ES2370432B1 - PROCEDURE, SET OF PRIMERS AND KIT FOR DETECTION OF INFECTIOUS PANCREATIC NECROSIS VIRUS (IPNV) IN ASYMPTOMATIC FISH. - Google Patents

Abstract

Procedure, set of primers and kit for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish, using the combination of RT-PCR, nested RT-PCR and dot-blot hybridization. Specific primers designed to amplify a 599 base pair fragment of the VP2 precursor region have been designed for RT-PCR. For the nested RT-PCR, internal primers that amplify a 191 bp fragment have been designed. For hybridization, a double-stranded DNA probe labeled with digoxigenin has been designed from the purified products of nested RT-PCR. Optimum conditions have been established for the different RNA amplification steps, as well as for dot-blot hybridization. The invention allows detecting this virus in fish tissues, including blood samples, quickly, reliably and effectively. The procedure described is applicable in the aquaculture sector.

Description

Procedure, set of primers and kit for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish.

Technical sector

The present invention relates to a set of primers, method and kit for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish, more specifically to the application of a non-destructive or bloody procedure comprising the combination of RT- PCR, nested RT-PCR, and dot blot hybridization; to the primers used in said procedure; and to the kit that allows performing said procedure. The present invention is applicable in the aquaculture sector, and more particularly in the control of the state of health of fish, both natural and cultivated populations.

State of the art

Infectious pancreatic necrosis disease (IPN) is an acute, highly contagious viral disease with a very wide geographic distribution that affects farmed and wild fish, both freshwater and marine environments, as well as diadromous species. This disease, which can occur as epizootic outbreaks reaching a high morbidity and mortality rate, has been described in the five continents, being more frequent in Europe, in the Far East, and in North and South America (Rodríguez et al., 2003 Adv. Virus Res., 62: 113-165).

The causative agent of this disease has been called IPNV, a virus belonging to the genus Aquabirnavirus, family Birnaviridae. This virus has a naked icosahedral virion with two segments of double stranded RNA as genetic material. The main proteins of the nucleocapsid are VP2 (major protein) and VP3 (internal capsid protein) (Dobos, 1995. Ann. Rev. Dis., 5: 25-54). The use of immunological techniques has demonstrated the existence of various serogroups and serotypes within the IPNV, although these techniques do not typify isolated marine fish origins (Riji John and Richards, 1999. J. Gen. Virol., 80: 2061-2065) . For these reasons, molecular techniques have recently been applied for the classification of aquabirnaviruses (Blake et al., 2001. Dis. Aquat. Org., 45: 89-102). Six genoprupos (I to VI) comprising 10 genotypes (I.1, I.2, II, III.1, II.2, IV.1, IV.2, V.1, V.2 and VI) have been established based on restriction analysis (RFLP) and sequencing of the gene encoding the virus capsid VP2 protein (Cutrin et al., 2004. Appl. Environ. Microbiol., 70: 1059-1067). Subsequently, a seventh genogroup (VII) has been established based on the sequence of the binding region between the VP2 and VP4 genes (Nishizawa et al., 2005. J. Gen. Virol., 86: 1973-1978).

IPNV causes clinical infections in fry and juveniles of wild and farmed fish, and survivors of the infection may become asymptomatic carriers of the virus (Imajoh et al., 2005. Fish Shell fi sh Immunol., 18: 163-177). The standard analysis method recommended by the World Organization for Animal Health (OIE) in 2003 is based on the following points:

1. Isolation of IPNV in cell culture from the tissues of diseased fish. Identification of the isolated IPNV in cell culture, by neutralization with specific antibodies or by indirect immuno fl uorescence

or by an enzyme immunoassay.

2. Direct detection of IPNV in fish tissues affected by indirect immuno fl uorescence, enzyme immunoassay, coagglutination or immunohistochemistry.

As can be seen, the recommended method involves tissue destruction and death of the analyzed fish, and much time is required for the analyzes. There is a demand, by companies in the Aquaculture sector, for the development of rapid and effective systems for the detection of IPNV and to avoid the slaughter of fish in order to establish a plan for sanitation of non-transparent carriers.

The most effective systems for detecting microorganisms by molecular methods, which do not require their culture, are based on the polymerase chain reaction (PCR) (Mullis and Faloona, 1987. Meth. Enzymol. 155: 335350). The present invention refers to a protocol based on PCR techniques for the detection of the IPNV virus, as well as the primers specific for it. In addition, it is combined with a dot-blot hybridization protocol, using a double-stranded DNA probe (cDNA), to increase the sensitivity of the technique and as a tool for the confirmation of amplification products. This method, being non-destructive, would prevent the consequent valuable loss of fish culture, such as breeding specimens.

