SN/T 5325.6-2020Quantitative detection of food-borne viruses in exported food. Digital PCR method - Part 6: Coxsackie virus (English PDF)

3.7 Abbreviations

3.7.1 CV (Coxsackie virus). Coxsackie virus.

3.7.2 CV-A6 (Coxsackie virus-A6). Coxsackie virus A6.

3.7.3 CV-A10 (Coxsackie virus-A10). Coxsackie virus A10.

3.7.4 CV-A16 (Coxsackie virus-A16). Coxsackie virus A16.

3.7.5 CV-B2 (Coxsackie virus-B2). Coxsackie virus B2.

3.7.6 PC (process control). Process quality control.

3.7.7 EC RNA (external control RNA). External control RNA.

3.7.8 pfu (plaque forming unit). plaque forming unit.

8.1 Selection of quality control substances

8.1.1 PC substance

Choose MS2 phage or other equivalent substances as the PC substance for CV detection. In vitro culture to obtain accurate titer (concentration), Reach 108 pfu/mL~1011 pfu/mL, and store at -80 °C after aliquoting. When used, it is diluted to 107 pfu/mL~109 pfu/mL.

8.1.2 EC RNA

Use the RNA containing the target fragment for CV detection as the detection EC RNA, and store it at -80°C. Can also be purchased containing CV for testing purposes Certified reference material for fragment RNA.

8.2 Virus enrichment in different food matrices

8.2.1 Soft fruits

8.2.1.1 Cut 25 g of soft fruits or raw vegetables into small pieces about 2.5 cm × 2.5 cm × 2.5 cm (if the fruit or vegetable is smaller than the body Product, don't cut).

8.2.1.2 Add 10 µL of PC substance to the surface of soft fruits or raw vegetables, and let stand at room temperature for 30 min.

8.2.1.3 Move the sample pieces to a sample bag with a 400 mL mesh filter bag, and add 40 mL TGB E solution (soft fruit samples, Need to add 30 U A.niger pectinase or 1 140 U A.aculeatus pectinase).

8.2.1.4 60 r/min at room temperature, shaking for 20 min. For acidic soft fruits, the pH should be checked every 10 minutes during the shaking process, such as pH when the value is lower than 9.0, use 1 mol/L NaOH to adjust the pH value to 9.5, and each time the pH value is adjusted, extend the shaking time by 10 min.

8.2.1.5 Transfer the shaking solution to a 50 mL centrifuge tube at 4°C, 10 000 r/min, and centrifuge for 30 min. Transfer the supernatant to a clean centrifuge tube in the medium, adjust the pH to 7.0 with 1 mol/L HCl.

8.2.1.6 Add 0.25 times the volume of 5 × PEG/NaCl solution, shake well, and shake at 4 °C overnight or 60 r/min for 60 min. 4 °C, 10 000 r/min, Centrifuge for 10 min to compact the pellet, discard the supernatant, and add 500 µL PBS to suspend the pellet.

8.2.1.7 For some soft fruit samples with too much slurry, resuspend in PBS, add 500 µL of chloroform-n-butanol, vortex to mix, Incubate at room temperature for 5 min, 4 °C, 10 000 r/min, centrifuge for 15 min, and take the supernatant for RNA extraction.

8.2.2 Hard surface food

8.2.2.1 Add 10 µL of PC substance to the surface of hard-surfaced food with an area of no more than 100 cm2, and let it stand for 30 min at room temperature for adsorption.

8.2.2.2 After moistening the sterile cotton swab with PBS, wipe the surface of the food vigorously (<= 100 cm2). Record the wiped area.

8.2.2.3 Immerse the cotton swab in 490 µL PBS buffer, press the sterile cotton, and repeat the immersion and squeeze 3 to 4 times to ensure the squeeze Squeeze out the largest amount of virus, measure and record the number of liquid mL, for subsequent RNA extraction.

8.2.3 Shellfish

8.2.3.1 Wear protective gloves and use a sterile shellfish peeler to open at least 10 shellfish.

8.2.3.2 Use sterile scissors, surgical forceps or other equivalent instruments to dissect out the digestive glands in the shellfish soft tissue on the rubber pad, and place them in the dry in a net petri dish. Collect 2.0 g ± 0.2 g digestive glands.

8.2.3.3 Add 10 µL of PC substance to the collected digestive glands, and let stand at room temperature for 30 min.

8.2.3.4 Use a sterile blade or equivalent homogenizer to homogenize the digestive glands and transfer to a centrifuge tube. Add 2.0 mL of proteinase K solution and mix well.

8.2.3.5 Use a constant temperature shaker or equivalent device, 37 °C, 320 times/min, shaking for 60 minutes.

8.2.3.6 Put the centrifuge tube into a water bath or equivalent device at 60°C for 15 minutes. Centrifuge at 3 000 r/min at room temperature for 5 min, take the supernatant, Used for subsequent RNA extraction.

