WO2010097490A1 - GENOME SEQUENCES OF PRIMERS AND A PROBE FOR THE QUANTIFICATION OF PORCINE ADENOVIRUSES (PAdV) - Google Patents

GENOME SEQUENCES OF PRIMERS AND A PROBE FOR THE QUANTIFICATION OF PORCINE ADENOVIRUSES (PAdV) Download PDF

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WO2010097490A1
WO2010097490A1 PCT/ES2010/000077 ES2010000077W WO2010097490A1 WO 2010097490 A1 WO2010097490 A1 WO 2010097490A1 ES 2010000077 W ES2010000077 W ES 2010000077W WO 2010097490 A1 WO2010097490 A1 WO 2010097490A1
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seq
sample
padv
chain reaction
primers
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French (fr)
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Rosa GIRONÈS LLOP
Ayalkibet Hundesa Gonfa
Carlos Maluquer De Motes Porta
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Universidad De Barcelona
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the invention describes a method for the detection of PAdV in environmental samples, water and food.
  • bacterial indicators have been used including Escherichia coli and fecal coliform bacteria to monitor water quality and safety.
  • these monitoring are not often correlated with viral pathogens or protozoan parasites and therefore leave the problem of real-time detection unresolved.
  • Faecal contamination can originate from point or non-point sources.
  • point sources of faecal contamination include discrete sources such as discharges from large animal husbandry operations, treated and untreated effluents from sewage, storm water, and combined sewage.
  • Non-point sources are diffuse and include agricultural sources and animal waste applied to agricultural fields.
  • the identification of sources of microbiological contamination plays a very important role in obtaining effective handling and remediation strategies, and is known as traceability of microbial source sources (MST).
  • MST includes a group of methodologies that aim to identify, and in some cases quantify, the dominant sources of fecal contamination in the environment and, more specifically, in water resources (cf. Stoeckel et al. "Performance, Design, and Analysis in Microbial Source Tracking Studies "ADPI.
  • hepatitis E virus is a human viral pathogen that causes acute hepatitis and has been described as highly prevalent in young pigs and frequently present in faecal samples, as well as in sewage and sludge produced in slaughterhouses that process pigs (cf. Clement- Casares et al., "Hepatitis E virus epidemiology in industrialized countries” Emerqinq Infectious Diseases 2003, vol. 9, pp. 448-54).
  • the remaining problem is the positive identification of fecal contamination of porcine origin, which allows to accurately locate the source of contamination for remediation processes and evaluate the potential public health impact of fecal contamination in water.
  • PCR polymerase chain reaction
  • qPCR real-time quantitative PCR
  • PCR and nested PCR (nPCR) protocols are typically used because of their high sensitivity, but are not quantitative and are subject to a high probability of cross-contamination by DNA, which is usually solved by sequencing the PCR products.
  • Real-time qPCR has emerged as a valuable technique because it successfully reduces the likelihood of contamination in the laboratory and time-consuming manipulations, and allows rapid and sensitive quantification of small amounts of target DNA in biological and environmental samples.
  • the Adenoviridae family is the only known group of enteric viruses that have double-stranded DNA genomes. This represents an advantage due to the conservation and stability of the sequences.
  • the family Polvomaviridae also includes DNA viruses that produce persistent infections and that are frequently excreted in the urine, and for these reasons both viruses have been proposed as markers of fecal contamination.
  • Human adenoviruses (HAdVs) are highly prevalent in wastewater as well as in river water and bivalve molluscs with fecal contamination (cf. Fong et al., "Enteric viruses of humans and animate in aquatic environments: health risks, detection, and potential water quality assessment tools "Microbiol. Mol. Biol. Rev. 2005, vol. 69, pp.
  • porcine adenoviruses belong to the genus Mastadenovirus. They are divided into six serotypes and have been described as abundant and highly prevalent in feces, slaughterhouse wastewater and sludge (cf.
  • a method for the quantification of contamination by PAdV in a sample suspected of containing it develops a qPCR assay with a set of specific primers / probe capable of detecting and quantifying PAdV in excreta, porcine sludge and different types of water.
  • the present invention relates to a method for the detection and / or quantification of porcine adenoviruses (PAdV) in a sample, which it comprises the extraction of the PAdV nucleic acid from the sample; The amplification by chain reaction of the nucleic acid polymerase extracted in the presence of at least one primer selected from SEQ ID NO: 1 or a functional variant thereof, SEQ ID NO: 2 or a functional variant thereof, and SEQ ID NO : 3 or a functional variant thereof, or its complementary chains; and the detection and / or quantification of PAdV evaluating the result of the polymerase chain reaction.
  • PAdV porcine adenoviruses
  • Real-time qPCR is a molecular technique that allows the quantification of a small number of genomic copies. Therefore, in a particular embodiment of the invention, this polymerase chain reaction is a quantitative polymerase chain reaction (qPCR).
  • qPCR quantitative polymerase chain reaction
  • the qPCR is performed in the presence of a specific probe for the PAdV genome sequence, and in a more particular embodiment, the qPCR is performed in the presence of a probe comprising the sequence identified as SEQ ID NO: 3 or a functional variant thereof, or its complementary chain.
  • a qPCR probe consists of a nucleotide sequence that contains a fluorophore group at one end and a blocking agent at the other end. The arrival of the polymerase enzyme will release the probe and separate the blocking agent, so that each amplification cycle results in the emission of fluorescence.
  • Another embodiment of the invention is a qPCR made in the presence of the SEQ ID NO: 3 probe or its complementary chain, carrying a fluorophore group and a blocking agent.
  • the parameters necessary for the evaluation of the efficiency of the test were analyzed. Among them, the optimal concentrations of primers and probe were adjusted to obtain acceptable values for the correlation coefficient, Ct, and the slope of the standard regression line.
  • the real-time qPCR requires the automatic comparison of the emitted fluorescence with standard values at all times. Therefore, another embodiment of the invention is the evaluation of the amount of virus including a comparison of the quantification of the result of the polymerase chain reaction with at least one control sample.
  • control sample comprises a positive control taken from at least one curve standard generated by the involvement of the polymerase chain reaction in the presence of the primers corresponding to SEQ ID NO: 1 and SEQ ID NO: 2, or their complementary chains, in a 612 bp sequence of the PAdV3 hexon gene at different concentrations.
  • a polymerase chain reaction for the detection of PAdV in samples in the presence of the described primers can be comprised in a chain reaction of the nested polymerase. Also, it could be possible for the detection of PAdV a semi-nested PCR using a set of primers including SEQ ID NO: 1 or SEQ ID NO: 2 as the first primer, and SEQ ID NO: 3 as the other primer. Accordingly, another embodiment of the invention comprises the polymerase chain reaction being a polymerase chain reaction in the presence of one of the primers identified as SEQ ID NO: 1 or SEQ ID NO: 2 or a variant functional thereof, and of the sequence SEQ ID NO: 3 or a functional variant thereof, or its complementary chains.
  • oligonucleotides may be subject to modifications without deviating from the invention provided herein. It is intended that fragments and / or functional variants of the oligonucleotides provided herein be included in the present invention. It is contemplated that fragments and / or functional variants of the primers and / or the probe described herein can be used in accordance with the methodologies proposed herein to achieve the objectives of the present invention. For example, a fragment or an extension or a functional variant of a primer or a probe that retains the same function that could be used in the quantification of PAdV is included in the present invention.
  • a fragment may include a sequence comprising any number of nucleotides less than those found in a nucleic acid sequence of interest provided herein.
  • An extension may include a sequence comprising any number of nucleotides greater than those found in a nucleic acid sequence of interest provided herein.
  • the specificity of an oligonucleotide refers to the ability of the product, that is, a primer or a probe, to hybridize with the target region or sequence under suitable conditions to carry out a polymerase chain reaction.
  • the functional variant of any of the primers or probe provided herein refers to the ability of the oligonucleotide to hybridize with the target region or sequence under suitable conditions to carry out a polymerase chain reaction. It is fully contemplated that the tools and methodologies for the detection of nucleic acids can be adapted for use in accordance with all the biological samples described.
  • the method of the invention is capable of detecting viruses in a wide spectrum of samples.
  • the study of non-pathogenic PAdV as a method for traceability of sources of human and animal fecal contamination in environmental samples should provide data for the improvement of the manipulation and control of water quality.
  • An embodiment of the invention applies the method in an environmental sample.
  • Another embodiment of the invention applies the method in water samples, and in a more particular embodiment, the water sample is a waste water sample. They are considered included as samples of wastewater, urban and farm wastewater.
  • the sample suspected of containing PAdV is a sample of slaughterhouse waste, which includes samples of water and sludge.
  • the method is also applied in faecal samples.
  • the invention is also widely applicable in the industrial sector.
  • Another embodiment applies the method of the invention to food samples.
  • the invention is also applicable for the detection of the presence of viruses in commercial articles, such as biosolids derived from the composting industry.
  • Another particular embodiment of the invention is the application of the method in fertilizer samples.
  • Another aspect of the present invention relates to oligonucleotides identified by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, or their complementary chains.
  • Another aspect refers to the use of oligonucleotides identified as SEQ ID NO: 1 and SEQ ID NO: 2 or their complementary chains as specific primers for the PAdV genome, and the oligonucleotide identified as SEQ ID NO: 3 or its complementary chain as specific probe for the PAdV genome sequence.
  • a primer comprising SEQ ID NO: 1 or a functional variant thereof and a reverse primer one comprising SEQ ID NO: 2 or a functional variant thereof is preferred as a direct primer.
  • Another aspect of the invention relates to the use of the primers and the probe described above, or a functional variant thereof, for the detection of PAdV using the polymerase chain reaction. In a preferred embodiment, this polymerase chain reaction is the qPCR.
  • kits for carrying out the method of the invention comprising reagents suitable for the detection and / or quantification of PAdV in a sample
  • a particular embodiment of the invention is that this kit includes at least one of the primers identified as SEQ ID NO: 1 and SEQ ID NO: 2 and / or the probe identified as SEQ ID NO: 3, or its complementary chains.
  • FIG. 1 shows the amplification graphs obtained with serial dilutions 1: 10 of the cloned viral DNA of PAdV.
  • the amplification graphs show the log of dRn (reported fluorescence signal) in relation to the number of cycles.
  • FIG. 2 shows the standard curve obtained with serial dilutions 1: 10 of the cloned viral DNA of PAdV.
  • the log of the initial amount of viral DNA is represented with respect to the threshold cycle (Ct values), calculated as the fractional cycle number in which a significant increase in Rn exceeds a certain threshold (horizontal line).
  • FIG. 3 shows the evaluation of the inhibition of the qPCR assay for PAdV in environmental samples according to the method of extraction of AN used.
  • the figure shows the PAdV levels previously added to the AN extractions obtained with the NucliSens® nucleic acid extraction kit from Biomérieux (Bx) and the method previously described by Boom et al. (B), as detected in dilutions 1: 10 (in gray), 1: 100 (in white) and undiluted (in black).
  • Three different types of environmental samples were studied: slaughterhouse wastewater (SW), urban wastewater (US), and pig sludge (S).
  • FIG. 4 shows the results obtained in the qPCR assays for HAdV and PAdV.
  • the qPCR procedure was applied to the quantification of porcine adenoviruses in: 38 stool samples grouped from 18 pig farms in various areas in the Basque Country (northern Spain, Cantabrian Coast) and Catalonia (northeastern Spain, Mediterranean coast) ; 8 samples of wastewater from a slaughterhouse that processes pigs in Catalonia; 6 river water samples obtained below an important agricultural area; and 9 samples of urban wastewater from Catalonia, which were also analyzed for the presence of HAdV.
  • the pellet was eluted with 4 ml of 0.25 N glycine buffer (pH 9.5), and the suspended solids were separated by centrifugation at 12,000 xg for 15 min.
  • the viruses were finally concentrated by ultracentrifugation (110,000 xg for 1 h at 4 0 C), resuspended in 100 ⁇ l of PBS, and stored at -80 0 C.
  • the protocol used for the processing of the river samples was a combination of the EPA method (EPA 600 / 4-84 / 013 (N 14)), with minor modifications, and the method based on ultracentrifugation and elution in 0.25 N glycine buffer ( pH 9.5) (cf. Albinana-Gimenez et al., "Distribution of polyomaviruses, adenoviruses and hepatitis E virus in the environmental and na Drinking-Water treatment plant" Environ. Sci. Techno. 2006, vol. 40, pp. 7416 -22).
