ES2572031B1 - Method of detection of human respiratory syncytial virus type A, human respiratory syncytial virus type B and human metapneumovirus - Google Patents
Method of detection of human respiratory syncytial virus type A, human respiratory syncytial virus type B and human metapneumovirus Download PDFInfo
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- ES2572031B1 ES2572031B1 ES201431573A ES201431573A ES2572031B1 ES 2572031 B1 ES2572031 B1 ES 2572031B1 ES 201431573 A ES201431573 A ES 201431573A ES 201431573 A ES201431573 A ES 201431573A ES 2572031 B1 ES2572031 B1 ES 2572031B1
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Abstract
Método de detección del virus sincitial respiratorio humano tipo A, del virus sincitial respiratorio humano tipo B y del metapneumovirus humano.#Método de detección simultánea del virus sincitial respiratorio humano A, del virus sincitial respiratorio humano B y del metapneumovirus humano en muestras biológicas, cebadores, sondas, composiciones kit y sus usos.Method of detection of human respiratory syncytial virus type A, human respiratory syncytial virus type B and human metapneumovirus. # Simultaneous detection method of human respiratory syncytial virus A, human respiratory syncytial virus B and human metapneumovirus in biological samples, primers , probes, kit compositions and their uses.
Description
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DESCRIPCIONDESCRIPTION
Metodo de deteccion del virus sincitial respiratorio humano tipo A, del virus sincitial respiratorio humano tipo B y del metapneumovirus humanoMethod of detection of human respiratory syncytial virus type A, human respiratory syncytial virus type B and human metapneumovirus
CAMPO DE LA INVENCIONFIELD OF THE INVENTION
La presente invencion se encuentra dentro del campo de la Biotecnologia. Particularmente, se refiere a un metodo que permite la amplification genomica en tiempo real del virus sincitial respiratorio humano tipo A (VRS A), del virus sincitial respiratorio humano tipo B (VRS B) y del metapneumovirus humano (hMPV) en una muestra biologica aislada. Por tanto, el metodo facilita la deteccion de estos virus en una muestra del paciente.The present invention is within the field of Biotechnology. Particularly, it refers to a method that allows real-time genomic amplification of human respiratory syncytial virus type A (RSV A), human respiratory syncytial virus type B (RSV B) and human metapneumovirus (hMPV) in an isolated biological sample . Therefore, the method facilitates the detection of these viruses in a patient sample.
ANTECEDENTES DE LA INVENCIONBACKGROUND OF THE INVENTION
Los tres virus mas importantes implicados en procesos de bronquiolitis agudas en lactantes son: el VRS A, VRS B y hMPV.The three most important viruses involved in acute bronchiolitis processes in infants are: RSV A, RSV B and hMPV.
El Virus Respiratorio Sincitial (VRS) es un mixovirus de ARN del genero Pneumovirus que pertenece a la familia de los Paramyxoviridae. Este virus es altamente contagioso y se difunde con las secreciones nasofaringeas de los individuos infectados por contacto directo o a traves de las gotas de saliva. El VRS puede causar grandes epidemias de bronquiolitis y neumomas, que afectan especialmente a ninos pequenos de todo el mundo. Se estima que el VRS solo en los Estados Unidos es responsable de 100.000 hospitalizaciones al ano (Tang y Crowe, 2007. Manual of clinical microbiology. 9a Edition vol. 2). Concretamente en Espana, se estima que las infecciones por VRS originan anualmente entre 15.000-20.000 visitas pediatricas de urgencia (Datos de la Asociacion Espanola de Pediatria).Respiratory Syncytial Virus (RSV) is an RNA mixovirus of the genus Pneumovirus that belongs to the Paramyxoviridae family. This virus is highly contagious and spreads with the nasopharyngeal secretions of infected individuals by direct contact or through saliva drops. RSV can cause major epidemics of bronchiolitis and pneumomas, which especially affect young children around the world. It is estimated that RSV alone in the United States is responsible for 100,000 hospitalizations per year (Tang and Crowe, 2007. Manual of clinical microbiology. 9th Edition vol. 2). Specifically in Spain, it is estimated that RSV infections originate annually between 15,000-20,000 emergency pediatric visits (Data from the Spanish Association of Pediatrics).
En base a sus diferencias antigenicas se identifican dos grupos principales de VRS: A y B, que se diferencian sobre todo en la glicoprotema G. Las diferentes secuencias de la protema G dan lugar a 6 subgrupos del A y 3 subgrupos en el B. No se han demostrado diferencias clmicas ni epidemiologicas entre ambos grupos, aunque es probable que haya unas cepas mas virulentas que otras.Based on their antigenic differences, two main groups of RSV are identified: A and B, which differ mainly in the G glycoprotem. The different sequences of the G protein give rise to 6 subgroups of A and 3 subgroups in B. No It has been shown that there are no clinical or epidemiological differences between the two groups, although it is likely that there are more virulent strains than others.
El diagnostico rapido y preciso de la infection por VRS es crucial para el manejo del paciente y el control de la infeccion (Woo et al., 1997. J. Clin. Microbiol. 35, 1579-1581;The rapid and accurate diagnosis of RSV infection is crucial for patient management and infection control (Woo et al., 1997. J. Clin. Microbiol. 35, 1579-1581;
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Adcock et al., 1997. Pediatr. Infect. Dis. J. 16, 842-846; Doherty et al., 1998. J. Hosp. Infect. 38, 203-206). La infeccion por VRS se identifica por metodos de diagnostico rapido basados en la inmunofluorescencia e inmunoensayo enzimatico en muestras de moco nasal. La sensibilidad de estos metodos esta entre el 80-90%. Tambien se puede identificar por aislamiento del virus en cultivos celulares de secreciones respiratorias aunque requiere de 3 a 5 dias. Actualmente las lmeas de investigation estan dirigidas a optimizar pruebas de reaction en cadena de la polimerasa que aumentan la sensibilidad y especificidad de la prueba.Adcock et al., 1997. Pediatr. Infect Dis. J. 16, 842-846; Doherty et al., 1998. J. Hosp. Infect 38, 203-206). RSV infection is identified by rapid diagnostic methods based on immunofluorescence and enzyme immunoassay in nasal mucus samples. The sensitivity of these methods is between 80-90%. It can also be identified by virus isolation in cell cultures of respiratory secretions although it requires 3 to 5 days. Currently, the investigation lines are aimed at optimizing polymerase chain reaction tests that increase the sensitivity and specificity of the test.
El metapneumovirus humano es un virus ARN del genero Metapneumovirus que pertenece a la familia de los Paramyxoviridae. Este virus puede provocar enfermedades respiratorias de cierta gravedad, sobre todo en ninos (incluido cuadros de bronquiolitis aguda semejante a los producidos por los VRS).Human metapneumovirus is an RNA virus of the genus Metapneumovirus that belongs to the family of Paramyxoviridae. This virus can cause respiratory diseases of a certain severity, especially in children (including acute bronchiolitis symptoms similar to those caused by RSV).
El hMPV posee caracteristicas propias muy similares al VRS, al igual que lo es su epidemiologia, su distribution estacional y sus manifestaciones clmicas. Al igual que el VRS, el hMPV se presenta principalmente en los meses de invierno (aunque tambien suele prolongarse durante los meses de primavera). Ademas, se ha encontrado hMPV en el 70% de los pacientes que eran ventilados en unidades de cuidados intensivos pediatricos debido a VRS (Greensill et al., 2003. Emerg. Infect. Dis. 9, 372-375). Por otro lado, se ha descrito que la bronquiolitis fue la manifestation primaria de infeccion por hMPV mas habitual (62%) en los pacientes.The hMPV has its own characteristics very similar to RSV, as is its epidemiology, seasonal distribution and weather manifestations. Like RSV, hMPV occurs mainly in the winter months (although it also usually lasts during the spring months). In addition, hMPV has been found in 70% of patients who were ventilated in pediatric intensive care units due to RSV (Greensill et al., 2003. Emerg. Infect. Dis. 9, 372-375). On the other hand, it has been described that bronchiolitis was the most common primary manifestation of hMPV infection (62%) in patients.
