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SN/T 5226-2019Detection of turtle-derived components in exported foods Real-time fluorescence PCR method (English PDF)

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Issued by

General Administration of Customs

Level / Type

National · Recommended

Issue date

January 1, 1970

Implementation date

July 1, 2020

Scope

SN/T 5226-2019 (Detection of turtle-derived components in exported foods Real-time fluorescence PCR method) is available as an English-translated PDF.

SN/T 5226-2019 — This standard specifies the real-time fluorescent PCR detection method for turtle-derived components in exported food.

This standard is only applicable to Chinese soft-shelled turtle, yellow-sand soft-shelled turtle, black soft-shelled turtle, pearl soft-shelled turtle, mountain turtle, spiny soft-shelled turtle, small soft-shelled turtle, Indian small-headed soft-shelled turtle and Qualitative detection of the components of the Burmese marginal soft-shelled turtle and other soft-shelled turtles.

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Document preview — SN/T 5226-2019

National Standard of the People's Republic of China

ICS: 67.050
Classification: C 53

Issued by: General Administration of Customs

Foreword
1 Scope
2 Normative references
3 Terms and definitions
4 Abbreviations
5 Method summary
6 Reagents and materials
6.1 Primers and probes for detection
6.2 Reagents
7 Apparatus and equipment
8 Inspection steps
8.1 DNA extraction
8.2 Determination of DNA concentration and purity
8.3 Real-time fluorescent PCR detection
9 Quality control
10 Judgment and expression of results
10.1 Judgment of results
10.2 Presentation of results
11 Anti-pollution measures
12 Detection limit

Foreword

This standard was drafted in accordance with the rules given in GB/T 1.1-2009.

1 Scope

This standard specifies the real-time fluorescent PCR detection method for turtle-derived components in exported food.

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated reference documents, the latest version (including all amendments) is applicable to this document.

GB/T 6682 Analytical laboratory water specifications and test methods GB 19489 Laboratory Biosafety General Requirements GB/T 27403-2008 Laboratory Quality Control Specification Food Molecular Biology Testing

3 Terms and definitions

The following terms and definitions apply to this document.

Trionyx Sinensis

It is a class of amphibians and reptiles in the subfamily Trionyx subfamily of the Reptilidae Turtles. Its meat is rich in nutrients and is a good nourishing product.

Turtles are also used as medicinal materials.

Polymerase chain reaction; PCR the template gene sequence is first denatured by high temperature to become single-stranded. Under the action of DNA polymerase and suitable reaction conditions, according to the template sequence the two designed primers anneal to the corresponding complementary sequences on the two strands of the template DNA, and then bind to each other.

Under the action of synthase, four kinds of deoxyribonucleic acid (dNTP) are used as substrates to extend the primers, and then repeat denaturation, annealing and Extend this cycle, so that the gene fragments to be amplified will be amplified geometrically.

Real-time fluorescent PCR Fluorescent groups are added to the PCR reaction system, and the accumulation of fluorescent signals is used to monitor the entire PCR process in real time.

Ct value cycle threshold the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold.

4 Abbreviations

The following abbreviations apply to this document.

DNA. deoxyribonuleic acid, deoxyribonucleic acid.

PCR. polymerase chain reaction, polymerase chain reaction.

dNTP. deoxyribonuleoside triphosphate, deoxyribonuleoside triphosphate.

dATP. deoxyadenosine triphosphate, deoxyadenosine triphosphate.

dCTP. deoxycytidine triphosphate, deoxycytidine triphosphate.

dGTP. deoxyguanosine triphosphate, deoxyguanosine triphosphate.

dTTP. deoxythymidine triphosphate, deoxythymidine triphosphate.

Tris. Tris (Hydroxymethyl) aminomethane.

EDTA. Ethylene diaminetetraacetic acid, ethylene diamine tetraacetic acid.

Taq. Thermus aquaticus, Thermus aquaticus.

OD. optical density, optical density.

FAM. Carboxyfluorescein,.

BHQ1.Black hole quencher 1, black hole quencher group 1.

5 Method summary

After the sample is ground, DNA is extracted, the DNA is used as a template, and the specific detection primers for the mitochondrial cytochrome b gene of the soft-shelled turtle and Probe, perform real-time fluorescent PCR amplification, observe the amplification phenomenon of real-time fluorescent PCR, and judge whether there are turtle components in the sample.

6 Reagents and materials

Unless otherwise specified, all reagents are analytical reagents or biochemical reagents. The experimental water meets the requirements of GB/T 6682.All reagents are Dispense with DNase-free containers.

6.1 Primers and probes for detection

The primers and probes for the amplification of soft-shelled turtle components are shown in Table 1.See Appendix A for the target sequence amplified by the turtle primer.

6.2 Reagents

6.2.1 Tissue lysate. 1 mol/L Tris-HCl (pH 8.0); 0.5 mol/L EDTA (pH 8.0); 10% (mass concentration) SDS.

6.2.2 Proteinase K solution (20 mg/mL).

6.2.3 Saturated phenol.

6.2.4 Trichloromethane.

6.2.5 Isoamyl alcohol.

6.2.6 75% ethanol.

