ES2567056T3 - Secuencias para la detección e identificación de Staphylococcus aureus resistentes a meticilina - Google Patents

Secuencias para la detección e identificación de Staphylococcus aureus resistentes a meticilina Download PDF

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ES2567056T3
ES2567056T3 ES14168417.5T ES14168417T ES2567056T3 ES 2567056 T3 ES2567056 T3 ES 2567056T3 ES 14168417 T ES14168417 T ES 14168417T ES 2567056 T3 ES2567056 T3 ES 2567056T3
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primers
mrep
sequences
methicillin
detection
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Ann Huletsky
Valery Rossbach
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GeneOhm Sciences Canada Inc
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GeneOhm Sciences Canada Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Physics & Mathematics (AREA)
  • Immunology (AREA)
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  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract

El ácido nucleico de SEQ ID NO: 43, 44, 45, 46 o 51, o el complemento de dicho ácido nucleico.

Description

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Descripción de las figuras
La figura 1 es un diagrama que ilustra la posición de los cebadores desarrollados por Hiramatsu et al. (patente de los EE.UU. n.º 6 156 507) en la unión del extremo derecho de SCCmec-cromosoma para detectar e identificar SARM.
La figura 2 es un diagrama que ilustra la posición de los cebadores seleccionados en la presente invención en la 5 unión del extremo derecho de SCCmec-orfX para detectar eidentificar SARM.
La figura 3 es un diagrama que ilustra la posición de los cebadores seleccionados en la presente invención para secuenciar los nuevos tipos de MREP.
La figura 4 ilustra un alineamiento de secuencias de nueve tipos de MREP.
Leyendas de las figuras
10 Figura 1. Organización esquemática de los tipos I, II y III de las uniones en el extremo derecho de SCCmec-orfX, y localización de los cebadores (SEQ ID n.º 52-63) descritos por Hiramatsu et al. para detectar e identificar SARM. El tamaño de los amplicones se describe en la tabla 7.
Figura 2. Organización esquemática delos MREP de tipos i, ii, iii, iv, v, vi, vii, v y ix, y localización de los cebadores y la sonda fluorescible que están dirigidos selectivamente a todos los tipos de MREP (SEQ ID n.º 20, 64, 66, 67, 79,
15 80, 84, 112, 115, 116, 84, 163 y 164) que se desarrollaron, incluidos los de la invención. El tamaño de los amplicones se describe en latabla 7.
Figura 3. Organización esquemática de las uniones en el extremo derecho de SCCmec-cromosoma, y localización de los cebadores (SEQ ID n.º 65, 68, 69, 70, 77, 96, 118, 126, 132, 150 y 158) desarrollados para secuenciar los MREP de tipos iv, v, vi, vii, viii, ix y x.
20 Figura 4. Alineamiento múltiple de secuencias de representantes de nueve tipos de MREP (representados por trozos de las SEQ ID n.º 1, 2, 104, 51, 50, 171, 165, 167 y 168 para los tiposi, ii, iii, iv, v, vi, vii, v y ix, respectivamente).
Descripción de los anexos
Los anexos muestran las estrategias utilizadas para seleccionar cebadores y sondas internos:
El anexo I ilustra la estrategia para seleccionar los cebadores a partir de las secuencias de SCCmec y de orfX 25 específicasdelosSCCmec delos tiposI yII.
El anexo II ilustra la estrategia para seleccionar las sondas fluorescibles específicas para detectar en tiempo real las uniones en el extremo derecho de SCCmec-orfX.
Tal y como se muestra en estos anexos, los cebadores para amplificación seleccionados pueden contener inosinas y/o bases ambiguas. La inosina es un análogo de nucleótido capaz de unirse específicamente a cualquiera de los 30 cuatro nucleótidos A, C, G o T. Otra posibilidad es que se utilicen oligonucleótidos degenerados que consisten en una mezcla de oligonucleótidos que tienen dos o más de los cuatro nucleótidos A, C, G o T en el sitio de las discordancias. La inclusión de la inosina y/o de degeneraciones en los cebadores para amplificación proporciona cierta tolerancia a los apareamientos erróneos, con lo que se permite la amplificación de un abanico más amplio de secuencias nucleotídicas diana (Dieffenbach y Dveksler, 1995, PCR Primer: A Laboratory Manual, Cold Spring
35 Harbor Laboratory Press, Plainview, Nueva York).
Ejemplos
EJEMPLO 1:
Los cebadores desarrollados por Hiramatsu et al. sólo pueden detectar las cepas de SARM que pertenecen alos MREP de tipos i, ii y iii, mientras que no detectan nuevos tipos deMREP prevalentes.
40 Tal y como se muestra en la figura 1, Hiramatsu et al. han desarrollado varios cebadores que pueden hibridarse específicamente al extremo derecho de los ADN de SCCmec de tipos I, II y III. Combinaron estos cebadores con cebadores específicos de la región del cromosoma de S. aureus localizada a la derecha del sitio de integración de SCCmec para detectar los SARM. Hiramatsu et al. demostraron que el conjunto de cebadores (SEQ ID n.º 22, 24 y 28 en la patente de los EE.UU. n.º 6 156 507 quecorrespondea las SEQ ID n.º 56, 58 y 60 en la presenteinvención)
45 eran los más específicos y ubicuos para detectar SARM. Este conjunto de cebadores ofrece productos de amplificación de 1,5 kb para el SCCmec del tipo I, de 1,6 kb para el SCCmec del tipo II y de 1,0 kb para el SCCmec de tipo III (tabla 7). Se ensayó la ubicuidad y la especificidad de este ensayo de PCR múltiplex con 39 cepas de SARM, 41 cepas de SASM, 9 cepas de SCNRM y 11 cepas de SCNSM (tabla 2). En una mezcla de reacción de PCR de 20 µl se amplificó 1 µl de una suspensión bacteriana estandarizada tratada o de una preparación de ADN
50 genómico bacteriano purificado de bacterias. Cada reacción de PCR contenía KCl a 50 mM, Tris-HCl a 10 mM (pH 9,0), Triton X-100 al 0,1%, MgCl2 a 2,5 mM, 0,4 µM de cada uno de los cebadores específicos de SCCmec y de orfX
20
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64/109c orfX/MREPdetipoix 369 64/204c orfX/MREPdetipovi 348 64/112c orfX/MREPdetipovii 214 64/113c orfX/MREPdetipovii 263 64/115c orfX/MREPdetipov 227 64/116c orfX/MREPdetipov 318
a La longitud del amplicón se da en pares de bases para los tipos de MREP amplificados por el conjunto de cebadores.
bConjunto de cebadores descritos por Hiramatsu et al. en la patente de los EE.UU. n.º 6 156 507.
5 cConjuntodecebadoresdesarrolladosenlapresentememoria.
d orfSA0022 hace referencia a la designación del marco abierto de lectura del número de acceso de GenBank AP003129 (SEQ ID n.º 231).
Tabla 8. Otros cebadores desarrollados, incluidos los de la invención
ADN de origen SEQ ID n.º Diana Posicióna SEQ ID n.º 77 MREP de tipo iv 993 43 65 MREP de tipo v 636 47 70 orfX 1796 3 68 IS431 626 92 69 mecA 1059 78 96 mecA 1949 78 81 mecA 1206 78 114 MREP de tipo vii 629b 165 117 MREP de tipo ii 856 194 118 MREP de tipo ii 974b 194 119 MREP de tipo vii 404 189 120 MREP de tipo vii 477b 189 123 MREP de tipo vii 551 165 124 MREP de tipo ii 584 170 125 MREP de tipo ii 689b 170 126 orfSA0021 336 231 127 orfSA0021 563 231 128 orfSA0022d 2993 231 129 orfSA0022d 3467b 231 132 orfX 3700 231 145 MREP de tipo iv 988 51 146 MREP de tipo v 1386 51 147 MREP de tipo iv 891b 51 148 MREP de tipo ix 664 168 149 MREP de tipo ix 849b 168 150 MREP de tipo vii 1117b 165
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Claims (1)