Detailed description of the invention

The technical problem posed is to achieve a fast and reliable method that allows the specific detection of IPNV in fish blood samples. The proposed non-destructive procedure is based on the application of two molecular techniques, a nested RT-PCR and a dot-blot hybridization for the detection of IPNV from the blood of non-transparent fish carrying the virus. For RT-PCR, specific primers have been designed that amplify a 599 base pair (bp) fragment of the VP2 precursor region (majority viral capsid protein) within the viral gene encoding polyprotein. For the nested RT-PCR, internal primers have been designed that amplify a 191 bp fragment. Through this technique we have been able to detect IPNV in less than a day's work and without the need for cell cultures of the samples. From this 191 bp ampli fi cation product, a digoxigenin-labeled cDNA probe has been designed to hybridize the products obtained by the RT-PCR and nested RT-PCR reactions in dot-blot.

Thus, an object of the present invention is a method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish, including blood samples, comprising the following steps:

to.

Extraction and purification of the total RNA from the samples.

b.

RT-PCR ampli fi cation of a 599 bp sequence of the VP2 protein precursor region using a set of primers in which at least two of the primers of said set have sequences comprising SEQ ID NO 1 and SEQ ID NO 2 Preferably, said set is formed by a first primer whose sequence comprises SEQ ID NO 1 and a second primer whose sequence comprises SEQ ID NO 2. More preferably, the nucleotide sequence of the first primer is identical to SEQ ID NO1 and the nucleotide sequence of the second primer is identical to SEQ ID NO 2.

C.

Amplification of a 191 bp fragment of the products resulting from the first amplification by nested RT-PCR using a set of primers in which at least two of the primers of said set have sequences comprising SEQ ID NO 3 and SEQ ID NO 4 Preferably, said assembly is formed by a first primer whose sequence comprises SEQ ID NO 3 and a second primer whose sequence comprises SEQ ID NO 4. More preferably, the nucleotide sequence of the first primer is identical to SEQ ID NO 3 and The nucleotide sequence of the second primer is identical to SEQ ID NO 4.

d.

Obtaining double-stranded DNA probes from the products resulting from the second amplification (nested RT-PCR).

and.

Dot-blot hybridization of the products resulting from the second ampli fi cation (nested RT-PCR) using the double stranded DNA probes obtained in step (d).

F.

Detection of hybridized products by immunoassay.

Another set of the present invention are the sets of primers referred to above and used in ampli fi cations by means of RT-PCR and nested RT-PCR.

Another object of the present invention is a kit for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish, including blood samples, comprising at least a few sets of primers as described above.

Preferably the kit comprises:

to.

Set of primers for RT-PCR amplification of a 599 bp sequence of the VP2 protein precursor region in which at least two of the primers of said set have sequences comprising SEQ ID NO 1 and SEQ ID NO 2 ; preferably, said set is formed by a first primer whose sequence comprises SEQ ID NO 1 and a second primer whose sequence comprises SEQ ID NO 2; more preferably, the nucleotide sequence of the first primer is identical to SEQ ID NO 1 and the nucleotide sequence of the second primer is identical to SEQ ID NO 2;

b.

Set of primers for the amplification of a 191 bp fragment of the products resulting from the first amplification by nested RT-PCR in which at least two of the primers of said set have sequences comprising SEQ ID NO 3 and SEQ ID NO 4 ; preferably, said set is formed by a first primer whose sequence comprises SEQ ID NO 3 and by a second primer whose sequence comprises SEQ ID NO 4; more preferably, the nucleotide sequence of the first primer is identical to SEQ ID NO 3 and the nucleotide sequence of the second primer is identical to SEQ ID NO 4; Y

C.

a control sample for the detection of infectious pancreatic necrosis virus (IPNV).

Description of the drawings

Figure 1.-Alignment of the sequence of the products of the RT-PCR and RT-PCR nested with published sequences of isolates of IPNV: IPNV Ab (L40580), IPNV VR-299 (L40584), solevirus (EF493156), IPNV Sp (AF342728). The location of the primers designed for RT-PCR (SoleSegA-F2 and SoleSegA-R2) and the location of the primers for nested RT-PCR (R23 and R24) are indicated.

Figure 2.-Speci fi city of RT-PCR (A) and nested RT-PCR (B) for the detection of IPNV. M: 100 bp XIV molecular marker (Roche); 1: negative control, water-DEPC; 2: VNNV; 3: VHSV; 4: negative control, RNA from non-inoculated BF-2 cells; 5: IPNV Ab; 6: solevirus; 7: IPNV Sp; 8: IPNV VR-299.

