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Detection of enterovirus and hepatitis A virus RNA in mussels ( Mytilus spp.) by reverse transcriptase‐polymerase chain reaction
Author(s) -
Casas N.,
Suñén E.
Publication year - 2001
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2001.01221.x
Subject(s) - reverse transcriptase , rna , rna extraction , nucleic acid , biology , chromatography , reverse transcription polymerase chain reaction , microbiology and biotechnology , virology , chemistry , biochemistry , messenger rna , gene
N.CASAS AND E.SUÑÉN. 2001 . Aims: A simple and effective method of concentrating and purifying enteric viruses from mussel samples to be detected by nucleic acid extraction reverse transcriptase‐polymerase chain reaction (RT‐PCR) has been evaluated. Methods and Results: Seeded mussels were processed by alkaline elution, polyethylene glycol (PEG) precipitation, solvent extraction and PEG precipitation. Final concentrates were assayed by infectivity and RT‐PCR after nucleic acid extraction. Two RNA extraction methods were comparatively evaluated for removing inhibitory substances: guanidinium thiocyanate extraction and Purescript™ RNA isolation. Both procedures removed most inhibitors allowing the detection of viral RNA at inoculum levels as low as 4 pfu g –1 for poliovirus type 1 and 1·8–18 most probable number of cytopathogenic units g –1 for HAV. When inhibitors remained, they were efficiently removed by Sephadex column chromatography before RNA extraction. Conclusions: The described method is effective for the detection of enteric viruses in mussels by RT‐PCR. The use of Purescript™ RNA isolation makes the method faster, safer and very easy to perform.