  • One hundred liters of river water was filtered through Zeta Plus MK electropositive filters at 1 l / min using a peristaltic pump
  • Millipore The viruses retained in 900 ml of 0.25 N glycine buffer (pH 9.5) and 1% meat extract (Becton, Dickinson & Co., Sparks MD, USA) were eluted. by reverse flow using a Millipore peristaltic pump at 0.4 l / min for 45 min. For flocculation, 3% meat extract was added, the pH was adjusted to 3.5 using 5M HCI and the resulting suspension was magnetically stirred for 30 min, finally centrifuged at 12,800 xg for 25 min at 4 0 C. After this step, The sample was treated using the protocol previously applied for virus recovery from urban wastewater.
  • the qPCR procedure is based on a TaqMan assay and uses two primers and a fluorogenic probe that recognizes a 68 bp fragment in the PAdV genome hexon gene.
  • the primers and the probe were designed specifically for the amplification of PAdV.
  • GenBank DNA PAdV sequences were aligned using the ClustalW program (European Bioinformatics Institute, UK) and a conserved 68 bp fragment was selected using Primer Express software (Applied Biosystems). The homology of all oligonucleotides was finally verified using Blast Search.
  • the probe was labeled with FAM (6- carboxyfluorescein) as a fluorophore group at the 5 'end and BHQ-1 (Black-HoIe Quencher 1) as a blocking agent at the 3' end.
  • the sequence of the direct primer corresponds to SEQ ID NO: 1
  • the sequence of the reverse primer corresponds to SEQ ID NO: 2
  • Its location in the genome using as reference Ia strain PAdV serotype 3 is as follows: Direct primer Q-PAdV-F: between the base pair (bp) 20701 and 20718.
  • Inverse primer Q-PAdV-R between bp 20768 and 20751.
  • Probe Q-PAdV-P between pb 20722 and 20737.
  • Standard curves were generated by transformation of E. coli JM109 cells (Promega) with plasmid pGEM-T Easy (Promega) containing a 612 bp sequence of the PAdV-3 hexon. The transformation was carried out following the manufacturer's instructions.
  • Colonies carrying the desired plasmid were analyzed by PCR to contain the target DNA, which was obtained with the QIAGEN Plasmid Midi kit (QIAGEN, Inc.) following the manufacturer's instructions Once obtained and analyzed by Genequant Pro (Amersham Biosciences), according to the molecular weight of the plasmid, 10 ⁇ g of DNA was linearized with EcoRI, purified with the QUIAquick PCR purification kit (QIAGEN, Inc.) and subsequently quantified again, before obtaining serial dilutions of 10 '1 to 10 9 molecules of viral DNA per 10 ⁇ l in TE buffer. The standard dilutions aliquoted and stored at -80 0 C until use.
  • Nucleic acids (AN) from viral concentrates were extracted using a method based on the method Boom et al. (1990) using guanidino isothiocyanate to denature viral capsids and silica particles to bind nucleic acids until final elution in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) and subsequent storage at -80 0 C. Also used, according to the manufacturer's instructions, the NucliSens ® (Biomérieux) nucleic acid extraction kit, based on the same principles and containing silica-coated magnetic balls.
  • PCR mixture was prepared and dispensed in a dedicated work area Ia preparation of PCR reagents; The samples were loaded in a pre-PCR laboratory and, finally, the plate was transferred to a separate laboratory for the addition of the qPCR standards.
  • the amplifications were performed in a 25- ⁇ l reaction mixture containing 10 ⁇ l of DNA and 15 ⁇ l of TaqMan ® Universal PCR Master Mix, 0.9 ⁇ M of each primer (Q-PAdV-F and Q-PAdV-R) and 0.225 ⁇ M of fluorogenic probe (Q-PAdV-P).
  • the TaqMan ® Universal PCR Master Mix is supplied in a 2x concentration and contains AmpliTaq Gold ® DNA polymerase, dNTPs with dUTP, passive reference, optimized buffer components and AmpErase ® uracil-N-Glycosylase. After the activation of the uracil-N-GIclosilase (2 min, 50 0 C) and the activation of the AmpliTaq GoId for 10 min at 95 0 C, 45 cycles were performed (15 s at 95 0 C, 20 s at 55 0 C and 20 s at 60 0 C) in an MX3000P (Stratagene) detection system.
  • Nested PCR assays were performed for the amplification of PAdV. 10 ⁇ L of extracted nucleic acids and their 1: 10 dilution were analyzed in a 40 ⁇ L reaction mixture containing IxPCR Buffer, MgCI 2 at 1.5 mM, 0.025 mM of each dNTP, 25 pmol of primers and 2 units of Taq DNA polymerase (Bloron GmbH, Germany). The reaction conditions were: 94 0 C for 4 min, 30 cycles of 92 0 C for 60 s, 60 s at the corresponding hybridization temperature and extension at 72 0 C for 75 s. The amplification was completed with 7 min of extension at 72 0 C.
  • Ampllcones obtained after nPCR were purified by QIAquick PCR purification kit (QIAGEN, Inc.). The purified DNA was sequenced directly with the ABI PRISM TM Dye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 with Ampli Taq DNA polymerase ® FS (Applied Biosystems) following the manufacturer 's instructions. The conditions for the 25 sequencing cycles were: denaturation at 96 ° C for 10 s, hybridization for 5 s at 50 ° C and extension at 60 ° C for 4 min. The nested primers were used for sequencing at a concentration of 0.05 ⁇ M. The results were analyzed using the ABI PRISM 377 automatic sequencer (Perkin-Elmer, Applied Biosystems).
  • sequences obtained are compared to those of GenBank and EMBL (European Molecular Biology Library) using the NCBI basic program BLAST (The National Center for Biotechnoiogy Information, http://www.ncbi.nlm.nih.gov/BLAST/).
  • the sequence alignments were carried out through the EBI ClustalW program (European Bioinformatics Institute of the EMBL, http://www.ebi.ac.uk/clustalw/).
  • the quantification of HAdV in the samples was carried out by means of a protocol based on TaqMan ® , using the direct primer SEQ ID NO: 4, the reverse primer SEQ ID NO: 5 and the probe SEQ ID NO: 6, where "w “may be” a “or T,” k “may be” g “or T 1 " s “may be” g “or” c “, V may be” a “or” g “, and” y “may be “c” or "t”.
  • This assay detects the 51 described serotypes of human adenoviruses.
  • Quantifications were performed in a 25- ⁇ l reaction mixture containing 10 ⁇ l of DNA and 15 ⁇ l of TaqMan ® Universal PCR Master Mix (Applied Biosystems) containing 0.9 ⁇ M of each primer and 0.225 ⁇ M of fluorogenic probe.
  • a plasmid pBR322 containing the HAdV41 hexon sequence was used to construct the standard of 10 1 to 10 7 copies of DNA per 10 ⁇ l added to the PCR reaction. Each dilution of the DNA standard was analyzed in triplicate.
  • a set of primers / probe was designed that amplified a small amplicon.
  • concentration of the primers and the probe was optimized by analyzing concentrations of primers ranging between 0.4 and 0.9 ⁇ M and probe, between 0.225 and 0.9 ⁇ M, for each reaction. Hybridization temperatures were also optimized.
  • the assay identified between 1 and 10 viral DNA molecules (PAdV standard) as the limit of detection per reaction tube.
  • PAdV standard 1 and 10 viral DNA molecules
  • FIG. 1 A representative test of those carried out is shown in FIG. 1. In all trials, the average R-square value was 0.996 ⁇ 0.003, the slope values ranged from -3.461 to -3.492 (average value -3.481), and the estimated efficiency of them was 94.5%.
  • oligonucleotides The specificity of the oligonucleotides was verified using the Blast Search tool.
  • the set of primers / probe was tested with serum samples or cell culture of various species of HAdV. Serotypes 2, 5, 7, 35, 40 and 42 were analyzed at concentrations ranging from 10 2 to 10 8 viral particles / ml and no fluorogenic signal was detected.
  • bovine fecal samples were analyzed in which BAdV had been detected and which contained serotypes A and I and strains with 82% homology with BAdV 2 in concentrations ranging between 10 1 and 10 4 gc / g of stool No false positives caused by cross reactions between DNAs of viruses that infected the various hosts studied were detected. Test sensitivity
  • the assay was able to detect 1 to 10 genomic copies per reaction tube. According to the initial volumes of water and the amounts of fecal matter analyzed, the quantifications corresponded to 4.2 ml of wastewater, 1 I of water from the Ter River, and 0.1 g of feces. The sensitivity was stable even when large amounts of heterologous viral DNA were added but related to the reaction tubes: the addition of 10 8 copies of type 40 HAdV did not affect the detection of the PAdV DNA.
  • the Nucleospin ® NA extraction kit and the homemade nucleic acid extraction method based on silica and guanidino isothiocyanate particles were compared by analysis of porcine sludge and urban and slaughterhouse wastewater in the that known amounts of viral DNA (standard PAdV) had been added.
  • standard PAdV viral DNA
  • the Nucleospin ® kit detected greater amounts of DNA in the undiluted sample of pig sludge and slaughterhouse wastewater and in the 1: 10 dilution of urban wastewater.
  • the quantitative data of PAdV obtained from pig feces and environmental samples are shown in Table 2. Seventeen of the twenty-one pooled samples of pig feces collected in the Basque Country were positive for qPCR. No significant differences were observed between fattening and breeding animals or depending on the age of the animals. Be detected PAdV in swine faecal samples in high concentrations and high prevalence: 76.4% positive pooled samples, average concentration of 5.58x10 5 gc / g. The observed results were confirmed by applying nPCR. Discrepancies were observed between the 2 methods in only 2 samples that showed low concentrations of PAdV using qPCR and were negative for nPCR. However, these results are expected when samples with low virus concentrations are analyzed in repeated experiments. PAdV amplicons obtained by nPCR from a positive sample from each of the 9 farms studied were sequenced. The nucleotide sequences observed for all samples showed a similarity to PAdV-3 that ranged from 93 to 98%.
  • the qPCR assay was also used to quantify PAdV in seventeen pooled samples of pig feces collected in 9 farms in Catalonia that had been previously analyzed by nPCR as well as sequenced showing high similarity with PAdV-3 strains (cf. Maluquer de Motes et al., supra APPI. Environ. Microbiol. 2004). All samples were also positive for qPCR, showing an average concentration of PAdV of 7.25x10 5 gc / g, a value equivalent to the results observed in faecal samples collected in the Basque Country (5.58x10 5 gc / g).
  • the wastewater treatment plant selected for the study treats urban wastewater from a large population and no agricultural or agricultural activities have been described in the area. Consistent with this, no PAdV was detected in the samples analyzed while HAdV was identified in all samples with average values of 2.7x10 3 gc / ml.

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Abstract

The invention relates to a sensitive test for the detection of porcine adenoviruses in environmental samples, water and food. Specifically, the invention relates to a quantitative tool intended for the analysis of porcine adenoviruses as indicators of the presence of porcine contamination. The invention further relates to a kit comprising two sequences of primers and a probe sequence capable of detecting and quantifying the aforementioned virus by means of PCR. The test has been proven to have great specificity since it has not displayed positive results in samples containing human or bovine adenoviruses. In addition, the test detected porcine fecal contamination in samples that were highly diluted and had been collected at a considerable distance from the initial source.

Description

Secuencias qenómicas de cebadores y sonda para la cuantificación de adenovirus porcinos (PAdV) Qenomic sequences of primers and probe for the quantification of porcine adenoviruses (PAdV)
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La invención describe un método para Ia detección de PAdV en muestras medioambientales, agua y alimentos.The invention describes a method for the detection of PAdV in environmental samples, water and food.