El diagnostico en la actualidad de la infeccion de hMPV se realiza mediante cultivo celular, serologia y microscopia electronica. Pero las lmeas mas actuales estan dirigidas al diagnostico por RT-PCR que aumentarian la sensibilidad (Mackay et al., 2003. J. Clin. Microbiol. 41, 100-105).The diagnosis of hMPV infection is currently made by cell culture, serology and electron microscopy. But the most current lines are directed to the diagnosis by RT-PCR that would increase sensitivity (Mackay et al., 2003. J. Clin. Microbiol. 41, 100-105).
Con el objetivo principal de evitar las dificultades que se presentan en el diagnostico de la bronquiolitis, la cual es generada mayoritariamente por los virus descritos anteriormente, la presente invention se refiere a un metodo basado en la tecnica de RT-PCR multiple a tiempo real para asistir al facultativo en la diagnosis de la bronquiolitis cuando se detecta la presencia de VRS y hMPV.With the main objective of avoiding the difficulties that arise in the diagnosis of bronchiolitis, which is mostly generated by the viruses described above, the present invention relates to a method based on the technique of multiple real-time RT-PCR for assist the physician in the diagnosis of bronchiolitis when the presence of RSV and hMPV is detected.
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BREVE DESCRIPCION DE LA INVENCIONBRIEF DESCRIPTION OF THE INVENTION
Un primer aspecto de la invention se refiere a un metodo, de ahora en adelante primer metodo de la invencion, de detection del virus VRS A, del virus VRS B, del virus hMPV, o cualquiera de sus combinaciones, que comprende:A first aspect of the invention relates to a method, hereinafter the first method of the invention, of detection of VRS A virus, VRS B virus, hMPV virus, or any combination thereof, comprising:
i) extraer el ARN de una muestra biologica aislada de un individuo,i) extracting the RNA from an isolated biological sample of an individual,
ii) deteccion de las secuencias de nucleotidos que comprenden la SEQ ID NO: 3, SEQ ID NO: 6, y SEQ ID NO: 9.ii) detection of nucleotide sequences comprising SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.
En una realization preferida de este aspecto e la invencion, la deteccion se realiza de manera simultanea. Mas preferiblemente, se realiza mediante la amplification de las secuencias de nucleotidos recogidas en la SEQ ID NO: 3, SEQ ID NO: 6, y SEQ ID NO: 9 por RT-PCR. Aun mas preferiblemente, la deteccion se realiza empleando un conjunto de parejas de cebadores que comprenden las secuencias SEQ ID NO: 1 y SEQ ID NO: 2; SEQ ID NO: 4 y SEQ ID NO: 5; SEQ ID NO: 7 y SEQ ID NO: 8. Aun mas preferiblemente, la extraction del acido nucleico incluye una etapa de purification. Aun mas preferiblemente, la reaction de PCR es una reaction multiplex. Aun mas preferiblemente, el primer metodo de la invencion ademas comprende una etapa:In a preferred embodiment of this aspect and the invention, the detection is carried out simultaneously. More preferably, it is performed by amplifying the nucleotide sequences collected in SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9 by RT-PCR. Even more preferably, the detection is performed using a set of primer pairs comprising the sequences SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 7 and SEQ ID NO: 8. Even more preferably, the extraction of the nucleic acid includes a purification step. Even more preferably, the PCR reaction is a multiplex reaction. Even more preferably, the first method of the invention further comprises a step:
iii) revelado y comparacioniii) development and comparison
En otra realizacion preferida de este aspecto de la invencion, la muestra biologica es un aspirado nasal.In another preferred embodiment of this aspect of the invention, the biological sample is a nasal aspirate.
En otra realizacion preferida de este aspecto de la invencion, la muestra biologica es un lavado broncoalveolar, un broncoaspirado, un cepillado telescopado o un lavado nasofaringeo.In another preferred embodiment of this aspect of the invention, the biological sample is a bronchoalveolar lavage, a bronchoaspirate, a telescopic brush or a nasopharyngeal wash.
Un segundo aspecto de la invencion se refiere a los cebadores, de ahora en adelante cebadores de la invencion, de secuencia nucleotidica:A second aspect of the invention relates to the primers, hereafter primers of the invention, of nucleotide sequence:
- a) to)
- SEQ ID NO: 1, SEQ ID NO: 1,
- b) b)
- SEQ ID NO: 2, SEQ ID NO: 2,
- c) C)
- SEQ ID NO: 4, SEQ ID NO: 4,
- d) d)
- SEQ ID NO: 5, SEQ ID NO: 5,
- e) and)
- SEQ ID NO: 7, y SEQ ID NO: 7, and
- f) F)
- SEQ ID NO: 8. SEQ ID NO: 8.
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Un tercer aspecto de la invention se refiere a las sondas, de ahora en adelante sondas de la invencion, de secuencia nucleotidica:A third aspect of the invention relates to probes, hereinafter probes of the invention, of nucleotide sequence:
a) SEQ ID NO: 3,
a) SEQ ID NO: 3,
b) SEQ ID NO: 6, y
b) SEQ ID NO: 6, and
c) SEQ ID NO: 9.
c) SEQ ID NO: 9.
Un cuarto aspecto de la invencion se refiere al uso de los cebadores de la invencion, y/o las sondas de la invencion, para la detection del virus VRS A, del virus VRS B y del virus hMPV. En una realization preferida de este aspecto de la invencion, la deteccion es simultanea.A fourth aspect of the invention relates to the use of the primers of the invention, and / or the probes of the invention, for the detection of the VRS A virus, the VRS B virus and the hMPV virus. In a preferred embodiment of this aspect of the invention, the detection is simultaneous.
En otra realizacion preferida de este aspecto de la invencion, la pareja de cebadores SEQ ID NO: 1 y SEQ ID NO: 2, y la sonda SEQ ID NO: 3, se utilizan para la deteccion del VRS A.In another preferred embodiment of this aspect of the invention, the primer pair SEQ ID NO: 1 and SEQ ID NO: 2, and the probe SEQ ID NO: 3, are used for the detection of RSV A.
En otra realizacion preferida de este aspecto de la invencion, la pareja de cebadores SEQ ID NO: 4 y SEQ ID NO: 5, y la sonda SEQ ID NO: 6, se utilizan para la deteccion del VRS B.In another preferred embodiment of this aspect of the invention, the primer pair SEQ ID NO: 4 and SEQ ID NO: 5, and the probe SEQ ID NO: 6, are used for the detection of RSV B.
En otra realizacion preferida de este aspecto de la invencion, la pareja de cebadores SEQ ID NO: 7 y SEQ ID NO: 8, y la sonda SEQ ID NO: 9, se utilizan para la deteccion del hMPV.In another preferred embodiment of this aspect of the invention, the primer pair SEQ ID NO: 7 and SEQ ID NO: 8, and the probe SEQ ID NO: 9, are used for hMPV detection.
Un quinto aspecto de la invencion se refiere a una composition, de ahora en adelante composition de la invencion, que comprende la secuencia SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y/o SEQ ID NO: 9, o cualquiera de sus combinaciones.A fifth aspect of the invention relates to a composition, hereinafter the composition of the invention, comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 9, or any combination thereof.
En una realizacion preferida, la composicion de la invencion comprende las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 y SEQ ID NO: 8. En otra realizacion preferida, la composicion de la invencion comprende las sondas con secuencias que comprenden las mostradas en SEQ ID NO: 3, SEQ ID NO: 6 y SEQ ID NO: 9.In a preferred embodiment, the composition of the invention comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8. In Another preferred embodiment, the composition of the invention comprises probes with sequences comprising those shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
En otra realizacion preferida, la composicion de la invencion comprende simultaneamente las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y SEQ ID NO: 9.In another preferred embodiment, the composition of the invention simultaneously comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
Un sexto aspecto de la invencion se refiere al kit o dispositivo, kit o dispositivo de laA sixth aspect of the invention relates to the kit or device, kit or device of the
invencion, que comprende la secuencia SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQinvention, comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
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ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y/o SEQ ID NO: 9, o cualquiera de sus combinaciones.ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 9, or any combination thereof.
