WO2011061373A2

WO2011061373A2 - Quantification of bovine polyomavirus (bpyv) for tracing bovine contamination in the environment

Info

Publication number: WO2011061373A2
Application number: PCT/ES2010/070737
Authority: WO
Inventors: Rosa GIRONÈS LLOP; Ayalkibet Hundesa Gonfa; Sílvia HUNDESA GONFA
Original assignee: Universidad De Barcelona
Priority date: 2009-11-20
Filing date: 2010-11-17
Publication date: 2011-05-26
Also published as: WO2011061373A3; WO2011061373A8

Abstract

A quantitative PCR assay is provided specific for the bovine polyomavirus having the objective of determining the levels of excretion and the concentration of bovine polyomaviruses in urine and environmental samples, including urban waste water, slaughterhouse waste water and river water. The assay is specific and did not show positive results when samples were analysed containing contamination of human or porcine origin. The assay provides a quantitative method for tracing the origin of contamination for the analysis of bovine polyomaviruses as indicators of the presence of bovine contamination in environmental samples.

Description

Quantification of bovine polyomavirus (BPvV) to track bovine contamination in the environment

The invention describes a method for detecting bovine polyomavirus (BPyV) in water or food samples and in other types of samples.

environmental.

STATE OF THE TECHNIQUE

Contamination of fecal / urinary origin in aquatic systems may pose a risk to human health due to the consumption of contaminated food or water or its use for recreational purposes. In addition, this fact implies significant economic losses as well as significant changes in natural ecosystems. Identification and tracing of the origin of fecal / urinary contamination is necessary to guarantee water quality, assess possible health risks and determine appropriate remediation strategies.

Several pollution tracking techniques have been developed

microbial ("Microbial Source Tracking", MST) to discriminate between origins of fecal / urinary contamination of human and non-human origin and to distinguish pollution caused by different animal species. These methodologies are based on the detection of a microorganism present in the feces thus indicating the presence of contamination and, by

consequently, of the potential pathogens such as bacteria, viruses and parasites. Other methodologies are based on the detection of certain chemical products that may be associated with excretions of human and non-human origin (eg caffeine, sterols and fecal tin). Among the most widely used methods, the detection of Bacteroidales RNAr 16S type markers and the genotyping of bacteriophages of F-specific RNAs stand out.

In order to provide more adequate standards to guarantee water quality, the detection of enteric viruses has led to

promising tool due to the high specificity of the host, the stability in different environments and the prevalence of these viruses in different geographical areas. Techniques based on the use of polymerase chain reaction (PCR) have been conventionally applied to assess the presence or absence of genomic copies of viruses

Pollution indicators However, the recent development of real-time quantitative PCR (qPCR) techniques allows detection and quantification with high specificity and small sensitivity

target molecule concentrations.

Adenoviruses and polyomaviruses constitute two different families of DNA genome viruses that are excreted in high concentrations by the human population and by animals. The detection of some viruses of these families has proven to be a promising and useful tool to track the origin of contamination in environmental samples.

In a previous study, (Hundesa, A. 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) a preliminary analysis of the prevalence in the environment and the potential applicability of human and animal adenoviruses and polyomaviruses as tracking tools was carried out. Detection by PCR (nested PCR) designed to specifically detect human adenoviruses ("human adenovirus", HAdV), porcine adenovirus ("porcine adenovirus", PAdV), bovine adenovirus ("bovine adenovirus", BAdV) and bovine polyomavirus ("bovine" polyomavirus ", BPyV) was applied in samples

environmental. PAdV and BPyV were detected in a high percentage of samples potentially affected by fecal contamination of porcine or bovine origin, respectively. For the detection of BPyV, two different sets of primers were used that amplified two different regions of the genome: primers based on the VP1 region designed by Wang et al., ("Detection and molecular characterization of bovine polyomavirus in bovine sera in New Zealand ", New Zealand Veterinary Journal 2005, vol. 53, pp. 26-30) and other primers designed to amplify an agnoprotein region. VP1-based primers amplified a large region of the BPyV genome. On the other hand, previous studies have demonstrated the applicability of HAdV and JC polyomavirus ("JC polyomavirus", JCPyV) as markers of human fecal contamination. Like the use of HAdV and JCPyV in the tracking of human contamination, PAdVs have been described as useful tools for detecting swine fecal contamination. The PAdV were initially proposed as indicators of fecal contamination in the environment together with the BAdV. However, the

Applicability of BAdV has been hindered by the difficulty in designing molecular methods sufficiently sensitive due to their high genetic diversity and, probably as a consequence, their low prevalence detected by molecular methods.