ESTADO DE LA TÉCNICA ANTERIORSTATE OF THE PREVIOUS TECHNIQUE
La contaminación microbiológica del medio ambiente supone un riesgo significativo para Ia salud humana a través de exposiciones recreacionales o consumo de agua o alimentos contaminados. De acuerdo con los requerimientos de Ia Directiva Marco para el Agua y Ia Ley para Aguas Limpias de los Estados Unidos de América, ha existido un cambio en las normativas sobre Ia calidad de los efluentes desde el enfoque tradicional sobre una fuente puntual hacia un enfoque de captura más amplio (cfr. Stapletone et al., "Microbial Source tracking: a forensic technique for microbial source identification" J. Environ. Monit. 2007, vol. 9, pp. 427-39). Dado este nuevo enfoque, existe Ia necesidad de nueva información en las dinámicas microbianas de las áreas de captación de aguas para un control efectivo de Ia calidad de las aguas en el punto de uso. Históricamente, se han usado indicadores bacterianos incluyendo Escherichia coli y bacterias coliformes fecales para monitorear Ia calidad y seguridad de las aguas. Sin embargo, estas monitorizaciones no están a menudo correlacionadas con patógenos virales o parásitos protozoos y por Io tanto dejan sin resolver el problema de Ia detección en tiempo real.Microbiological contamination of the environment poses a significant risk to human health through recreational exposures or consumption of contaminated food or water. In accordance with the requirements of the Water Framework Directive and the Clean Water Law of the United States of America, there has been a change in the regulations on the quality of effluents from the traditional approach to a point source towards a focus on broader capture (cf. Stapletone et al., "Microbial Source tracking: a forensic technique for microbial source identification" J. Environ. Monit. 2007, vol. 9, pp. 427-39). Given this new approach, there is a need for new information on the microbial dynamics of the water catchment areas for effective control of the quality of the water at the point of use. Historically, bacterial indicators have been used including Escherichia coli and fecal coliform bacteria to monitor water quality and safety. However, these monitoring are not often correlated with viral pathogens or protozoan parasites and therefore leave the problem of real-time detection unresolved.
La contaminación fecal se puede originar desde fuentes puntuales o no puntuales. Generalmente, las fuentes puntuales de contaminación fecal incluyen fuentes discretas como vertidos procedentes de grandes operaciones de cría de animales, efluentes tratados y no tratados de aguas residuales, agua de tormentas, y alcantarilado combinado. Las fuentes no puntuales son difusas e incluyen fuentes agrícolas y residuos animales aplicados a los campos agrícolas. La identificación de las fuentes de contaminación microbiológica juega un papel muy importante en Ia obtención de estrategias de manipulación y remediación efectivas, y se conoce como trazabilidad de fuentes de contaminación microbianas ("microbial source tracking", MST). MST incluye un grupo de metodologías que tienen el fin de identificar, y en algunos casos cuantificar, las fuentes dominantes de contaminación fecal en el ambiente y, más específicamente, en recursos hídricos (cfr. Stoeckel et al. "Performance, Design, and Analysis in Microbial Source Tracking Studies" ADPI. Environ. Microbiol. 2007, vol. 73, pp. 2405- 15). La contaminación medioambiental y de las aguas por residuos generados en granjas porcinas es una fuente de contaminación química y microbiológica. El virus de Ia hepatitis E es un patógeno viral humano que causa hepatitis aguda y se ha descrito como altamente prevalente en cerdos jóvenes y frecuentemente presente en muestras fecales, así como en aguas residuales y lodos producidos en mataderos que procesan cerdos (cfr. Clemente-Casares et al., "Hepatitis E virus epidemiology in industrialised countries" Emerqinq Infectious Diseases 2003, vol. 9, pp. 448-54). El problema remanente es Ia identificación positiva de contaminación fecal de origen porcino, que permita localizar con exactitud Ia fuente de contaminación para procesos de remediación y evaluar el impacto potencial sobre Ia salud pública de Ia contaminación fecal en el agua.Faecal contamination can originate from point or non-point sources. Generally, point sources of faecal contamination include discrete sources such as discharges from large animal husbandry operations, treated and untreated effluents from sewage, storm water, and combined sewage. Non-point sources are diffuse and include agricultural sources and animal waste applied to agricultural fields. The identification of sources of microbiological contamination plays a very important role in obtaining effective handling and remediation strategies, and is known as traceability of microbial source sources (MST). MST includes a group of methodologies that aim to identify, and in some cases quantify, the dominant sources of fecal contamination in the environment and, more specifically, in water resources (cf. Stoeckel et al. "Performance, Design, and Analysis in Microbial Source Tracking Studies "ADPI. Environ. Microbiol. 2007, vol. 73, pp. 2405-15). Environmental and water pollution from waste generated in pig farms is a source of chemical and microbiological contamination. The hepatitis E virus is a human viral pathogen that causes acute hepatitis and has been described as highly prevalent in young pigs and frequently present in faecal samples, as well as in sewage and sludge produced in slaughterhouses that process pigs (cf. Clement- Casares et al., "Hepatitis E virus epidemiology in industrialized countries" Emerqinq Infectious Diseases 2003, vol. 9, pp. 448-54). The remaining problem is the positive identification of fecal contamination of porcine origin, which allows to accurately locate the source of contamination for remediation processes and evaluate the potential public health impact of fecal contamination in water.
Se han descrito varios métodos moleculares basados en Ia reacción en cadena de Ia polimerasa (PCR) para Ia detección específica de virus como adenovirus humanos (HAdV) y poliomavirus (JCPyV y BKPyV), adenovirus porcinos (PAdV) y poliomavirus bovinos (BPyV) como indicadores de contaminación de origen animal o humano en aguas y en moluscos bivalvos (cfr. Bofill-Mas et al., "Quantification and stability of human adenoviruses and polyomavirus JCPyV ¡n wastewater matrices" Appl. Environ. Microbiol. 2006, vol. 72, pp. 7894-6; Formiga-Cruz et al., "Evaluation of potential indicators of viral contamination ¡n shellfish with applicability to diverse geographical áreas" Appl. Environ. Microbiol. 2003. vol. 69, pp. 1556-63; Hundesa et al., "Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment" Appl. Environ. Microbiol. 2006. vol. 72, pp. 7886-93; Pina et al., "Viral pollution ¡n the environment and shellfish: human adenovirus detection by PCR as an Índex of human viruses" APPI. Environ. Microbiol. 1998, vol. 64, pp. 3376-82; Puig et al., "Detection of adenoviruses and enteroviruses in polluted waters by nested-PCR amplification" Appl. Environ. Microbiol. 1994, vol. 60, pp. 2963- 70; Maluquer et al. "Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination" Appl. Environ. Microbiol. 2004, vol. 70, pp. 1448-54).Several molecular methods based on the polymerase chain reaction (PCR) for the specific detection of viruses such as human adenoviruses (HAdV) and polyomavirus (JCPyV and BKPyV), porcine adenovirus (PAdV) and bovine polyomavirus (BPyV) have been described as indicators of contamination of animal or human origin in waters and bivalve molluscs (cf. Bofill-Mas et al., "Quantification and stability of human adenoviruses and polyomavirus JCPyV ¡n wastewater matrices" Appl. Environ. Microbiol. 2006, vol. 72 , pp. 7894-6; Formiga-Cruz et al., "Evaluation of potential indicators of viral contamination in a shellfish with applicability to diverse geographic areas" Appl. Environ. Microbiol. 2003. vol. 69, pp. 1556-63; Hundesa et al., "Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment" Appl. Environ. Microbiol. 2006. vol. 72, pp. 7886-93; Pina et al., "Viral pollution ¡n the environment and shell fish: human adenovirus detection by PCR as an Index of human viruses "APPI. Environ. Microbiol 1998, vol. 64, pp. 3376-82; Puig et al., "Detection of adenoviruses and enteroviruses in polluted waters by nested-PCR amplification" Appl. Environ. Microbiol 1994, vol. 60, pp. 2963-70; Maluquer et al. "Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination "Appl. Environ. Microbiol. 2004, vol. 70, pp. 1448-54).
Otros estudios se relacionan con métodos similares para enterovirus bovinos y tescovirus (cfr. Jiménez-Clavero et al., "Teschoviruses as indicators of porcine fecal contamination of surface water" APPI. Environ. Microbiol. 2003. vol. 69, pp. 6311-5; and "Survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription- PCR" APPI. Environ. Microbiol. 2005, vol. 71 , pp. 3536-43).Other studies are related to similar methods for bovine enteroviruses and tescoviruses (cf. Jiménez-Clavero et al., "Teschoviruses as indicators of porcine fecal contamination of surface water" APPI. Environ. Microbiol. 2003. vol. 69, pp. 6311- 5; and "Survey of bovine enterovirus in biological and environmental samples by a highly sensitive real-time reverse transcription-PCR" APPI. Environ. Microbiol. 2005, vol. 71, pp. 3536-43).
La elevada estabilidad de los virus en el medio ambiente, Ia especificidad de huésped y Ia elevada prevalencia de algunas infecciones víricas a Io largo del año en Ia población apoya considerablemente el uso de técnicas de PCR cuantitativa a tiempo real (qPCR) para Ia identificación y cuantificación de virus específicos que pueden ser usados como herramientas de trazabilidad de Ia fuente de contaminación. Para Ia identificación de contaminación fecal de origen porcino, Ia cuantificación de virus de DNA excretados persistentemente a Io largo del año puede permitir el desarrollo de protocolos de coste efectivo con cuantificaciones de mayor exactitud de las fuentes de contaminación en comparación con virus de RNA; esto se debe a Ia mayor exactitud de Ia cuantificación por qPCR y a Ia menor sensibilidad a inhibidores, puesto que Ia transcriptasa reversa no es usada cuando se amplifican virus DNA.The high stability of the viruses in the environment, the host specificity and the high prevalence of some viral infections throughout the year in the population considerably supports the use of real-time quantitative PCR (qPCR) techniques for the identification and quantification of specific viruses that can be used as traceability tools of the source of contamination. For the identification of fecal contamination of porcine origin, the quantification of DNA viruses persistently excreted throughout the year may allow the development of cost effective protocols with quantifications of greater accuracy of the sources of contamination compared to RNA viruses; This is due to the greater accuracy of the quantification by qPCR and the lower sensitivity to inhibitors, since the reverse transcriptase is not used when DNA viruses are amplified.
Los protocolos de PCR y PCR anidada (nPCR) se usan típicamente debido a su elevada sensibilidad, pero no son cuantitativos y están sujetos a una alta probabilidad de contaminación cruzada por DNA, que normalmente se soluciona mediante secuenciación de los productos de PCR. La qPCR a tiempo real ha emergido como una técnica valiosa porque reduce exitosamente las probabilidades de contaminación en el laboratorio y las manipulaciones que consumen tiempo, y permite Ia cuantificación rápida y sensible de pequeñas cantidades de DNA diana en muestras biológicas y medioambientales.The PCR and nested PCR (nPCR) protocols are typically used because of their high sensitivity, but are not quantitative and are subject to a high probability of cross-contamination by DNA, which is usually solved by sequencing the PCR products. Real-time qPCR has emerged as a valuable technique because it successfully reduces the likelihood of contamination in the laboratory and time-consuming manipulations, and allows rapid and sensitive quantification of small amounts of target DNA in biological and environmental samples.
La familia Adenoviridae es el único grupo conocido de virus entéricos que presenta genomas DNA de doble cadena. Esto representa una ventaja debido a Ia conservación y estabilidad de las secuencias. La familia Polvomaviridae incluye también virus DNA que producen infecciones persistentes y que son frecuentemente excretados en Ia orina, y por estos motivos ambos virus han sido propuestos como marcadores de Ia contaminación fecal. Los adenovirus humanos (HAdVs) son altamente prevalentes en aguas residuales así como en agua de río y moluscos bivalvos con contaminación fecal (cfr. Fong et al., "Enteric viruses of humans and animáis in aquatic environments: health risks, detection, and potential water quality assessment tools" Microbiol. Mol. Biol. Rev. 2005, vol. 69, pp. 357-71 ; and "Molecular assays for targeting Human and Bovine viruses in coastal water and their application for Library-lndependent source tracking" Appl. Environ. Microbiol. 2005, vol. 71 , pp. 2070-8) y están distribuidos de forma conservada en diversas áreas geográficas. Además, los adenovirus son más estables que los enterovirus a Ia radiación UV y a Ia fluoración. Como un grupo específico dentro de Ia familia, los adenovirus porcinos (PAdV) pertenecen al género Mastadenovirus. están divididos en seis serotipos y se han descrito como abundantes y altamente prevalentes en heces, aguas residuales de matadero y lodos (cfr. Hundesa et al., "Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment" Aopl. Environ. Microbiol. 2006, vol. 72, pp. 7886-93). Otra ventaja de los PAdV es que están ampliamente diseminados en Ia población porcina y no producen enfermedades severas clínicamente. Estos datos focalizan PAdV como indicador de contaminación fecal porcina y muestran claramente su aplicabilidad para Ia identificación y cuantificación de Ia contaminación fecal porcina en el medio ambiente, demostrando su validez como herramienta para Ia trazabílidad de contaminación fecal microbiana.The Adenoviridae family is the only known group of enteric viruses that have double-stranded DNA genomes. This represents an advantage due to the conservation and stability of the sequences. The family Polvomaviridae also includes DNA viruses that produce persistent infections and that are frequently excreted in the urine, and for these reasons both viruses have been proposed as markers of fecal contamination. Human adenoviruses (HAdVs) are highly prevalent in wastewater as well as in river water and bivalve molluscs with fecal contamination (cf. Fong et al., "Enteric viruses of humans and animate in aquatic environments: health risks, detection, and potential water quality assessment tools "Microbiol. Mol. Biol. Rev. 2005, vol. 69, pp. 357-71; and" Molecular assays for targeting Human and Bovine viruses in coastal water and their application for Library-lndependent source tracking "Appl. Environ. Microbiol. 2005, vol. 71, pp. 2070-8) and are conservedly distributed in different geographical areas. In addition, adenoviruses are more stable than enteroviruses at UV radiation and fluorination. As a specific group within the family, porcine adenoviruses (PAdV) belong to the genus Mastadenovirus. they are divided into six serotypes and have been described as abundant and highly prevalent in feces, slaughterhouse wastewater and sludge (cf. Hundesa et al., "Identification of human and animal adenoviruses and polyomaviruses for determination of sources of fecal contamination in the environment "Aopl. Environ. Microbiol. 2006, vol. 72, pp. 7886-93). Another advantage of the PAdV is that they are widely disseminated in the pig population and do not produce clinically severe diseases. These data focus PAdV as an indicator of swine fecal contamination and clearly show its applicability for the identification and quantification of swine fecal contamination in the environment, demonstrating its validity as a tool for the traceability of microbial fecal contamination.