En una realization preferida, la composition de la invention comprende las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 y SEQ ID NO: 8. En otra realizacion preferida, la composicion de la invencion comprende las sondas con secuencias que comprenden las mostradas en SEQ ID NO: 3, SEQ ID NO: 6 y SEQ ID NO: 9.In a preferred embodiment, the composition of the invention comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8. In Another preferred embodiment, the composition of the invention comprises probes with sequences comprising those shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
En otra realizacion preferida, la composicion de la invencion comprende simultaneamente las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y SEQ ID NO: 9.In another preferred embodiment, the composition of the invention simultaneously comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
Un septimo aspecto de la invencion se refiere al uso de la composicion de la invencion, o el kit o dispositivo de la invencion, para la detection del virus VRS A, del virus VRS B y del virus hMPV, o cualquiera de sus combinaciones, en una muestra biologica.A seventh aspect of the invention relates to the use of the composition of the invention, or the kit or device of the invention, for the detection of the VRS A virus, the VRS B virus and the hMPV virus, or any combination thereof, in A biological sample.
En una realizacion preferida de este aspecto de la invencion, la deteccion se realiza de manera simultanea.In a preferred embodiment of this aspect of the invention, the detection is performed simultaneously.
Un octavo aspecto de la invencion se refiere a un medio de almacenamiento legible por un ordenador que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos del metodo de la invencion.An eighth aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of the method of the invention.
En una realizacion preferida de este aspecto de la invencion, el medio de almacenamiento legible comprende al menos una secuencia comprendida en cualquiera de las sondas de la invencion.In a preferred embodiment of this aspect of the invention, the readable storage medium comprises at least one sequence comprised in any of the probes of the invention.
En otra realizacion preferida de este aspecto de la invencion que comprende oligonucleotidos o microarreglos de canal unico disenados a partir de al menos una secuencia conocida o un ARNm comprendida cualquiera de las sondas de la invencion.In another preferred embodiment of this aspect of the invention comprising oligonucleotides or single channel microarrays designed from at least one known sequence or an mRNA comprising any of the probes of the invention.
Un noveno aspecto de la invencion se refiere a una senal transmisible que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos del metodo de la invencion.A ninth aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer carry out the steps of the method of the invention.
Un decimo aspecto de la invencion se refiere a un programa de ordenador almacenado enA tenth aspect of the invention relates to a computer program stored in
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el medio legible por un ordenador de la invention, que comprende codigo de software adaptado para llevar a cabo los pasos de cualquiera de los metodos de la invencion.the computer-readable medium of the invention, comprising software code adapted to carry out the steps of any of the methods of the invention.
DESCRIPCION DETALLADA DE LA INVENCIONDETAILED DESCRIPTION OF THE INVENTION
Los autores de la presente invencion han analizado el material genetico de los virus mas relevantes relacionados con la bronquiolitis y han generado un metodo para la detection de los mismos.The authors of the present invention have analyzed the genetic material of the most relevant viruses related to bronchiolitis and have generated a method for their detection.
La presente invencion se refiere a un metodo, cebadores, sondas, composiciones, kits para su uso en la deteccion del VRS A, del VRS B y del hMPV en muestras biologicas.The present invention relates to a method, primers, probes, compositions, kits for use in the detection of RSV A, RSV B and hMPV in biological samples.
Por tanto, un primer aspecto de la invencion se refiere a un metodo, de ahora en adelante primer metodo de la invencion, de deteccion del virus VRS A, del virus VRS B, del virus hMPV, o cualquiera de sus combinaciones, que comprende:Therefore, a first aspect of the invention relates to a method, hereinafter the first method of the invention, of detecting the VRS A virus, the VRS B virus, the hMPV virus, or any combination thereof, comprising:
i) extraer el ARN de la muestra biologica aislada de un individuo,i) extract the RNA from the isolated biological sample of an individual,
ii) deteccion de las secuencias de nucleotidos que comprenden la SEQ ID NO: 3, SEQ ID NO: 6, y SEQ ID NO: 9.ii) detection of nucleotide sequences comprising SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.
En una realization preferida de este aspecto e la invencion, la deteccion se realiza de manera simultanea. Mas preferiblemente, se realiza mediante la amplification de las secuencias de nucleotidos recogidas en la SEQ ID NO: 3, SEQ ID NO: 6, y SEQ ID NO: 9 por RT-PCR. Aun mas preferiblemente, la deteccion se realiza empleando un conjunto de parejas de cebadores que comprenden las secuencias SEQ ID NO: 1 y SEQ ID NO: 2; SEQ ID NO: 4 y SEQ ID NO: 5; SEQ ID NO: 7 y SEQ ID NO: 8. Aun mas preferiblemente, la extraction del ARN/ADN incluye una etapa de purification. Aun mas preferiblemente, la reaction de PCR es una reaction multiplex. Aun mas preferiblemente, el primer metodo de la invencion ademas comprende una etapa:In a preferred embodiment of this aspect and the invention, the detection is carried out simultaneously. More preferably, it is performed by amplifying the nucleotide sequences collected in SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9 by RT-PCR. Even more preferably, the detection is performed using a set of primer pairs comprising the sequences SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 5; SEQ ID NO: 7 and SEQ ID NO: 8. Even more preferably, RNA / DNA extraction includes a purification step. Even more preferably, the PCR reaction is a multiplex reaction. Even more preferably, the first method of the invention further comprises a step:
iii) revelado y comparacioniii) development and comparison
En una realizacion preferida, el paso iii) se realiza con un termociclador a tiempo real que lee la fluorescencia emitida por diferentes canales, con su correspondiente longitud de onda. En una realizacion aun mas preferida se utilizan 5 canales: 3 para los virus, 1 para el control de reaccion y 1 para el control comparador con ruido de fondo.In a preferred embodiment, step iii) is performed with a real-time thermal cycler that reads the fluorescence emitted by different channels, with their corresponding wavelength. In an even more preferred embodiment, 5 channels are used: 3 for viruses, 1 for reaction control and 1 for comparator control with background noise.
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En otra realization preferida de este aspecto de la invention, la muestra biologica es un aspirado nasal.In another preferred embodiment of this aspect of the invention, the biological sample is a nasal aspirate.
En otra realizacion preferida de este aspecto de la invencion, la muestra biologica es un lavado broncoalveolar, un broncoaspirado, un cepillado telescopado o un lavado nasofarigeo.In another preferred embodiment of this aspect of the invention, the biological sample is a bronchoalveolar lavage, a bronchoaspirate, a telescopic brush or a nasopharyngeal wash.
Los pasos (ii) y/o (iii) de los metodos descritos anteriormente pueden ser total o parcialmente automatizados, por ejemplo, por medio de un equipo robotico sensor para la detection de la cantidad en el paso (ii) o la comparacion computerizada en el paso (iii).Steps (ii) and / or (iii) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (ii) or the computerized comparison in step (iii).
Una "muestra biologica aislada” incluye, pero sin limitarnos a, celulas, tejidos y/o fluidos biologicos de un organismo, obtenidos mediante cualquier metodo conocido por un experto en la materia. Preferiblemente, la muestra biologica aislada de un individuo del paso (i) es el un aspirado nasal.An "isolated biological sample" includes, but is not limited to, cells, tissues and / or biological fluids of an organism, obtained by any method known to a person skilled in the art. Preferably, the biological sample isolated from an individual of the step (i ) is a nasal aspirate.
El termino "individuo”, tal y como se utiliza en la description, se refiere a animales, preferiblemente mamiferos, y mas preferiblemente, humanos. El termino "individuo” no pretende ser limitativo en ningun aspecto, pudiendo ser este de cualquier edad, sexo y condition fisica.The term "individual", as used in the description, refers to animals, preferably mammals, and more preferably, humans. The term "individual" is not intended to be limiting in any aspect, and may be of any age, sex and physical condition.
