WO2016066878A1

WO2016066878A1 - Method for detecting the human respiratory syncytial virus type a, the human respiratory syncytial virus type b and the human metapneumovirus

Info

Publication number: WO2016066878A1
Application number: PCT/ES2015/070774
Authority: WO
Inventors: Alberto TENORIO ABREU
Original assignee: Servicio Andaluz De Salud
Priority date: 2014-10-27
Filing date: 2015-10-27
Publication date: 2016-05-06
Also published as: ES2572031B1; ES2572031R2; ES2572031A2

Abstract

Method for simultaneously detecting the human respiratory syncytial virus type A, the human respiratory syncytial virus type B and the human metapneumovirus in biological samples, primers, probes, compositions, kit and the uses thereof.

Description

METHOD OF DETECTION OF HUMAN RESPIRATORY SYNCTIAL VIRUS TYPE A, HUMAN RESPIRATORY SYNTHETIC VIRUS TYPE B AND

HUMAN METAPNEUMOVIRUS

DESCRIPTION

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

The three most important viruses involved in acute bronchiolitis processes in infants are: RSV A, RSV B and hMPV.

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 through direct contact or through saliva drops. RSV can cause major epidemics of bronchiolitis and pneumonia, which especially affect young children around the world. RSV is estimated that in the US alone is responsible for 100,000 hospitalizations annually (Tang and Crowe, 2007. Manual of Clinical Microbiology. 9 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).

Based on their antigenic differences, two main groups of RSV are identified: A and B, which differ mainly in glycoprotein G. The different sequences of the G protein give rise to 6 subgroups of A and 3 subgroups in B. No they have demonstrated clinical and epidemiological differences between the two groups, although it is likely that there are more virulent strains than others.

The rapid and accurate diagnosis of RSV infection is crucial for patient management and infection control (Woo et al., 1997. J. Clin. Microbio !. 35, 1579-

1581; Adcock et al., 1997. Pediatr. I infected. Dis. J. 16, 842-846; Doherty et al., 1998. J. Hosp. I infected 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 research lines are aimed at optimizing polymerase chain reaction tests that increase the sensitivity and specificity of the test. Human metapneumovirus is an RNA virus of the genus Meta pneumovi rus that belongs to the Paramyxoviridae family. This virus can cause respiratory diseases of a certain severity, especially in children (including acute bronchiolitis symptoms similar to those caused by RSV). The hMPV has its own characteristics very similar to RSV, as is its epidemiology, seasonal distribution and clinical 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. Infecí

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.

The diagnosis of hMPV infection is currently made by cell culture, serology and electron microscopy. But the most current lines are aimed at diagnosis by RT-PCR that would increase sensitivity (Mackay et ai, 2003. J. Clin. Microbio !. 41, 100-105). 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 real-time multiple RT-PCR technique for assist the physician in the diagnosis of bronchiolitis when the presence of RSV and hMPV is detected.

BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the invention relates to a method, hereinafter the first method of the invention, for detecting VRS A virus, VRS B virus, hMPV virus, or any combination thereof, comprising:

i) extracting the RNA from an isolated biological sample of an individual,

ii) detection of nucleotide sequences comprising SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9.

In a preferred embodiment of this aspect of 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) development and comparison

In another preferred embodiment of this aspect of the invention, the biological sample is a nasal aspirate.

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 lavage. A second aspect of the invention relates to the primers, hereafter primers of the invention, of nucleotide sequence:

a) SEQ ID NO: 1,

b) SEQ ID NO: 2,

c) SEQ ID NO: 4,

d) SEQ ID NO: 5,

e) SEQ ID NO: 7, and

f) SEQ ID NO: 8.

A third aspect of the invention relates to probes, hereinafter probes of the invention, of nucleotide sequence:

a) SEQ ID NO: 3,

b) SEQ ID NO: 6, and

c) SEQ ID NO: 9.

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.

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.

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.

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.

A fifth aspect of the invention relates to a composition, hereinafter 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. 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.

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.

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.

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.

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 VRS A virus, VRS B virus and hMPV virus, or any combination thereof, in A biological sample

In a preferred embodiment of this aspect of the invention, the detection is performed simultaneously. An eighth aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer carry out the steps of the method of the invention. 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.

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.

A ninth aspect of the invention relates to a transmissible signal comprising program instructions capable of causing a computer to carry out the steps of the method of the invention.

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.

DETAILED DESCRIPTION OF THE INVENTION

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.

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. Therefore, a first aspect of the invention relates to a method, hereinafter the first method of the invention, for detecting VRS A virus, VRS B virus, hMPV virus, or any combination thereof, comprising:

i) extracting the RNA from the isolated biological sample of an individual,

ii) detection of nucleotide sequences comprising SEQ ID NO: 3,

SEQ ID NO: 6, and SEQ ID NO: 9.

In a preferred embodiment of this aspect of 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) development and comparison

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. In another preferred embodiment of this aspect of the invention, the biological sample is a nasal aspirate.

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.

