected organism produces. The 0: 3 and 0: 9 antigens of Yersinia enterocolitica are identified simultaneously and separately.
Descripción breve brief description
El método de diagnóstico de la presente invención consiste en: la toma de una porción representativa de la muestra, su dispersión en un diluyente específico, la aplicación de esta dispersión de la muestra en un dispositivo inmunocromatográfico específico, la formación de complejos entre los anticuerpos del dispositivo y los antígenos de la muestra y la visualización de estos complejos en la membrana del dispositivo. The diagnostic method of the present invention consists in: taking a representative portion of the sample, its dispersion in a specific diluent, the application of this dispersion of the sample in a specific immunochromatographic device, the formation of complexes between the antibodies of the device and sample antigens and visualization of these complexes on the device membrane.
El dispositivo inmunocromatográfico utilizado es de un solo uso. No se requiere ningún tipo de instrumentación para llevar a cabo el método o interpretar el resultado. Como muestra se utilizan heces o cultivos de enriquecimiento. The immunochromatographic device used is for single use. No instrumentation is required to carry out the method or interpret the result. As a sample, feces or enrichment cultures are used.
El tiempo de preparación de la muestra son unos dos minutos y el tiempo de desarrollo de la prueba es de 5 a 10 minutos, lo que significa entre 10 y 20 veces menos que los ensayos EIA (ELISA), mucho más complejos de llevar a cabo y necesitan de instrumentación de laboratorio. Por su sencillez la prueba se puede llevar a cabo en la consulta del médico, e incluso, con las instrucciones adecuadas, por el propio paciente cuando la muestra es fecal. The sample preparation time is about two minutes and the test development time is 5 to 10 minutes, which means between 10 and 20 times less than the EIA (ELISA) tests, much more complex to carry out and they need laboratory instrumentation. Because of its simplicity, the test can be carried out in the doctor's office, and even, with the appropriate instructions, by the patient himself when the sample is faecal.
Además de con muestras de heces, el dispositivo de la invención también se puede utilizar para la identificación de Yersinia enterocolítica a partir de cultivos o coprocultivos en medios de enriquecimiento ya sean selectivos o no y se realicen en medio líquido o semisólido (agar). En particular resulta adecuada la utilización del medio CIN (Cefsulodina-Irgasan-Novobiocina) cultivado entre 25 °C y 37 °C durante 24-48h. Cuando el cultivo se realiza en placas de agar, como en este caso, se procede a dispersar una o varias colonias en el diluyente antes de adicionarlo al dispositivo inmunocromatográfico de la invención. En este caso el procedimiento debería realizarse en un laboratorio con las medidas de seguridad y la experiencia adecuadas. In addition to stool samples, the device of the invention can also be used for the identification of Yersinia enterocolitica from cultures or coprocultures in enrichment media, whether selective or not, and are made in liquid or semi-solid medium (agar). In particular, the use of the CIN (Cefsulodine-Irgasan-Novobiocin) medium grown between 25 ° C and 37 ° C for 24-48h is appropriate. When the culture is carried out on agar plates, as in this case, one or more colonies are dispersed in the diluent before adding it to the immunochromatographic device of the invention. In this case, the procedure should be performed in a laboratory with the appropriate safety measures and experience.
Descripción de la figuras Description of the figures
Figura 1 : Esquema de las etapas del método de diagnóstico. El dispositivo o vial específico para la toma de muestras fecales (Figura 1 A) es un vial de plástico de unos 3 ml_ de capacidad, en el que previamente se ha dispensado 1 mi de diluyente, con un tapón a rosca que dispone de un vástago cuyo extremo, diseñado al efecto, permite la recolección de una muestra de heces (Figura 1 B) sólidas o semisólidas y su introducción en el vial. Tras esto, se cierra y se agita vigorosamente (Figura 1 C) hasta la completa dispersión de la muestra. Por último, se rompe la parte opuesta del dispositivo (Figura 1 D) y se añaden unas gotas sobre el lugar de aplicación de la muestra del dispositivo (Figura 1 E). Figure 1: Scheme of the stages of the diagnostic method. The specific device or vial for the collection of faecal samples (Figure 1 A) is a plastic vial of about 3 ml_ capacity, in which 1 ml of diluent has been previously dispensed, with a screw cap that has a stem whose end, designed for this purpose, allows the collection of a stool sample (Figure 1 B) solid or semi-solid and its introduction into the vial. After this, it is closed and stirred vigorously (Figure 1 C) until complete dispersion of the sample. Finally, the opposite part of the device is broken (Figure 1 D) and a few drops are added on the place of application of the device sample (Figure 1 E).