El termino "comparacion”, tal y como se utiliza en la descripcion, se refiere pero no se limita, a la comparacion del resultado de la amplification de la muestra biologica a analizar, tambien llamada muestra biologica problema, con una amplificacion de una o varias muestras de referencia deseable. La muestra de referencia puede ser analizada, por ejemplo, simultanea o consecutivamente, junto con la muestra biologica problema. La comparacion descrita en el apartado (iii) del metodo de la presente invencion puede ser realizada manualmente o asistida por ordenador.The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the result of the amplification of the biological sample to be analyzed, also called the problem biological sample, with an amplification of one or more Desirable reference samples The reference sample can be analyzed, for example, simultaneously or consecutively, together with the biological problem sample.The comparison described in section (iii) of the method of the present invention can be performed manually or assisted by computer .
El termino "PCR” corresponde a las siglas de reaction en cadena de la polimerasa mediante la cual se pueden obtener millones de copias de las regiones de ADN deseadas. Se caracteriza por el empleo de parejas de cebadores que acotan la region de la que se realizaran millones de copias, lo que tambien se conoce como "amplificar ADN" durante la PCR. La PCR esta compuesta por un numero determinado de ciclos, compuestos a su vez por tres fases en las que las hebras de ADN se separan, se unen los cebadores y se elongan las nuevas hebras de ADN. En cada ciclo, si la eficiencia de la reaccion es delThe term "PCR" corresponds to the acronym of polymerase chain reaction whereby millions of copies of the desired DNA regions can be obtained. It is characterized by the use of primer pairs that delimit the region from which they will be made million copies, which is also known as "amplifying DNA" during PCR.The PCR is composed of a certain number of cycles, in turn composed of three phases in which the strands of DNA separate, the primers bind and the new strands of DNA are elongated. In each cycle, if the reaction efficiency is
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100% se produce un crecimiento exponencial de los fragmentos de ADN objeto de la amplification.100% exponential growth of the DNA fragments subject to amplification occurs.
En el caso de la invention, el termino “RT-PCR” corresponde a las siglas de reaction en cadena de la polimerasa con transcriptasa inversa. Es una variante de PCR y tambien se usa para generar una gran cantidad de copias de ADN (amplificacion). En la RT-PCR, sin embargo, una hebra de ARN es retrotranscripta en ADN complementary (ADNc) usando una enzima llamada transcriptasa inversa, y el resultado, se amplifica en una PCR tradicional. La amplificacion exponencial mediante PCR en Transcription Reversa supone una tecnica altamente sensible, que puede detectar un numero de copias de ARN muy bajo. Puede utilizarse como metodo de detection molecular de genes y para estudiar el genoma de virus de ARN como los retrovirus. Otro de sus usos se relaciona con la cuantificacion de la expresion genica, mediante la combination de esta tecnica con el analisis de Northern blot. Una de las caracteristicas mas importantes es que en el proceso de RT-PCR, el ADNc generado ya no lleva los intrones que si tendria el ADN original. De este modo, al expresar el ADNc producto de la RT-PCR, se generara un ARNm formado exclusivamente por exones.In the case of the invention, the term "RT-PCR" corresponds to the acronym of polymerase chain reaction with reverse transcriptase. It is a variant of PCR and is also used to generate a large number of copies of DNA (amplification). In RT-PCR, however, an RNA strand is retrotranscribed in complementary DNA (cDNA) using an enzyme called reverse transcriptase, and the result is amplified in a traditional PCR. Exponential amplification by PCR in Reverse Transcription is a highly sensitive technique, which can detect a very low number of RNA copies. It can be used as a method of molecular detection of genes and to study the genome of RNA viruses such as retroviruses. Another of its uses is related to the quantification of genetic expression, by combining this technique with Northern blot analysis. One of the most important characteristics is that in the RT-PCR process, the generated cDNA no longer carries the introns that the original DNA would have. Thus, by expressing the cDNA product of the RT-PCR, an mRNA formed exclusively by exons will be generated.
En la presente invencion se entiende por "Reaccion de amplificacion multiplex" a la reaccion PCR en la cual se amplifica mas de una secuencia de ADN en una misma reaccion, mediante el empleo de dos o mas parejas de cebadores en unico tubo junto con el resto de los reactivos de la reaccion con el fin de amplificar simultaneamente multiples secuencias de ADN.In the present invention, "multiplex amplification reaction" is understood as the PCR reaction in which more than one DNA sequence is amplified in the same reaction, by using two or more pairs of primers in a single tube together with the rest. of the reaction reagents in order to simultaneously amplify multiple DNA sequences.
El termino "oligonucleotido" hace referencia a la secuencia de bases de nucleotidos unidos por enlaces fosfodiester, habitualmente no mayores de 50 nucleotidos.The term "oligonucleotide" refers to the sequence of nucleotide bases linked by phosphodiester bonds, usually not greater than 50 nucleotides.
Las "condiciones de amplificacion" o "condiciones de extension" se refieren indistintamente a condiciones bajo las cuales una polimerasa puede anadir nucleotidos al extremo 3' de un polinucleotido. Dichas condiciones de amplificacion o extension son muy conocidas en la tecnica (Sambrook y Russell, 2001. Molecular Cloning: A Laboratory Manual, 3a Edition, Cold Spring Harbor Laboratory Press; Ausubel et al., Current Protocols in Molecular Biology, 1987-2007, John Wiley & Sons.)The "amplification conditions" or "extension conditions" refer interchangeably to conditions under which a polymerase can add nucleotides to the 3 'end of a polynucleotide. Such amplification or extension conditions are well known in the art (Sambrook and Russell, 2001. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press; Ausubel et al., Current Protocols in Molecular Biology, 1987-2007, John Wiley & Sons.)
Por “virus” en esta memoria, se entienden todos los agentes infecciosos microscopicos acelulares que solo pueden multiplicarse dentro de las celulas de otros organismos.By "virus" herein, all acellular microscopic infectious agents are understood that can only multiply within the cells of other organisms.
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El VRS pertenece al genera Pneumovirus, que se engloba dentro del Dominio Acytota, Grupo V: Virus ARN monocatenario negativo, Orden Mononegavirales, Familia Paramyxoviridae, Subfamilia Pneumovirinae.The RSV belongs to the Pneumovirus gene, which is included in the Acytota Domain, Group V: Negative single-stranded RNA virus, Mononegavirales Order, Paramyxoviridae Family, Pneumovirinae Subfamily.
El hMPV pertenece al genero Metapneumovirus, que se engloba dentro del Dominio Acytota, Grupo V: Virus ARN monocatenario negativo, Orden Mononegavirales, Familia Paramyxoviridae, Subfamilia Pneumovirinae.The hMPV belongs to the genus Metapneumovirus, which is encompassed within the Acytota Domain, Group V: negative single-stranded RNA virus, Mononegavirales Order, Paramyxoviridae Family, Pneumovirinae Subfamily.
La familia Paramyxoviridae esta formada por virus parecidos a los ortomixovirus, pero con diferencias que justifican la separation de familias. Los virus de la parainfluenza, el sarampion, la pariotiditis, el virus sincitial respiratorio y el metapneumovirus forman parte de esta familia, y afectan principalmente a la poblacion infantil.The Paramyxoviridae family consists of orthomyxovirus-like viruses, but with differences that justify the separation of families. The viruses of parainfluenza, measles, mumps, respiratory syncytial virus and metapneumovirus are part of this family, and mainly affect the child population.
