Detection and identification of aquatic birnaviruses by PCR assay

Abstract

A reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection and identification of aquatic birnaviruses. The four sets of primers (PrA, PrB, PrC, and PrD) that we used are specific for regions of cDNA coded by genome segment A of aquatic birnaviruses. PrA identifies a large fragment (1,180 bp) within the pVP2-coding region, and PrB identifies a 524-bp fragment within the sequence amplified by PrA. Primer set PrC frames a genome fragment (339 bp) within the NS-VP3-coding region, and PrD identifies a 174-bp sequence within the fragment identified by PrC. PrB and PrD amplified cDNAs from all nine recognized serotypes of aquatic birnavirus serogroup A as well as the N1 isolate that may represent a 10th serotype. These results indicate that these three primer sequences are highly conserved and can be used in PCR assays for group identification of these viruses. PrA routinely produced amplification products from eight serotypes but exhibited variable results with one serotype, and primer PrC identified 6 of the 11 virus isolates tested. The qualitative sensitivity of the RT-PCR assay was evaluated by comparison of the results with those of cell culture isolation assays. With the exception of one sample, the RT-PCR assay with primer PrD was as accurate as cell culture isolation for detecting virus in kidney and spleen tissues from naturally infected, asymptomatic carrier fish. These results indicate that the RT-PCR assay can be a rapid and reliable substitute for cell culture methods for the detection of aquatic birnaviruses.

Isolation in cell culture and detection by PCR‐based technology of IPNV‐like virus from leucocytes of carrier turbot, Scophthalmus maximus (L.)

“…In recent years, RT-PCR has become a commonly used tool for the detection of IPNV, in both cell culture (Rimstad, Hornes, Olsvik & Hyllseth 1990;López-Lastra, Gonzalez, Jashes & Sandino 1994;Wang, Wi & Lee 1997) and infected salmonid fish (Blake, Schill, McAllister, Lee, Singer & Nicholson 1995;Novoa, Blake, Nicholson & Figueras 1995;Rodriguez et al 2001;Taksdal, Dannevig & Rimstad 2001), even in the carrier state (Rodriguez et al 2001;Taksdal et al 2001), providing significant improvement in speed, sensitivity and repetitiveness compared with the traditional detection methods (Dopazo & Barja 2002). The use of nested PCR increases significantly the sensitivity achieved by RT-PCR (Rimstad et al 1990;López-Lastra et al 1994) and also facilitates sequencing and other approaches to RT-PCR amplicon characterization.…”

Virulence Comparison of Three Buhl-Subtype Isolates of Infectious Pancreatic Necrosis Virus in Brook Trout Fry

“…Primers.-Primers to be used for the reverse transcription and polymerase chain reactions were constructed at the Oregon State University (OSU) Center for Gene Research on the basis of primers described by Blake et al (1995) for use with the Jasper isolate of IPNV. The 5Ј primer designed was 5Ј TGA GAT CCA TTA TGC TTC CCG A 3Ј.…”

Genetic analysis of infectious pancreatic necrosis virus from Scotland

“…RNA extracted from the kidney and cell cultures, pass 1, 2 and 3 were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) as described by Smail et al (2006), with the following modification. An 1180 bp portion of the VP2 region of segment A was amplified using primers A1 (5¢-TGAGATCCATTATGCTTCCAGA-3¢) and A2 (5¢-GACAGGATCATCTTGGCATAGT-3¢) (Blake, Schill, McAllister, Lee, Singer & Nicholson 1995). Amplification reactions were carried out on a Techne Genius thermocycler with heated lid, programmed to perform 1 cycle of 94°C for 2 min followed by 45 cycles of denaturing at 94°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1 min followed by a single extension step of 5 min at 72°C.…”