Comparison of methods for detection of norovirus in oysters
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Comparison of methods for detection of norovirus in oysters. / Schultz, Anna Charlotte; Saadbye, Peter; Hoorfar, Jeffrey; Nørrung, Birgit.
I: International Journal of Food Microbiology, Bind 114, Nr. 3, 20.03.2007, s. 352-6.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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 -
ID: 46987017