Note. The sample processing should generally be transported in an environment below 4 °C. The laboratory should conduct the test as soon as possible after receiving the sample. If the test cannot be performed temporarily, it should Store the samples in a refrigerator at -80°C and thaw them before testing. Sample processing and PCR reaction should be carried out in a separate work area or room. Each the samples can be set up for 2 to 3 parallel processing.

8.3 Viral RNA extraction and purification

8.3.1 Virus cracking

Add the virus extract to the centrifuge tube, add Trizol reagent equal volume to the virus extract, mix well, shake vigorously, and place at room temperature 5 min, add 0.2 times volume of chloroform, vortex vigorously and mix for 30 s (not too strong to avoid emulsification layer, you can also mix upside down by hand Centrifuge for 5 min at 4 °C, 12 000 r/min, and transfer the upper water phase to a new centrifuge tube, and the middle layer cannot be sucked out.

8.3.2 Viral RNA extraction

Add an equal volume of isopropanol to the centrifuge tube, mix by inversion, place at room temperature for 5 min, 4 °C, 12 000 r/min, centrifuge for 5 min, discard Put the clear liquid upside down on absorbent paper, soak the liquid (different samples must be dried in different places on the absorbent paper).

8.3.3 Viral RNA purification

8.3.3.1 Add an equal volume of 75% ethanol each time, and wash the RNA precipitate twice by inversion.

8.3.3.2 Centrifuge for 10 min at 4 °C, 12 000 r/min, carefully discard the supernatant, put it on absorbent paper, and dry the liquid (not the same the product must be soaked in different places with absorbent paper), the supernatant remaining in the centrifuge tube is sucked dry with a micropipette, and one sample is replaced with a pipette tip.

Do not touch the tip of the precipitate, and dry it at room temperature for 3 minutes. It should not be too dry to avoid insoluble RNA.

8.3.3.3 Add 100 µL of RNase-free ultrapure water, mix gently to dissolve the RNA on the tube wall, 2 000 r/min, centrifuge for 5 s, on ice Save it for later use.

Note. Viral RNA can be manually extracted and purified, or commercial viral RNA extraction and purification kits can be used. Wear disposable rubber during operation Rubber or latex gloves and change frequently. The extracted RNA is reacted immediately, or stored at 4 °C for less than 8 h. If long-term storage is built it is recommended to store at -80 °C.

8.4 Quality control

8.4.1 Blank control, use RNase-free ultrapure water as the blank control.

8.4.2 Negative control, take food samples without CV and RNA extracted as negative control.

8.4.3 Positive control. EC RNA with a known nucleic acid concentration is used as a positive control.

8.4.4 PC substance, the recovery rate of MS2 RNA in food is used to express the recovery rate of CV RNA in food, which is used to evaluate the detection process.

Validity and calculation of the actual content of CV in the sample.

8.5 Digital PCR amplification

8.5.1 RNA template dilution

After the viral RNA is extracted, choose an appropriate dilution factor to dilute the RNA template according to the digital PCR platform used.

The solution is used for subsequent quantitative detection.

8.5.2 Establishment of PCR reaction system

Set 3 parallels for each sample, set the reaction system to dual, do dual digital PCR for A6 and A10 respectively; do A16, B2 do Double digital PCR. The system is. one-step digital RT-PCR reaction master mix 8 µL, 10 µmol/L upstream and downstream primers each 1.5 µL, each 10 µmol/L probe is 0.5 µL, RNA template is 2 µL, and the final volume of the reaction is 20 µL.

8.5.3 Micro-reaction system generation, digital PCR reaction amplification and result reading

According to the requirements of the instrument, add the prepared digital PCR reaction mixture into the sample hole of the micro-reaction system generator, and press the instrument operation instructions generate a micro-reaction system. Place the micro-reaction system in a PCR amplification instrument and perform amplification according to the following parameters. 60 °C, 30 min (1 °C/s), 1 cycle; 95 °C, 5 min (1 °C/s), 1 cycle; 94 °C, 30 s, 55 °C, 1 min (1 °C/s), 40 cycles; 98 °C, 10 min (1 °C/s), 1 cycle; 12 °C store the reaction product. Fluorescence detection of the micro reaction system.

9.1 Threshold setting

Set the fluorescence threshold limit according to the end-point fluorescence value of the negative segmentation system in the digital PCR result. Threshold limits need to be negative and positive the amplification results are clearly distinguished.

9.2 Control group setting

There is no amplification in the negative control group and the blank control group, and the detection value of EC RNA in the positive control should not exceed its value.

25% of the theoretical value, and the recovery rate of the PC control is > 1%. If there is a non-compliance, the experiment should be repeated.

Calculate the recovery rate according to formula (1).