  1. imagen1
ES14168417.5T 2001-06-04 2002-06-04 Secuencias para la detección e identificación de Staphylococcus aureus resistentes a meticilina Expired - Lifetime ES2567056T3 (es)

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CA002348042A CA2348042A1 (en) 2001-06-04 2001-06-04 Sequences for detection and identification of methicillin-resistant staphylococcus aureus

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ES10181536T Expired - Lifetime ES2526518T5 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo ix resistentes a meticilina
ES10181533T Expired - Lifetime ES2536887T5 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo v resistentes a meticilina
ES09174581T Expired - Lifetime ES2389467T3 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de staphyloccocus aureus resistente a meticilina
ES14168420T Expired - Lifetime ES2568072T5 (es) 2001-06-04 2002-06-04 Secuencias para la detección e identificación de Staphylococcus aureus resistente a meticilina
ES10181534T Expired - Lifetime ES2526517T5 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo vi resistentes a meticilina
ES10181535.5T Expired - Lifetime ES2526410T3 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo viii resistentes a meticilina
ES02740158T Expired - Lifetime ES2223310T3 (es) 2001-06-04 2002-06-04 Secuencias para la deteccion y la identificacion de staphyloccocus aureus resistente a la meticilina.
ES14168417.5T Expired - Lifetime ES2567056T3 (es) 2001-06-04 2002-06-04 Secuencias para la detección e identificación de Staphylococcus aureus resistentes a meticilina

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ES10181536T Expired - Lifetime ES2526518T5 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo ix resistentes a meticilina
ES10181533T Expired - Lifetime ES2536887T5 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo v resistentes a meticilina
ES09174581T Expired - Lifetime ES2389467T3 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de staphyloccocus aureus resistente a meticilina
ES14168420T Expired - Lifetime ES2568072T5 (es) 2001-06-04 2002-06-04 Secuencias para la detección e identificación de Staphylococcus aureus resistente a meticilina
ES10181534T Expired - Lifetime ES2526517T5 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo vi resistentes a meticilina
ES10181535.5T Expired - Lifetime ES2526410T3 (es) 2001-06-04 2002-06-04 Métodos y secuencias para la detección e identificación de cepas de Staphylococcus aureus MREJ tipo viii resistentes a meticilina
ES02740158T Expired - Lifetime ES2223310T3 (es) 2001-06-04 2002-06-04 Secuencias para la deteccion y la identificacion de staphyloccocus aureus resistente a la meticilina.

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AT (1) ATE447623T1 (es)
AU (6) AU2002315592B2 (es)
CA (7) CA2348042A1 (es)
DE (2) DE02740158T1 (es)
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DK2322655T3 (en) 2014-12-15
DK2322663T3 (en) 2015-05-04
CA2448975C (en) 2011-07-26
AU2011202388B2 (en) 2012-03-01

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