Document Detail


Absolute quantitation of infectious salmon anaemia virus using different real-time reverse transcription PCR chemistries.
MedLine Citation:
PMID:  18789975     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Routine laboratory diagnosis of infectious salmon anaemia virus (ISAV) infection is primarily by reverse transcription polymerase chain reaction (RT-PCR) because of the high sensitivity and rapid turnaround time of the test. This paper describes methods for highly reproducible absolute viral load measurements using external standard curves generated with either ISAV recombinant plasmid DNA (pDNA) standards or transcribed RNA standards prepared by in vitro transcription with T7 RNA polymerase, and using a two tube real-time or quantitative (q)RT-PCR with SYBR Green I chemistry and a single tube qRT-PCR with TaqMan probe chemistry. When applied to virus samples of known virus titer for the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-085-1, both methods showed a 100-fold lower detectable titer for RPC/NB-04-085-1 but with a higher number of viral RNA molecules compared to NBISA01. Overall, the SYBR Green I method overestimated copy numbers in samples having equivalent Ct values with the TaqMan probe method. Taken together, the findings suggest that the TaqMan probe method with the in vitro transcribed RNA standard curve is the preferred method for reliable and rapid quantitation of ISAV in samples.
Authors:
Samuel T Workenhe; Molly J T Kibenge; Tokinori Iwamoto; Frederick S B Kibenge
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-09-26
Journal Detail:
Title:  Journal of virological methods     Volume:  154     ISSN:  0166-0934     ISO Abbreviation:  J. Virol. Methods     Publication Date:  2008 Dec 
Date Detail:
Created Date:  2008-11-25     Completed Date:  2009-02-05     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8005839     Medline TA:  J Virol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  128-34     Citation Subset:  IM    
Affiliation:
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, P.E.I., Canada C1A 4P3.
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MeSH Terms
Descriptor/Qualifier:
Animals
Isavirus / genetics,  isolation & purification*
Organic Chemicals
Orthomyxoviridae Infections / veterinary*,  virology
Reverse Transcriptase Polymerase Chain Reaction / methods*
Staining and Labeling / methods
Viral Load*
Chemical
Reg. No./Substance:
0/Organic Chemicals; 163795-75-3/SYBR Green I

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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