Infectious Salmon Anemia Virus
A small project about ISAVirus brought to you by a group of Year 2 MBIO students from Ngee Ann Polytechnic. Click on the pastel orange box on the left for the names of the students involved in this project. Thank you. :) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Detection of ISAV Thursday, 26 January 2012 @ 12:39
Infectious Salmon Anemia Virus (ISAV) can be quantitatively detected by using a one-tube real-time reverse transcription–polymerase chain reaction (RT–PCR) that utilizes SYBR Green. Primers targeted at ISAV RNA segment 8 is used and ISAV isolate U5575-1 is utilized as a template. The process includes one cycle of reverse transcription, 50 cycles of cDNA amplification as well as data analysis took only 80 minutes. The melting curve and gel electrophoresis analysis 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. Materials & Methods:
-Fish tissues are obtained to extract total RNA by macerating and homogenizing the sample. - 1:1 dilution in sterile PBS is then carried out. - centrifugation at 3000rpm for 15 minutes and supernatant is used for RNA extraction. -TRIZOL reagent is used to purify virus
-Infectious Salmon Anemia Virus undergoes centrifugation at 4430 rpm for 30 minutes at 4˚C. -Ammonium sulphate added to concentrate the viral supernatant -RNA from purified virus extracted using TRIZOL reagent
-viral RNA diluted 10 times in RNase-free PCR grade water -Sample quantitated using spectrophotometry Table 1. Optimized reagent concentrations in 20 μl reaction volume for ISAV SYBR Green real-time RT–PCR
a: Reagents are part of the RNA Amplification Kit SYBR Green I (Roche Diagnostics GmbH). b: Primers have been previously used in ISAV RT–PCR assays ( [Devold et al., 2000] and [Kibenge et al., 2000]). Table 2. Thermal profile for ISAV SYBR Green real-time RT–PCR
Melting curve was performed from 70 to 95 °C in 0.1 °C/s increments. The tissue samples from fish that survived an experimental ISAV infection were tested to demonstrate the use of SYBR Green real-time RT–PCR assay for fish tissues with different amounts of ISAV RNA. Both conventional one-tube RT–PCR and SYBR Green-based real-time RT–PCR assays were compared on these samples to determine which of these assays work best in detecting ISAV RNA. The real-time RT–PCR assay detected ISAV RNA in more tissues that the conventional one-tube RT–PCR assay.This is expected of the result as real-time RT PCR shows that it is 100 times more sensitive than the conventional one-tube RT-PCR. Real-time RT-PCR assay also proves that it is able to obtain a relative amount of viral RNA in the sample from the Ct values. Since all the fish tested were survivors of a lethal experimental ISAV challenge, the results demonstrate that the real-time RT–PCR assay would be useful in detecting subclinical ISAV infections in fish. Table 3. Detection of ISAV RNA segment 8 by conventional RT–PCR and SYBR Green real-time RT–PCR in different tissues of fish at 76 days post-ISAV challenge
a: + Denotes positive by conventional one-tube RT–PCR or real-time RT–PCR detection format. b: − Denotes negative by either agarose gel electrophoresis for conventional one-tube RT–PCR or real-time RT–PCR detection format. c: Ct value. d: ISAV RNA concentration (ng/μl) extrapolated from the standard curve e: NC denotes not calculated | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||