CMS2×A×V1

C0×V2

×100% (1)

Where.

B (%)-recovery rate, that is, the recovery rate of PC substance detection.

CMS2 - PC substance concentration, that is, the copy concentration of PC substance detection target fragment in the diluted RNA template, which is determined by PC After the substance's digital PCR detection value is multiplied by the volume of the digital PCR reaction system, it is divided by the digital PCR reaction system the volume of the RNA template is calculated in units of copies per microliter.

A - The dilution factor of the RNA template.

V1-The volume of the RNA template after extraction, in microliters.

C0-The original concentration of the PC substance added to the sample, in units of copies per microliter.

V2 --The volume of PC substance added to the sample, in microliters.

9.3 Analysis and expression of test results

9.3.1 Analysis and expression of sample results

When the relative standard deviation (%) of the three repeated test results of each sample does not exceed 25%, the average of the three repeated test results the average value is used as the test result of the sample and is recorded as CMean (copy per microliter).

9.3.2 Calculation of test results

9.3.2.1 There is no CV specific fluorescence signal, there is no amplification in the negative control group and the blank control group, the positive control the detection value of EC RNA should not exceed 25% of its theoretical value. If the recovery rate of PC control is > 1%, it is considered that the sample to be tested does not contain CV, the test result is expressed as "Coxsackie virus not detected".

9.3.2.2 If the sample to be tested has a significant CV fluorescence signal, and the three parallel RSDs are <= 25%, then calculate according to formula (2).

Coxsackie virus content = CMean×A×V1 (2) the test result is expressed as "Coxsackie virus detected", and the content is.

Appendix A

(Normative)

Preparation of PC substance A.1 Summary this standard uses MS2 phage as the PC substance, and other equivalent viruses that do not cross-react with CV can also be used as the PC substance.

The MS2 phage is a single-stranded sense RNA virus with a genome containing 3 569 nucleotides. MS2 phage can infect F (male strain) large Enterobacter Escherichia, which can be cultured and purified, is resistant to Dnase or Rnase digestion, and can be used to count plaques.

Determine the level of potency. Commercial MS2 phage reagents, MS2 phage certified standard materials, or PC materials in the kit can also be used.

A.2 Reagents and instruments A.2.1 MS2 phage (ATCC 15597-B1).

A.2.2 Escherichia coli (ATCC 15597). Escherichia coli (ATCC 15597) is the MS2 phage (ATCC 15597-B1) host bacteria, it is recommended to use nutrient broth and nutrient agar for recovery, separation, purification and propagation.

A.2.3 Culture medium.

a) Bottom nutrient agar. ordinary nutrient agar

b) Upper nutrient agar. tryptone 10 g, NaCl 5.0 g, glucose 1.0 g, agar powder 6.0 g, 2.5 mol/L CaCl2 reserve 10 mL of liquid, 1,000 mL of distilled water, adjust the pH to 7.2.

A.2.4 Reagents and instruments. PBS, chloroform, biological safety cabinet, constant temperature incubator, etc.

A.3 MS2 phage culture and titer determination A.3.1 Amplification culture of MS2 phage Escherichia coli (ATCC 15597) freeze-dried powder was inoculated into 5.0 mL nutrient broth, cultured at 37 °C for 18 h-24 h, then streaked Plant on ordinary nutrient agar plates, culture at 37 °C for 18 h-24 h, pick a single colony and streak to inoculate on ordinary nutrient agar plates, 37 °C Cultivate for 18 h to 24 h, then pick a single colony and inoculate it in 5.0 mL nutrient broth, incubate at 37 °C for 18 h to 24 h for later use.

Using the double-layer agar culture method, 0.4 mL of Escherichia coli (ATCC 15597) suspension was dissolved with 0.1 mL of MS2 phage Mix the solution, incubate at 37 °C for 5 min, add 4 mL of the upper nutrient agar cooled to about 50 °C, and pour it into the lower layer after mixing.

Pave the nutrient agar on top, solidify the upper nutrient agar, incubate inverted at 37 °C for 17 to 24 hours, observe the plaque. Scrape the upper agar, mash it, Add 5.0 mL PBS to a sterile centrifuge tube, add a few drops of chloroform, 37 °C, 220 r/min, incubate for 1 h, 5 000 r/min, centrifuge for 15 min, the supernatant is the MS2 phage suspension. Repeat the above method to achieve mass proliferation of MS2 phage. After sterilization with 0.22 µm microporous filter, Divide into 1 mL/tube and store at -80 °C.

A.3.2 Determination of phage titer 10-fold dilution of MS2 phage suspension, inoculate 3 plates at each dilution, and cultivate according to A.3.1 double-layer agar culture method from 17 h to 24 h, take the plate with the number of plaques between 30 and 300 to calculate the MS2 phage titer. titer = average number of pfu ÷...