Detection of infectious salmon anaemia virus by real-time RT-PCR.
A one-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) using LightCycler technology and SYBR Green chemistry that quantitatively detects infectious salmon anaemia virus (ISAV) in biological samples is described. The assay utilized primers targeting ISAV RNA segment 8, with ISAV isolate U5575-1 as template. The entire optimized assay, including one cycle of reverse transcription, 50 cycles of complementary DNA amplification, and data acquisition and analysis took only 80min. The melting curve and gel electrophoresis analyses of real-time RT-PCR showed harmony with each other as a virus-specific single melting peak and a product of the expected size of 211 bp were obtained. A regression line between the mean threshold cycle (Ct) values and viral template concentrations over a 1:10(5) dilution range with an r(2) value of 0.962 and a slope of -3.771 indicated that the assay was highly reproducible. This assay was 100 times more sensitive than the conventional one-tube RT-PCR assay when compared on the same sample. Analysis of different tissues from fish that survived an ISAV experimental infection further confirmed that real-time RT-PCR was more sensitive than regular RT-PCR for detection of ISAV nucleic acids. Temporal analysis of ISAV-infected TO cell cultures showed that the amount of the specific viral RNA increased more than 100-fold within 32 h post-inoculation (p.i.) and over 1200-fold by 144 h p.i. The melting curve analysis throughout the duration of the infection sampled had a single melting peak suggesting that the virus population was uniform in the targeted region. Quantitative analysis of CHSE-214 cell cultures infected with different ISAV isolates indicated that ISAV isolates, based on their ability to replicate and cause cytopathic effects in CHSE-214 cells, may be differentiated into three CHSE phenotypes: replicating cytopathic, replicating non-cytopathic, and non-replicating. Thus, the SYBR Green real-time RT-PCR is a sensitive, rapid, and highly reproducible assay that can be used to quantitate ISAV in biological samples.
Munir
Khalid
K
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PEI, Canada C1A 4P3.
Kibenge
Frederick S B
FS
D000818:Animals; D004196:Disease Outbreaks / Q000662:veterinary; D005393:Fish Diseases / Q000821:virology*; D017686:Oncorhynchus mykiss / Q000821:virology; D009975:Orthomyxoviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009976:Orthomyxoviridae Infections / Q000662:veterinary* / Q000821:virology; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D012474:Salmon / Q000821:virology*
D012367:RNA, Viral