GB 4789.42-2025 National food safety standard - Food microbiological examination - Norovirus
1 Scope
This standard specifies a real-time fluorescence RT-PCR examination method for Norovirus in food.
This standard is applicable to the detection of norovirus in shellfish, raw vegetables, hard surface foods, soft fruits and packaged drinking water.
2 Equipment and materials
In addition to conventional sterilization and incubation equipment used in microbiological laboratories, other equipment and materials required are as follows:
2.1 Real-time fluorescence PCR.
2.2 Cryogenic centrifuge: with centrifugal force >12,000×g, temperature ≤4℃; the casing volume is suitable for 1.5mL/50mL centrifuge tube.
2.3 Sterile blade or equivalent homogenizer.
2.4 Vortex mixer.
2.5 Electronic balances: with sensitivities of 0.1g and 0.1mg, respectively.
2.6 Full temperature oscillator or equivalent device: with a temperature range of 2℃ to 60℃.
2.7 Constant temperature water bath or constant temperature metal bath: with a temperature range of room temperature ~100℃.
2.8 Centrifuge: with centrifugal force >5,000×g; the casing volume is suitable for 50mL/15mL centrifuge tube.
2.9 Cryogenic freezer: -80℃.
2.10 Micropipette.
2.11 pH meter or precision pH test paper.
2.12 Aseptic homogeneous bag: 400mL (volume), with a filter screen.
2.13 Sterile cotton swabs.
2.14 Sterile shellfish shucker.
2.15 Rubber pad.
2.16 Sterile scissors.
2.17 Sterile tweezers.
2.18 Aseptic culture dish.
2.19 RNase-free glassware, RNase-free centrifuge tube, RNase-free pipette tip, RNase-free spatula, RNase-free PCR thin-walled tube: See E.1 of Annex E.
2.20 Ultrafiltration concentration centrifuge tube (with a cut-off protein molecular weight of 50kD or 100kD).
2.21 Sterile mixed cellulose membrane (with a pore size of 0.45μm, and a diameter of 47mm).
3 Culture media and reagents
Unless otherwise specified, analytically-pure reagents shall be adopted for all experiments; and RNase-free ultrapure water (see E.2.1) shall be adopted for the experiment.
3.1 Primers and probes of GI and GII of norovirus: See Annex A.
3.2 Process control virus primers and probes: See Annex C.
3.3 Process control virus: Mengovirus or escherichia coli phage MS2, See Annex C.
3.4 External amplification control RNA: See Annex D.
3.5 Tris/glycine/beef extract (TGBE) buffer: See E.2.2.
3.6 5×PEG/NaCl solution: See E.2.3.
3.7 Phosphate buffer solution (PBS): See E.2.4.
3.8 Chloroform/n-butanol mixture: See E.2.5.
3.9 Proteinase K solution: See E.2.6.
3.10 75% ethanol: See E.2.7.
3.11 Trizol reagent: See E.2.8.
3.12 Hydrochloric acid (HCl) solution (6mol/L): See E.2.9.
3.13 Sodium hydroxide (NaOH) solution (1mol/L): See E.2.10.
Foreword i
1 Scope
2 Equipment and materials
3 Culture media and reagents
4 Examination procedures
5 Operation steps
6 Results and report
7 Others
Annex A Primers and probes for real-time fluorescence RT-PCR
Annex B Reaction system and parameters of real-time fluorescence RT-PCR
Annex C Process control virus culture as well as primers and probes used
Annex D Preparation of external amplification control RNA
Annex E Removal of RNase and preparation of RNase-free solutions