Figure 3.-Specific detection of different IPNV genotypes by dot-blot hybridization technique from the products of (A) RT-PCR and (B) nested RT-PCR. C1 and C2 are negative controls consisting of the PCR and RNA reagent extracted from uninoculated BF-2 cells, respectively.

Figure 4. Percentages of detection of IPNV from blood samples of asymptomatic specimens of letters and snappers. The samples were analyzed by nested RT-PCR and combination of nested RT-PCR and dot-blot hybridization.

Embodiments of the invention

An embodiment of the invention will be described in detail below and without limitation.

Extraction and purification of total RNA from samples

A commercial kit (QIAamp® RNA Blood Mini Kit, QIAGEN) was used to extract the total RNA.

Detailed description of the PCR protocol

The primers used were designed with the help of the OLIGO 6.7.1 software. The information of the primers is summarized in Table 1. For the specific detection of IPNV, the pair of external primers SoleSegA-F2 and SoleSegA-R2 were designed, which are located at positions 832 and 1431, respectively, of the segment sequence. A of IPNV Sp (genotype III. 1, GenBank accession number AF342728) (Fig. 1). These primers amplify a 599 bp fragment of the gene that encodes the polyprotein, specifically in the sequence corresponding to the VP2 protein precursor (pVP2). The internal primers, R23 and R24, were designed according to the sequence of the segment A of the solevirus (GenBank accession no. EF493156), and generate an amplicon of 191 bp.

RT-PCR is performed in one step using the SuperScriptTM III One-Step RT-PCR System Platinum® Taq DNA polymerase (Invitrogen). The reaction mixture (50 μl, final volume) contains 25 μl of 2x Reaction Mix [0.2 mM deoxynucleotides (dNTPs), 1.6 mM MgSO4], 0.45 pmol / μl of each primer, and 2 μl of the SuperScriptTM III RT / Platinum Taq Mix enzyme. 1 μg of virus RNA is used as template. The negative controls used were total RNA extracted from non-inoculated BF-2 cells, and water treated with diethyl pyrocarbonate (DEPC).

TABLE 1

Primers designed for the specific detection of IPNV by the technique of RT-PCR and nested RT-PCR

The RT-PCR reactions consisted of a first step of cDNA synthesis at 45 ° C for 30 min, followed by a denaturation at 94 ° C for 2 min. Next, 35 cycles of amplification were carried out consisting of a denaturation at 94 ° C for 15 s, banding at 58 ° C for 30 s and extension at 68 ° C for 30 s. Finally, an extension cycle was performed at 68 ° C for 10 min.

To carry out nested RT-PCR reactions, 2 μl of the products resulting from the first ampli fi cation were used as a template. The reactions were carried out in a mixture of 50 μl (final volume) containing: 5 μl of 0.2 mM dNTPs (Roche); 1x buffer (10 mM Tris HC1, 1.5 mM MgCl2, 50 mM KC1, pH 8.3 20 ° C); 0.3 pmol / μl of each primer, and 2.5 U of the enzyme Taq DNA Polymerase (Roche). The second ampli fi cations consisted of an initial phase of denaturation at 94 ° C for 5 min, followed by 35 cycles composed of a denaturation at 94 ° C for 30 s, banding at 58 ° C for 30 s, and extension at 72 ° C for 30 s. The final extension was performed at 72 ° C for 5 min.

The amplification products were visualized by electrophoresis in 2% agarose gels stained with ethidium bromide. The molecular marker used was 100 bp DNA MWXIV (Roche). The confirmation of the amplification products was carried out by direct sequencing thereof, using the ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) and the specific primers of each reaction.

In order to evaluate the specificity of the techniques developed for the detection of IPNV, ampli fi cations were made in which total RNA extracted from cell cultures inoculated with isolates belonging to different IPNV genogroups was used, as well as cell cultures inoculated with VHSV viruses. (viral haemorrhagic septicemia virus) and VNNV genotype SJNNV (viral nerve necrosis virus). The results are specified in Fig. 2.

For the determination of the sensitivity limit, the IPNV titer was determined, for this purpose the total RNA was extracted from the titrated viral lysate, and batteries of serial dilutions were used that were used as template in various RT-PCR reactions. The sensitivity limit of the technique varies from 10-100 Plate Forming Units (PFU) / ml for RT-PCR and from 1-10 PFU / ml for nested RT-PCR.

Obtaining specific DNA probes

The products resulting from ampli fi cation by nested RT-PCR of solevirus RNA were cloned into the vector pCR®4-TOPO® and subsequently sequenced. The mapping of the probes with digoxigenin (DIG) was carried out by PCR reactions using these plasmids as a template in amplification reactions containing DIG-11-dUTP (Roche).