A nested PCR assay has been proposed to detect bovine enterovirus ("BEV") as a potential tool to track bovine fecal contamination (Fong TT et al., "Molecular assays targeting human and enteric viruses in coastal waters and their application for library- independent source tracking "Applied and Environmental Microbioloqy 2005, vol. 71, pp. 2070-8). However, enteroviruses have a seasonal distribution in environmental samples being more prevalent in the autumn-winter months, which represents an important drawback for their use as indicators. In addition, they are RNA viruses, which makes their quantification in environmental samples by PCR much more expensive and less reliable than the quantification of DNA viruses due to the extra retranscription step necessary prior to cDNA amplification.

Bovine polyomavirus (BPyV) belongs to the genus Polvomavirus of the family Polvomaviridae. The first isolated BPyV type virus was described as a contaminant of macaque kidney cell cultures. International studies of hundreds of sera derived from cattle from different groups of animals showed that about 60% of the sera had anti-BPyV antibodies. The importance of this virus cannot be underestimated in the pharmaceutical industry since the presence of the virus in serum batches represents a high risk of contamination of therapeutic products for human and animal use.

Patent application WO 2006/94238 describes a method in which several primers useful for studying the presence of different viruses are used. (some members of the Polvomaviridae family) in samples. The primers are directed to the T antigen (TAg) region of various members of the Polvomaviridae family and once this region is amplified, it is then studied by mass spectrometry.

The comparison of currently available MST techniques using blind samples showed that there is no technique that alone can ensure a source of contamination (Field, KG et al., "A comparative study of culture-independent, library-independent genotypic methods of fecal source tracking ", J. Water Health 2003, vol. 1, pp. 181-94).

The health risk assessment associated with fecal / urinary contamination requires appropriate indicators and a rapid quantification method. Thus, there is a continuing need for new indicators and tests to quantify contamination derived from different animal origins and specifically of bovine origin.

EXPLANATION OF THE INVENTION

The present invention provides a quantitative assay for urine samples of cattle and other environmental samples for the evaluation of the concentration of a host-specific and widely excreted DNA virus, bovine polyomavirus (BPyV). In addition to the detection of BPyV in samples, the assay allows quantification.

The results described support the usefulness of the qPCR assay directed at BPyV for its quantification in bovine urine, and the applicability of BPyV as a tool in studies to trace the origin of the

contamination in a wide variety of samples from various geographical areas because the BPyV genome is highly conserved between different strains. The trial is a reliable tool for health risk analysis and facilitates the evaluation of the effect of possible interventions aimed at improving the quality of water and the environment.

Therefore, in a first aspect the present invention provides a method for the detection and / or quantification of BPyV in a sample, which it comprises: (a) the extraction of the BPyV nucleic acid from the sample; (b) at least one PCR amplification of the nucleic acid extracted in the presence of at least one primer selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and a functional variant thereof, and their complementary chains; and (c) the detection and / or quantification of BPyV evaluating the result of the PCR.

In a particular embodiment of the invention, the PCR is a quantitative PCR (qPCR), which allows quantifying a small number of genomic copies. More particularly, the qPCR is performed in the presence of a probe specific for the BPyV genome sequence. 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 sequence.

A qPCR probe consists of a nucleotide sequence that contains a fluorophore group at one end and a blocking group at the other end. The polymerase that moves, releases the probe so that fluorescence is emitted in each amplification cycle. Thus, in a particular embodiment of the invention, the probe carries a fluorophore group and a blocking agent.