EXPLICACIÓN DE LA INVENCIÓNEXPLANATION OF THE INVENTION
Se proporciona aquí un método para Ia cuantificación de contaminación por PAdV en una muestra sospechosa de contener el mismo. La presente invención desarrolla un ensayo de qPCR con un set de cebadores/sonda específicos capaz de detectar y cuantificar PAdV en excreta, lodos porcinos y diferentes tipos de agua.A method is provided here for the quantification of contamination by PAdV in a sample suspected of containing it. The present invention develops a qPCR assay with a set of specific primers / probe capable of detecting and quantifying PAdV in excreta, porcine sludge and different types of water.
La presente invención se refiere a un método para Ia detección y/o cuantificación de adenovirus porcinos (PAdV) en una muestra, que comprende la extracción del ácido nucleico de PAdV de Ia muestra; Ia amplificación por reacción en cadena de Ia polimerasa del ácido nucleico extraído en presencia de al menos un cebador seleccionado entre SEQ ID NO: 1 o una variante funcional del mismo, SEQ ID NO: 2 o una variante funcional del mismo, y SEQ ID NO: 3 o una variante funcional del mismo, o sus cadenas complementarias; y Ia detección y/o cuantificación de PAdV evaluando el resultado de Ia reacción en cadena de Ia polimerasa.The present invention relates to a method for the detection and / or quantification of porcine adenoviruses (PAdV) in a sample, which it comprises the extraction of the PAdV nucleic acid from the sample; The amplification by chain reaction of the nucleic acid polymerase extracted in the presence of at least one primer selected from SEQ ID NO: 1 or a functional variant thereof, SEQ ID NO: 2 or a functional variant thereof, and SEQ ID NO : 3 or a functional variant thereof, or its complementary chains; and the detection and / or quantification of PAdV evaluating the result of the polymerase chain reaction.
La qPCR a tiempo real es una técnica molecular que permite Ia cuantificación de un pequeño número de copias genómicas. Por tanto, en una realización particular de Ia invención, esta reacción en cadena de Ia polimerasa es una reacción en cadena de Ia polimerasa cuantitativa (qPCR). En otra realización, Ia qPCR se realiza en presencia de una sonda específica para Ia secuencia del genoma de PAdV, y en una realización más particular, Ia qPCR se realiza en presencia de una sonda que comprende Ia secuencia identificada como SEQ ID NO: 3 o una variante funcional de Ia misma, o su cadena complementaria.Real-time qPCR is a molecular technique that allows the quantification of a small number of genomic copies. Therefore, in a particular embodiment of the invention, this polymerase chain reaction is a quantitative polymerase chain reaction (qPCR). In another embodiment, the qPCR is performed in the presence of a specific probe for the PAdV genome sequence, and in a more particular embodiment, the qPCR is performed in the presence of a probe comprising the sequence identified as SEQ ID NO: 3 or a functional variant thereof, or its complementary chain.
Una sonda de qPCR está constituida por una secuencia de nucleótidos que contiene un grupo fluoróforo en uno de sus extremos y un agente bloqueador en el otro extremo. La llegada de Ia enzima polimerasa liberará Ia sonda y separará el agente bloqueador, de forma que cada ciclo de amplificación resulta en Ia emisión de fluorescencia. Otra realización de Ia invención es una qPCR realizada en presencia de Ia sonda SEQ ID NO: 3 o su cadena complementaria, llevando un grupo fluoróforo y un agente bloqueador.A qPCR probe consists of a nucleotide sequence that contains a fluorophore group at one end and a blocking agent at the other end. The arrival of the polymerase enzyme will release the probe and separate the blocking agent, so that each amplification cycle results in the emission of fluorescence. Another embodiment of the invention is a qPCR made in the presence of the SEQ ID NO: 3 probe or its complementary chain, carrying a fluorophore group and a blocking agent.
Para el desarrollo del ensayo de qPCR para PAdV, se analizaron los parámetros necesarios para Ia evaluación de Ia eficiencia del ensayo. Entre ellos, se ajustaron las concentraciones óptimas de cebadores y sonda para obtener valores aceptables para el coeficiente de correlación, Ct, y Ia pendiente de Ia recta de regresión del estándar. La qPCR a tiempo real requiere Ia comparación automática de Ia fluorescencia emitida con valores estándar en cada momento. Por Io tanto, otra realización de Ia invención es Ia evaluación de Ia cantidad de virus incluyendo una comparación de Ia cuantificación del resultado de Ia reacción en cadena de Ia polimerasa con al menos una muestra control. En una realización particular de Ia invención, Ia muestra control comprende un control positivo tomado de al menos una curva estándar generada por Ia implicación de Ia reacción en cadena de Ia polimerasa en Ia presencia de los cebadores correspondientes a SEQ ID NO: 1 y SEQ ID NO: 2, o sus cadenas complementarias, en una secuencia de 612 pb del gen del hexon de PAdV3 a diferentes concentraciones.For the development of the qPCR test for PAdV, the parameters necessary for the evaluation of the efficiency of the test were analyzed. Among them, the optimal concentrations of primers and probe were adjusted to obtain acceptable values for the correlation coefficient, Ct, and the slope of the standard regression line. The real-time qPCR requires the automatic comparison of the emitted fluorescence with standard values at all times. Therefore, another embodiment of the invention is the evaluation of the amount of virus including a comparison of the quantification of the result of the polymerase chain reaction with at least one control sample. In a particular embodiment of the invention, the control sample comprises a positive control taken from at least one curve standard generated by the involvement of the polymerase chain reaction in the presence of the primers corresponding to SEQ ID NO: 1 and SEQ ID NO: 2, or their complementary chains, in a 612 bp sequence of the PAdV3 hexon gene at different concentrations.
Una reacción en cadena de Ia polimerasa para Ia detección de PAdV en muestras en Ia presencia de los cebadores descritos puede estar comprendida en una reacción en cadena de Ia polimerasa anidada. También, podría ser posible para Ia detección de PAdV una PCR semi-anidada usando un set de cebadores incluyendo SEQ ID NO: 1 o SEQ ID NO: 2 como primer cebador, y SEQ ID NO: 3 como el otro cebador. De acuerdo con esto, otra realización de Ia invención comprende la reacción en cadena de Ia polimerasa siendo una reacción en cadena de Ia polimerasa en Ia presencia de uno de los cebadores identificado como SEQ ID NO: 1 o SEQ ID NO: 2 o una variante funcional de los mismos, y de Ia secuencia SEQ ID NO: 3 o una variante funcional de Ia misma, o sus cadenas complementarias.A polymerase chain reaction for the detection of PAdV in samples in the presence of the described primers can be comprised in a chain reaction of the nested polymerase. Also, it could be possible for the detection of PAdV a semi-nested PCR using a set of primers including SEQ ID NO: 1 or SEQ ID NO: 2 as the first primer, and SEQ ID NO: 3 as the other primer. Accordingly, another embodiment of the invention comprises the polymerase chain reaction being a polymerase chain reaction in the presence of one of the primers identified as SEQ ID NO: 1 or SEQ ID NO: 2 or a variant functional thereof, and of the sequence SEQ ID NO: 3 or a functional variant thereof, or its complementary chains.
Está previsto que los oligonucleótidos descritos puedan estar sujetos a modificaciones sin desviarse de Ia invención aquí proporcionada. Está previsto que se incluyan en la presente invención fragmentos y/o variantes funcionales de los oligonucleótidos aquí proporcionados. Se contempla que fragmentos y/o variantes funcionales de los cebadores y/o Ia sonda descritos aquí pueden ser usados de acuerdo con las metodologías aquí propuestas para alcanzar los objetivos de Ia presente invención. Por ejemplo, se incluye en Ia presente invención un fragmento o una extensión o una variante funcional de un cebador o una sonda que retiene Ia misma función tal que pudiera ser empleada en Ia cuantificación de PAdV. Un fragmento puede incluir una secuencia que comprenda cualquier número de nucleótidos menor a los que encontrados en una secuencia del ácido nucleico de interés aquí proporcionada. Una extensión puede incluir una secuencia que comprenda cualquier número de nucleótidos superior a los encontrados en una secuencia del ácido nucleico de interés aquí proporcionada. La especificidad de un oligonucleótido se refiere a la capacidad del producto, es decir de un cebador o una sonda, para hibridar con Ia región o secuencia diana bajo condiciones adecuadas para llevar a cabo una reacción en cadena de Ia polimerasa. La variante funcional de cualquiera de los cebadores o Ia sonda aquí proporcionados se refiere a Ia capacidad del oligonucleótido para hibridar con la región o secuencia diana bajo condiciones adecuadas para llevar a cabo una reacción en cadena de Ia polimerasa. Está completamente contemplado que las herramientas y metodologías para Ia detección de ácidos nucleicos puedan ser adaptadas para el uso de acuerdo con todas las muestras biológicas descritas.It is envisioned that the described oligonucleotides may be subject to modifications without deviating from the invention provided herein. It is intended that fragments and / or functional variants of the oligonucleotides provided herein be included in the present invention. It is contemplated that fragments and / or functional variants of the primers and / or the probe described herein can be used in accordance with the methodologies proposed herein to achieve the objectives of the present invention. For example, a fragment or an extension or a functional variant of a primer or a probe that retains the same function that could be used in the quantification of PAdV is included in the present invention. A fragment may include a sequence comprising any number of nucleotides less than those found in a nucleic acid sequence of interest provided herein. An extension may include a sequence comprising any number of nucleotides greater than those found in a nucleic acid sequence of interest provided herein. The specificity of an oligonucleotide refers to the ability of the product, that is, a primer or a probe, to hybridize with the target region or sequence under suitable conditions to carry out a polymerase chain reaction. The functional variant of any of the primers or probe provided herein refers to the ability of the oligonucleotide to hybridize with the target region or sequence under suitable conditions to carry out a polymerase chain reaction. It is fully contemplated that the tools and methodologies for the detection of nucleic acids can be adapted for use in accordance with all the biological samples described.
El método de Ia invención es capaz de detectar virus en un amplio espectro de muestras. El estudio de PAdV no patógenos como método para Ia trazabilidad de fuentes de contaminación fecal humana y animal en muestras medioambientales debería proporcionar datos para Ia mejora de la manipulación y el control de Ia calidad del agua. Una realización de Ia invención aplica el método en una muestra medioambiental. Otra realización de Ia invención aplica el método en muestras de agua, y en una realización más particular, Ia muestra de agua es una muestra de agua residual. Se consideran incluidas como muestras de agua residual, agua residual urbana y de granja. En otra realización de Ia invención Ia muestra sospechosa de contener PAdV es una muestra de residuos de matadero, que incluye muestras de agua y lodos. El método también se aplica en muestras fecales. La invención es, además, de amplia aplicación en el sector industrial. Otra realización aplica el método de Ia invención a muestras de alimentos. La invención es también aplicable para Ia detección de Ia presencia de virus en artículos comerciales, tales como biosólidos derivados de Ia industria del compostaje. Otra realización particular de Ia invención es Ia aplicación del método en muestras de fertilizantes.The method of the invention is capable of detecting viruses in a wide spectrum of samples. The study of non-pathogenic PAdV as a method for traceability of sources of human and animal fecal contamination in environmental samples should provide data for the improvement of the manipulation and control of water quality. An embodiment of the invention applies the method in an environmental sample. Another embodiment of the invention applies the method in water samples, and in a more particular embodiment, the water sample is a waste water sample. They are considered included as samples of wastewater, urban and farm wastewater. In another embodiment of the invention, the sample suspected of containing PAdV is a sample of slaughterhouse waste, which includes samples of water and sludge. The method is also applied in faecal samples. The invention is also widely applicable in the industrial sector. Another embodiment applies the method of the invention to food samples. The invention is also applicable for the detection of the presence of viruses in commercial articles, such as biosolids derived from the composting industry. Another particular embodiment of the invention is the application of the method in fertilizer samples.