Figura 2: Vistas del dispositivo inmunocromatográfico. Figure 2: Views of the immunochromatographic device.
Figura 2A: Vista del dispositivo inmunocromatográfico en formato tira. Dicha tira incluye los siguientes elementos: un material absorbente (1 ), una membrana porosa (2) con una línea de control (3), una línea para la detección de Yersinia enterocolítica 0:3 (4) y otra línea para la detección de Yersinia enterocolítica 0:9 (5), un lugar de aplicación de la muestra (6) y un lugar para el conjugado anticuerpo-marcador (7). Figura 2B: Vista superior del dispositivo de diagnóstico abierto en la que se observan las dos tiras inmunocromatográficas, una para 0:3 y otra para 0:9, en colocación paralela sobre la parte inferior del dispositivo. Figure 2A: View of the immunochromatographic device in strip format. Said strip includes the following elements: an absorbent material (1), a porous membrane (2) with a control line (3), a line for the detection of Yersinia enterocolitica 0: 3 (4) and another line for the detection of Yersinia enterocolitica 0: 9 (5), a place of application of the sample (6) and a place for the antibody-marker conjugate (7). Figure 2B: Top view of the open diagnostic device in which the two immunochromatographic strips are observed, one for 0: 3 and one for 0: 9, in parallel placement on the bottom of the device.
Figura 2C: Vista superior del dispositivo de diagnóstico, en la que se observan las ventanas de resultados (A, B) y los puntos de aplicación de la muestra (S) en la cara superior del dispositivo. Figure 2C: Top view of the diagnostic device, in which the result windows (A, B) and the sample application points (S) are observed on the upper face of the device.
Descripción detallada Detailed description
Los dispositivos inmunocromatográficos (IC) para la detección tanto de antígenos como de anticuerpos están descritos en el estado de la técnica (por ejemplo, en la patente EP 12481 12). Immunochromatographic (IC) devices for the detection of both antigens and antibodies are described in the state of the art (for example, in EP 12481 12).
El dispositivo de la presente invención se puede realizar en una sola tira inmunocromatográfica (IC) o bien en dos tiras inmunocromatográficas. The device of the present invention can be performed in a single immunochromatographic (IC) strip or in two immunochromatographic strips.
Cuando se usa una sola tira IC se trata de una prueba de tres líneas: una de control, otra para la detección de Yersinia enterocolítica 0:3 y otra para la detección de Yersinia enterocolítica 0:9 . When a single IC strip is used, it is a three-line test: one for control, another for the detection of Yersinia enterocolitica 0: 3 and another for the detection of Yersinia enterocolitica 0: 9.
En la versión de dos tiras IC, una tira se usa para la detección de Yersinia enterocolítica 0:3 y otra tira para la detección de Yersinia enterocolítica 0:9 según se representa en la figura 2B. Existe también la posibilidad de utilizar una sola de estas tiras para detectar 0:3 u 0:9 según se desee. In the version of two IC strips, one strip is used for the detection of Yersinia enterocolitica 0: 3 and another strip for the detection of Yersinia enterocolitica 0: 9 as shown in Figure 2B. There is also the possibility of using only one of these strips to detect 0: 3 or 0: 9 as desired.