Said PCR reactions were performed in a final volume of 100 μl. The reaction mixture is composed of 10 μl of the DIG Labeling Mix (Roche) PCR solution (dATP, dCTP, 0.2 mM dGTP; 0.19 mM dTTP; 0.01 mM DIG-11-dUTP), 0.5x Colorless GoTaq® Flexi Buffer, 4.5 mM MgCl2, 0.15 pmol / μl of the specific internal primers (Table 1), 2.5 U of GoTaq® DNA Polymerase (Promega), and 0.5 μg of plasmid DNA.

The amplification profile is constituted by a cycle of denaturation at 95 ° C for 2 min, followed by 35 cycles consisting of a denaturation at 95 ° C for 1 min, 1 min of banding at 53 ° C and 10 min of extension at 72 ° C. Finally, an extension of 10 min at 72 ° C was carried out and the reactions were maintained for 5 min at 25 ° C.

The ampli fi cation products were visualized on 2% agarose gels stained with ethidium bromide, and were used as probes without further purification. The labeled probes were stored at -20 until use.

Dot blot hybridization

A dot-blot hybridization method was developed using double-stranded DNA probes (cDNA) obtained as indicated above. This technique has been applied in combination with the nested RT-PCR or RT-PCR described above in order to increase the sensitivity in the detection of the viral genome.

10 μl of the previously denatured RT-PCR or nested RT-PCR products were analyzed by boiling for 5 min. These samples were rapidly cooled on ice and placed, using the MilliBlot-D or MilliBlot-S (Millipore) equipment, on nylon membranes (Hybond-N +, Amersham Biosciences). The membranes were previously moistened with sterile double-distilled water and 5x sodium salt citrate buffer (SSC) (1x SSC: 50 mM NaCl; 15 mM sodium citrate; pH 7). The fixation was carried out with UV light (2 pulses of 700 μJ / cm2) (Ultraviolet Crosslinker, Amersham Biosciences).

The optimization of the dot-blot hybridization protocol included the evaluation of different prehybridization and hybridization temperatures (45, 51, 57 and 68 ° C), as well as the need to use 50% formamide in the hybridization buffer. The optimal hybridization conditions were 57 ° C and 50% formamide in the hybridization buffer, to guarantee the total elimination of possible unexpectedities caused by the use of RNA extracted from uninoculated BF-2 cells as a template for RT-based reactions. PCR Prehybridization was performed for 1-6 h with hybridization buffer (5x SSC; 0.1% Sarcosyl; 0.02% SDS; 2% Stock Blocking -Roche; 50% formamide-Roche) preheated to the test temperature. The probe was denatured by boiling for 5 min. Once cooled rapidly on ice, it was diluted in preheated hybridization buffer. Hybridization was carried out at the temperature tested in a hybridization oven (Amersham Pharmacia, Biotech) for 16 h with stirring. Once finalized, the diluted probe was collected and stored at -20 ° C for later use.

Subsequently, two successive washes were carried out in continuous agitation with buffers supplemented with different concentrations of salts (SSC 2x and SSC 1x at room temperature for 5 and 15 min, respectively, and 0.1x SSC at the hybridization temperature for 15 min).

Detection of hybridized products

The detection of the hybridized products was performed by immunoassay following the instructions of the marking systems (DIG-11-dUTP, Roche) and detection with DIG (anti-DIG-AP, Roche) used. The development of the alkaline phosphatase (AP) activity in the membrane was carried out by chemiluminescence, using CSPD (Roche) as the substrate of the AP, and the Hyper fi lm ECL radiographic film (Amersham, Biosciences). The film was exposed to the luminescent reaction in a cassette (HypercassetteTM, Amersham Biosciences) during different times of overexposure (between 4 and 30 min).

To evaluate the specificity of the hybridization system developed, the designed ADNbc probe was hybridized with amplification products obtained by means of nested RT-PCR and RT-PCR, using RNA extracted from BF-2 and SSN-1 cells inoculated with VHSV and VNNV as template. SJNNV genotype), respectively. The specificity of the probe designed for the detection of IPNV was evaluated using genogroup II (IPNV Ab), and genotypes I.2 (IPNV VR-299) and III.1 (IPNV Sp). The results are expressed in Fig. 3.

The sensitivity of the technique was estimated according to the viral titer according to the methodology described in the sensitivity section of RT-PCR and nested RT-PCR. As samples, the amplification products of nested RT-PCR or RT-PCR reactions were used. The sensitivity of dot-blot hybridization is similar to PCR-based methods, with a sensitivity limit of 10-100 PFU / ml for dot-blot hybridization from RT-PCR reactions and 1-10 PFU / ml for hybridization in dot-blot from the nested RT-PCR reactions.

Example 1

The present invention is further illustrated by the following application example.