Otro aspecto de Ia presente invención se refiere a los oligonucleótidos identificados por una secuencia de ácidos nucleicos seleccionada del grupo que consiste de SEQ ID NO: 1 , SEQ ID NO: 2 y SEQ ID NO: 3, o sus cadenas complementarias. Otro aspecto se refiere al uso de los oligonucleótidos identificados como SEQ ID NO: 1 y SEQ ID NO: 2 o sus cadenas complementarias como cebadores específicos para el genoma de PAdV, y del oligonucleótido identificado como SEQ ID NO: 3 o su cadena complementaria como sonda especifica para Ia secuencia del genoma de PAdV. Se prefiere como cebador directo un cebador que comprenda SEQ ID NO: 1 o una variante funcional del mismo y como cebador inverso uno que comprenda SEQ ID NO: 2 o una variante funcional del mismo. Otro aspecto de Ia invención se relaciona con el uso de los cebadores y de Ia sonda descritos arriba, o una variante funcional de los mismos, para Ia detección de PAdV usando Ia reacción en cadena de Ia polimerasa. En una realización preferida, esta reacción en cadena de Ia polimerasa es Ia qPCR.Another aspect of the present invention relates to oligonucleotides identified by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, or their complementary chains. Another aspect refers to the use of oligonucleotides identified as SEQ ID NO: 1 and SEQ ID NO: 2 or their complementary chains as specific primers for the PAdV genome, and the oligonucleotide identified as SEQ ID NO: 3 or its complementary chain as specific probe for the PAdV genome sequence. A primer comprising SEQ ID NO: 1 or a functional variant thereof and a reverse primer one comprising SEQ ID NO: 2 or a functional variant thereof is preferred as a direct primer. Another aspect of the invention relates to the use of the primers and the probe described above, or a functional variant thereof, for the detection of PAdV using the polymerase chain reaction. In a preferred embodiment, this polymerase chain reaction is the qPCR.
Otro aspecto de Ia invención se refiere a un kit para realizar el método de Ia invención que comprende reactivos adecuados para Ia detección y/o cuantificación de PAdV en una muestra, y una realización particular de Ia invención es que este kit incluya al menos uno de los cebadores identificados como SEQ ID NO: 1 y SEQ ID NO: 2 y/o Ia sonda identificada como SEQ ID NO: 3, o sus cadenas complementarias.Another aspect of the invention relates to a kit for carrying out the method of the invention comprising reagents suitable for the detection and / or quantification of PAdV in a sample, and a particular embodiment of the invention is that this kit includes at least one of the primers identified as SEQ ID NO: 1 and SEQ ID NO: 2 and / or the probe identified as SEQ ID NO: 3, or its complementary chains.
A Io largo de Ia descripción y las reivindicaciones Ia palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en Ia materia, otros objetos, ventajas y características de Ia invención se desprenderán en parte de Ia descripción y en parte de Ia práctica de Ia invención.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 characteristics of the invention will emerge partly from the description and partly from the practice of the invention.
BREVE DESCRICIÓN DE LOS DIBUJOSBRIEF DESCRIPTION OF THE DRAWINGS
La FIG.1 muestra los gráficos de amplificación obtenidos con diluciones seriadas 1 :10 del ADN viral clonado de PAdV. Los gráficos de amplificación muestran el log de dRn (señal de fluorescencia reportada) en relación con el número de ciclos.FIG. 1 shows the amplification graphs obtained with serial dilutions 1: 10 of the cloned viral DNA of PAdV. The amplification graphs show the log of dRn (reported fluorescence signal) in relation to the number of cycles.
La FIG. 2 muestra Ia curva estándar obtenida con diluciones seriadas 1 :10 del ADN viral clonado de PAdV. El log de Ia cantidad inicial de DNA viral se representa respecto al ciclo umbral (valores Ct), calculado como el número ciclo fraccional en el que un incremento significativo de Rn excede un determinado umbral (línea horizontal).FIG. 2 shows the standard curve obtained with serial dilutions 1: 10 of the cloned viral DNA of PAdV. The log of the initial amount of viral DNA is represented with respect to the threshold cycle (Ct values), calculated as the fractional cycle number in which a significant increase in Rn exceeds a certain threshold (horizontal line).
La FIG. 3 muestra Ia evaluación de Ia inhibición del ensayo de qPCR para PAdV en muestras medioambientales de acuerdo con el método de extracción de AN usado. La figura muestra los niveles de PAdV añadidos previamente a las extracciones de AN obtenidos con el kit de extracción de ácidos nucleicos NucliSens® de Biomérieux (Bx) y el método previamente descrito por Boom et al. (B), como detectados en las diluciones 1 :10 (en gris), 1 :100 (en blanco) y no diluida (en negro). Se estudiaron tres tipos diferentes de muestras medioambientales: aguas residuales de matadero (SW), agua residual urbana (US), y lodos porcinos (S).FIG. 3 shows the evaluation of the inhibition of the qPCR assay for PAdV in environmental samples according to the method of extraction of AN used. The figure shows the PAdV levels previously added to the AN extractions obtained with the NucliSens® nucleic acid extraction kit from Biomérieux (Bx) and the method previously described by Boom et al. (B), as detected in dilutions 1: 10 (in gray), 1: 100 (in white) and undiluted (in black). Three different types of environmental samples were studied: slaughterhouse wastewater (SW), urban wastewater (US), and pig sludge (S).
La FIG. 4 muestra los resultados obtenidos en los ensayos de qPCR para HAdV y PAdV.FIG. 4 shows the results obtained in the qPCR assays for HAdV and PAdV.
EJEMPLOSEXAMPLES
Obtención de muestrasSample collection
Se aplicó el procedimiento de qPCR a Ia cuantificación de adenovirus porcinos en: 38 muestras de heces agrupadas de 18 granjas de cerdos de diversas áreas en el País Vasco (norte de España, Costa del Cantábrico) y Cataluña (noreste de España, costa mediterránea); 8 muestras de agua residual procedente de un matadero que procesa cerdos en Cataluña; 6 muestras de agua de río obtenidas por debajo de una importante área granjera; y 9 muestras de agua residual urbana de Cataluña, que fueron analizadas también para Ia presencia de HAdV.The qPCR procedure was applied to the quantification of porcine adenoviruses in: 38 stool samples grouped from 18 pig farms in various areas in the Basque Country (northern Spain, Cantabrian Coast) and Catalonia (northeastern Spain, Mediterranean coast) ; 8 samples of wastewater from a slaughterhouse that processes pigs in Catalonia; 6 river water samples obtained below an important agricultural area; and 9 samples of urban wastewater from Catalonia, which were also analyzed for the presence of HAdV.
Se recogieron treinta y ocho muestras fecales agrupadas de cerdo en diferentes periodos en diechiocho granjas localizadas en dos regiones diferentes en España: Cataluña (9 granjas) y el País Vasco (9 granjas). Las muestras se recogieron de animales de engorde y de cría, y se agruparon de acuerdo con Ia edad y el objetivo comercial de los individuos. Se concentraron los virus mediante un procedimiento basado en elución y ultracentrifugación de los virus (cfr. Maluquer de Motes et al., supra Appl. Environ. Microbiol. 2004). Brevemente, 1 g de cada grupo fue eluído en 3.5 mi de tampón glicina 0.25 N (pH 9.5), mantenido en hielo durante 30 min y luego centrifugado (9,200 x g durante 15 min). Finalmente, se concentró el sobrenadante por ultracentrifugación (110,000 x g durante 1 h a 40C) y las partículas víricas fueron resuspendidas en 100 μl de PBS y almacenadas a -80 0C.Thirty-eight pooled fecal samples of pigs were collected at different periods in eighteen farms located in two different regions in Spain: Catalonia (9 farms) and the Basque Country (9 farms). The samples were collected from fattening and breeding animals, and were grouped according to the age and commercial objective of the individuals. Viruses were concentrated by a procedure based on elution and ultracentrifugation of the viruses (cf. Maluquer de Motes et al., Supra Appl. Environ. Microbiol. 2004). Briefly, 1 g of each group was eluted in 3.5 ml of 0.25 N glycine buffer (pH 9.5), kept on ice for 30 min and then centrifuged (9,200 xg for 15 min). Finally, the supernatant was concentrated by ultracentrifugation (110,000 xg for 1 h at 4 0 C) and the viral particles were resuspended in 100 µl of PBS and stored at -80 0 C.
Se recogieron nueve muestras de agua residual urbana en Ia entrada de una planta de tratamiento de agua residual localizada en Sant Adriá del Besos, (Barcelona, Cataluña), que procesa 670,000 m3 de agua residual al día procedente de una población equivalente aproximadamente a 1.8 millones de habitantes de una área urbana sin ninguna actividad granjera o agrícola. Todas las muestras fueron recogidas en containers estériles de polietileno de 500 mi y mantenidas a 40C hasta 8 h antes de su análisis y los virus fueron concentrados a partir de alícuotas de 40 mi (cfr. Puig et al., supra Appl. Environ. Microbiol. 1994). Brevemente, se ultracentrifugaron 40 mi de agua residual (110,000 x g durante 1 h a 4 0C) para sedimentar todas las partículas víricas junto con material en suspensión. Se eluyó el sedimento con 4 mi de tampón glicina 0.25 N (pH 9.5), y los sólidos suspendidos se separaron por centrifugación a 12,000 x g durante 15 min. Los virus se concentraron finalmente por ultracentrifugación (110,000 xg durante 1 h a 4 0C), resuspendidos en 100 μl de PBS, y almacenados a -80 0C.Nine samples of urban wastewater were collected at the entrance of a wastewater treatment plant located in Sant Adriá del Besos, (Barcelona, Catalonia), which processes 670,000 m 3 of wastewater per day from a population equivalent to approximately 1.8 million inhabitants of an urban area without any agricultural or agricultural activity. All samples were collected in sterile 500 ml polyethylene containers and kept at 4 0 C until 8 h before analysis and the viruses were concentrated from 40 ml aliquots (cf. Puig et al., Supra Appl. Environ Microbiol. 1994). Briefly, 40 ml of residual water (110,000 xg for 1 h at 4 0 C) was ultracentrifuged to sediment all viral particles together with suspended material. The pellet was eluted with 4 ml of 0.25 N glycine buffer (pH 9.5), and the suspended solids were separated by centrifugation at 12,000 xg for 15 min. The viruses were finally concentrated by ultracentrifugation (110,000 xg for 1 h at 4 0 C), resuspended in 100 µl of PBS, and stored at -80 0 C.
Se recogieron ocho muestras de agua residual de un matadero que procesa bovino y en mayor número, porcino de 5 y 6 meses de edad. Se recogieron las muestras en containers estériles de polietileno de 500 mi y se mantuvieron a 40C hasta 8 h antes de su análisis. Se concentraron los virus de las muestras siguiendo el mismo procedimiento descrito para agua residual urbana.Eight samples of residual water were collected from a slaughterhouse that processes cattle and in greater numbers, pigs of 5 and 6 months of age. Samples were collected in sterile 500 ml polyethylene containers and kept at 4 0 C until 8 h before analysis. Sample viruses were concentrated following the same procedure described for urban wastewater.
Se recogieron seis muestras de agua del río Ter por debajo de una importante zona de granjas porcinas en Cataluña con diferentes pueblos que descargan en el río los efluentes resultantes del tratamiento de aguas residuales. Las muestras de agua se recogieron en un periodo de 4 meses, las muestras se concentraron en el punto de recogida y los filtros con los concentrados se mantuvieron a 40C hasta 8 horas antes de su análisis.Six samples of water were collected from the Ter River below an important area of pig farms in Catalonia with different villages that discharge the effluents resulting from wastewater treatment into the river. Water samples were collected over a period of 4 months, the samples were concentrated at the point of collection and the filters with the concentrates were kept at 0 ° C until 8 hours before their analysis.