Para la detección de los antígenos 0:3 y 0:9 se utilizan anticuerpos monoclonales específicos para cada uno de ellos y que están disponibles comercialmente. Con estos anticuerpos monoclonales, se preparan, por separado, conjugados con micropartículas coloreadas. Estas micropartículas inmunorreactivas son depositadas en uno de los materiales del dispositivo inmunocromatográfico y actúan como fase móvil. Los mismos anticuerpos son inmovilizados por separado sobre la membrana porosa que actúa como fase fija y en la que se produce la captura inmunológica por separado de los antígenos 0:3 y 0:9 de Yersinia enterocolítica. La membrana y el material con el conjugado son montados con la ayuda de otros materiales sobre un soporte plástico y éste se corta en forma tira. Para determinar la presencia de Yersinia enterocolitica en muestras de heces se resuspende la muestra de heces en el diluyente de dispersión de la muestra en una proporción aproximada 1 /10. Si la muestra es líquida 100 μΙ_ de muestra se mezclan con 1 ml_ del diluyente. Si la muestra es sólida se separan 100 mg de muestra con la ayuda de una espátula y se dispersan en 1 ml_ de diluyente. Resulta muy adecuado la utilización del vial de toma de muestra en el que se introduce la muestra con la ayuda de un vástago tras lo que se cierra con el tapón a rosca y se agita vigorosoramente para, posteriormente, romper la parte superior del tapón y añadir 4-5 gotas sobre el punto o puntos de aplicación de la muestra del dispositivo inmunocromatográfico. De esta manera la manipulación de la muestra resulta un procedimiento sencillo rápido e higiénico. For the detection of 0: 3 and 0: 9 antigens, specific monoclonal antibodies are used for each of them and are commercially available. With these monoclonal antibodies, conjugates with colored microparticles are prepared separately. These immunoreactive microparticles are deposited in one of the materials of the immunochromatographic device and act as a mobile phase. The same antibodies are immobilized separately on the porous membrane that acts as a fixed phase and in which the immunological capture occurs separately from the 0: 3 and 0: 9 antigens of Yersinia enterocolitica. The membrane and the material with the conjugate are mounted with the help of other materials on a plastic support and this is cut in strip form. In order to determine the presence of Yersinia enterocolitica in stool samples, the stool sample is resuspended in the dispersion diluent of the sample in an approximate proportion 1/10. If the sample is liquid, 100 μΙ of the sample is mixed with 1 ml of the diluent. If the sample is solid, 100 mg of sample are separated with the help of a spatula and dispersed in 1 ml of diluent. It is very appropriate to use the sampling vial in which the sample is introduced with the help of a stem after which it is closed with the screw cap and vigorously shaken to subsequently break the top of the cap and add 4-5 drops on the point or points of application of the immunochromatographic device sample. In this way the manipulation of the sample is a quick and hygienic simple procedure.
Tras la aplicación de la muestra se esperan 10 minutos para visualizar las líneas y se interpreta el resultado. En la versión de una tira, el test se considera negativo si solo aparece una sola línea, la de control (por ejemplo, verde) en la ventana de resultados; positivo para Yersinia enterocolitica 0:3 si además de la línea de control aparece una línea en la posición de Yersinia enterocolitica 0:3 (por ejemplo, azul); positivo para Yersinia enterocolitica 0:9 si además de la línea de control aparece una línea en la posición de la Yersinia enterocolitica 0:9 (por ejemplo, roja); y positivo para ambas (0:3 y 0:9) si aparecen tres líneas (por ejemplo, una verde, una roja y una azul). Con el dispositivo de 2 tiras se interpreta cada tira por separado de forma similar. After the application of the sample, 10 minutes are expected to visualize the lines and the result is interpreted. In the version of a strip, the test is considered negative if only one line appears, the control line (for example, green) in the results window; positive for Yersinia enterocolitica 0: 3 if in addition to the control line a line appears in the position of Yersinia enterocolitica 0: 3 (for example, blue); positive for Yersinia enterocolitica 0: 9 if in addition to the control line a line appears in the position of Yersinia enterocolitica 0: 9 (for example, red); and positive for both (0: 3 and 0: 9) if three lines appear (for example, one green, one red and one blue). With the 2-strip device, each strip is interpreted separately in a similar way.
Para determinar la presencia de Yersinia enterocolitica en cultivo de enriquecimiento, el cultivo en medio CIN (para coprocultivo) es adecuado para el aislamiento de Yersinia spp. (condiciones aerobias, 24 h, 37 °C). Tras 24 horas de incubación en agar CIN (agar Cefsulodin-Irgasan-Novobiocin) las potenciales colonias de Yersinia enterocolitica aparecen con el centro hundido de color rojo rodeadas de un borde claro-transparente dando la apariencia de un "ojo de buey". Para llevar a cabo el método de la invención se examinan las placas de CIN tras la incubación y se seleccionan 3-4 colonias pequeñas (1 -2mm de diámetro) con el centro rojo hundido y con el borde más claro, sin color, muy definido e intacto. Se abre el vial para dilución de muestras y con el vástago se pican las 3 ó 4 colonias sospechosas de Yersinia. Se cierra el vial, se agita y después de romper la parte superior del tapón, se añaden 4-5 gotas sobre el punto o puntos de aplicación de la muestra del dispositivo inmunocromatográfico. To determine the presence of Yersinia enterocolitica in enrichment culture, the culture in CIN medium (for coproculture) is suitable for the isolation of Yersinia spp. (aerobic conditions, 24 h, 37 ° C). After 24 hours of incubation in CIN agar (Cefsulodin-Irgasan-Novobiocin agar) the potential colonies of Yersinia enterocolitica appear with the sunken red center surrounded by a clear-transparent edge giving the appearance of an "ox eye". To carry out the method of the invention, the CIN plates are examined after incubation and 3-4 small colonies (1-2 mm in diameter) are selected with the sunken red center and with the lighter edge, no color, very defined and intact The vial for sample dilution is opened and the 3 or 4 suspicious Yersinia colonies are chopped with the stem. The vial is closed, shaken and after breaking the part top of the cap, 4-5 drops are added on the point or points of application of the immunochromatographic device sample.
