Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification

Millard, Paul J.; Bickerstaff, Lee E.; LaPatra, Scott E.; Kim, Carol H.

doi:10.1111/j.1365-2761.2006.00705.x

Cited by 12 publications

(15 citation statements)

References 26 publications

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“…Since the commencement of the present study, ISAV detection procedures have been developed utilising real-time RT-PCR with MGB probe detection (Plarre et al 2005), and rolling circle amplification with molecular padlock probe detection (Millard et al 2006 Table 3. Intra-assay and inter-assay variation of real-time NASBA for detection of ISAV.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, Monis & Griglio (2006) detected a single product when using SG in melt curve analysis, but multiple products in agarose gel electrophoresis, with potential implications when using SG for fluorescent detection in routine virus diagnosis. However, the use of SG for fluorescent detection can also be advantageous, since its binding efficiency is not affected by nucleotide sequence variation at probe recognition sites, which could theoretically impair the efficiency of the molecular beacon used in the ISAV NASBA assay.Since the commencement of the present study, ISAV detection procedures have been developed utilising real-time RT-PCR with MGB probe detection (Plarre et al 2005), and rolling circle amplification with molecular padlock probe detection (Millard et al 2006 Table 3. Intra-assay and inter-assay variation of real-time NASBA for detection of ISAV.…”

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confidence: 99%

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

Starkey¹,

Smail²,

Bleie³

et al. 2006

Dis. Aquat. Org.

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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)...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Starkey¹,

Smail²,

Bleie³

et al. 2006

Dis. Aquat. Org.

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“…Linear MPP can display a high degree of selectivity for target nucleic acid sequences (Baner et al, 1998;Millard et al, 2006). To apply the surface-associated RCA technique to multiplex array formats it is necessary to examine the specificity of surfaceassociated primers for MPP.…”

Section: Evaluation Of the Selectivity Of The Technique For Applicatimentioning

confidence: 99%

“…The sensitivity of the technique was evaluated based on a "best case" scenario in which all linear probes used in the formation of cMPP are circularized; prior work (Millard et al, 2006) has shown this assumption to be valid. Circularized MPP stock concentration is therefore, at maximum, 100 nM.…”

Section: Evaluation Of the Sensitivity Of The Technique And Limiting mentioning

confidence: 99%

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Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification

McCarthy

Bickerstaff

Cunha

et al. 2007

Biosensors and Bioelectronics

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Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes

et al. 2006

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Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (ISAV) were detected and identified using a combination of surface-associated molecular padlock DNA probes (MPPs) and rolling circle amplification (RCA) in microcapillary tubes. DNA oligonucleotides with base sequences identical to RNA obtained from IHNV or ISAV were recognized by MPPs. Circularized MPPs were then captured on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA produced DNA concatamers, which were labeled with fluorescent SYBR Green II nucleic acid stain, and measured by microfluorimetry. Molecular padlock probes, combined with this method of surface-associated isothermal RCA, exhibited high selectivity without the need for thermal cycling. This method is applicable to the design of low-power field sensors capable of multiplex detection of viral, bacterial, and protozoan pathogens within localized regions of microcapillary tubes.

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Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification

Cited by 12 publications

References 26 publications

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

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

Nucleic acid sensing by regenerable surface-associated isothermal rolling circle amplification

Detection and identification of IHN and ISA viruses by isothermal DNA amplification in microcapillary tubes

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