IPNV detection from asymptomatic fish

The diagnostic techniques developed in the present invention have been applied to the detection of asymptomatic carriers in snapper and rta populations captured from the natural environment and established as pre-reproducers. For this purpose, blood was drawn from a representative number of animals. The blood was immediately mixed with heparin (0.1%, final concentration) and stored at 4 ° C until processing (no more than 24 h).

Total RNA from the blood samples was extracted using the commercial QIAamp® RNA Blood Mini Kit (QIAGEN) from 200 µl of blood treated with heparin following the instructions of the commercial house. RNA was quantified by spectrophotometry, at 260 nm, using ND-1000 (NanoDrop), and stored at -80 ° C until analysis by combining the dot-blot amplification and hybridization techniques described in the present invention. The blood samples were analyzed by combining a RT-PCR, nested RT-PCR and dot-blot hybridization of the nested RT-PCR products with the developed probe.

None of the blood samples analyzed by RT-PCR were positive for the detection of IPNV. Figure 4 shows the percentages of individuals carrying IPNV according to the detection method used (nested RT-PCR or combination of nested RT-PCR and dot-blot hybridization of the amplification products of nested RT-PCR) .

The application of nested RT-PCR allowed the detection of IPNV in 53.1% of the analyzed erts. Assays based on the combination of nested RT-PCR and dot-blot hybridization of the ampli fi cation products have allowed the detection of the IPNV genome in all the analyzed samples of urta (Figure 4). In the case of the snapper population, the use of nested RT-PCR allowed the detection of IPNV in up to 77.8% of animals analyzed. The combination of nested RT-PCR and hybridization allowed the detection of IPNV in 94.4% of the analyzed samples.

Claims (17)

one.

Set of primers for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish characterized in that at least two of the primers of said set have sequences identical to SEQ IDNO 1 and SEQ ID NO 2.

2.

Set of primers for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to the preceding claim characterized in that said set is formed by a first primer whose sequence is identical to SEQ ID NO 1 and by a second primer whose sequence It is identical to SEQ ID NO 2.

3.

Set of primers for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish characterized in that at least two of the primers of said set have sequences identical to SEQ IDNO 3 and SEQ ID NO 4.

Four.

Set of primers for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to the preceding claim characterized in that said set is formed by a first primer whose sequence is identical to SEQ ID NO 3 and by a second primer whose sequence It is identical to SEQ ID NO 4.

5.

Procedure for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish comprising:

to.

Extraction and purification of the total RNA from the samples,

b.

RT-PCR ampli fi cation of a 599 bp sequence of the VP2 protein precursor region using a set of primers according to claim 1 or 2.

C.

Amplification of a 191 bp fragment of the products resulting from the first amplification by nested RT-PCR using a set of primers according to claim 3 or 4.

d.

Obtaining double-stranded DNA probes from the products resulting from the second amplification (nested RT-PCR).

and.

Dot-blot hybridization of the products resulting from the second ampli fi cation (nested RT-PCR) using the double stranded DNA probes obtained in step (d).

F.

Detection of hybridized products by immunoassay.

6.

Method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to the preceding claim characterized in that the RT-PCR reaction (b) is carried out under the conditions of: 30 minutes at 45 ° C, 2 minutes at 94 ° C, 35 cycles of 15 seconds at 94 ° C -30 seconds at 58 ° C -30 seconds at 68 ° C, and 10 minutes at 68 ° C.

7.

Method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to claim 5 or 6 characterized in that the nested RT-PCR reaction (c) is carried out under the conditions of: 5 minutes at 94 ° C, 35 cycles of 30 seconds at 94ºC -30 seconds at 58ºC -30 seconds at 72ºC, and 5 minutes at 72ºC.

8.

Method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to any of claims 5 to 7, characterized in that the probes obtained are labeled with digoxigenin (DIG).

9.

Method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to any of claims 5 to 8 characterized in that dot-blot hybridization comprises:

to.

Denaturation of amplification products;

b.

Filtration of denatured products through nylon membranes;

C.

Fixing with ultraviolet light;

d.

Incubation at 57 ° C for 1-6 hours in hybridization buffer with 50% formamide; and

and.

Hybridization at 57 ° C for 16 hours with the denatured cDNA probe labeled with digoxigenin.

10. Method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to any of claims 5 to 9 characterized in that the detection of the hybridized products by immunoassay comprises:

to.

Successive washings in buffers with 2xSSC at room temperature (5 minutes), and repeat the process with 1xSSC for 15 minutes at room temperature;

b.

Washes at 57 ° C 15 minutes with 0.1xSSC;

C.

Detection by immunoassay, using an anti-DIG-AP antibody; Y

d.

Development of AP activity by chemiluminescence with the CSPD substrate.

eleven.