Tras la aplicación de la muestra se esperan 10 minutos para visualizar las líneas y se interpreta el resultado como se ha descrito anteriormente. After application of the sample, 10 minutes are expected to visualize the lines and the result is interpreted as described above.
Para una mayor facilidad y conveniencia, el dispositivo de diagnóstico y el vial con el diluyente pueden agruparse, junto las instrucciones de uso, en forma de kit para la realización de una o más pruebas diagnósticas. For greater ease and convenience, the diagnostic device and the vial with the diluent can be grouped, together with the instructions for use, in the form of a kit for performing one or more diagnostic tests.
Ejemplos de realización: Examples of realization:
Ejemplo 1 : Example 1 :
Preparación de los conjugados Conjugate Preparation
Se prepara un conjugado del anticuerpo monoclonal anti- Yersinia enterocolitica 0:3 con micropartículas de poliestireno. Se utilizan partículas coloreadas con grupos carboxilo en su superficie de 300 nm de diámetro nominal (K1 030 de la marca Estapor, Merck, Darmstadt, Alemania). 1 ml_ de partículas al 10 % (p/v) se lavan por centrifugación y resuspensión en tampón MES (ácido 2-(N- morfolino)etanosulfónico) 10 mM de pH 6 y se le añade EDC (1 -etil-3-(3- dimetilaminopropil)-carbodiimida) hasta una concentración de 5 mM. Se incuba 1 h a 37 grados centígrados y se retira el exceso de reactivo por centrifugación. Las partículas así activadas se resuspenden en tampón MES 10 mM de pH 6 y se les añade el anticuerpo monoclonal anti- Yersinia enterocolitica 0:3 hasta una concentración superficial de 2 mg/m2 tras lo que se incuban 18 h a 4 °C y posteriormente se lavan en Tween-20 al 0,1 % (p/v). De la misma manera, se prepara un conjugado del anticuerpo monoclonal anti- Yersinia enterocolitica 0:9 con micropartículas de poliestireno del mismo o distinto color. A conjugate of the 0: 3 anti-Yersinia enterocolitica monoclonal antibody is prepared with polystyrene microparticles. Colored particles with carboxyl groups on their surface with a nominal diameter of 300 nm are used (K1 030 of the Estapor brand, Merck, Darmstadt, Germany). 1 ml_ of 10% (w / v) particles are washed by centrifugation and resuspension in 10 mM MES (2- (N-morpholino) ethanesulfonic acid) pH 6 buffer and EDC (1-ethyl-3- ( 3- dimethylaminopropyl) -carbodiimide) to a concentration of 5 mM. Incubate 1 h at 37 degrees Celsius and remove excess reagent by centrifugation. The particles thus activated are resuspended in 10 mM MES buffer of pH 6 and 0: 3 anti-Yersinia enterocolitica monoclonal antibody is added to a surface concentration of 2 mg / m 2 after which they are incubated 18 h at 4 ° C and subsequently washed in 0.1% Tween-20 (w / v). In the same way, a conjugate of the 0-9 anti-Yersinia enterocolitica monoclonal antibody with polystyrene microparticles of the same or different color is prepared.
Los conjugados con partículas para línea de control se obtienen de la misma manera pero utilizando streptavidina (S4762, Sigma-Aldrich) a una concentración superficial final de 1 mg/m2. Conjugates with control line particles are obtained in the same way but using streptavidin (S4762, Sigma-Aldrich) at a final surface concentration of 1 mg / m 2 .
