Detection of infectious salmon anaemia virus by real-time nucleic acid sequence based amplification

Abstract

We have developed a real-time nucleic acid sequence based amplification (NASBA) procedure for detection of infectious salmon anaemia virus (ISAV). Primers were designed to target a 124 nucleotide region of ISAV genome segment 8. Amplification products were detected in real-time with a molecular beacon (carboxyfluorescin [FAM]-labelled and methyl-red quenched) that recognised an internal region of the target amplicon. Amplification and detection were performed at 41°C for 90 min in a Corbett Research Rotorgene. The real-time NASBA assay was compared to a conventional RT-PCR for ISAV detection. From a panel of 45 clinical samples, both assays detected ISAV in the same 19 samples. Based on the detection of a synthetic RNA target, the real-time NASBA procedure was approximately 100× more sensitive than conventional RT-PCR. These results suggest that real-time NASBA may represent a useful diagnostic procedure for ISAV.KEY WORDS: Infectious salmon anaemia virus · Orthomyxovirus · NASBA · Diagnostics · Fish · Nucleic acid amplification Resale or republication not permitted without written consent of the publisherDis Aquat Org 72: [107][108][109][110][111][112][113] 2006 Sensitive and specific diagnostic procedures are essential for the effective control of ISA. Methods currently used for the detection of ISAV include virus isolation in SHK-1, ASK, or CHSE-214 cells (Cipriano & Miller 2003), in situ hybridization (Gregory 2002), indirect fluorescent antibody testing (Falk & Dannevig 1995), and RT-PCR (Mjaaland et al. 1997, Rimstad et al. 1999, Devold et al. 2000. A real-time RT-PCR method for the diagnosis of ISAV utilising SyBr Green I detection of amplification products has been described by Munir & Kibenge (2004).Nucleic acid sequence based amplification (NASBA) is an isothermal nucleic acid amplification procedure based on the activity of 3 enzymes; reverse transcriptase, RNase H, and T7 RNA polymerase (Compton 1991). Real-time detection in NASBA can be performed using molecular beacon probes (Leone et al. 1998). NASBA detection methods have been described for several viruses including human immunodeficiency virus type 1 (de Baar et al. 1999 (Starkey et al. 2004) and SARS-associated coronavirus (Keightley et al. 2005).In the present study we report on the development of a real-time NASBA procedure for detection of ISAV. The real-time NASBA assay was compared to a conventional RT-PCR assay for ISAV using previously described primers (Mjaaland et al. 1997). MATERIALS AND METHODSClinical samples. A panel of 45 clinical samples (Atlantic salmon kidney) obtained from outbreaks of ISA in Scotland, the Faroe Islands and Norway was used in this study (see Table 1). Samples 1 to 20 were archival, and following collection were stored at -70°C. Samples 21 to 45 were obtained from Atlantic salmon during an outbreak of ISA, and were stored in RNAlater (Ambion) at -20°C.RNA isolation. Isolation of RNA from tissue samples for use in both RT-PCR and NASBA was performed using the Nucleospin procedure (Machery Nagel)...

Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish

“…Currently, molecular techniques based on the amplification of genetic material in samples such as qPCR have been developed and validated for detection of other fish viruses (Ciulli et al., ; Dalla Valle et al., ; Starkey et al., ). A one step, real‐time RT‐PCR was developed to detect the RNA1 genomic sequence of the betanodavirus infection in various fish species including tilapia (Baud et al., ).…”

"Pelacakan Gen Env-TM Virus Penyakit Jembrana Galur Tabanan 1995 dengan Metode Nucleic Acid Sequence Based Amplificaton (DETECTION ENV-TM GENE OF JEMBRANA DISEASE VIRUS OF TABANAN 1995 STRAIN BY NUCLEIC ACID SEQUENCE BASED AMPLIFICATION METHOD)"

“…Metode NASBA telah diaplikasikan untuk deteksi berbagai penyakit seperti flu burung/ Avian Influenza Virus (AIV), penyakit mulut dan kuku/Foot and Mouth Disease Virus (FMD), penyakit tetelo/New Castle Disease Virus (NDV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), dan Infectious Salmon Anaemia Virus/ISAV (Starkey et al, 2006). Metode NASBA sebagai alternatif selain PCR mampu melacak gen env-TM VPJ galur Tabanan 1995 secara dini pada sampel jaringan dan darah.…”