The primers and the probe of the invention are specific for BPyV and do not detect other polyomaviruses described. The essay provides a

optimal specificity, sensitivity and reliability as demonstrated by the correlation coefficient, Ct values and the slope of the straight regression pattern (see section below). The specificity of the trial was tested in real scenarios and no cross reaction was observed when analyzing samples containing contamination from other sources (human and pig) or when analyzing samples without bovine contamination of urinary origin (bovine feces).

Likewise, the limit of detection of the assay (10 ° -10 ¹ GC / reaction) was constant even when high concentrations of viral DNA from various sources were added, as is the case when analyzing real samples. High specificity and sensitivity are crucial requirements to track the origin of the contamination. Contrary to what happens in the case of BAdV, BPyVs do not have a high genetic diversity. All amplicons obtained were sequenced and identities of> 98% were observed. In addition, these amplicons had an identity> 94% with BPyV sequences present in the available databases (Schuurman, R. et al., "The complete nucleotide sequence of bovine polyomavirus", J. Gen. Virol. 1990, vol. 71, pp. 1723-35). This is a clear advantage for the use of BPyV as environmental indicators.

Real-time qPCR requires a timely and automatic comparison of emitted fluorescence with standard values. Thus, a particular embodiment of the invention is the evaluation of the amount of virus, which comprises a comparison of the quantification of the PCR result 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 standard curve generated by a PCR in the presence of primers with sequence SEQ ID NO: 1 and SEQ ID NO: 2, or their complementary sequences, performed on a sequence of 77 bp of the VP1 gene of BPyV at different concentrations.

In another embodiment of the invention, the detection of BPyV in samples is carried out by semi-nested PCR in the presence of one of the oligonucleotides selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and variants functional thereof, and its complementary sequences, and in the presence of the oligonucleotide with SEQ ID NO: 3, or a functional variant thereof, or its complementary sequence.

It is envisioned that the described oligonucleotides may be subject to modifications without departing from the scope of matter provided herein. The present invention includes fragments and / or functional variants of the oligonucleotides provided herein. Fragments and / or functional variants are those that retain a similar function to be

employees in the quantification of BPyV. The similar function is characterized by the ability of the oligonucleotide to hybridize with the target region or sequence under conditions suitable for carrying out a PCR. The method of the invention is capable of detecting the virus in a wide spectrum of samples. The study of non-pathogenic BPyV as a method for tracing the origin of human and animal fecal contamination in environmental samples should provide data for the improvement of

handling and control of water quality. An embodiment of the invention applies the method in an environmental sample. Another embodiment applies the method in a water sample, and in a 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 BPyV is a sample of slaughterhouse waste, which includes samples of water and sludge. In other embodiments the samples are faecal samples, food samples, as well as commercial items such as biosolids derived from the composting industry. Another particular embodiment of the invention is the application of the method in fertilizer samples.

One aspect of the invention relates to an oligonucleotide with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and functional variants thereof, and their complementary sequences. In another aspect, the invention relates to the use of oligonucleotides identified as SEQ ID NO: 1 or SEQ ID NO: 2, or functional variants thereof, or their sequences

complementary, as specific primers for the detection of BPyV. Another aspect is related to the use of the oligonucleotide identified as SEQ ID NO: 3, or a functional variant thereof, or its sequence

complementary, as a specific probe for the detection of BPyV. A primer comprising SEQ ID NO: 1, or a functional variant thereof is preferably a direct primer. A primer comprising SEQ ID NO: 2, or a functional variant thereof is preferably a reverse primer.

In another aspect, the invention provides a kit for carrying out the method of the invention, comprising suitable reagents for this. A preferred embodiment of the invention is this kit comprising at least one oligonucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and functional variants thereof, and their sequences complementary. Unless defined otherwise, all the technical and scientific terms used here have the same meaning as those commonly understood by a person skilled in the field of the invention. Methods and materials similar or equivalent to those described herein may be used in the practice of the present invention. Throughout the description and the

claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following particular embodiments and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers all possible combinations of the particular and preferred embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amplification graphs (FIG. 1A) and the standard curve (FIG. 1 B) obtained with 1: 10 serial dilutions of the cloned viral DNA (from 10 ¹ to 10 ⁶ genomic copies in a qPCR assay) from BPyV. The amplification graphs show the log of dRn (reported fluorescence signal, "F") in relation to the number of cycles ("C"). In the standard curve, the log of the initial amount of viral DNA ("lq") is represented with respect to the threshold cycle ("Te", in Ct values), calculated as the fractional cycle number in which a significant increase in Rn exceeds a certain threshold (horizontal line), y = -3.573 LOG (x) + 43.18 (r = 0.997)

DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

Sample collection

Twenty-six urine samples were collected from different groups of 2 to 4 year old cattle from different areas of Catalonia (northeastern Spain, Mediterranean coast) and kept in sterile polyethylene containers at 4 ° C for a maximum of 8 hours before being processed. The viral particles were concentrated from 7 ml samples of urine per ultracentrifugation (1 10,000 xg, 1 h at 4 ° C). The sediments were resuspended in 100 μΙ of PBS, and stored at -80 ° C.

At the same time, ten stool samples from the same animals were collected from their rectum. The viruses present in these samples were concentrated by a procedure based on elution and subsequent ultracentrifugation of the virus (Maluquer de Motes et al., "Detection of Bovine and Porcine Adenovirus for Tracing the Source of fecal Contamination", Appl. Environ. Microbiol. 2004, vol. 70, pp. 1448-54). 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 x g for 15 min). The resulting supernatant was concentrated by ultracentrifugation (1 10,000 x g for 1 h at 4 ° C) and the viral particles were resuspended in 100 µΙ of PBS and stored at -80 ° C.

Eleven samples of wastewater from slaughterhouses were collected in Catalonia where pigs and cattle were processed, as well as horses and sheep. 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 ° C until 8 h before analysis and the viruses were concentrated from 40 ml aliquots (Puig et al., "Detection of adenoviruses and enteroviruses in polluted waters by nested-PCR

amplification ", Appl. Environ. Microbiol. 1994, vol. 60, pp. 2963-70).

Briefly, 40 ml of residual water (1 10,000 x g for 1 h at 4 ° C) was ultracentrifuged to sediment all the 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 x g for 15 min. The viruses were finally concentrated by ultracentrifugation (1 10,000 xg for 1 h at 4 ° C), resuspended in 100 μΙ of PBS, and stored at -80 ° C.

Six samples of 1 liter of river water were collected from the Ter River in an area of important activity of cattle and pig farming in Catalonia and where the treated wastewater from different municipalities is discharged. The samples were collected over a period of 3 months in 2008.

The protocol used for the processing of river water samples was a combination of the EPA method (EPA 600 / 4-84 / 013 (N14)), with

minor modifications, and the method based on ultracentrifugation and elution in 0.25 N glycine buffer (pH 9.5) (Albinana-Gimenez et al., "Distribution of polyomaviruses, adenoviruses and hepatitis E virus in the environmental and in a Drinking-Water treatment plant" , Environ. Sci. Techno. 2006, vol. 40, pp. 7416-22). One hundred liters of river water were filtered through filters

Zeta Plus MK electropositive at 1 l / min using a Millipore peristaltic pump. 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, the resulting suspension was magnetically stirred for 30 min, and finally centrifuged at 12,800 x g for 25 min at 4 ° C. The sediment material was eluted in 42 ml of PBS. After this step, the sample was treated using the previously applied protocol for virus recovery from urban wastewater.

Design of the primer / probe set for the qPCR assay for BPyV

The qPCR procedure is based on a TaqMan assay and uses two primers and a fluorogenic probe that recognizes a 77 bp fragment in the VP1 gene of the BPyV genome. BPyV DNA sequences of the GenBank were aligned using the ClustalW program (European Bioinformatics

Institute, UK) and a conserved fragment of 77 bp was selected using Primer Express software (Applied Biosystems). In order to obtain a sufficiently specific and sensitive assay, the primers and the probe were finally chosen based on the alignments with the sequences available in databases, and based on experimental data. The homology and specificity of all oligonucleotides was finally verified using a Blast search. The probe was labeled with FAM (6- carboxyfluorescein) as a fluorophore group at the 5 'end and BHQ-1 (Black-Hole Quencher 1) as a blocking agent at the 3' end. Sequence of the direct primer corresponds to SEQ ID NO: 1, the reverse primer sequence corresponds to SEQ ID NO: 2, and the probe sequence, with SEQ ID NO: 3. Its location in the BPyV genome According to access number D13942 in GenBank it is as follows:

Direct primer QB-F1 -1: between nucleotide positions 2122 and 2144. Reverse primer QB-R1 -1: between nucleotide positions 2198 and 2177. Probe QB-P1 -2: between nucleotide positions 2150 and 2172.