Ejemplo 2: Preparación del test de una tira IC. Example 2: Test preparation of an IC strip.
Una mezcla de los tres conjugados con partículas descritos en concentraciones de 0,02%, 0,04% y 0,05% respectivamente, se diluyen en una solución que contiene sacarosa 10%, caseína bovina 2%, seroalbúmina bovina 0,5 % y Tween-20 2% en tampón TRIS (tris(hidroximetil)aminometano) de pH 8,4. Esta solución se deposita a razón de 14 μί/οηι en un material bobinado de fibras de poliéster no entretejidas de entre 20-30 mm de ancho que se seca en corriente de aire a 45 °C tras la deposición (5 min) y durante 24 h en una cámara a 30 grados y 20 % de humedad relativa. A mixture of the three conjugates with particles described in concentrations of 0.02%, 0.04% and 0.05% respectively, are diluted in a solution containing 10% sucrose, 2% bovine casein, 0.5% bovine serum albumin and 2% Tween-20 in TRIS buffer (tris (hydroxymethyl) aminomethane) of pH 8.4. This solution is deposited at a rate of 14 μί / οηι in a winding material of non-interwoven polyester fibers between 20-30 mm wide that is dried in an air stream at 45 ° C after deposition (5 min) and for 24 h in a chamber at 30 degrees and 20% relative humidity.
Los anticuerpos anti- Yersinia enterocolítica 0:3 , anti- Yersinia enterocolítica 0:9 y anti-streptavidina (para la línea de control) se dializan en PBS, se llevan a una concentración entre 0,5 y 1 mg/mL y se depositan linealmente de forma paralela sobre una membrana de nitrocelulosa laminada (Millipore Hi-Flow Plus) de 25 mm de ancho y de tamaño de poro entre 10 y 30 μηι a razón de 1 μΐ/cm tras lo cual se seca en corriente de aire a 45 °C tras la deposición (2 min) e inmediatamente después durante 24 h en una cámara a 30 °C y 20 % de humedad relativa. The anti-Yersinia enterocolitica 0: 3, anti-Yersinia enterocolitica 0: 9 and anti-streptavidin antibodies (for the control line) are dialyzed in PBS, brought to a concentration between 0.5 and 1 mg / mL and deposited linearly in parallel on a laminated nitrocellulose membrane (Millipore Hi-Flow Plus) 25 mm wide and pore size between 10 and 30 μηι at a rate of 1 μΐ / cm after which it is dried in a stream of air at 45 ° C after deposition (2 min) and immediately afterwards for 24 hours in a chamber at 30 ° C and 20% relative humidity.
El material con el conjugado, la membrana y el material absorbente se montan conforme indica la figura 2A sobre un soporte plástico con una lámina adhesiva y las tiras son cortadas transversalmente a su montaje a una anchura de 4 mm. The material with the conjugate, the membrane and the absorbent material are assembled as indicated in Figure 2A on a plastic support with an adhesive sheet and the strips are cut transversely to their assembly to a width of 4 mm.
Ejemplo 3: Example 3:
Preparación del dispositivo con dos tiras IC. Preparation of the device with two IC strips.
La tira para la detección de Yersinia enterocolítica 0:3, se prepara de la misma forma que se ha descrito anteriormente pero sin utilizar el anticuerpo anti- Yersinia enterocolítica 0:9 en la membrana y sin depositar el coloide anti- Yersinia enterocolítica 0:9 en el absorbente. La tira para la de detección de Yersinia enterocolítica 0:9 se prepara de la misma manera pero sin el anticuerpo anti- Yersinia enterocolítica 0:3 en la membrana y sin el coloide anti- Yersinia enterocolítica 0:3 en el absorbente. The strip for the detection of Yersinia enterocolitica 0: 3, is prepared in the same way as described above but without using the anti-Yersinia enterocolitic antibody 0: 9 on the membrane and without depositing the colloid anti-Yersinia enterocolitica 0: 9 in the absorbent. The strip for the detection of Yersinia enterocolitica 0: 9 is prepared in the same manner but without the anti-Yersinia enterocolitic antibody 0: 3 in the membrane and without the anti-Yersinia enterocolytic colloid 0: 3 in the absorbent.
