Detection of norovirus genotype I.3b and II.4 in bioaccumulated blue mussels using different virus recovery methods.
Noroviruses (NoVs) are the most common non bacterial human pathogens associated with shellfish borne gastroenteritis. Norovirus detection is based on molecular procedures such as reverse transcriptase (RT)-PCR. A variety of methods have been developed to extract viral RNA from complex shellfish matrixes and to reduce the level of RT-PCR inhibitors. The present study had three objectives: 1) Determine the most appropriate sample treatment protocol for detection of NoVs in mussels, 2) Examine whether there is a variation of the binding affinity between a NoV GI and a GII strain to mussel digestive tissue and how this influences the detection sensitivity, 3) Establish an internal control for sample processing and virus detection. Three RNA extraction methods were evaluated on extracts from blue mussels (Mytilus edulis) spiked with NoV GII.4. The most efficient RNA extraction method was subsequently used for evaluation of three virus recovery methods of blue mussels bio accumulated with NoV GI.3b and GII.4. Mengovirus was evaluated as an internal process control and TaqMan RT-PCRs were used for virus detection. Elution of the two viruses from shellfish tissue differed, indicating a difference in binding affinities. Only a method based upon Proteinase K digestion followed by NucliSenseasyMAG was able to detect both NoV GI.3b and GII.4 (3.0% and 3.5% recovery respectively). The results show that the processing method influences the possibility to detect different variants of NoV.
Comelli
Heidi Lange
HL
Department of Food Safety and Infection Biology, The Norwegian School of Veterinary Science, 0033 Oslo, Norway. Heidi.Comelli@veths.no
Rimstad
Espen
E
Larsen
Stig
S
Myrmel
Mette
M
D000818:Animals; D017250:Caliciviridae Infections / Q000175:diagnosis / Q000821:virology; D003257:Consumer Product Safety; D019286:Endopeptidase K / Q000378:metabolism; D005243:Feces / Q000821:virology; D005506:Food Contamination / Q000032:analysis*; D005759:Gastroenteritis / Q000821:virology; D005838:Genotype; D006801:Humans; D049880:Mytilus edulis / Q000821:virology*; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*; D019914:Taq Polymerase / Q000378:metabolism
D012367:RNA, Viral; D019914:Taq Polymerase; D019286:Endopeptidase K