El protocolo usado para el procesamiento de las muestras de río fue una combinación del método EPA (EPA 600/4-84/013 (N 14)), con modificaciones menores, y el método basado en ultracentrifugación y elución en tampón glicina 0.25 N (pH 9.5) (cfr. Albinana-Gimenez et al., "Distribution of polyomaviruses, adenoviruses and hepatitis E virus in the environmental and ¡n a Drinking-Water treatment plant" Environ. Sci. Techno. 2006, vol. 40, pp. 7416-22). Se filtraron cien litros de agua de río a través de filtros electropositivos Zeta Plus MK a 1 l/min usando una bomba peristálticaThe protocol used for the processing of the river samples was a combination of the EPA method (EPA 600 / 4-84 / 013 (N 14)), with minor modifications, and the method based on ultracentrifugation and elution in 0.25 N glycine buffer ( pH 9.5) (cf. Albinana-Gimenez et al., "Distribution of polyomaviruses, adenoviruses and hepatitis E virus in the environmental and na Drinking-Water treatment plant" Environ. Sci. Techno. 2006, vol. 40, pp. 7416 -22). One hundred liters of river water was filtered through Zeta Plus MK electropositive filters at 1 l / min using a peristaltic pump
Millipore. Se eluyeron los virus retenidos en 900 mi de tampón glicina 0.25 N (pH 9.5) y extracto de carne 1 % (Becton, Dickinson & Co., Sparks MD, USA) mediante flujo reverso usando una bomba peristáltica Millipore a 0.4 l/min durante 45 min. Para Ia floculación se añadió extracto de carne al 3%, se ajustó el pH a 3.5 usando HCI 5M y se agitó magnéticamente Ia suspensión resultante durante 30 min, finalmente se centrifugó a 12,800 x g durante 25 min a 40C. Tras este paso, Ia muestra se trató usando el protocolo previamente aplicado para Ia recuperación de virus a partir de agua residual urbana.Millipore The viruses retained in 900 ml of 0.25 N glycine buffer (pH 9.5) and 1% meat extract (Becton, Dickinson & Co., Sparks MD, USA) were eluted. by reverse flow using a Millipore peristaltic pump at 0.4 l / min for 45 min. For flocculation, 3% meat extract was added, the pH was adjusted to 3.5 using 5M HCI and the resulting suspension was magnetically stirred for 30 min, finally centrifuged at 12,800 xg for 25 min at 4 0 C. After this step, The sample was treated using the protocol previously applied for virus recovery from urban wastewater.
Diseño del set de cebadores/sonda para el ensayo de αPCR para PAdVDesign of the primer / probe set for the αPCR test for PAdV
El procedimiento de qPCR se basa en un ensayo TaqMan y usa dos cebadores y una sonda fluorogénica que reconoce un fragmento de 68 bp en el gen del hexon del genoma de PAdV. Los cebadores y Ia sonda fueron diseñados específicamente para Ia amplificación de PAdV. Se alinearon secuencias de PAdV DNA del GenBank usando el programa ClustalW (European Bioinformatics Institute, UK) y se seleccionó un fragmento conservado de 68 bp mediante el software Primer Express (Applied Biosystems). La homología de todos los oligonucleótidos fue finalmente verificada usando Blast Search. Se marcó la sonda con FAM (6- carboxifluoresceína) como grupo fluoróforo en el extremo 5' y BHQ-1 (Black- HoIe Quencher 1 ) como agente bloqueador en el extremo 3'. La secuencia del cebador directo se corresponde con SEQ ID NO: 1 , la secuencia del cebador inverso corresponde a SEQ ID NO: 2, y Ia secuencia de Ia sonda, a SEQ ID NO: 3. Su localización en el genoma usando como referencia Ia cepa PAdV serotipo 3 (número de acceso AJ237815 GenBank) es como sigue: Cebador directo Q-PAdV-F: entre el par de bases (pb) 20701 y 20718. Cebador inverso Q-PAdV-R: entre pb 20768 y 20751. Sonda Q-PAdV-P: entre pb 20722 y 20737.The qPCR procedure is based on a TaqMan assay and uses two primers and a fluorogenic probe that recognizes a 68 bp fragment in the PAdV genome hexon gene. The primers and the probe were designed specifically for the amplification of PAdV. GenBank DNA PAdV sequences were aligned using the ClustalW program (European Bioinformatics Institute, UK) and a conserved 68 bp fragment was selected using Primer Express software (Applied Biosystems). The homology of all oligonucleotides was finally verified using Blast Search. The probe was labeled with FAM (6- carboxyfluorescein) as a fluorophore group at the 5 'end and BHQ-1 (Black-HoIe Quencher 1) as a blocking agent at the 3' end. The sequence of the direct primer corresponds to SEQ ID NO: 1, the sequence of the reverse primer corresponds to SEQ ID NO: 2, and the sequence of the probe, to SEQ ID NO: 3. Its location in the genome using as reference Ia strain PAdV serotype 3 (accession number AJ237815 GenBank) is as follows: Direct primer Q-PAdV-F: between the base pair (bp) 20701 and 20718. Inverse primer Q-PAdV-R: between bp 20768 and 20751. Probe Q-PAdV-P: between pb 20722 and 20737.
Construcción de los estándares de qPCR para PAdVConstruction of qPCR standards for PAdV
Se generaron curvas estándar mediante transformación de células E. coli JM109 (Promega) con el plásmido pGEM-T Easy (Promega) que contenía una secuencia de 612 pb del hexon de PAdV-3. La transformación se llevó a cabo siguiendo las instrucciones del fabricante. Las colonias llevando el plásmido deseado se analizaron por PCR para contener el DNA diana, que fue obtenido con el kit QIAGEN Plasmid Midi kit (QIAGEN, Inc.) siguiendo las instrucciones del fabricante. Una vez obtenidos y analizados por Genequant Pro (Amersham Biosciences), de acuerdo con el peso molecular del plásmido, se linearizó 10 μg de DNA con EcoRI, purificado con el QUIAquick PCR purification kit (QIAGEN, Inc.) y posteriormente cuantificado de nuevo, antes de obtener diluciones seriadas de 10'1 a 109 moléculas de DNA viral por 10 μl en tampón TE. Las diluciones del estándar se separaron en alícuotas y se almacenaron a -80 0C hasta su uso.Standard curves were generated by transformation of E. coli JM109 cells (Promega) with plasmid pGEM-T Easy (Promega) containing a 612 bp sequence of the PAdV-3 hexon. The transformation was carried out following the manufacturer's instructions. Colonies carrying the desired plasmid were analyzed by PCR to contain the target DNA, which was obtained with the QIAGEN Plasmid Midi kit (QIAGEN, Inc.) following the manufacturer's instructions Once obtained and analyzed by Genequant Pro (Amersham Biosciences), according to the molecular weight of the plasmid, 10 μg of DNA was linearized with EcoRI, purified with the QUIAquick PCR purification kit (QIAGEN, Inc.) and subsequently quantified again, before obtaining serial dilutions of 10 '1 to 10 9 molecules of viral DNA per 10 μl in TE buffer. The standard dilutions aliquoted and stored at -80 0 C until use.
Extracción de ácidos nucleicosNucleic acid extraction
Los ácidos nucleicos (AN) de los concentrados virales se extrajeron usando un procedimiento basado en el método Boom et al. (1990) que usa isotiocianato de guanidino para desnaturalizar las cápsides víricas y partículas de sílice para unir los ácidos nucleicos hasta su elución final en tampón TE (Tris 10 mM, EDTA 0.1 mM, pH 7.4) y posterior almacenamiento a -80 0C. También se usó, de acuerdo con las instrucciones del fabricante, el kit de extracción de ácidos nucleicos NucliSens® (Biomérieux), basado en los mismos principios y que contiene bolas magnéticas cubiertas de sílice.Nucleic acids (AN) from viral concentrates were extracted using a method based on the method Boom et al. (1990) using guanidino isothiocyanate to denature viral capsids and silica particles to bind nucleic acids until final elution in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) and subsequent storage at -80 0 C. Also used, according to the manufacturer's instructions, the NucliSens ® (Biomérieux) nucleic acid extraction kit, based on the same principles and containing silica-coated magnetic balls.
Ensayo de αPCR para PAdVΑPCR assay for PAdV
Para todos los experimentos Ia mezcla de PCR se preparó y se dispensó en un área de trabajo dedicada Ia preparación de reactivos de PCR; las muestras se cargaron en un laboratorio pre-PCR y, finalmente, Ia placa se transfirió a un laboratorio separado para Ia adición de los estándares de qPCR. Las amplificaciones se realizaron en una mezcla de reacción de 25-μl que contenía 10 μl de DNA y 15 μl de TaqMan® Universal PCR Master Mix, 0.9 μM de cada cebador (Q-PAdV-F y Q-PAdV-R) y 0.225 μM de sonda fluorogénica (Q-PAdV-P). La TaqMan® Universal PCR Master Mix se suministra en una concentración 2x y contiene AmpliTaq Gold® DNA polymerase, dNTPs con dUTP, referencia pasiva, componentes tamponadores optimizados y AmpErase® uracil-N-Glicosilasa. Tras Ia activación de Ia uracil-N-GI¡closilasa (2 min, 50 0C) y Ia activación de Ia AmpliTaq GoId durante 10 min a 95 0C, se realizaron 45 ciclos (15 s a 95 0C, 20 s a 55 0C y 20 s a 60 0C) en un sistema de detección MX3000P (Stratagene). Se analizaron dos diluciones 1 :10 (1 :10 y 1 :100) del DNA extraído en duplicado (4 análisis/muestra) para el análisis de muestras medioambientales, mientras que se analizaron diluciones seriadas 1 :10 del estándar de qPCR por triplicado cuando se cuantificaron copias del genoma viral (ge). En todas las qPCR llevadas a cabo Ia cantidad de DNA se definió como media de los datos obtenidos. Se añadió en cada ensayo un control sin molde. La inhibición enzimática de las muestras se analizó por análisis de diversas diluciones de las muestras así como por adición de cantidades conocidas de DNA diana a las muestras medioambientales.For all experiments the PCR mixture was prepared and dispensed in a dedicated work area Ia preparation of PCR reagents; The samples were loaded in a pre-PCR laboratory and, finally, the plate was transferred to a separate laboratory for the addition of the qPCR standards. The amplifications were performed in a 25-μl reaction mixture containing 10 μl of DNA and 15 μl of TaqMan ® Universal PCR Master Mix, 0.9 μM of each primer (Q-PAdV-F and Q-PAdV-R) and 0.225 μM of fluorogenic probe (Q-PAdV-P). The TaqMan ® Universal PCR Master Mix is supplied in a 2x concentration and contains AmpliTaq Gold ® DNA polymerase, dNTPs with dUTP, passive reference, optimized buffer components and AmpErase ® uracil-N-Glycosylase. After the activation of the uracil-N-GIclosilase (2 min, 50 0 C) and the activation of the AmpliTaq GoId for 10 min at 95 0 C, 45 cycles were performed (15 s at 95 0 C, 20 s at 55 0 C and 20 s at 60 0 C) in an MX3000P (Stratagene) detection system. Two dilutions 1: 10 (1: 10 and 1: 100) of the DNA extracted in duplicate (4 analyzes / sample) were analyzed for the analysis of environmental samples, while serial dilutions 1: 10 of the qPCR standard were analyzed in triplicate when Copies of the viral genome (ge) were quantified. In all the qPCR carried out, the amount of DNA was defined as the average of the data obtained. A control without mold was added in each test. Enzymatic inhibition of the samples was analyzed by analysis of various dilutions of the samples as well as by adding known amounts of target DNA to the environmental samples.
Ensayo de nPCR para PAdVNPCR test for PAdV
Se realizaron ensayos de PCR anidada para Ia amplificación de PAdV. Se analizaron 10 μL de ácidos nucleicos extraídos y su dilución 1 :10 en una mezcla de reacción de 40 μL que contenía IxPCR Buffer, MgCI2 a 1.5 mM, 0.025 mM de cada dNTP, 25 pmol de cebadores y 2 unidades de Taq DNA polymerase (Bloron GmbH, Germany). Las condiciones de reacción fueron: 94 0C durante 4 min, 30 ciclos de 92 0C durante 60 s, 60 s a Ia temperatura de hibridación correspondiente y extensión a 72 0C durante 75 s. Se completó Ia amplificación con 7 min de extensión a 72 0C. Tras Ia primera PCR, se añadió 1 μL del producto de Ia primera PCR a 49 μL de mezcla de nPCR que contenía los mismos componentes que Ia mezcla de Ia primera PCR pero con 0.16 mM de cada cebador anidado. Se llevó a cabo una segunda amplificación de 30 ciclos idéntica a Ia anterior.Nested PCR assays were performed for the amplification of PAdV. 10 μL of extracted nucleic acids and their 1: 10 dilution were analyzed in a 40 μL reaction mixture containing IxPCR Buffer, MgCI 2 at 1.5 mM, 0.025 mM of each dNTP, 25 pmol of primers and 2 units of Taq DNA polymerase (Bloron GmbH, Germany). The reaction conditions were: 94 0 C for 4 min, 30 cycles of 92 0 C for 60 s, 60 s at the corresponding hybridization temperature and extension at 72 0 C for 75 s. The amplification was completed with 7 min of extension at 72 0 C. After the first PCR, 1 μL of the product of the first PCR was added to 49 μL of nPCR mixture containing the same components as the mixture of the first PCR but with 0.16 mM of each nested primer. A second amplification of 30 cycles identical to the previous one was carried out.