Los Paramyxovirus miden entre 156-300nm, tienen cubierta lipidica, genoma de ARN de una cadena negativa, doble membrana lipidica derivada de la celula huesped, glucoprotemas de superficie que forman proyecciones HN con action neuroaminidasa y hemaglutinacion, y una F que participa en la penetration viral y estimula la fusion de membranas. Ademas la protema de membrana M que forma parte de la cubierta lipidica y la ribonucleoprotema NP que es antigeno fijador del complemento. Estos virus abordan las vias respiratorias y se instalan con mas frecuencia en la parte altas de estas vias, donde invaden las celulas epiteliales, se replican intensamente, generan la formation de celulas gigantes y producen citolisis, pero no producen estados viremicos (salvo excepcionalmente), por lo que la infection circunscribe al sistema respiratorio. Cuando invade regiones mas bajas produce bronquitis, bronquiolitis y neumoma. (Microbiologia y Parasitologia Humana. Bases etiologicas de las enfermedades infecciosas y parasitarias. 3a Edition. Editorial Medica Panamericana)Paramyxoviruses measure between 156-300nm, have lipid cover, RNA genome of a negative chain, double lipid membrane derived from the host cell, surface glycoprotems that form HN projections with neuroaminidase and hemagglutination action, and an F that participates in penetration viral and stimulates the fusion of membranes. In addition, the M membrane membrane that is part of the lipid shell and the NP ribonucleoprotem that is complement fixative antigen. These viruses address the respiratory tract and install themselves more frequently in the upper part of these pathways, where they invade epithelial cells, replicate intensely, generate giant cell formation and produce cytolysis, but do not produce viremic states (except exceptionally), So the infection circumscribes the respiratory system. When it invades lower regions it produces bronchitis, bronchiolitis and pneumoma. (Microbiology and Human Parasitology. Etiological bases of infectious and parasitic diseases. 3rd Edition. Editorial Panamericana Medica)
Un segundo aspecto de la invention se refiere a los cebadores, de ahora en adelante cebadores de la invencion, de secuencia nucleotidica:A second aspect of the invention relates to the primers, hereafter primers of the invention, of nucleotide sequence:
a) SEQ ID NO: 1,
a) SEQ ID NO: 1,
b) SEQ ID NO: 2,
b) SEQ ID NO: 2,
c) SEQ ID NO: 4,
c) SEQ ID NO: 4,
d) SEQ ID NO: 5,
d) SEQ ID NO: 5,
e) SEQ ID NO: 7, y
e) SEQ ID NO: 7, and
f) SEQ ID NO: 8.
f) SEQ ID NO: 8.
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En la presente invention se entiende por "cebador" o "primer" a la secuencia de nucleotidos a partir de la cual la ADN polimerasa inicia la smtesis de una molecula nueva de ADN. Los cebadores son secuencias nucleotidicas cortas, aproximadamente de 15-24 nucleotidos de longitud que se pueden alinear con una hebra de ADN diana gracias a la complementariedad de bases para formar un hibrido entre el cebador y la hebra diana de ADN. Despues, el enzima ADN polimerasa puede extender el cebador a lo largo de la hebra diana de ADN. Los metodos para preparar y usar cebadores se describen, por ejemplo en Sambrook et al. (2001) y Ausubel et al. (1999).In the present invention, "primer" or "first" is understood as the nucleotide sequence from which DNA polymerase starts the synthesis of a new DNA molecule. The primers are short nucleotide sequences, approximately 15-24 nucleotides in length that can be aligned with a strand of target DNA thanks to the complementarity of bases to form a hybrid between the primer and the target strand of DNA. Then, the enzyme DNA polymerase can extend the primer along the DNA strand. Methods for preparing and using primers are described, for example in Sambrook et al. (2001) and Ausubel et al. (1999).
En el contexto de la presente invencion se entiende "extracto de ARN/ADN", cuando tras someter la muestra biologica a un procedimiento de extraction, separation, purification o donation de acidos nucleicos, entre otros, se obtiene como resultado ya sea en seco, en solution, unido o no a otras moleculas, adherido o no a diversas sustancias o lechos, materia en la que el ARN/ADN se encuentra en mayor proportion relativa respecto al resto de moleculas presentes, en comparacion con la muestra biologica de partida.In the context of the present invention, "RNA / DNA extract" is understood when, after subjecting the biological sample to a procedure of extraction, separation, purification or donation of nucleic acids, among others, it is obtained as a result either dry, in solution, bound or not to other molecules, adhered or not to various substances or beds, matter in which the RNA / DNA is in a greater relative proportion with respect to the rest of the molecules present, in comparison with the biological starting sample.
Los cebadores de la invencion comprenden las siguientes secuencias:The primers of the invention comprise the following sequences:
SEQ ID NO: 1: 5'-AGCAAATCAATGTCACTAACACC-3'SEQ ID NO: 1: 5'-AGCAAATCAATGTCACTAACACC-3 '
SEQ ID NO: 2: 5'-TGTAGTACCATTGTGTGTTGTG-3'SEQ ID NO: 2: 5'-TGTAGTACCATTGTGTGTTGTG-3 '
SEQ ID NO: 4: 5'-GACATGTGTTTATTACCATTTTAG-3'SEQ ID NO: 4: 5'-GACATGTGTTTATTACCATTTTAG-3 '
SEQ ID NO: 5: 5'-GGTGTTGTCGTTTGTAGTGCT-3'SEQ ID NO: 5: 5'-GGTGTTGTCGTTTGTAGTGCT-3 '
SEQ ID NO: 7: 5'-ACAATGGCAACTTTGCTTAAAGAA-3'SEQ ID NO: 7: 5'-ACAATGGCAACTTTGCTTAAAGAA-3 '
SEQ ID NO: 8: 5'- TGCTGAAGGCCTCTGATTTTG-3'SEQ ID NO: 8: 5'- TGCTGAAGGCCTCTGATTTTG-3 '
Asi, en las realizaciones preferidas de la invencion las parejas de cebadores usadas comprenden SEQ ID NO: 1 y SEQ ID NO: 2 y/o SEQ ID NO: 4 y SEQ ID NO: 5 y/o SEQ ID NO: 7 y SEQ ID NO: 8.Thus, in preferred embodiments of the invention the pairs of primers used comprise SEQ ID NO: 1 and SEQ ID NO: 2 and / or SEQ ID NO: 4 and SEQ ID NO: 5 and / or SEQ ID NO: 7 and SEQ ID NO: 8.
En el contexto de la presente invencion se entiende por "pareja de cebadores" o "primer pair", al conjunto de dos cebadores que, empleados en una misma reaction de amplification o PCR, permiten obtener multiples copias de una secuencia diana de ADN. Cada uno de los cebadores hibrida con la secuencia diana, de manera que se amplifica la secuencia de nucleotidos acotada mediante cada pareja de cebadores. La extension de los cebadores durante los ciclos de PCR determina la multiplication exponencial 2N de la secuencia de nucleotidos acotada por los cebadores, siendo N el numero de ciclos de la reaccion PCR.In the context of the present invention, "primer pair" or "first pair" is understood as the set of two primers which, when used in the same amplification or PCR reaction, allow multiple copies of a DNA target sequence to be obtained. Each of the primers hybridizes with the target sequence, so that the bounded nucleotide sequence is amplified by each pair of primers. The extension of the primers during the PCR cycles determines the exponential multiplication 2N of the nucleotide sequence bounded by the primers, N being the number of cycles of the PCR reaction.
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Un tercer aspecto de la invencion se refiere a las sondas, de ahora en adelante sondas de la invencion, de secuencia nucleotidica:A third aspect of the invention relates to the probes, hereinafter probes of the invention, of nucleotide sequence:
a) SEQ ID NO: 3,
a) SEQ ID NO: 3,
b) SEQ ID NO: 6, y
b) SEQ ID NO: 6, and
c) SEQ ID NO: 9.
c) SEQ ID NO: 9.
En el contexto de la presente invencion, el termino "sonda” se define como un oligonucleotido que es complementary o sustancialmente complementary a la secuencia del amplicon, dentro de los limites de los cebadores. En la presente forma de realizacion preferida, la sonda de captura no se encuentra provista de la adicion de una cola, pero podria anadirse una cola, con desoxirribonucleotidos o ribonucleotidos. La sonda se puede producir preferiblemente sinteticamente o como un subconjunto de ADN obtenido naturalmente, preparados por metodologias estandar (por ejemplo, mediante digestion por endonucleasas de restriccion).In the context of the present invention, the term "probe" is defined as an oligonucleotide that is complementary or substantially complementary to the sequence of the amplicon, within the limits of the primers. In the present preferred embodiment, the capture probe it is not provided with the addition of a tail, but a tail could be added, with deoxyribonucleotides or ribonucleotides.The probe can preferably be produced synthetically or as a subset of naturally obtained DNA, prepared by standard methodologies (for example, by endonuclease digestion of restriction).
Por lo tanto, no existe, excepto para la funcion pretendida, ninguna diferencia fundamental entre un "cebador", un "oligonucleotido" o una "sonda" segun la invencion.Therefore, there is, except for the intended function, no fundamental difference between a "primer", an "oligonucleotide" or a "probe" according to the invention.