6.2.7 NaAc solution (3 mol/L).

6.2.8 TE buffer (Tris-HCl, EDTA buffer). 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8.0).

6.2.9 Real-time fluorescent PCR reaction mixture. 12.5 µL reaction system includes 1U~3 U Taq enzyme, 1×PCR buffer, 2.5 mmol/L~ 4.0 mmol/L Mg2, 0.2 mmol/L dNTP. If there are equivalent commercial kits, these equivalent products can also be used.

7 Apparatus and equipment

7.1 Real-time fluorescent PCR machine.

7.2 Centrifuge. the centrifugal speed is not less than 12 000 r/min.

7.3 Constant temperature water bath.

7.4 Balance. Sensitivity 0.1 g, 0.01 g.

7.5 Micropipette. 10 µL, 100 µL,.200 µL, 1000 µL.

7.6 Nucleic acid protein analyzer or ultraviolet spectrophotometer.

7.7 Vortex oscillator.

7.8 Mortar and crushing device.

7.9 pH meter.

7.10 Refrigerator 2 °C~8 °C, -20 °C.

8 Inspection steps

8.1 DNA extraction

Weigh.200 g of the sample to be tested, grind and mix, and then weigh 100 mg of the above sample into a 1.5 mL centrifuge tube, and add 700 µL of pretreatment Hot (56 °C) tissue lysate and 20 µL proteinase K solution (20 mg/mL), mix well with a vortex shaker, and bath in 56 °C water for 2 h.

Shake and mix well during the period; add 700 µL saturated phenol to each tube, mix upside down, centrifuge at 12,000 r/min 4 °C for 10 min; transfer the supernatant in a clean centrifuge tube, add 1/2 volume of the supernatant volume of saturated phenol and 1/2 volume of the supernatant volume of chloroform. isoamyl alcohol (24.1) upside down Mix well, centrifuge at 12 000 r/min 4 °C for 10 min; transfer the supernatant to a clean centrifuge tube, add an equal volume of chloroform. isoamyl alcohol (24.1) up and down Mix by inversion, centrifuge at 12 000 r/min 4 °C for 10 min; transfer the supernatant to a clean centrifuge tube, add an equal volume of chloroform, 12 000 r/min Centrifuge at 4 °C for 10 min; take about 400 µL of supernatant, add 2.5 times volume of cold absolute ethanol and 1/10 volume of NaAc solution (3 mol/L), Precipitate at -20 °C for 2 h or overnight; centrifuge at 12 000 r/min at 4 °C for 30 min, discard the solution, add 1 000 µL of 75% cold ethanol, Centrifuge at 12 000 r/min 4 °C for 10 min; discard the supernatant, and add 50 µL TE buffer (pH 8.0) to dissolve the DNA precipitate after natural drying.

Store at -20 °C for later use.

Note. An equivalent DNA extraction kit can also be used to extract sample DNA.

8.2 Determination of DNA concentration and purity

Use a nucleic acid protein analyzer or an ultraviolet spectrophotometer to detect the absorbance values A260 and A280 at 260 nm and 280 nm, respectively. DNA the concentration is calculated according to formula (1).

8.3 Real-time fluorescent PCR detection

8.3.1 Real-time fluorescent PCR reaction system

Prepare the real-time fluorescent PCR reaction system according to Table 2.Two parallels should be set for each sample to be tested.

8.3.2 Real-time PCR reaction parameters

Real-time fluorescent PCR reaction conditions vary slightly with different instruments, generally. 95 °C/10 min; 95 °C/15 s, 58 °C/60 s, 40 Cycles.

8.3.3 Experimental control

A positive control, a negative control, and a blank control should be set up during the inspection process. Use a sample known to contain turtle ingredients as a positive control, Use a sample that is known to contain no turtle components as a negative control, and use an equal volume of double-distilled water instead of template DNA as a blank control.

9 Quality control

9.1 Blank control. There is no logarithmic increase in fluorescence, and the corresponding Ct value is greater than 40.0.

9.2 Negative control. There is no logarithmic increase in fluorescence, and the corresponding Ct value is greater than 40.0.

9.3 Positive control. There is a logarithmic increase in fluorescence, and a typical amplification curve appears in the fluorescence channel, and the corresponding Ct value is less than or equal to 30.0.

10 Judgment and expression of results

10.1 Judgment of results

In the case of compliance with Chapter 9, the following result judgments can be made.

a) If the Ct value is less than or equal to 35.0, the test sample is judged to be positive;

b) If the Ct value is greater than or equal to 40.0, the test sample is judged to be negative;

c) If it is greater than 35.0 and less than 40.0, repeat it once. If the Ct value is still less than 40.0 after re-amplification, the test sample is judged to be positive;

If the Ct value after re-amplification is greater than or equal to 40.0, the test sample is judged to be negative.

10.2 Presentation of results

10.2.1 If the result is positive, it shall be expressed as "turtle component detected".

10.2.2 If the result is negative, it is stated as "no turtle component detected".

11 Anti-pollution measures

The measures to prevent cross-contamination during the testing process shall be implemented in accordance with the provisions in Appendix D of GB/T 27403-2008.

12 Detection limit

The minimum detection limit specified by this method is 0.01% (mass percentage).

Appendix A

(Informative appendix)

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This preview omits tables, figures, formulas and parts of the technical clauses. The complete document — 7 pages — is available in the English PDF.

Referenced standards

Normative references

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