Method for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish according to any of claims 5 to 10 characterized in that the samples are blood samples.

12.

Kit for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish by the method defined in any of claims 5 to 11, comprising:

to.

Set of primers according to any of claims 1 or 2,

b.

Set of primers according to any of claims 3 or 4, and

C.

Show control.

SEQUENCE LIST <110> University of Malaga, Agricultural and Fisheries Research and Training Institute <120> Procedure, set of primers and kit for the detection of infectious pancreatic necrosis virus (IPNV) in asymptomatic fish <130> APPLICATION_UMA_IFAPA <160> 4 <210> 1 <211> 21 <212> DNA <213> Artificial sequence <223> SoleSegA-F2 <400> 1 <210> 2 <211> 19 <212> DNA <213> Artificial sequence <223> SoleSegA-R2 <400> 2 <210> 3 <211> 27 <212> DNA <213> Artificial sequence <223> R23 <400> 3 <210> 4 <211> 27 <212> DNA <213> Artificial sequence <223> R24 <400> 4 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200901759 SPAIN Date of submission of the application: 06.08.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C12Q1 / 68 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

X

GARCÍA-ROSADO, E., CANO, I., MARTÍN-ANTONIO, B. et al. Co-occurrence of viral and bacterial pathogens in disease outbreaks affecting newly cultured sparid fish. International Microbiology September 2007, Vol. 10, No. 3, pages 193-199. ISSN 1139-6709. <Doi: 10.2436 / 20.1501.01.27> 1-4,12

Y

5-11

Y

CANO, I., FERRO, P., ALONSO, M. C. et al. Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species. Journal of Applied Microbiology. January 2007, Vol. 102, No. 1, pages 32-40. ISSN 1364-5072. <Doi: 10.1111 / j.1365-2672.2006.03066.x> 5-11

X

RODRÍGUEZ, S., ALONSO, M., PÉREZ-PRIETO, S. I. Detection of infections pancreatic necrosis virus (IPNV) from leukocytes of carrier rainbow trout Oncorhynchus mykiss. Fish Pathology September 2001, Vol. 36, No. 3, pages 139-146. ISSN 0388-788X. 1-4,12

Y

5-11

Y

PHROMJAI, J., BOONSAENG, V., WITHYACHUMNARNKUL, B. et al. Detection of hepatopancreatic parvovirus in Thai shrimp Penaeus monodon by in situ hybridization, dot blot hybridization and PCR amplification. Diseases of Aquatic Organisms. October 2002, Vol. 51, No. 3, pages 227-232. ISSN 0177-5103. 5-11

X

LOPEZ-LASTRA, M., GONZÁLEZ, M., JASHES, M. et al. A detection method for infectious pancreatic necrosis virus (IPNV) based on reverse transcription (RT) -polymerase chain reaction (PCR). Journal of Fish Diseases. 1994, Vol. 17, No. 3, pages 269-282. ISSN 0140-7775. 1-4,12

Y

5-11

Y

WONGTEERASUPAYA, C., TONGCHUEA, W., BOONGSAENG, V. et al. Detection of yellowhead virus (YHV) of Penaeus monodon by RT-PCR amplification. Diseases of Aquatic Organisms. December 1997, Vol. 31, No. 3, pages 181-186. ISSN 0177-5103. 5-11

TO

CUTRÍN, J. M., LÓPEZ-VÁZQUEZ, C., OLVEIRA, J. G. et al. Isolation in cell culture and detection by PCR-based technology of IPNV-like virus from leucocytes of carrier turbot, Scophthalmus maximus (L.). Journal of Fish Diseases. December 2005, Vol. 28, No. 12, pages 713-722. ISSN 0140-7775. 5,8-11

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 14.11.2011

Examiner E. Relaño Reyes Page 1/6

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200901759 Minimum documentation searched (classification system followed by classification symbols) C12Q Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, EMBLall, REGISTRY, HCAPLUS, MEDLINE, BIOSIS, EMBASE, NPL, COMPNDX, INSPEC, XPESP State of the Art Report Page 2/6  WRITTEN OPINION Application number: 200901759 Date of the Written Opinion: 14.11.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 3-12 Claims 1, 2 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-12 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/6  WRITTEN OPINION Application number: 200901759 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

GARCÍA-ROSADO, E. et al. International Microbiology September 2007, Vol. 10, No. 3, pages 193-199. 09.2007

D02

CANO, I. et al. Journal of Applied Microbiology. January 2007, Vol. 102, No. 1, pages 32-40. 01.2007

D03

RODRÍGUEZ, S. et al. Fish Pathology September 2001, Vol. 36, No. 3, pages 139-146. 09.2001

D04

PHROMJAI, J. et al. Diseases of Aquatic Organisms. October 2002, Vol. 51, No. 3, pages 227-232. 04.10.2002

