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J Appl Microbiol. 2001 Jan;90(1):89-95.

Detection of enterovirus and hepatitis A virus RNA in mussels (Mytilus spp.) by reverse transcriptase-polymerase chain reaction.

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1
Departamento de Inmunología, Microbiología y Parasitología, Facultad de Farmacia, Universidad del Pais Vasco, Vitoria-Gasteiz, Spain.

Abstract

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 Purescripttrade mark 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.

CONCLUSION:

The described method is effective for the detection of enteric viruses in mussels by RT-PCR. The use of Purescripttrade mark RNA isolation makes the method faster, safer and very easy to perform.

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