Steps (ii) and / or (iii) of the methods described above can be fully or partially automated, for example, by means of robotic sensor equipment for the detection of the quantity in step (ii) or the computerized comparison in step (iii).

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 isolated biological sample of an individual from step (i) is a nasal aspirate.

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.

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 biological problem 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 problem biological sample. The comparison described in section (iii) of the method of the present invention can be performed manually or assisted by a computer.

The term "PCR" corresponds to the polymerase chain reaction acronym whereby millions of copies of the desired DNA regions can be obtained. It is characterized by the use of pairs of primers that limit the region from which millions of copies will be made, which is also known as "amplify

DNA "during the 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 efficiency of the reaction is 100%, exponential growth of the DNA fragments subject to amplification occurs.

In the case of the invention, the term "RT-PCR" corresponds to the acronym for 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 into 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 gene detection and to study the genome of RNA viruses such as retroviruses. Another of its uses is related to the quantification of gene 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.

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. The term "oligonucleotide" refers to the sequence of nucleotide bases linked by phosphodiester bonds, usually not greater than 50 nucleotides.

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.)

By "virus" herein, all acellular microscopic infectious agents are understood that can only multiply within the cells of other organisms.

RSV belongs to the genus Pneumovirus, which is encompassed within the Domain Acytota, Group V: Single-stranded negative RNA virus, Mononegavirales Order, Paramyxoviridae Family, Pneumovirinae Subfamily.

The hMPV belongs to the genus Meta pneumovi rus, which is encompassed within the Acytota Domain, Group V: Negative single stranded RNA virus, Mononegavirales Order, Paramyxoviridae Family, Pneumovirinae Subfamily.

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.

Paramyxoviruses measure between 156-300nm, have lipid covering, RNA genome of a negative chain, double lipid membrane derived from the host cell, surface glycoproteins that form HN projections with neuroaminidase and hemagglutination action, and an F that participates in penetration viral and stimulates membrane fusion. In addition, the M membrane protein that is part of the lipid envelope and the NP ribonucleoprotein which is complement fixation antigen. These viruses address the airways and are installed 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 viral conditions (except exceptionally), So the infection circumscribes the respiratory system. When it invades lower regions it produces bronchitis, bronchiolitis and pneumonia. (Microbiology and Parasitology Human. Etiological basis of infectious and parasitic diseases. 3rd Edition. Médica Panamericana Editorial)

A second aspect of the invention relates to the primers, hereafter primers of the invention, of nucleotide sequence:

a) SEQ ID NO: 1,

b) SEQ ID NO: 2,

c) SEQ ID NO: 4,

d) SEQ ID NO: 5,

e) SEQ ID NO: 7, and f) SEQ ID NO: 8.

In the present invention, "primer" or "first" is understood as the nucleotide sequence from which DNA polymerase initiates 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 DNA polymerase enzyme 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).

In the context of the present invention, "RNA / DNA extract" is understood when, after subjecting the biological sample to a process of extraction, separation, purification or cloning 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. The primers of the invention comprise the following sequences:

SEQ ID NO: 1: 5 ^' -AGCAAATCAATGTCACTAACACC-3 ^'

SEQ ID NO: 2: 5 ^' -TGTAGTACCATTGTGTGTTGTG-3 ^'

SEQ ID NO: 4: 5 ^' -G ACATGTGTTTATTACCATTTTAG-3 ^'

SEQ ID NO: 5: 5 ^' -GGTGTTGTCGTTTGTAGTGCT-3 ^'

SEQ ID NO: 7: 5 ^' -ACAATGGCAACTTTGCTTAAAGAA-3 ^'

SEQ ID NO: 8: 5 ^' - TGCTGAAGGCCTCTGATTTTG-3 ^'

Thus, in the 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.

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 target sequence to be obtained of DNA. Each of the primers hybridizes with the target sequence, so that the bounded nucleotide sequence is amplified by each pair of primers. The extent of the primers during the PCR cycles determines the 2 ^N exponential multiplication of the nucleotide sequence bounded by the primers, N being the number of cycles of the PCR reaction.

a) SEQ ID NO: 3,

b) SEQ ID NO: 6, and

c) SEQ ID NO: 9.

In the context of the present invention, the term "probe" is defined as an oligonucleotide that is complementary or substantially complementary to the amplicon sequence, within the limits of the primers. In the present preferred embodiment, the capture probe 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 restriction endonuclease digestion). Therefore, there is, except for the intended function, no fundamental difference between a "primer", an "oligonucleotide" or a "probe" according to the invention.

Preferably, the probes of the invention comprise the following sequences: SEQ ID NO: 3: 5 ^' -TCAGCCGACCCAACCA-3 ^'

SEQ ID NO: 6: 5 ^' -ACTCACCTAATCAATCAA-3 ^'

SEQ ID NO: 9: 5 ^' -CATCAGGCAATATTC-3 ^'

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.

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 of the hMPV virus. In a preferred embodiment of this aspect of the invention, the detection is simultaneous.

A fifth aspect of the invention relates to a composition, hereinafter 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.

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.