D05

LOPEZ-LASTRA, M. et al. Journal of Fish Diseases. 1994, Vol. 17, No. 3, pages 269-282. 1994

D06

WONGTEERASUPAYA, C. et al. Diseases of Aquatic Organisms. December 1997, Vol. 31, No. 3, pages 181-186. 30.12.1997

D07

CUTRÍN, J. M. et al. Journal of Fish Diseases. December 2005, Vol. 28, No. 12, pages 713-722. 12.2005

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the present application is a procedure for the detection of pancreatic necrosis virus (IPNV) comprising: a) the extraction and purification of RNA from blood samples; b) amplification of the 599 bp sequence between positions 831 and 1431 of VP2, by RT-PCR with sequence primers SEQ ID NO 1 and 2; c) amplification by nested RT-PCR of the above product, using sequence primers SEQ ID NO 3 and 4; d) obtaining probes labeled with digoxigenin from the product resulting from nested RT-PCR; e) hybridization by "dot blot" of the products of the second amplification, using the probes obtained in step d); and f) the detection of the hybridized by immunoassay (claims 5 to 11). In addition, the pairs of sequence primers SEQ ID NO 1 and 2 (claims 1 and 2), and SEQ ID NO 3 and 4 (claims 3 and 4) are also included, as well as the kit comprising these four primers together with a control sample (claim 12). In D01 the possible causes of microbiological infections in snappers, thefts and sargos are investigated. For this, various methods of detection of different bacteria and viruses are developed, including the IPNV. In order to detect the presence of IPNV, a nested PCR was designed, in which the first pair of primers (F2 and R2) have a 100% identity with the primers of SEQ ID NO 1 and 2 of the application. D02 demonstrates that the combination of the PCR technique with membrane hybridization increases its sensitivity. To this end, he conducted a study on the detection of the LCDV virus by PCR and "dot blot" (also called "slot blot"). The probe was the amplified sequence itself, labeled with digoxigenin. In D03, a method of IPNV detection by nested RT-PCR is anticipated. The first PCR amplifies nucleotide region 162 to 1321 of VP2, and the second from position 568 to 1181. With this method, using blood as a sample, the presence of IPNV in asymptomatic fish is detected. D04 develops an HPV virus detection method by PCR and subsequent hybridization. Hybridization by "southern blot" with the DNA amplified and labeled with digoxigenin, decreases the detection limit of the technique one hundred times. In D05 five nested VP2 PCRs are disclosed, in order to detect the presence of IPNV, the five pairs of primers being able to detect IPNV and specific of said virus. In this document, it is noted that nested PCRs increase sensitivity to PCRs. Although “southern blots” of the PCRs are performed, they are used to check their specificity, and not to increase their sensitivity. D06 sets up a YHV detection procedure using RT-PCR and subsequent southern blotting. This last step increases the sensitivity of the method one hundred times. In D07 the possibility of the detection of IPNV from blood samples in asymptomatic fish is studied. For this, a RT-PCR of VP1 was performed, improving the sensitivity of the technique, both by means of southern blot with a probe labeled with digoxigenin, and by means of a nested RT-PCR. State of the Art Report Page 4/6  WRITTEN OPINION Application number: 200901759 1.NOVEDAD (Art. 6.1 LP 11/1986)  1.1. CLAIMS 1 AND 2 The object of claims 1 and 2 is the set of primers for the detection of IPNV of SEQ sequences. ID. NO. 1 and 2. D01 discloses a method of detection of IPNV in fish by a nested RT-PCR, in which primers F2 and R2 have 100% identity respectively with the SEQ. ID. NO. 1 and 2. Consequently, the object of claims 1 and 2, has been unequivocally disclosed in D01, so that these claims do not meet the requirement of novelty (Art. 6.1 LP 11/1986).

1.2.

 CLAIMS FROM 3 TO 12

Claims 3 to 12 are new (Art. 6.1 LP 11/1986).

2.

 INVENTIVE ACTIVITY (Art. 8.1 LP 11/1986)