Montaje del dispositivo. Las dos tiras IC (0:3 y 0:9) se disponen en el interior de una carcasa de material plástico que se ha diseñado de tal forma que presenta un alojamiento para cada una de las tiras, dos ventanas diferentes para la adición de las muestras y otras dos ventanas para la visualización de los resultados. La carcasa plástica puede ir marcada o impresa para la correcta indicación al usuario de la posición de cada tira. Device mounting The two IC strips (0: 3 and 0: 9) are arranged inside a plastic housing that has been designed in such a way that it has a housing for each of the strips, two different windows for the addition of the samples and two other windows for viewing the results. The plastic housing can be marked or printed for the correct indication to the user of the position of each strip.
Ejemplo 4. Example 4
Diluyente de la muestra. Diluent of the sample.
Se prepara una solución para la dispersión de las muestras de heces consistente en una disolución acuosa de cloruro de sodio 150 mM, Tritón X-100 al 1 %, anticuerpos IgG inespecíficos de ratón 250 μg/mL y anticuerpos IgG inespecíficos de conejo a 100 μg/mL en un tampón TRIS 200 mM a un pH de 9,1 . Esta preparación se dispensa en viales para la toma de muestra a razón de 1 mL/vial. Estos viales se utilizan para recogida y preparación de la muestra. A solution is prepared for the dispersion of stool samples consisting of an aqueous solution of 150 mM sodium chloride, 1% Triton X-100, nonspecific mouse IgG antibodies 250 μg / mL and nonspecific rabbit IgG antibodies at 100 μg / mL in a 200 mM TRIS buffer at a pH of 9.1. This preparation is dispensed in vials for sampling at a rate of 1 mL / vial. These vials are used to collect and prepare the sample.

Claims

REIVINDICACIONES
1 . Dispositivo inmunocromatográfico para la detección de Yersinia enterocolítica en muestras biológicas caracterizado por que comprende una ventana de aplicación de la muestra, otra ventana de resultados y: i) una tira inmunocromatográfica la cual comprende una zona (7) con conjugados de los anticuerpos monoclonales anti- Yersinia enterocolítica 0:3 y 0:9 con micropartículas inmunorreactivas y una zona de membrana porosa (2) que comprende anticuerpos anti- Yersinia enterocolítica 0:3 y 0:9 inmovilizados dispuestos por separado (3, 4), o ii) dos tiras inmunocromatográficas, donde una tira comprende una zona (7) con conjugados del anticuerpo monoclonal anti- Yersinia enterocolítica 0:3 con micropartículas inmunorreactivas, y una zona de membrana porosa (2) que comprende anticuerpos anti- Yersinia enterocolítica 0:3 inmovilizados, y otra tira comprende una zona (7) con conjugados del anticuerpo monoclonal anti- Yersinia enterocolítica 0:9 con micropartículas inmunorreactivas, y una zona de membrana porosa (2) que comprende anticuerpos anti- Yersinia enterocolítica 0:9 inmovilizados. one . Immunochromatographic device for the detection of enterocolytic Yersinia in biological samples characterized in that it comprises a sample application window, another result window and: i) an immunochromatographic strip which comprises a zone (7) with conjugates of the anti-monoclonal antibody Yersinia enterocolitic 0: 3 and 0: 9 with immunoreactive microparticles and a porous membrane zone (2) comprising anti-Yersinia enterocolitic antibodies 0: 3 and 0: 9 immobilized separately arranged (3, 4), or ii) two strips immunochromatographic, where a strip comprises a zone (7) with conjugates of the monoclonal anti-Yersinia enterocolitic antibody 0: 3 with immunoreactive microparticles, and a porous membrane zone (2) comprising immobilized anti-Yersinia enterocolytic 0: 3 antibodies, and another strip comprises a zone (7) with conjugates of the anti-Yersinia enterocolitic monoclonal antibody 0: 9 with immunoreactive microparticles, and a porous membrane zone (2) comprising immobilized anti-Yersinia enterocolytic antibodies 0: 9.
2. Dispositivo de acuerdo con la reivindicación anterior donde las micropartículas inmunorreactivas son coloreadas. 2. Device according to the preceding claim wherein the immunoreactive microparticles are colored.
3. Dispositivo de acuerdo con una cualquiera de las reivindicaciones anteriores donde las micropartículas inmunorreactivas son de poliestireno. 3. Device according to any one of the preceding claims wherein the immunoreactive microparticles are made of polystyrene.