Different concentrations of primers (final concentrations in the reaction of 0.05 to 0.9 μΜ) and probe (final concentrations in the reaction of 0.05 to 0.25 μΜ) were tested in independent experiments and those that provided the best results as well as suitable hybridization temperatures were selected.

Construction of qPCR standards for BPyV

Standard curves were generated by transformation of E. coli JM109 cells (Promega, Madison, Wl, USA) with the plasmid pGEM-T Easy

(Promega, Madison, Wl, USA) containing a 416 bp sequence of the BP1 VP1 gene. The transformation was carried out following the

manufacturer's instructions After verification by sequencing, approximately 10 g of the DNA was linearized with Ncol, 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 μΙ in TE buffer. Standard dilutions were separated in aliquots and stored at -80 ° C until use.

Nucleic acid extraction

Nucleic acids from viral concentrates were extracted using the NucliSens ^® (Biomérieux) nucleic acid extraction kit containing silica coated magnetic beads. In previous studies this method proved to be very useful for obtaining inhibitor-free nucleic acids from environmental samples (Hundesa, A. et al., "Development of a qPCR assay for the quantification of porcine adenoviruses as an MST tool for swine fecal contamination in the environment ", J. Virol. Meth. 2009, vol. 158, pp. 130-135). The nucleic acids obtained were stored at -80 ° C until later use.

QPCR test for BPvV

The PCR mixtures were prepared and dispensed in a dedicated work area for the 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 qPCR standards. The amplifications were performed in a 25-μΙ reaction mixture containing 10 μΙ of DNA and 15 μΙ of TaqMan ^® Universal PCR Master Mix, 0.4 μΜ of each primer (QB-F1 -1 and QB-R1 -1) and 0.12 μΜ of fluorogenic probe (QB-P1 -2). The TaqMan ^® Universal PCR Master Mix was supplied in a 2x concentration and contained AmpliTaq Gold ^® DNA polymerase, dNTPs with dUTP, passive reference, optimized buffer components and AmpErase ^® uracil-N-glycosylase. After activation of uracil-N-glycosylase (2 min, 50 ° C) and activation of AmpliTaq Gold for 10 min at 95 ° C, 45 cycles were performed (15 s at 95 ° C, 30 s at 60 ° C) in an MX3000P (Stratagene) detection system.

Two dilutions (1: 10 and 1: 100) of the extracted DNA were analyzed in duplicate (4 analyzes / sample) for the analysis of environmental samples, while serial dilutions 1: 10 of the qPCR standard containing 10 were analyzed in triplicate. ¹ to 10 ⁶ copies of DNA when copies of the viral genome (GC) were quantified. In all the qPCR carried out the amount of DNA was defined as the average of the data obtained. A control without DNA template was added in each assay. Enzymatic inhibition of the samples was analyzed with various dilutions of the samples as well as by adding known amounts of target DNA to the samples.

environmental.

NPCR assay for BPvV and sequencing

Nested PCR (nPCR) assays for BPyV amplification were based on the previously described assays (Wang, J. et al., 2005 supra). The amplicons 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 primers were used for sequencing at a concentration of 0.05 μΜ. The results were analyzed using the ABI PRISM 377 automatic sequencer (Perkin-Elmer, Applied Biosystems). The sequences obtained were compared with those of GenBank and EMBL (European Molecular Biology Library) using the NCBI basic program BLAST (The National Center for Biotechnology 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/).

QPCR assay for the quantification of JCPyV and PAdV

The quantification of JCPyV and PAdV in the samples was performed using a protocol based on TaqMan ^® and the primers described by Pal, A. et al. ("Real-time PCR assays for the detection of virus-specific DNA in samples with mixed populations of polyomaviruses" J. Virol. Methods 2006, vol. 135, pp. 32-42), and Bofill-Mas, S. et al ., ("Quantification and stability of human adenoviruses and polyomavirus JCPyV in wastewater matrices", Appl.

