TY - JOUR

T1 - Comparison of methods for detection of norovirus in oysters

AU - Schultz, Anna Charlotte

AU - Saadbye, Peter

AU - Hoorfar, Jeffrey

AU - Nørrung, Birgit

PY - 2007/3/20

Y1 - 2007/3/20

N2 - In the absence of culture methods for noroviruses, detection in foods relies on molecular techniques such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR) on extracted viral RNA followed by PCR product confirmation by hybridisation and/or sequencing. However, in order to obtain a successful detection it is of great importance to remove the tissue inhibitors during the viral RNA extraction. To select the most efficient extraction procedure of oysters we have compared four protocols. A pool of digestive gland material from oyster samples was divided into 1.5 g portions and spiked with 10-fold dilutions of human faecal samples containing norovirus genogroup II. The samples were tested on three different occasions using four different sample treatment protocols. The protocols were assessed with regard to their ability to recover viral RNA and detect norovirus in spiked oysters and for their in-house reproducibility. One method using viral elution by a Mixer Mill Cell Disrupter resulted in a 10-fold better recovery than the other three protocols when an RT-seminested PCR (G2SKR/COG2F and G2SKR/G2SKF) detection approach was applied. Although less distinctive this was also the case when NoV was detected by a single round RT-PCR approach using the primers JV13i and JV12y. The second most efficient method was a method using chloroform extraction and polyethylene precipitation.

AB - In the absence of culture methods for noroviruses, detection in foods relies on molecular techniques such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR) on extracted viral RNA followed by PCR product confirmation by hybridisation and/or sequencing. However, in order to obtain a successful detection it is of great importance to remove the tissue inhibitors during the viral RNA extraction. To select the most efficient extraction procedure of oysters we have compared four protocols. A pool of digestive gland material from oyster samples was divided into 1.5 g portions and spiked with 10-fold dilutions of human faecal samples containing norovirus genogroup II. The samples were tested on three different occasions using four different sample treatment protocols. The protocols were assessed with regard to their ability to recover viral RNA and detect norovirus in spiked oysters and for their in-house reproducibility. One method using viral elution by a Mixer Mill Cell Disrupter resulted in a 10-fold better recovery than the other three protocols when an RT-seminested PCR (G2SKR/COG2F and G2SKR/G2SKF) detection approach was applied. Although less distinctive this was also the case when NoV was detected by a single round RT-PCR approach using the primers JV13i and JV12y. The second most efficient method was a method using chloroform extraction and polyethylene precipitation.

KW - Animals

KW - Chemical Precipitation

KW - Chloroform

KW - Consumer Product Safety

KW - Feces

KW - Food Contamination

KW - Food Microbiology

KW - Humans

KW - Norovirus

KW - Ostreidae

KW - Polyethylene

KW - RNA, Viral

KW - Reproducibility of Results

KW - Reverse Transcriptase Polymerase Chain Reaction

KW - Sensitivity and Specificity

KW - Shellfish

KW - Faculty of Health and Medical Sciences

KW - Norovirus

KW - Oyster

KW - RNA

KW - Sample

KW - Virus

U2 - 10.1016/j.ijfoodmicro.2006.09.028

DO - 10.1016/j.ijfoodmicro.2006.09.028

M3 - Journal article

C2 - 17182147

VL - 114

SP - 352

EP - 356

JO - International Journal of Food Microbiology

JF - International Journal of Food Microbiology

SN - 0168-1605

IS - 3

ER -