4. Uso de un dispositivo según una cualquiera de las reivindicaciones anteriores para la detección de Yersinia enterocolítica en una muestra biológica. 4. Use of a device according to any one of the preceding claims for the detection of Yersinia enterocolitica in a biological sample.
5. Uso según la reivindicación anterior donde la muestra biológica es una muestra de heces. 5. Use according to the preceding claim wherein the biological sample is a stool sample.
6. Uso según la reivindicación 4 donde la muestra biológica es un cultivo de enriquecimiento. 6. Use according to claim 4 wherein the biological sample is an enrichment culture.
7. Método de diagnóstico de las enfermedades causadas por Yersinia enterocolítica caracterizado por que comprende las siguientes etapas: 7. Method of diagnosis of diseases caused by Yersinia enterocolitica characterized by comprising the following stages:
a. dispersión de una muestra biológica de heces o de un cultivo de enriquecimiento en un diluyente,  to. dispersion of a biological stool sample or an enrichment culture in a diluent,
b. aplicación de la dispersión de a) en la ventana de muestra de un dispositivo según una cualquiera de las reivindicaciones 1 a 3,  b. application of the dispersion of a) in the sample window of a device according to any one of claims 1 to 3,
c. interpretación del resultado según la aparición de bandas en la ventana de resultados.  C. interpretation of the result according to the appearance of bands in the results window.
8. Kit para llevar a cabo el método de diagnóstico de acuerdo con la reivindicación 7 caracterizado por que comprende:  8. Kit for carrying out the diagnostic method according to claim 7 characterized in that it comprises:
a. un dispositivo inmunocromatográfico según una cualquiera de las reivindicaciones 1 -3,  to. an immunochromatographic device according to any one of claims 1-3,
b. un diluyente para la dispersión de la muestra biológica,  b. a diluent for the dispersion of the biological sample,
c. un vial para la toma de muestra y su manipulación.  C. a vial for sampling and handling.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012017125A1 (en) * 2010-08-05 2012-02-09 Certest Biotec, S.L. Method and device for rapid diagnosis of hepatitis a in faecal samples
WO2012052586A1 (en) * 2010-10-19 2012-04-26 Certest Biotec,S.L. Method and device for the rapid diagnosis of diseases in faecal samples

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012017125A1 (en) * 2010-08-05 2012-02-09 Certest Biotec, S.L. Method and device for rapid diagnosis of hepatitis a in faecal samples
WO2012052586A1 (en) * 2010-10-19 2012-04-26 Certest Biotec,S.L. Method and device for the rapid diagnosis of diseases in faecal samples

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FERNANDEZ-LAGO, L. ET AL.: "Rapid serotyping of enteropathogenic Yersinia enterocolitica strains by co-agglutination.", ANNALES DE L'INSTITUT PASTEUR MICROBIOLOGY., vol. 139, no. 4, July 1988 (1988-07-01), pages 461 - 471 *
LI, Q. ET AL.: "A rapid method for detection of Yersinia enterocolitica serotype 0:3 in pig feces using monoclonal antibodies.", VETERINARY MICROBIOLOGY., vol. 32, no. 1, July 1992 (1992-07-01), pages 29 - 41 *
MINGRONE, M. G. ET AL.: "Characteristics of Yersinia enterocolitica isolated from children with diarrhea in Italy.", JOURNAL OF CLINICAL MICROBIOLOGY., vol. 25, no. 7, July 1987 (1987-07-01), pages 1301 - 1304 *
SCHMIDT, M. ET AL.: "Production and characterization of monoclonal antibodies specific for pathogenic serogroups 0:3, 0:8, and 0:9 of Yersinia enterocolitic Zentralblatt fuer Bakteriologie, Mikrobiologie, und Hygiene.", SERIES A, MEDICAL MICROBIOLOGY, INFECTIOUS DISEASES, VIROLOGY, PARASITOLOGY., vol. 265, no. 1-2, June 1987 (1987-06-01), pages 113 - 123 *
WAUTERS G ET AL.: "Somatic and flagellar antigens of Yersinia enterocolitica", ANNALES DE L'INSTITUT PASTEUR., vol. 120, no. 5, May 1971 (1971-05-01), pages 631 - 642 *
WAUTERS, G. ET AL.: "Supplement to the antigenic schema of Yersinia enterocolitica", ANNALES DE L'INSTITUT PASTEUR., vol. 122, no. 5, May 1972 (1972-05-01), pages 951 - 956 *

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