Environ. Microbiol 2006, vol. 72, pp. 7894-6) for JCPyV, and by Hundesa et al., 2009 (supra), for PAdV.

Positive control samples

When the study was conducted, no strain of BPyV was available for use as a positive control. Consequently, the assays were optimized using the fragment obtained from both environmental and bovine urine samples that had given positive amplification by nPCR and that had subsequently been sequenced and used in phylogenetic analyzes. A 416 bp amplicon corresponding to a fragment of the gene encoding VP1 was cloned into a pGEM-T Easy vector. This vector was used as a validation control for nPCR experiments and as a standard in quantitative qPCR assays. Test specificity

The specificity of the oligonucleotides was verified by

Blast search tool. Additionally, the set of primers and probe was tested with different samples collected from areas where contamination of bovine origin was not expected: 8 samples of urban wastewater previously positive for HAdV and JCPyV by nPCR and qPCR; 5 samples of swine feces previously positive for PAdV by nPCR and qPCR, and 10 samples of pig urine, two of which had been positive for PAdV by nPCR. No fluorescent signal was observed when these samples were analyzed in independent experiments using the primer / probe set designed to quantify BPyV. In addition, 10 stool samples of bovine origin were analyzed to determine if BPyV could be excreted in bovine feces and the assay could detect the BAdV commonly present in this type of samples. No BPyV was detected in these feces.

No porcine or human viruses were detected by the qPCR assay designed for BPyV.

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 7 ml of urine, 4.2 ml of wastewater and 1 ml of river water. The sensitivity was stable even when large amounts of related heterologous viral DNA were added to the reaction tubes: the presence of 10 ⁵ copies of HAdV and JCPyV did not affect the detection of BPyV DNA. The samples analyzed by qPCR were also analyzed by nPCR as shown in TABLE 1. All bovine urine samples positive for qPCR were for nPCR. In wastewater from slaughterhouses and river water, the qPCR test showed a slightly smaller number of positive samples compared to that performed by nPCR. TABLE 1: Results obtained in the qPCR and nPCR tests for the detection of BPyV in bovine urine and in environmental samples. ^(a) the slaughterhouse wastewater samples analyzed in Catalonia were selected from previously positive samples by nPCR.

The presence of inhibitory substances in PCR assays in environmental samples is a recognized problem that was taken into account in this study. All samples were quantified by qPCR assays in duplicate of both undiluted and decimally diluted nucleic acid extracts, producing more reliable results and minimizing the effect of inhibitors potentially present in the samples. To estimate the sensitivity of the qPCR in the analyzed samples, concentrations of 1x10 ² , 1 x10 ³ and 1 x10 ⁴ plasmid copies were added to the

Nucleic acid extractions and duplicate test results were compared with purified plasmids as standards. In undiluted extractions very low levels of inhibitors were observed.

BPyV excretion levels in bovine urine

A collection of bovine urine was analyzed by qPCR in order to evaluate the excretion of BPyV by these animals. The quantitative data obtained are presented in TABLE 1. BPyV was detected in 8 of 26 urine samples collected in different farms in Catalonia at an average concentration of 2.21 x10 ⁴ GC / I (range between 3.08x10 ³ - 7.58x10 ⁴ ). They were not observed

significant differences between animals 2 and 4 years old. These results were confirmed by nPCR and only positive samples. by qPCR they were also by nPCR. The sequencing of the amplicons obtained by nPCR resulted in nucleotide sequences very similar to the BPyV sequences existing in databases presenting homologies greater than 98% between them.

Detection and quantification of BPyV in environmental samples

The wastewater treatment plant selected in this study treats urban wastewater of a large population and there is no evidence of the development of livestock activities in that area.