In a preferred embodiment of this aspect of the invention, the detection is performed simultaneously.

An eighth aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of having a computer carry out the steps of the method of the invention. 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.

Thus, for example, oligonucleotide sequences can be constructed on the surface of a chip by sequential elongation of 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.

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.

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 can 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 non-standard modified base moieties, derivatized, including, but not limited to, N ⁶ -methyl-adenine, N ⁶ -terc-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- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1- methylguanine, 2,2-methylguanine, 1-methylguanine, 1-methylguanine -dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5'-methoxycarboxymethyl 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pesudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 2-thiouracil, 4-thiouracil - methyluracil (ie thymine), uracil-5-oxyacetic acid methyl ester, 3- (3-amino-3-N-2-carbox ipropil) 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. 6,001.61 1; 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.

Throughout the description and the 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 features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES 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 an amplification curve.

EXAMPLES OF THE INVENTION Experimental Design

A design of probes and primers was performed for the detection of the three viruses through the computer application primers express, purifying and perfecting the oligonucleotides manually. The result is collected in the following tables:

I KNOW THAT

Content ID VRS B Tm sequences

in GC (%)

NO:

Forward

4 5 ^' -G ACATGTGTTTATTACCATTTTAG-3 ^' 56.6 29 (FVRSB)

Reverse

5 5 ^' -GGTGTTGTCG III GTAGTGCT-3 ^' 59.5 48 (RVRSB)

Probe

6 5 ^' -ACTCACCTAATCAATCAA-3 ^'

MGB Each reaction was carried out in a total volume of 25 μΙ (12.5 μΙ master mix, 6 μΙ of primers including the three pairs of each virus to be detected, 1.5 μΙ dilution of the three probes, 1 μΙ enzyme taq polymerase and 4 μΙ of eluted problem with the nucleic acid extract of 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 retrotranscription (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.

Results

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 RSV 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, 1 1 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.

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. Diagnostic tests. VRS BinaxNow rapid test (Confidence level: 95%)

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.

An additional test of specificity and discrimination in case of coinfection has also been performed, testing in a test 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.

A specificity test for 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.

Preliminary sensitivity tests have been performed, consisting of 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 performed by nanodrop of amplicons PCR reaction product (may contain other impurities causing interference). We are currently waiting to be able to perform the sensitivity tests by the reference method consisting of cloning the amplicons in E. coli strains for further purification and quantification, making serial dilutions to obtain the exact limit of detection for each of the virus.

Claims

1. - Method of detection of human respiratory syncytial virus A (RSV A), human respiratory syncytial virus B (RSV B) and human metapneumovirus (hMPV) or any combination thereof, comprising:

i. extract the RNA from an isolated biological sample from an individual,

I. detection of nucleotide sequences comprising SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 9.

2. - The method according to the preceding claim, wherein the detection is performed simultaneously.

3. - The method according to claims 1-2, wherein the amplification of the nucleotide sequences collected in SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9 is performed by RT-PCR.

4. - The method according to claims 1-3, wherein 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.

5. - The method according to claims 1-4, wherein the DNA extraction includes a purification step.

6. - The method according to claims 1-5, wherein the PCR reaction is a multiplex reaction.

7. - The method according to claims 1-6, wherein the method further comprises a step Ni:

Neither. revealed and comparison

8. - The method according to claims 1-7, characterized in that the sample (i) is nasal aspirated.

9. - The method according to claims 1-7, characterized in that the sample (i) is a bronchoalveolar lavage, a bronchoaspirate, a telescopic brushing or a nasopharyngeal lavage.

10. - Nucleotide sequence primers:

a) SEQ ID NO: 1

b) SEQ ID NO: 2

c) SEQ ID NO: 4

d) SEQ ID NO: 5

e) SEQ ID NO: 7, and

f) SEQ ID NO: 8.

1 1. - Nucleotide sequence probes

a) SEQ ID NO: 3

b) SEQ ID NO: 6, and

c) SEQ ID NO: 9.

12. - Use of the primers according to claim 10, and / or the probes according to claim 1, for the detection of the VRS A virus, the VRS B virus and the hMPV virus.

13. - The use of the primers and probes according to the preceding claim, wherein the detection of the viruses is simultaneous.

14. - The use of the primers and probes according to claim 12, wherein the pair of primers SEQ ID NO: 1 and SEQ ID NO: 2, and the probe SEQ ID NO: 3, are used for the detection of the virus VRS A .

15. - The use of the primers and probes according to claim 12, wherein the pair of primers SEQ ID NO: 4 and SEQ ID NO: 5, and the probe SEQ ID NO: 6, are used for the detection of the VRS B virus .

16. - The use of the primers and probes according to claim 12, wherein the pair of primers SEQ ID NO: 7 and SEQ ID NO: 8, and the probe SEQ ID NO: 9, are used for the detection of hMPV virus.

17.- Composition that 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.

18.- Kit or device that 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 .

19. Use of the composition according to claim 17, or of the kit or device according to claim 18, for the simultaneous detection of the VRS A virus, the VRS B virus and the hMPV virus.