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SN/T 5201-2020 English PDF

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SN/T 5201-2020: (Technical specifications for species identification of forest musk deer)
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Basic data

Standard ID SN/T 5201-2020 (SN/T5201-2020)
Description (Translated English) (Technical specifications for species identification of forest musk deer)
Sector / Industry Commodity Inspection Standard (Recommended)
Classification of Chinese Standard B41
Classification of International Standard 65.020.01
Word Count Estimation 12,149
Date of Issue 2020-12-30
Date of Implementation 2021-07-01
Regulation (derived from) General Administration of Customs Announcement No. 136 [2020]
Issuing agency(ies) General Administration of Customs

SN/T 5201-2020: (Technical specifications for species identification of forest musk deer)

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Protocol for species identification of moschus berezovskii The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards Replace SN/T 1162-2002 Issued by the General Administration of Customs of the People's Republic of China 2019-12-30 released 2021-07-01 implementation

Foreword

This document is drafted in accordance with GB/T 1.1-2020. This document was proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this document. Chinese Academy of Inspection and Quarantine Sciences, Northeast Forestry University. The main drafters of this document. Lv Jizhou, Wu Shaoqiang, Lin Xiangmei, Yuan Xiangfen, Wang Xiaolong, Wang Caixia. Technical specifications for species identification of forest musk deer

1 Scope

This document specifies the morphology and PCR identification methods of forest musk deer species. This document is applicable to species identification of forest musk deer and its products.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document. GB/T 6682 Analytical laboratory water specifications and test methods GB 19489 Laboratory Biosafety General Requirements GB/T 27403 Laboratory Quality Control Specification Food Molecular Biology Testing 3 Terms, definitions and abbreviations There are no terms and definitions that need to be defined in this document. The following abbreviations apply to this document. COI. cytochrome c oxidase subunit 1 (cytochrome c oxidase subunit 1); DNA. deoxyribonucleic acid (deoxyribonucleic acid); dNTP. deoxy-ribonucleoside triphosphate; PCR. polymerase chain reaction (polymerase chain reaction); SDS. sodium dodecyl sulfate (sodium dodecyl sulfate).

4 Morphological characteristics

See Appendix A for the typical morphological characteristics of forest musk deer.