Secuenciación de PAdVPAdV sequencing
Los ampllcones obtenidos tras nPCR se purificaron mediante QIAquick PCR purification kit (QIAGEN, Inc.). El DNA purificado se secuenció directamente con el ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction kit versión 3.1 con Ampli Taq® DNA polymerase FS (Applied Biosystems) siguiendo las instrucciones del fabricante. Las condiciones para los 25 ciclos de secuenciación fueron: desnaturalización a 96 ° C durante 10 s, hibridación durante 5 s a 50 ° C y extensión a 60 ° C durante 4 min. Los cebadores anidados se usaron para Ia secuenciación a concentración de 0.05 μM. Los resultados se analizaron usando el secuenciador automático ABI PRISM 377 (Perkin-Elmer, Applied Biosystems). Las secuencias obtenidas se compararon con las del GenBank y EMBL (European Molecular Biology Library) usando el programa básico BLAST del NCBI (The National Center for Biotechnoiogy Information, http://www.ncbi.nlm.nih.gov/BLAST/). Los alineamientos de las secuencias se llevaron a cabo por medio del programa ClustalW del EBI (European Bioinformatics lnstitute of the EMBL, http://www.ebi.ac.uk/clustalw/).Ampllcones obtained after nPCR were purified by QIAquick PCR purification kit (QIAGEN, Inc.). The purified DNA was sequenced directly with the ABI PRISM ™ Dye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 with Ampli Taq DNA polymerase ® FS (Applied Biosystems) following the manufacturer 's instructions. The conditions for the 25 sequencing cycles were: denaturation at 96 ° C for 10 s, hybridization for 5 s at 50 ° C and extension at 60 ° C for 4 min. The nested primers were used for sequencing at a concentration of 0.05 μM. The results were analyzed using the ABI PRISM 377 automatic sequencer (Perkin-Elmer, Applied Biosystems). The sequences obtained are compared to those of GenBank and EMBL (European Molecular Biology Library) using the NCBI basic program BLAST (The National Center for Biotechnoiogy Information, http://www.ncbi.nlm.nih.gov/BLAST/). The sequence alignments were carried out through the EBI ClustalW program (European Bioinformatics Institute of the EMBL, http://www.ebi.ac.uk/clustalw/).
Ensayo de αPCR para HAdVΑPCR assay for HAdV
La cuantificación de HAdV en las muestras se llevó a cabo mediante un protocolo basado en TaqMan®, usando el cebador directo SEQ ID NO: 4, el cebador inverso SEQ ID NO: 5 y Ia sonda SEQ ID NO: 6, en donde "w" puede ser "a" o T, "k" puede ser "g" o T1 "s" puede ser "g" o "c", V puede ser "a" o "g", y "y" puede ser "c" o "t". Este ensayo detecta los 51 serotipos descritos de adenovirus humanos. Las cuantificaciones se realizaron en una mezcla de reacción de 25-μl que contenía 10 μl de DNA y 15 μl de TaqMan® Universal PCR Master Mix (Applied Biosystems) que contenía 0.9 μM de cada cebador y 0.225 μM de sonda fluorogénica. Se usó un plásmido pBR322 que contenía Ia secuencia del hexon de HAdV41 para construir el estándar de 101 a 107 copias de DNA por 10 μl añadidos a Ia reacción de PCR. Cada dilución del estándar de DNA se analizó por triplicado.The quantification of HAdV in the samples was carried out by means of a protocol based on TaqMan ® , using the direct primer SEQ ID NO: 4, the reverse primer SEQ ID NO: 5 and the probe SEQ ID NO: 6, where "w "may be" a "or T," k "may be" g "or T 1 " s "may be" g "or" c ", V may be" a "or" g ", and" y "may be "c" or "t". This assay detects the 51 described serotypes of human adenoviruses. Quantifications were performed in a 25-μl reaction mixture containing 10 μl of DNA and 15 μl of TaqMan ® Universal PCR Master Mix (Applied Biosystems) containing 0.9 μM of each primer and 0.225 μM of fluorogenic probe. A plasmid pBR322 containing the HAdV41 hexon sequence was used to construct the standard of 10 1 to 10 7 copies of DNA per 10 μl added to the PCR reaction. Each dilution of the DNA standard was analyzed in triplicate.
Tras Ia activación de Ia uracil-N-Glicosilasa (2 min, 50 0C) y activación de la AmpliTaq GoId durante 10 min a 95 0C, se realizaron 45 ciclos (15 s a 95 0C y 1 min a 60 0C) en un sistema de detección MX3000P (Stratagene). Se analizaron dos diluciones 1 :10 (1 :10 y 1 :100) del DNA extraído en duplicado (4 análisis/muestra) para el análisis de muestras medioambientales, mientras que se analizaron diluciones seriadas 1 :10 del estándar de qPCR por triplicado cuando se cuantificaron copias del genoma viral (ge). En todos las qPCR llevadas a cabo Ia cantidad de DNA se definió como media de los datos obtenidos. Se añadió un control sin molde en cada ensayo.After the activation of uracil-N-Glycosylase (2 min, 50 0 C) and activation of the AmpliTaq GoId for 10 min at 95 0 C, 45 cycles were performed (15 s at 95 0 C and 1 min at 60 0 C) in an MX3000P (Stratagene) detection system. Two dilutions 1: 10 (1: 10 and 1: 100) of the DNA extracted in duplicate (4 analyzes / sample) were analyzed for the analysis of environmental samples, while serial dilutions 1: 10 of the qPCR standard were analyzed in triplicate when Copies of the viral genome (ge) were quantified. In all the qPCR carried out the amount of DNA was defined as the average of the data obtained. A moldless control was added in each test.
Muestras de control positivoPositive control samples
Cuando el ensayo fue llevado a cabo, ninguna cepa de PAdV estaba disponible para uso como control positivo. Consecuentemente, se optimizaron los ensayos usando el fragmento obtenido tanto de muestras medioambientales como heces porcinas que había dado amplificación positiva por PCR anidada y que había sido posteriormente secuenciado y usado en análisis filogenéticos. Se clonó en un vector pGEM-T Easy un fragmento de 612 bp correspondiente a un fragmento interno del gen que codifica para el hexon que fue usado como control de validación para los experimentos de nPCR y como estándar en los ensayos cuantitativos de qPCR.When the test was carried out, no strain of PAdV was available for use as a positive control. Consequently, the assays were optimized using the fragment obtained from both samples environmental as swine feces that had given positive amplification by nested PCR and that had subsequently been sequenced and used in phylogenetic analyzes. A 612 bp fragment corresponding to an internal fragment of the gene coding for the hexon that was used as a validation control for the nPCR experiments and as standard in the quantitative qPCR assays was cloned into a pGEM-T Easy vector.
Diseño de cebadores/sonda v optimización del ensayo de qPCRPrimer / probe design and qPCR assay optimization
Para optimizar Ia especificidad y sensibilidad, se diseñó un set de cebadores/sonda que amplificaba un amplicón pequeño. La concentración de los cebadores y Ia sonda se optimizó mediante el análisis de concentraciones de cebadores que oscilaban entre 0.4 y 0.9 μM y de sonda, entre 0.225 y 0.9 μM, para cada reacción. Las temperaturas de hibridación se optimizaron también.To optimize the specificity and sensitivity, a set of primers / probe was designed that amplified a small amplicon. The concentration of the primers and the probe was optimized by analyzing concentrations of primers ranging between 0.4 and 0.9 μM and probe, between 0.225 and 0.9 μM, for each reaction. Hybridization temperatures were also optimized.
Usando las condiciones descritas para Ia qPCR, el ensayo identificó entre 1 y 10 moléculas de DNA viral (estándar de PAdV) como límite de detección por tubo de reacción. Un ensayo representativo de los llevados a cabo se muestra en Ia FIG. 1. En todos los ensayos el valor medio de R-cuadrado fue 0.996±0.003, los valores de pendiente oscilaron entre -3.461 y -3.492 (valor medio -3.481), y Ia eficiencia estimada de ellos fue del 94.5%.Using the conditions described for the qPCR, the assay identified between 1 and 10 viral DNA molecules (PAdV standard) as the limit of detection per reaction tube. A representative test of those carried out is shown in FIG. 1. In all trials, the average R-square value was 0.996 ± 0.003, the slope values ranged from -3.461 to -3.492 (average value -3.481), and the estimated efficiency of them was 94.5%.
Especificidad del ensayoTest specificity
La especificidad de los oligonucleótidos se verificó usando Ia herramienta Blast Search. Además, el set de cebadores/sonda fue ensayado con muestras de suero o cultivo celular de diversas especies de HAdV. Se analizaron los serotipos 2, 5, 7, 35, 40 y 42 en concentraciones que oscilaban entre 102 y 108 partículas víricas/ml y no se detectó señal fluorogénica. De Ia misma manera, se analizaron muestras fecales bovinas en las que se habían detectado BAdV y que contenían los serotipos A y I y cepas con 82% de homología con el BAdV 2 en concentraciones que oscilaban entre 101 y 104 gc/g de heces. No se detectaron falsos positivos causados por reacciones cruzadas entre DNA de los virus que infectaban los diversos huéspedes estudiados. Sensibilidad del ensayoThe specificity of the oligonucleotides was verified using the Blast Search tool. In addition, the set of primers / probe was tested with serum samples or cell culture of various species of HAdV. Serotypes 2, 5, 7, 35, 40 and 42 were analyzed at concentrations ranging from 10 2 to 10 8 viral particles / ml and no fluorogenic signal was detected. In the same way, bovine fecal samples were analyzed in which BAdV had been detected and which contained serotypes A and I and strains with 82% homology with BAdV 2 in concentrations ranging between 10 1 and 10 4 gc / g of stool No false positives caused by cross reactions between DNAs of viruses that infected the various hosts studied were detected. Test sensitivity
El ensayo fue capaz de detectar de 1 a 10 copias genómicas por tubo de reacción. De acuerdo con los volúmenes iniciales de agua y las cantidades de materia fecal analizada, las cuantificaciones correspondían a 4.2 mi de agua residual, 1 I de agua del río Ter, y 0.1 g de heces. La sensibilidad fue estable incluso cuando se añadían grandes cantidades de DNA vírico heterólogo pero relacionado a los tubos de reacción: Ia adición de 108 copias de HAdV tipo 40 no afecto Ia detección del DNA de PAdV.The assay was able to detect 1 to 10 genomic copies per reaction tube. According to the initial volumes of water and the amounts of fecal matter analyzed, the quantifications corresponded to 4.2 ml of wastewater, 1 I of water from the Ter River, and 0.1 g of feces. The sensitivity was stable even when large amounts of heterologous viral DNA were added but related to the reaction tubes: the addition of 10 8 copies of type 40 HAdV did not affect the detection of the PAdV DNA.
Cuando se analizaron muestras medioambientales, se asumió Ia presencia de sustancias inhibitorias de PCR en las reacciones puesto que Ia mayoría de muestras no diluidas no mostró señal fluorogénica. Para proporcionar una preparación de ácidos nucleicos limpia, se compararon el Nucleospin® NA extraction kit y el método casero de extracción de ácidos nucleicos basado en partículas de sílice y isotiocianato de guanidino mediante el análisis de lodos porcinos y aguas residuales urbanas y de matadero en las que se habían añadido cantidades conocidas de DNA viral (PAdV estándar). Cuando se usó el método casero de extracción, no se identificó DNA de PAdV en Ia muestra no diluida y las cantidades añadidas de DNA se detectaron tan solo en las diluciones 1 :10 e incluso 1 :100. (FIG. 2). Contrariamente, el kit Nucleospin® detectó cantidades mayores de DNA en Ia muestra no diluida de lodos porcinos y de agua residual de matadero y en Ia dilución 1 :10 de agua residual urbana.When environmental samples were analyzed, the presence of PCR inhibitory substances in the reactions was assumed since the majority of undiluted samples showed no fluorogenic signal. To provide a clean nucleic acid preparation, the Nucleospin ® NA extraction kit and the homemade nucleic acid extraction method based on silica and guanidino isothiocyanate particles were compared by analysis of porcine sludge and urban and slaughterhouse wastewater in the that known amounts of viral DNA (standard PAdV) had been added. When the home extraction method was used, no PAdV DNA was identified in the undiluted sample and the added amounts of DNA were detected only in dilutions 1: 10 and even 1: 100. (FIG. 2). On the contrary, the Nucleospin ® kit detected greater amounts of DNA in the undiluted sample of pig sludge and slaughterhouse wastewater and in the 1: 10 dilution of urban wastewater.