Consequently, BPyV was not detected in urban wastewater samples as shown in TABLE 2 where JCPyV was consistently detected in all samples. The slaughterhouse wastewater samples analyzed were obtained from slaughterhouses where cattle were processed. In a previous study, these samples had been positive for BPyV by nPCR (Hundesa, A. et al. 2006 supra). Consequently, BPyV was detected in all samples with average values of 2.95x10 ³ GC / I (range between 1.72x10 ² - 1 .60x10 ⁴ ). This concentration was similar to that observed for PAdV (mean value of 1.56x10 ³ ).

Contamination of bovine, porcine and human origin detected in samples collected from the Ter River is shown in TABLE 2. BPyV was detected in 3/6 analyzed samples (5.22x10 ¹ - 5.46x10 ² , mean value 3.06x10 ² GC / I) ; 2 samples were positive for JCPyV (2.35x10 ² and 2.79x10 ² , average value 2.57x10 ² GC / I) and 6 for PAdV (range between 2.59x10 ^or - 1 .86x10 ¹ , medium range 8.37x10 ° GC / I). Samples were collected over a period of 3 months and all viruses were detected at different sampling times.

TABLE 2. Results obtained in the qPCR tests for BPyV, PAdV and JCPyV in environmental samples. NT means "not tested."

The results show that BPyV (or PAdV) was not detected in urban wastewater where only fecal contamination of human origin (HAdV and JCPyV) was detected. In contrast, BPyV (and PAdV) was detected in all samples of slaughterhouse wastewater analyzed. These samples did not contain fecal contamination of human origin and no HAdV was detected in them. The Ter River samples were collected from a rural area with high agricultural and livestock activity and with numerous municipalities located above the sampling point. Consequently, fecal contamination of both animal and human origin was detected in these samples.

Claims

one . Method for the detection and / or quantification of bovine polyomavirus

("bovine polyomavirus", BPyV) in a sample, comprising:

a) the extraction of the BPyV nucleic acid from the sample;

b) at least one polymerase chain reaction (PCR) amplification of the nucleic acid extracted in the presence of at least one primer selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 , and functional variants thereof, and their complementary chains; Y

c) the detection and / or quantification of BPyV evaluating the result of the PCR.

2. Method according to claim 1, wherein the PCR is a quantitative PCR (qPCR).

3. Method according to claim 2, wherein the qPCR is performed in the presence of a specific probe for the BPyV genome sequence.

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. Method according to claim 4, wherein the probe carries a fluorophore group and a blocking agent.

6. Method according to claim 1, wherein the evaluation of step (c) comprises a comparison of the quantification of the PCR result with at least one control sample.

7. Method according to claim 6, wherein the control sample comprises a positive control taken from at least one standard curve generated by a PCR in the presence of primers with sequence SEQ ID NO: 1 and SEQ ID NO: 2, or their complementary sequences , performed on a sequence of 77 bp of the VP1 gene of BPyV at different concentrations.

8. Method according to claim 1, wherein the PCR is a semi-nested PCR in the presence of one of the oligonucleotides selected from the group that consists of SEQ ID NO: 1 and SEQ ID NO: 2, and functional vacancies thereof, and their complementary sequences, and in the presence of

oligonucleotide with sequence SEQ ID NO: 3, or a functional variant thereof, or its complementary sequence.

9. Method according to any of claims 1-8, wherein the sample is an environmental sample.

10. Method according to any of claims 1-9, wherein the sample is a water sample.

eleven . Method according to claim 10, wherein the sample is a sample of wastewater.

12. Method according to any of claims 1-8, wherein the sample is a sample of slaughterhouse waste.

13. Method according to any of claims 1-8, wherein the sample is a food sample.

14. Method according to any of claims 1 to 8, wherein the sample shows a fertilizer sample.

15. Oligonucleotide with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and functional variants thereof, and their complementary sequences.

16. Oligonucleotide according to claim 15, selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

17. Use of oligonucleotides with sequence SEQ ID NO: 1 or SEQ ID NO: 2, or functional variants thereof, or their complementary sequences, as specific primers for the detection of BPyV.

18. Use of the oligonucleotide with sequence SEQ ID NO: 3, or a functional variant thereof, or its complementary sequence, as a specific probe for the detection of BPyV.

19. Kit for carrying out a method as defined in any of claims 1-14, comprising at least one oligonucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and functional variants thereof, and their complementary sequences.