Preferiblemente, las sondas de la invencion comprenden las siguientes secuencias:Preferably, the probes of the invention comprise the following sequences:
SEQ ID NO: 3: 5'-TCAGCCGACCCAACCA-3'SEQ ID NO: 3: 5'-TCAGCCGACCCAACCA-3 '
SEQ ID NO: 6: 5'-ACTCACCTAATCAATCAA-3'SEQ ID NO: 6: 5'-ACTCACCTAATCAATCAA-3 '
SEQ ID NO: 9: 5'-CATCAGGCAATATTC-3'SEQ ID NO: 9: 5'-CATCAGGCAATATTC-3 '
Asi, las sondas pueden ser de entre 10 y 200 nucleotidos, mas preferiblemente, de entre 12 y 50 nucleotidos, y aun mas preferiblemente del numero de nucleotidos de las sondas de la invencion.Thus, the probes can be between 10 and 200 nucleotides, more preferably, between 12 and 50 nucleotides, and even more preferably the number of nucleotides of the probes of the invention.
Un cuarto aspecto de la invencion se refiere al uso de los cebadores de la invencion, y/o las sondas de la invencion, para la deteccion del virus VRS A, del virus VRS B y del virus hMPV. En una realizacion preferida de este aspecto de la invencion, la deteccion es simultanea.A fourth aspect of the invention relates to the use of the primers of the invention, and / or the probes of the invention, for the detection of the VRS A virus, the VRS B virus and the hMPV virus. In a preferred embodiment of this aspect of the invention, the detection is simultaneous.
En otra realizacion preferida de este aspecto de la invencion, la pareja de cebadores SEQ ID NO: 1 y SEQ ID NO: 2, y la sonda SEQ ID NO: 3, se utilizan para la deteccion del VRS A.In another preferred embodiment of this aspect of the invention, the primer pair SEQ ID NO: 1 and SEQ ID NO: 2, and the probe SEQ ID NO: 3, are used for the detection of RSV A.
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En otra realization preferida de este aspecto de la invention, la pareja de cebadores SEQ ID NO: 4 y SEQ ID NO: 5, y la sonda SEQ ID NO: 6, se utilizan para la detection del VRS B.In another preferred embodiment of this aspect of the invention, the primer pair SEQ ID NO: 4 and SEQ ID NO: 5, and the probe SEQ ID NO: 6, are used for the detection of RSV B.
En otra realizacion preferida de este aspecto de la invencion, la pareja de cebadores SEQ ID NO: 7 y SEQ ID NO: 8, y la sonda SEQ ID NO: 9, se utilizan para la deteccion del hMPV.In another preferred embodiment of this aspect of the invention, the primer pair SEQ ID NO: 7 and SEQ ID NO: 8, and the probe SEQ ID NO: 9, are used for hMPV detection.
Un quinto aspecto de la invencion se refiere a una composition, de ahora en adelante composition de la invencion, que comprende la secuencia SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y/o SEQ ID NO: 9, o cualquiera de sus combinaciones.A fifth aspect of the invention relates to a composition, hereinafter the composition of the invention, comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 9, or any combination thereof.
En una realizacion preferida, la composicion de la invencion comprende las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 y SEQ ID NO: 8. En otra realizacion preferida, la composicion de la invencion comprende las sondas con secuencias que comprenden las mostradas en SEQ ID NO: 3, SEQ ID NO: 6 y SEQ ID NO: 9.In a preferred embodiment, the composition of the invention comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8. In Another preferred embodiment, the composition of the invention comprises probes with sequences comprising those shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
En otra realizacion preferida, la composicion de la invencion comprende simultaneamente las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,In another preferred embodiment, the composition of the invention simultaneously comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y SEQ ID NO: 9.SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
Un sexto aspecto de la invencion se refiere al kit o dispositivo, kit o dispositivo de la invencion, que comprende la secuencia SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y/o SEQ ID NO: 9, o cualquiera de sus combinaciones.A sixth aspect of the invention relates to the kit or device, kit or device of the invention, comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and / or SEQ ID NO: 9, or any combination thereof.
En una realizacion preferida, la composicion de la invencion comprende las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 y SEQ ID NO: 8. En otra realizacion preferida, la composicion de la invencion comprende las sondas con secuencias que comprenden las mostradas en SEQ ID NO: 3, SEQ ID NO: 6 y SEQ ID NO: 9.In a preferred embodiment, the composition of the invention comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8. In Another preferred embodiment, the composition of the invention comprises probes with sequences comprising those shown in SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.
En otra realizacion preferida, la composicion de la invencion comprende simultaneamente las secuencias SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,In another preferred embodiment, the composition of the invention simultaneously comprises the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 y SEQ ID NO: 9.SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.
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El kit de la invention puede incluir controles positivos y/o negativos. El kit ademas puede contener, sin ningun tipo de limitation, tampones, soluciones de extraction de protemas, agentes para prevenir la contamination, inhibidores de la degradation de las protemas, etc. Por otro lado el kit puede incluir todos los soportes y recipientes necesarios para su puesta en marcha y optimization. Preferiblemente, el kit comprende ademas las instrucciones para llevar a cabo los metodos de la invencion.The kit of the invention may include positive and / or negative controls. The kit may also contain, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out the methods of the invention.
Un septimo aspecto de la invencion se refiere al uso de la composition de la invencion, o el kit o dispositivo de la invencion, para la detection del virus VRS A, del virus VRS B y del virus hMPV, o cualquiera de sus combinaciones, en una muestra biologica.A seventh aspect of the invention relates to the use of the composition of the invention, or the kit or device of the invention, for the detection of the VRS A virus, the VRS B virus and the hMPV virus, or any combination thereof, in A biological sample.
En una realization preferida de este aspecto de la invencion, la deteccion se realiza de manera simultanea.In a preferred embodiment of this aspect of the invention, the detection is performed simultaneously.
Un octavo aspecto de la invencion se refiere a un medio de almacenamiento legible por un ordenador que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos del metodo de la invencion.An eighth aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer perform the steps of the method of the invention.
En una realizacion preferida de este aspecto de la invencion, el medio de almacenamiento legible comprende al menos una secuencia comprendida en cualquiera de las sondas de la invencion.In a preferred embodiment of this aspect of the invention, the readable storage medium comprises at least one sequence comprised in any of the probes of the invention.
En otra realizacion preferida de este aspecto de la invencion que comprende oligonucleotidos o microarreglos de canal unico disenados a partir de al menos una secuencia conocida o un ARNm comprendida cualquiera de las sondas de la invencion.In another preferred embodiment of this aspect of the invention comprising oligonucleotides or single channel microarrays designed from at least one known sequence or an mRNA comprising any of the probes of the invention.
Un noveno aspecto de la invencion se refiere a una senal transmisible que comprende instrucciones de programa capaces de hacer que un ordenador lleve a cabo los pasos del metodo de la invencion.A ninth aspect of the invention relates to a transmissible signal comprising program instructions capable of having a computer carry out the steps of the method of the invention.
Asi, por ejemplo, las secuencias de oligonucleotidos pueden ser construidas en la superficie un chip mediante el elongamiento secuencial de una cadena en crecimiento con un solo nucleotido utilizando fotolitografia. Asi, los oligonucleotidos son anclados por el extremo 3' mediante un metodo de activacion selectiva de nucleotidos, protegidos por un reactivo fotolabil, mediante la incidencia selectiva de luz a traves de una fotomascara. La fotomascara puede ser fisica o virtual.Thus, for example, oligonucleotide sequences can be constructed on the surface of a chip by sequentially elongating a growing chain with a single nucleotide using photolithography. Thus, the oligonucleotides are anchored at the 3 'end by a method of selective activation of nucleotides, protected by a photolabile reagent, by the selective incidence of light through a photomask. The photomask can be physical or virtual.
