Detection of infectious haematopoietic necrosis virus and infectious salmon anaemia virus by molecular padlock amplification.
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.
Millard
P J
PJ
Laboratory for Surface Science and Technology, Department of Chemical and Biological Engineering,University of Maine, Orono, 04469, USA. paul.millard@maine.edu
Bickerstaff
L E
LE
LaPatra
S E
SE
Kim
C H
CH
D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology*; D005398:Fisheries / Q000379:methods; D029142:Infectious hematopoietic necrosis virus / Q000235:genetics / Q000302:isolation & purification*; D046829:Isavirus / Q000235:genetics / Q000302:isolation & purification*; D007668:Kidney / Q000821:virology; D015336:Molecular Probe Techniques / Q000662:veterinary*; D017686:Oncorhynchus mykiss / Q000821:virology; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D012474:Salmon / Q000821:virology; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012367:RNA, Viral