2.1. CLAIMS FROM 5 TO 11 The purpose of the present application is, according to claims 5 and 8, a method for the detection of IPNV in asymptomatic fish, comprising: a) RNA extraction and purification b) VP2 RT-PCR with sequence primers SEQ ID NO 1 and 2 c) RT-PCR nested with sequence primers SEQ ID NO 3 and 4 d) Obtaining labeled DNA probes with digoxigenin from the products obtained in the nested RT-PCR e) Hybridization in dot blot of the products obtained in the nested RT-PCR with the probes of step d) f) Detection of the hybridized by immunoassay. D01, D03 and D05, disclose various methods of detection of IPNV in fish, through nested RT-PCRs from different areas of VP2. In the case of D01, in the first PCR the same region is amplified as in the RT-PCR of section b) and the same primers are used (F2 has 100% identity with SEQ ID NO 1 and R2 100% of identity with SEQ. ID. NO. 2), an overlapping region being amplified in the nested PCR with that of the request (from nucleotide 941 to 1130 in the application, and from 1061 to 1177 in D01). In D03, the region amplified in the RT-PCR is from position 162 to 1321, and in the nested PCR from 568 to 1181. Therefore, the nested PCR sequence includes the entire region amplified in the stage. c) of the request (nucleotides from 941 to 1130). Finally, in D05 five nested RT-PCRs from different areas of VP2 are disclosed, among which there are two RT-PCRs that amplify overlapping sequences with the request's RT-PCR (regions 419-1076 and 571-1012) . In view of these documents, it is considered that different methods of IPNV are known in the state of the art in which they are amplified by nested RT-PCRs, various regions of VP2. Therefore, and given that the region amplified in the application does not present a surprising technical effect, the choice for amplification of one or another area of this gene is not considered relevant, but one of the alternatives that would be considered by an expert in the matter at the time of developing said procedure. In addition, in general, the design of the primers once the target to be amplified is not considered of special technical difficulty. Consequently, the fundamental difference between D01, D03 or D05 and the present application is the addition of some stages after the nested RT-PCR, in which the amplified region is marked, to be used as a probe in the hybridization with the product itself of said PCR. The technical effect is a greater sensitivity of the procedure, being able to determine the presence of IPNV in asymptomatic fish. Therefore, the problem to be solved is the development of more sensitive IPNV detection methods. State of the Art Report Page 5/6 WRITTEN OPINION Application number: 200901759 D02, D04 and D06 show that the detection of PCR products by hybridization with probes labeled with Digoxigenin, increases the sensitivity of the technique a hundredfold. In the case of D02 and D04, the PCR product itself is used as a probe, while in D06 a fragment of the cDNA. However, this last difference is not considered relevant. Consequently, it is understood that one skilled in the art would try to develop a method of detection of IPNV in asymptomatic fish, by means of a nested RT-PCR of VP2 (D01, D03 or D05) and the visualization by hybridization of the products amplified with a probe labeled with digoxigenin (D02, D04 or D06) with a reasonable expectation of success. Thus, in view of documents D01 and D02, D03 and D04, or D05 and D06, claims 5 and 8 are considered to have no inventive activity (Art. 8.1 LP 11/1986). Claim 6 specifies the temperatures and times of the RT-PCR, and the conditions of the nested PCR. One skilled in the art knows how to optimize a PCR by adjusting the different temperatures and times of its cycles, being part of the routine set-up of PCRs. Accordingly, claims 6 and 7 have no inventive activity (Art. 8.1 LP 11/1986). On the other hand, the object of claim 9 is the hybridization stages of the dot blot, and that of the detection of the hybridized products. It is understood that these stages and conditions are routine when performing this type of techniques, being able to do some variation in them due to the characteristics of each sequence. Thus, it is considered that claims 9 and 10 also do not meet the requirement of inventive activity (Art. 8.1 LP 11/1986). In claim 11 it is specified that the sample to be analyzed is blood. In the method disclosed in D03, the presence of IPNV in asymptomatic fish is detected, from peripheral blood. Although blood is not used as a sample in D01 and D05, the possibility of detecting IPNVs from blood samples, so the difference is not relevant. Consequently, claim 11 also does not present inventive activity (Art. 8.1 LP 11/1986). 2.2. CLAIMS FROM 1 TO 4 The purpose of the application is a set of primers for the detection of IPNV in asymptomatic fish, characterized in that they have the SEQ sequences. ID. NO. 1 and 2 (claims 1 and 2) or SEQ. ID. NO. 3 and 4 (claims 3 and 4). Given the arguments set forth in section 1.1., Claims 1 and 2 do not meet the requirement of inventive activity. In addition, as noted in section 2.1., Since differences in amplified regions are not considered relevant, none of the primers have inventive activity, since their design is a technique well known to a person skilled in the art. Therefore, claims 1 and 2 also do not have inventive activity in the light of documents D03 or D05, nor do claims 3 and 4 meet said requirement in view of documents D01, D03 or D05 (Art 8.1 LP 11 / 1986). 2.3 CLAIM 12 A claim 12 is intended as a kit comprising SEQ sequence primers. ID. NO. 1, 2, 3 and 4. Given the reasons set out in points 2.1 and 2.2, in view of D01, D03 or D05, claim 12 has no activity inventive (Art. 8.1 LP 11/1986). State of the Art Report Page 6/6