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La smtesis in situ sobre un soporte solido (por ejemplo, vidrio), podria hacerse mediante tecnolog^a chorro de tinta (ink-jet), lo que requiere sondas mas largas. Los soportes podrian ser, pero sin limitarse, filtros o membranas de NC o nylon (cargadas), silicio, o Portas de vidrio para microscopios cubiertos con aminosilanos, polilisina, aldehidos o epoxy. La sonda es cada una de las muestras del chip. El target es la muestra a analizar: RNA mensajero, RNA total, un fragmento de PCR, etc.Synthesis in situ on a solid support (for example, glass) could be done using ink-jet technology, which requires longer probes. The supports could be, but not limited to, filters or membranes of NC or nylon (charged), silicon, or glass slides for microscopes covered with aminosilanes, polylysine, aldehydes or epoxy. The probe is each of the chip samples. The target is the sample to be analyzed: messenger RNA, total RNA, a PCR fragment, etc.
Una secuencia de acido nucleico o polinucleotido puede comprender las cinco bases que aparecen biologicamente (adenina, guanina, timina, citosina y uracilo) y/o bases distintas de las cinco que aparecen biologicamente. Estas bases pueden servir para distintos propositos, por ejemplo, para estabilizar o desestabilizar la hibridacion; para estimular o inhibir la degradation de la sonda; o como puntos de union para restos detectables o restos de apantallamiento. Por ejemplo, un polinucleotido de la invention puede contener uno o mas restos de base modificados, no estandar, derivatizados, incluyendo, pero sin limitarse a, N6- metil-adenina, N6-terc-butil-bencil-adenina, imidazol, imidazoles sustituidos, 5-fluorouracilo, 5-bromouracilo, 5-clorouracilo, 5-yodouracilo, hipoxantina, xantina, 4-acetilcitosina, 5- (carboxihidroximetil) uracilo, 5-carboximetilaminometil-2-tiouridina, 5-A nucleic acid or polynucleotide sequence may comprise the five bases that appear biologically (adenine, guanine, thymine, cytosine and uracil) and / or bases other than the five that appear biologically. These bases may serve different purposes, for example, to stabilize or destabilize hybridization; to stimulate or inhibit probe degradation; or as junction points for detectable debris or screening debris. For example, a polynucleotide of the invention may contain one or more modified, non-standard, derivatized base moieties, including, but not limited to, N6-methyl-adenine, N6-tert-butyl-benzyl-adenine, imidazole, substituted imidazoles , 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-
carboximetilaminometiluracilo, dihidrouracilo,beta-D-galactosilqueosina, inosina, N6- isopenteniladenina,1- metilguanina, 1-metilinosina, 2,2-dimetilguanina, 2-metiladenina, 2- metilguanina, 3-metilcitosina, 5-metilcitosina, N6-metiladenina, 7-metilguanina, 5- metilaminometiluracilo, 5-metoxiaminometil-2-tiouracilo, beta-D-manosilqueosina, 5'- metoxicarboximetiluracilo, 5-metoxiuracilo, 2-metiltio-N6-isopenteniladenina, acido uracil-5- oxiacetico, wybutoxosina, pesudouracilo, queosina, 2-tiocitosina, 5-metil-2-tiouracilo, 2- tiouracilo, 2-tiouracilo, 4-tiouracilo, 5-metiluracilo (es decir, timina), ester metflico del acido uracil-5-oxiacetico, 3-(3-amino-3-N-2-carboxipropil)uracilo, (acp3)w, 2,6-diaminopurina, y 5- propinil pirimidina. Otros ejemplos de restos de bases modificados, no estandar, o derivatizados pueden encontrarse en las Patentes de EEUU Nos. 6.001.611; 5.955.589; 5.844.106; 5.789.562; 5.750.343; 5.728.525; y 5.679.785. Ademas, una secuencia de acido nucleico o polinucleotido puede comprender uno o mas restos de azucares modificados incluyendo, pero sin limitarse a, arabinosa, 2-fluoroarabinosa, xilulosa, y una hexosa.carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methylcytosine, N6 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- oxyacetic acid, wybutoracyl, pesy cheosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil (i.e., thymine), uracil-5-oxyacetic acid methyl ester, 3- (3 -amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, and 5- propynyl pyrimidine. Other examples of modified, non-standard, or derivatized base moieties can be found in US Pat. Nos. 6,001,611; 5,955,589; 5,844,106; 5,789,562; 5,750,343; 5,728,525; and 5,679,785. In addition, a nucleic acid or polynucleotide sequence may comprise one or more modified sugar moieties including, but not limited to, arabinose, 2-fluoroarabinous, xylulose, and a hexose.
Un decimo aspecto de la invencion se refiere a un programa de ordenador almacenado en el medio legible por un ordenador de la invencion, que comprende codigo de software adaptado para llevar a cabo los pasos de cualquiera de los metodos de la invencion.A tenth aspect of the invention relates to a computer program stored in the medium readable by a computer of the invention, comprising software code adapted to carry out the steps of any of the methods of the invention.
A lo largo de la description y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras caracteristicas tecnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y caracteristicas de la invention se desprenderan en parte de la descripcion y en parte de la practica de la invencion. Los 5 siguientes ejemplos y dibujos se proporcionan a modo de ilustracion, y no se pretende que sean limitativos de la presente invencion.Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following 5 examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCION DE LAS FIGURASDESCRIPTION OF THE FIGURES
10 Figura 1. Fundamento basico de la tecnica. Sonda TaqMan unida al ADN complementary con un fluoroforo en un extremo (azul) y un inhibidor de la fluorescencia en el otro (blanco). Al generar la hebra complementaria, la Taq polimerasa por su actividad exonucleasa al llegar al punto donde se encuentra unida la sonda, la digiere en nucleotidos y libera el fluoroforo emitiendo luz que es detectado y cuantificado representado por una curva de 15 amplification.10 Figure 1. Basic foundation of the technique. Complementary DNA-bound TaqMan probe with a fluorophore at one end (blue) and a fluorescence inhibitor at the other (white). When generating the complementary strand, Taq polymerase due to its exonuclease activity when it reaches the point where the probe is attached, digests it into nucleotides and releases the fluorophore emitting light that is detected and quantified represented by a 15 amplification curve.
EJEMPLOS DE LA INVENCIONEXAMPLES OF THE INVENTION
Diseno ExperimentalExperimental design
20 Se realizo un diseno de sondas y primers para la detection de los tres virus mediante la aplicacion informatica primers express, depurando y perfeccionando los oligonucleotidos de forma manual. El resultado se recoge en las siguientes tablas:20 A design of probes and primers was performed for the detection of the three viruses by means of the informative application primers express, purifying and perfecting the oligonucleotides manually. The result is collected in the following tables:
- SEQ ID NO: SEQ ID NO:
- VRS A Secuencias Tm Contenido en GC (%) RSV A Sequences Tm GC content (%)
- 1 one
- Forward (FVRSA) 5'-AGCAAATCAATGTCACTAACACC-3' 59,2 39 Forward (FVRSA) 5'-AGCAAATCAATGTCACTAACACC-3 '59.2 39
- 2 2
- Reverse (RVRSA) 5'-TGTAGTACCATTGTGTGTTGTG-3' 58,4 41 Reverse (RVRSA) 5'-TGTAGTACCATTGTGTGTTGTG-3 '58.4 41
- 3 3
- Sonda MGB 5'-TCAGCCGACCCAACCA-3' 63 MGB probe 5'-TCAGCCGACCCAACCA-3 '63
- SEQ ID NO: SEQ ID NO:
- VRS B Secuencias Tm Contenido en GC (%) VRS B Tm Sequences GC content (%)
- 4 4
- Forward (FVRSB) 5'-GACATGTGTTTATTACCATTTTAG-3' 56,6 29 Forward (FVRSB) 5'-GACATGTGTTTATTACCATTTTAG-3 '56.6 29
- 5 5
- Reverse (RVRSB) 5'-GGTGTTGTCGTTTGTAGTGCT-3' 59,5 48 Reverse (RVRSB) 5'-GGTGTTGTCGTTTGTAGTGCT-3 '59.5 48
- 6 6
- Sonda MGB 5'-ACTCACCTAATCAATCAA-3' MGB probe 5'-ACTCACCTAATCAATCAA-3 '
Cada reaccion se llevo a cabo en un volumen total de 25 pl (12,5 pl master mix, 6 pl de primers incluidos los tres pares de cada virus a detectar, 1,5 pl de dilucion de las tres sondas, 1 pl de enzima taq polimerasa y 4 pl de eluido problema con el extracto de acidos 5 nucleicos de la muestra). La cantidad de sonda en cada reaccion fue de 3-4 pmoles y la de los primers fue de 10 pmoles. El termociclador a tiempo real se programo con una primera fase de retrotranscripcion (RT) que consistio en un ciclo de 40°C durante 45 minutos para que todo el ARN pasase a ADN, y una segunda fase de PCR, con 40 ciclos de 95°C a 55°C.Each reaction was carried out in a total volume of 25 pl (12.5 pl master mix, 6 pl of primers including the three pairs of each virus to be detected, 1.5 pl of dilution of the three probes, 1 pl of enzyme taq polymerase and 4 pl of eluted problem with the extract of 5 nucleic acids from the sample). The amount of probe in each reaction was 3-4 pmoles and that of the primers was 10 pmoles. The real-time thermal cycler was programmed with a first back-transcription (RT) phase that consisted of a 40 ° C cycle for 45 minutes for all RNA to pass to DNA, and a second PCR phase, with 40 cycles of 95 ° C at 55 ° C.