Las muestras analizadas por qPCR se analizaron también por nPCR y los resultados confirmaron que Ia especificidad y Ia sensibilidad de ambas técnicas eran equivalentes.The samples analyzed by qPCR were also analyzed by nPCR and the results confirmed that the specificity and sensitivity of both techniques were equivalent.
PAdV en heces porcinasPAdV in pig feces
Los datos cuantitativos de PAdV obtenidos de heces porcinas y muestras medioambientales se muestran en Ia Tabla 2. Diecisiete de las veintiuna muestras agrupadas de heces porcinas recogidas en el País Vasco fueron positivas por qPCR. No se observaron diferencias significativas entre animales de engorde y de cría o en función de Ia edad de los animales. Se detectó PAdV en muestras fecales porcinas en altas concentraciones y alta prevalencia: 76.4% muestras agrupadas positivas, concentración media de 5.58x105 gc/g. Los resultados observados se confirmaron aplicando nPCR. Se observaron discrepancias entre los 2 métodos en tan solo 2 muestras que mostraban bajas concentraciones de PAdV usando qPCR y que fueron negativas por nPCR. Sin embargo, estos resultados son esperables cuando se analizan muestras con bajas concentraciones de virus en repetidos experimentos. Se secuenciaron los amplicones de PAdV obtenidos por nPCR de una muestra positiva procedente de cada una de las 9 granjas estudiada. Las secuencias de nucleótidos observadas para todas las muestras presentaron una similitud al PAdV-3 que oscilaba entre el 93 y el 98%.The quantitative data of PAdV obtained from pig feces and environmental samples are shown in Table 2. Seventeen of the twenty-one pooled samples of pig feces collected in the Basque Country were positive for qPCR. No significant differences were observed between fattening and breeding animals or depending on the age of the animals. Be detected PAdV in swine faecal samples in high concentrations and high prevalence: 76.4% positive pooled samples, average concentration of 5.58x10 5 gc / g. The observed results were confirmed by applying nPCR. Discrepancies were observed between the 2 methods in only 2 samples that showed low concentrations of PAdV using qPCR and were negative for nPCR. However, these results are expected when samples with low virus concentrations are analyzed in repeated experiments. PAdV amplicons obtained by nPCR from a positive sample from each of the 9 farms studied were sequenced. The nucleotide sequences observed for all samples showed a similarity to PAdV-3 that ranged from 93 to 98%.
El ensayo de qPCR se uso también para cuantificar PAdV en diecisiete muestras agrupadas de heces porcinas recogidas en 9 granjas de Cataluña que habían sido analizadas previamente por nPCR así como secuenciadas mostrando una alta similitud con cepas de PAdV-3 (cfr. Maluquer de Motes et al., supra APPI. Environ. Microbiol. 2004). Todas las muestras fueron también positivas por qPCR, mostrando una concentración media de PAdV de 7.25x105 gc/g, un valor equivalente a los resultados observados en muestras fecales recogidas en el País Vasco (5.58x105 gc/g).The qPCR assay was also used to quantify PAdV in seventeen pooled samples of pig feces collected in 9 farms in Catalonia that had been previously analyzed by nPCR as well as sequenced showing high similarity with PAdV-3 strains (cf. Maluquer de Motes et al., supra APPI. Environ. Microbiol. 2004). All samples were also positive for qPCR, showing an average concentration of PAdV of 7.25x10 5 gc / g, a value equivalent to the results observed in faecal samples collected in the Basque Country (5.58x10 5 gc / g).
PAdV en agua residual urbanaPAdV in urban wastewater
La planta de tratamiento de agua residual seleccionada para el estudio trata agua residual urbana procedente de una gran población y no se han descrito actividades agrícolas o granjeras en Ia zona. Consistentemente con ello, no se detectaron PAdV en las muestras analizadas mientras que se identificaron HAdV en todas las muestras con valores medios de 2.7x103 gc/ml.The wastewater treatment plant selected for the study treats urban wastewater from a large population and no agricultural or agricultural activities have been described in the area. Consistent with this, no PAdV was detected in the samples analyzed while HAdV was identified in all samples with average values of 2.7x10 3 gc / ml.
PAdV en aguas de ríoPAdV in river waters
En todas las aguas del río Ter existió contaminación fecal de origen humano y porcino, con mayores valores para HAdV (102 gc/l) que para PAdV, el cual estuvo presente en una concentración aproximada de 101 gc/l de agua. In all waters of the Ter River there was fecal contamination of human and porcine origin, with higher values for HAdV (10 2 gc / l) than for PAdV, which was present in an approximate concentration of 10 1 gc / l of water.

Claims

REIVINDICACIONES
1. Método para Ia detección y/o cuantificación de adenovirus porcinos (PAdV) en una muestra, dicho método comprendiendo: a) Ia extracción del ácido nucleico de PAdV de Ia muestra; b) Ia amplificación por reacción en cadena de Ia polimerasa del ácido nucleico extraído en presencia de al menos un cebador seleccionado entre SEQ ID NO: 1 o una variante funcional del mismo, SEQ ID NO: 2 o una variante funcional del mismo, y SEQ ID NO: 3 o una variante funcional del mismo, o sus cadenas complementarias; y c) Ia detección y/o cuantificación de PAdV evaluando el resultado de Ia reacción en cadena de Ia polimerasa.1. Method for the detection and / or quantification of porcine adenoviruses (PAdV) in a sample, said method comprising: a) the extraction of the PAdV nucleic acid from the sample; b) the amplification by chain reaction of the polymerase of the nucleic acid extracted in the presence of at least one primer selected from SEQ ID NO: 1 or a functional variant thereof, SEQ ID NO: 2 or a functional variant thereof, and SEQ ID NO: 3 or a functional variant thereof, or its complementary chains; and c) the detection and / or quantification of PAdV evaluating the result of the polymerase chain reaction.
2. Método según Ia reivindicación 1 , donde Ia reacción en cadena de Ia polimerasa es una reacción en cadena de Ia polimerasa cuantitativa (qPCR).2. Method according to claim 1, wherein the polymerase chain reaction is a quantitative polymerase chain reaction (qPCR).
3. Método según Ia reivindicación 2, donde Ia qPCR es en presencia de una sonda específica para Ia secuencia del genoma de PAdV.3. Method according to claim 2, wherein the qPCR is in the presence of a specific probe for the PAdV genome sequence.
4. Método según Ia reivindicación 3, donde Ia sonda comprende Ia secuencia identificada como SEQ ID NO: 3 o una variante funcional de Ia misma, o su secuencia complementaria.4. Method according to claim 3, wherein the probe comprises the sequence identified as SEQ ID NO: 3 or a functional variant thereof, or its complementary sequence.
5. Método según Ia reivindicación 4, donde Ia SEQ ID NO: 3 o su cadena complementaria lleva un grupo fluoróforo y un agente bloqueador.5. Method according to claim 4, wherein SEQ ID NO: 3 or its complementary chain carries a fluorophore group and a blocking agent.
6. Método según Ia reivindicación 1 , donde Ia evaluación comprende una comparación de Ia cuantificación del resultado de Ia reacción en cadena de polimerasa con al menos una muestra control.6. Method according to claim 1, wherein the evaluation comprises a comparison of the quantification of the result of the polymerase chain reaction with at least one control sample.
7. Método según Ia reivindicación 6, donde Ia muestra control comprende un control positivo tomado de al menos una curva estándar generada por una reacción en cadena de Ia polimerasa en presencia de los cebadores correspondientes a SEQ ID NO: 1 y SEQ ID NO: 2, o sus secuencias complementarias, en una secuencia de 612 pb del gen del hexon de PAdV-3 a diferentes concentraciones. 7. Method according to claim 6, wherein the control sample comprises a positive control taken from at least one standard curve generated by a polymerase chain reaction in the presence of the primers corresponding to SEQ ID NO: 1 and SEQ ID NO: 2 , or their complementary sequences, in a 612 bp sequence of the PAdV-3 hexon gene at different concentrations.
8. Método según Ia reivindicación 1 , donde Ia reacción en cadena de Ia polimerasa es una reacción en cadena de Ia polimerasa semi-anidada en presencia de uno de los cebadores identificados como SEQ ID NO: 1 o SEQ ID NO: 2 o una variante funcional de los mismos, y de Ia secuencia SEQ ID NO: 3 o una variante funcional de Ia misma, o sus secuencias complementarias.8. Method according to claim 1, wherein the polymerase chain reaction is a semi-nested polymerase chain reaction in the presence of one of the primers identified as SEQ ID NO: 1 or SEQ ID NO: 2 or a variant functional thereof, and the sequence SEQ ID NO: 3 or a functional variant thereof, or its complementary sequences.
9. Método según cualquiera de las reivindicaciones 1 a 8, donde Ia muestra es una muestra medioambiental.9. Method according to any of claims 1 to 8, wherein the sample is an environmental sample.
10. Método según cualquiera de las reivindicaciones 1 a 9, donde Ia muestra es una muestra de agua.10. Method according to any of claims 1 to 9, wherein the sample is a water sample.
11. Método según Ia reivindicación 10, donde Ia muestra es una muestra de agua residual.11. Method according to claim 10, wherein the sample is a sample of wastewater.
12. Método según cualquiera de las reivindicaciones 1 a 8, donde Ia muestra es una muestra de residuos de matadero.12. Method according to any of claims 1 to 8, wherein the sample is a sample of slaughterhouse waste.
13. Método según cualquiera de las reivindicaciones 1 a 8, donde la muestra es una muestra de alimentos.13. Method according to any of claims 1 to 8, wherein the sample is a food sample.
14. Método según cualquiera de las reivindicaciones 1 a 8, donde la muestra una muestra de fertilizante.14. Method according to any of claims 1 to 8, wherein the sample shows a fertilizer sample.
15. Oligonucleótidos identificados por una secuencia de ácido nucleico seleccionada del grupo consistente en SEQ ID NO: 1 , SEQ ID NO: 2 y SEQ ID NO: 3, y sus secuencias complementarias.15. Oligonucleotides identified by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and their complementary sequences.
16. Uso de los oligonucleótidos identificados como SEQ ID NO: 1 y SEQ ID NO: 2 o sus secuencias complementarias como cebadores específicos para el genoma de PAdV, y del oligonucleótido identificado como SEQ ID NO: 3 o su secuencia complementaria como sonda específica para Ia secuencia del genoma de PAdV.16. Use of the oligonucleotides identified as SEQ ID NO: 1 and SEQ ID NO: 2 or their complementary sequences as specific primers for the PAdV genome, and the oligonucleotide identified as SEQ ID NO: 3 or its complementary sequence as a specific probe for The PAdV genome sequence.
17. Uso de los cebadores y Ia sonda según Ia reivindicación 16, o una variante funcional de los mismos, para Ia detección de PAdV usando Ia reacción en cadena de Ia polimerasa.17. Use of the primers and the probe according to claim 16, or a functional variant thereof, for the detection of PAdV using Ia polymerase chain reaction.
18. Kit para Ia realización del método definido en Ia reivindicación 1 , que comprende reactivos adecuados para Ia detección y/o cuantificación de PAdV en una muestra.18. Kit for carrying out the method defined in claim 1, comprising reagents suitable for the detection and / or quantification of PAdV in a sample.
19. Kit según Ia reivindicación 18, que comprende al menos uno de los cebadores identificados como SEQ ID NO: 1 y SEQ ID NO: 2 y/o Ia sonda identificada como SEQ ID NO: 3, o sus secuencias complementarias. 19. Kit according to claim 18, comprising at least one of the primers identified as SEQ ID NO: 1 and SEQ ID NO: 2 and / or the probe identified as SEQ ID NO: 3, or its complementary sequences.
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