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Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock
P J Millard1, L E Bickerstaff, S E LaPatra
1Laboratory for Surface Science and Technology, Department of Chemical and Biological Engineering,University of Maine, Orono, 04469, USA. paul.millard@maine.edu
Abstract:
A new method for the molecular detection of the fish pathogens, infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV), is described. By employing molecular padlock probe (MPP) technology combined with rolling circle amplification (RCA) and hyperbranching (Hbr), it is possible to detect RNA target sequence from these viruses at levels comparable with those detected by the polymerase chain reaction (PCR), but without prior reverse transcription. The use of MPP technology combined with RCA and Hbr for the detection of IHNV and ISAV in fish exhibited selectivity comparable with that of PCR while potentially reducing the time and cost required for analysis. The method described was used to detect as few as 10(4) DNA oligonucleotide targets and was sequence-specific at the single base level. Viral RNA could be detected directly, either alone or in the presence of non-viral RNA from fish tissue. This technology is applicable for detecting a variety of microbes, in addition to IHNV and ISAV, and is ideal for further integration into a biosensor platform for on-site diagnosis of pathogen infection in fish.
Insights
A novel molecular detection method using padlock probes (MPP) and rolling circle amplification (RCA) accurately identifies fish viruses like infectious salmon anaemia virus (ISAV) and infectious haematopoietic necrosis virus (IHNV). This cost-effective technique offers direct RNA detection without reverse transcription, suitable for rapid, on-site diagnostics.
Area of Science:
- Veterinary Virology
- Molecular Biology
- Aquaculture Pathology
Background:
- Infectious haematopoietic necrosis virus (IHNV) and infectious salmon anaemia virus (ISAV) are significant fish pathogens.
- Accurate and rapid detection methods are crucial for managing viral outbreaks in aquaculture.
- Existing methods like PCR often require pre-processing steps such as reverse transcription.
Purpose of the Study:
- To develop a novel molecular detection method for IHNV and ISAV.
- To evaluate the sensitivity, specificity, and efficiency of this new method.
- To explore its potential for direct viral RNA detection and integration into biosensor platforms.
Main Methods:
- Utilized molecular padlock probe (MPP) technology.
- Combined MPP with rolling circle amplification (RCA) and hyperbranching (Hbr).
- Tested detection of viral RNA directly from fish tissue, with and without non-viral RNA.
Main Results:
- Achieved molecular detection of IHNV and ISAV RNA at levels comparable to PCR.
- Demonstrated sequence specificity at the single base level.
- Successfully detected as few as 10^4 DNA oligonucleotide targets and viral RNA directly.
Conclusions:
- The MPP, RCA, and Hbr method provides sensitive and specific detection of IHNV and ISAV.
- This approach eliminates the need for prior reverse transcription, potentially reducing time and cost.
- The technology is versatile for detecting various microbes and suitable for on-site biosensor applications in fish health management.