- SEQ ID NO: SEQ ID NO:
- Hmpv Secuencias Tm Conten en GC ido (%) Hmpv Sequences Tm Count in GC gone (%)
- 7 7
- Forward (FMET) 5'- ACAATGGCAACTTTGCTTAAAGAA-3' 58,3 33 Forward (FMET) 5'- ACAATGGCAACTTTGCTTAAAGAA-3 '58.3 33
- 8 8
- Reverse (RMET) 5 i '-TGCTGAAGGCCTCTGATTTTG-3' 59,5 48 Reverse (RMET) 5 i '-TGCTGAAGGCCTCTGATTTTG-3' 59.5 48
- 9 9
- Sonda MGB 5'-CATCAGGCAATATTC-3' 69 40 MGB probe 5'-CATCAGGCAATATTC-3 '69 40
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ResultadosResults
Las primeras pruebas con resultados preliminares satisfactorios se detallan a continuation: Se han analizado un total de 121 muestras de aspirados nasales procedentes de pacientes con bronquiolitis. Con las tecnicas actuales de las que dispone el centro, se detectaron 53 15 VRS mediante el test rapido y 1 metapneumovirus mediante la tecnica de amplification genomica NASBA. Con la nueva RT-PCR en ensayo ademas de esos 53 VRS y 1 metapneumovirus, se detectaron 11 VRS y 2 metapneumovirus, en total 64 VRS (34 VRS A y 30 VRS B) y 3 metapneumovirus. Es decir, casi un 20% mas de VRS y un 66% mas de hMPV.The first tests with satisfactory preliminary results are detailed below: A total of 121 samples of nasal aspirates from patients with bronchiolitis have been analyzed. With the current techniques available to the center, 53 15 VRS were detected by the rapid test and 1 metapneumovirus by the NASBA genomic amplification technique. With the new RT-PCR under test in addition to those 53 RSV and 1 metapneumovirus, 11 RSV and 2 metapneumovirus were detected, in total 64 RSV (34 RSV A and 30 RSV B) and 3 metapneumovirus. That is, almost 20% more VRS and 66% more hMPV.
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Los calculos de validez de pruebas diagnosticas para el Test rapido BinaxNow RSV en comparacion a la nueva RT-PCR, realizado mediante el programa Epidad 3.1, son los siguientes:The validity calculations of diagnostic tests for the BinaxNow RSV Rapid Test compared to the new RT-PCR, performed using the Epidad 3.1 program, are as follows:
Tablal. Pruebas diagnosticas. Test rapido VRS BinaxNow (Nivel de confianza: 95%)Tablal. Diagnostic tests. Quick test VRS BinaxNow (Confidence level: 95%)
- Parametros Parameters
- Valores Intervalo confianza (IC 95%) Values Confidence interval (95% CI)
- Sensibilidad (%) Sensitivity (%)
- 82,81 72,79-92,84 82.81 72.79-92.84
- Especificidad (%) Specificity (%)
- 100 99,12-100 100 99.12-100
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- Indice de validez (%) Validity Index (%)
- 90,91 85,37-96,44 90.91 85.37-96.44
- Valor predictivo + (%) Predictive value + (%)
- 100 99,06-100 100 99.06-100
- Valor predictivo - (%) Predictive Value - (%)
- 83,82 74,34-93,31 83.82 74.34-93.31
- Prevalencia (%) Prevalence (%)
- 52,89 43,59-62,2 52.89 43.59-62.2
- Indice de Youden Youden Index
- 0,83 0,74-0,92 0.83 0.74-0.92
- Razon de verosimilitud + Likelihood Reason +
- - - - -
- Razon de verosimilitud - Likelihood Reason -
- 0,17 0,1-0,29 0.17 0.1-0.29
Los datos preliminares muestran una Sensibilidad del 82,8% y una Especificidad del 100% para la prueba rapida. Con la prueba rapida se pierde el diagnostico etiologico de casi un 20% de enfermos respecto a la nueva RT-PCR objeto de invention.Preliminary data shows a sensitivity of 82.8% and a specificity of 100% for the rapid test. With the rapid test, the etiological diagnosis of almost 20% of patients is lost compared to the new RT-PCR object of the invention.
Se ha realizado tambien una prueba adicional de especificidad y discrimination en caso de coinfection, ensayando en un analisis una mezcla de tres eluidos con cada uno de los tres virus diferentes que es capaz de detectar la RT-PCR. Siendo probado la capacidad de detectar sin interferencias una posible coinfeccion triple en una misma muestra y en un solo analisis.An additional specificity and discrimination test has also been carried out in the case of coinfection, testing in an analysis a mixture of three eluted with each of the three different viruses that is capable of detecting RT-PCR. Being able to detect without interference a possible triple coinfection in the same sample and in a single analysis.
Tambien se ha realizado una prueba de especificidad para el VRS-A, VRS-B y hMPV mediante secuenciacion de los amplices obtenidos por RT-PCR. Dichos amplicones mostraron una homologia del 100% con las bases de datos del Genbank.A specificity test for the RSV-A, RSV-B and hMPV has also been performed by sequencing the amplices obtained by RT-PCR. These amplicons showed 100% homology with the Genbank databases.
Se han realizado pruebas preliminares de sensibilidad, consistente en determinar el limite de detection del numero de copias para cada uno de los 3 virus estudiados. La prueba se realizo mediante cuantificacion por nanodrop, siendo el limite de deteccion menor de 10 copias/ml de muestra para cada uno de los virus. Esta sensibilidad es excelente, aunque como se ha mencionado son datos preliminares, ya que la cuantificacion se realizo por nanodrop de amplicones producto de reaction de PCR (pudiendo contener otras impurezas causantes de interferencias). Actualmente estamos a la espera de poder realizar las pruebas de sensibilidad por el metodo de referencia consistente en la donation de los amplicones en cepas de E. coli para posterior purification y cuantificacion, haciendo diluciones seriadas para obtener el limite exacto de deteccion para cada uno de los virus.Preliminary sensitivity tests have been carried out, consisting in determining the limit of detection of the number of copies for each of the 3 viruses studied. The test was performed by nanodrop quantification, the detection limit being less than 10 copies / ml of sample for each virus. This sensitivity is excellent, although as mentioned above are preliminary data, since the quantification was carried out by nanodrop of amplicons resulting from the PCR reaction (may contain other impurities causing interference). We are currently waiting to be able to perform the sensitivity tests by the reference method consisting of the donation of the amplicons in E. coli strains for subsequent purification and quantification, making serial dilutions to obtain the exact limit of detection for each of the virus.
Claims (18)
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| PCT/ES2015/070774 WO2016066878A1 (en) | 2014-10-27 | 2015-10-27 | Method for detecting the human respiratory syncytial virus type a, the human respiratory syncytial virus type b and the human metapneumovirus |
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