5 PCR detection

5.1 Instrument and equipment 5.1.1 Conventional PCR machine. 5.1.2 Tissue grinder. 5.1.3 Ultra-clean workbench. 5.1.4 Ice maker. 5.1.5 High-speed refrigerated centrifuge. 5.1.6 Water bath. 5.1.7 Desktop small centrifuge. 5.1.8 Conventional refrigerator. 5.1.9 Vortex oscillator. 5.1.10 Electrophoresis instrument. 5.1.11 Gel imaging system. 5.1.12 Micro adjustable pipette (10 μL, 100 μL, 1,000 μL) and matching tips. 5.2 Reagents and materials 5.2.1 Anhydrous ethanol. 5.2.2 Proteinase K (20 mmol/μL). 5.2.3 50×TAE buffer (see B.1 in Appendix B for reagent preparation). 5.2.4 Positive control (genomic DNA of forest musk deer). 5.2.5 Tirs-HCl buffer (see B.3 in Appendix B for reagent preparation). 5.2.6 Physiological saline (see B.4 in Appendix B for the preparation of reagents). 5.2.7 EDTA solution (see B.5 in Appendix B for reagent preparation). 5.2.8 TES buffer (see B.6 in Appendix B for reagent preparation). 5.2.9 10% SDS solution (see B.7 in Appendix B for reagent preparation). 5.2.10 Sodium chloride. 5.2.11 Trichloromethane. 5.2.12 Isopropanol. 5.2.13 Glacial acetic acid. 5.2.14 Tris saturated phenol. 5.2.15 70% ethanol. 5.2.16 Goldview nucleic acid dye. 5.2.17 Taq DNA polymerase. 5.2.18 dNTP (2.5 mmol/L). 5.2.19 Agarose. 5.2.20 Sterilized double-distilled water (should meet the specifications of first-grade water in GB/T 6682, see Appendix B, B.8). 5.3 Primer pair Unless otherwise specified, the reagents used in this document are of analytical grade, and the test water should meet the requirements of GB/T 6682. 5.4 Detection method 5.4.1 Sample processing 5.4.1.1 Take.200 μL of fresh, refrigerated or frozen samples directly for nucleic acid extraction. 5.4.1.2 Use physiological saline to wash off blood stains, take 100 mg～200 mg of sample and place it in liquid nitrogen to fully grind, and put the ground powder into 1.5 mL centrifuge tube is used for nucleic acid extraction. 5.4.2 Extraction of sample DNA Use the following methods to extract DNA, or use an equivalent commercial DNA extraction kit, and operate according to the operating instructions. a) Take the sample processed in 5.4.1, add 450 μL TES buffer and mix well, then add 50 μL SDS solution (10%), 5 μL Proteinase K (20 mg/mL), after mixing well, incubate at 56 ℃ for 4 h, shake once every 2 h. b) Add an equal volume of Tris saturated phenol (500 μL) and mix well. c) Take the upper water phase to a new tube and add an equal volume of phenol. chloroform. isoamyl alcohol (25.24.1) (see Appendix B for reagent preparation) Medium B.9) Mix well, centrifuge at 12,000 r/min for 5 min at room temperature. d) Take the upper water phase to a new tube, add an equal volume of chloroform to mix, and centrifuge at 12,000 r/min for 5 min at room temperature. e) Take the upper water phase, add 2.5 times the volume of absolute ethanol, mix well, and place at -20 ℃ for more than 2 h or overnight, 12 000 r/min Centrifuge at room temperature for 15 min. f) Discard the supernatant, add 1 mL of pre-cooled 70% ethanol, and centrifuge at 12,000 r/min for 5 min at room temperature. g) Absorb the remaining liquid, dry it at room temperature for about 10 minutes, dissolve it with 50 μL sterilized double-distilled water, and store it at -20 ℃ for later use. 5.4.3 PCR amplification detection 5.4.3.1 PCR reaction system Note. Other commercial PCR amplification kits can also be used, such as Promega GoTaq® G2 DNA Polymerase kit. When testing samples, a positive control and a blank control should be set at the same time. The positive control is genomic DNA extracted from known forest musk deer (see attached Record B.2), the blank control is sterilized double distilled water. 5.4.3.2 PCR program and reaction conditions The reaction parameters are 95 ℃ 1 min; 95 ℃ denaturation 30 s, 48 ℃ annealing 30 s, 72 ℃ extension 1 min, 38 cycles; then 72 Extend at ℃ for 10 min and store at 4 ℃ at last. Note. For PCR instruments without a hot lid, 40 μL of mineral oil must be added. 5.4.4 Electrophoresis detection of PCR products Prepare 1% agarose gel plates with TAE running buffer (final concentration of Goldview dye 0.5 μg/mL). Put the tablet into the horizontal In the swimming tank, add 1×TAE running buffer just above the surface of the gel, mix 5 μL of the PCR amplification product with the corresponding loading buffer They were added to the sample wells with a voltage of 80 V to 100 V, a current of 40 mA to 50 mA, and electrophoresis for 30 min to 45 min. 5.4.5 Observation of gel imaging system After the amplified products are electrophoresed, use a gel imager to observe the test results, take pictures, and record the test results. 5.5 Judgment of results 5.5.1 Preliminary screening of morphology According to the morphological characteristics of forest musk deer and its approximate species described in Appendix A, a preliminary screening of suspected samples of forest musk deer, pending further nucleic acid amplification, Sequencing detection and identification. 5.5.2 Judgment basis for the establishment of PCR experiment After agarose gel electrophoresis, only the amplified band size of the positive control primer was the same as expected (243 bp), and the negative pair Only when there is no corresponding fragment can the PCR amplification test be determined to be valid, otherwise the experiment will be invalid. 5.5.3 Judgment basis for suspected samples If the sample has amplified bands and the size is the same as expected, it can be determined that the sample is suspected of containing forest musk-derived components, and then the amplified bands will be obtained. The COI sequence is sequenced; if there is no target band in the sample, it is determined that the forest musk-derived component of the sample is negative. 5.5.4 Determination of forest musk deer species Compare the sequence obtained by sequencing with the reference COI sequence in Appendix C (you can also use the international DNA barcode identification database Boldsystems When the highest sequence belongs to the forest musk deer, and the similarity reaches more than 99%, it is judged as the positive result of the corresponding experiment, and then it is determined as the detection Forest musk-derived ingredients.

Appendix A

(Normative) Overview of Morphological Characteristics of Forest Musk Deer The morphological characteristics of forest musk deer are summarized in Table A.1

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