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SN/T 5200-2020: (Technical specification for identification of pangolin species)

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Protocol for species identification of Manis Spp 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 2020-12-30 release 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. Wu Shaoqiang, Lu Jizhou, Lin Xiangmei, Yuan Xiangfen, Wang Xiaolong. Technical specification for identification of pangolin species

1 Scope

This document specifies Chinese pangolins, Malay pangolins, Indian pangolins, South African pangolins, giant pangolins, long-tailed pangolins and pangolins. The morphological characteristics of tree pangolins and other species and the SYBR Green I real-time fluorescent PCR identification method, as well as the sequencing results of PCR products and Sequence analysis and comparison of methods to determine the species of pangolin. This document applies to Chinese pangolins, Malay pangolins, Indian pangolins, South African pangolins, giant pangolins, long-tailed pangolins and Species identification of tree pangolin 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 for 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. Cytb. Cytochrome b (cytochrome b) DNA. deoxyribonucleic acid dNTP. deoxy-ribonucleoside triphosphate Real-time PCR. real-time polymerase chain reaction (real-time polymerase chain reaction) SDS. sodium dodecyl sulfate (sodium dodecyl sulfate)

4 Morphological characteristics

Chinese pangolin, Malay pangolin, Indian pangolin, South African pangolin, large pangolin, long-tailed pangolin and adult tree pangolin See Appendix A for typical morphological characteristics. See Appendix B for the typical morphological characteristics of four pangolin scales. Chinese pangolin, Malay pangolin, tree pangolin and giant pangolin. Based on the above characteristics, the species of live pangolin or suspected pangolin scale samples can be preliminarily identified, and the precise identification should be further Nucleic acid amplification and sequencing to determine the species. 5 Real-time PCR detection 5.1 Instruments and equipment 5.1.1 Real-time thermal cycler. 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 mixer. 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 electrophoresis buffer. For reagent preparation, see C.1 in Appendix C. 5.2.4 Genomic DNA positive control of pangolin. For the preparation of positive materials, see C.2.in Appendix C. 5.2.5 Tirs-Hcl buffer. For reagent preparation, see C.3.in Appendix C. 5.2.6 Physiological saline (0.85% NaCl solution). For reagent preparation, see C.4 in Appendix C. 5.2.7 EDTA solution. see Appendix C C.5 for reagent preparation. 5.2.8 TES buffer. see Appendix C C.6 for reagent preparation. 5.2.9 10% SDS solution. see Appendix C C.7 for reagent preparation. 5.2.10 Sodium chloride. 5.2.11 Trichloromethane. 5.2.12 Isoamyl alcohol. 5.2.13 Glacial acetic acid. 5.2.14 Tris-saturated phenol. 5.2.15 70% ethanol. 5.2.16 SYBR Premix Ex Taq enzyme 1). 5.2.17 Sterilized double-distilled water. should meet the specifications of first-grade water in GB/T 6682.see C.8 in Appendix C. 5.3 Primer pair Unless otherwise specified, the reagents used in this standard are of analytical grade, and the test water should meet the requirements of GB/T 6682 and GB 19489. 1) TAKARA SYBR Fluorescence Quantitative Kit is an example of a suitable commercially available product. This information is given for the convenience of this standard The user does not mean the approval of this product. 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 (5.2.6) to wash away blood stains, take a sample of 100 mg ~.200 mg, and place it in liquid nitrogen to fully grind the ground powder Put it into a 1.5 mL centrifuge tube 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. 5.4.2.1 Take the processed sample (5.4.1), add 450 μL TES buffer (5.2.8) and mix well, then add 50 μL 10% SDS solution, 5 μL of proteinase K (5.2.2), mix well, incubate at 56 ℃ for 4 h, shake once every 2 h. 5.4.2.2 Add an equal volume of Tris-saturated phenol (500 μL) and mix well. 5.4.2.3 Take the upper water phase to a new tube, add an equal volume of phenol. chloroform (5.2.11). isoamyl alcohol (25.24.1) (see reagent preparation) C.9) Mix well, centrifuge at 12,000 r/min for 5 min at room temperature. 5.4.2.4 Take the upper water phase to a new tube, add an equal volume of chloroform (5.2.11) and mix well, and centrifuge at 12,000 r/min for 5 min at room temperature. 5.4.2.5 Take the upper aqueous phase, add 2.5 times the volume of absolute ethanol (5.2.1), mix, and place at -20 ℃ for more than 2 h or overnight. Centrifuge at 12 000 r/min for 15 min at room temperature. 5.4.2.6 Discard the supernatant, add 1 mL of pre-cooled 70% ethanol (5.2.15), and centrifuge at 12,000 r/min for 5 min at room temperature. 5.4.2.7 Aspirate and discard the remaining liquid, dry it at room temperature for about 10 minutes, dissolve it with 50 μL of sterilized double-distilled water (5.2.17), and store it at -20 ℃ for later use. 5.4.3 SYBR Green I real-time fluorescent PCR amplification detection 5.4.3.1 SYBR Green I real-time fluorescent PCR reaction system 5.4.3.1.1 SYBR Premix Ex Taq enzyme (2×), 12.5 μL. 5.4.3.1.2 MAF (20 pmol/μL), 2.0 μL. 5.4.3.1.3 MAR (20 pmol/μL), 1.0 μL. 5.4.3.1.4 Sample DNA template, 1 μL. 5.4.3.1.5 Sterilized double distilled water (5.2.17), 3.5 μL. The total volume is 20 μL of the system. Mix it with a vortex mixer, centrifuge it immediately, and use it at 4°C for later use. Note. Other commercial SYBR Green I real-time fluorescent PCR amplification kits can be used 2). When testing samples, a positive control (5.2.4) and a blank control should be set at the same time. The positive control is a genome extracted from a known pangolin DNA (see C.2 in Appendix C), the blank control is sterilized double distilled water (5.2.17). 5.4.3.2 SYBR Green I real-time fluorescent PCR program and reaction conditions The reaction parameters are 95 ℃ 2 min, 95 ℃ denaturation 15 s, 60 ℃ annealing/extension 30 s, 40 cycles. Annealing/extension in each cycle Read the fluorescence signal at the end of the extension step at 60 ℃; after the end of the total extension, add the melting curve analysis step (60 ℃, 2 min; at 0.1 ℃/s The rate of opening the temperature; up to 95 ℃; the whole process is continuous collection of fluorescence.) 5.5 Judgment of results 5.5.1 The basis for judging the validity of the experiment In the amplification kinetics graph, the positive control can amplify a typical S-shaped curve, the melting curve peak is single, and the Tm value of the amplified product Uniformity, see Appendix D for typical curves; at the same time, the negative control has neither a typical S-type amplification curve nor a melting curve peak. Zeyang 2) Such as QuantiNDva SYBRGreen PCR Kit from QIAGEN. The sex control and negative control were established, and the SYBR Green I real-time fluorescent PCR was successful. 5.5.2 Judgment basis for suspected samples If the sample has a typical S-shaped amplification curve, the Ct value is less than 35, and the melting curve peak is single and similar to the Tm value of the positive control amplification product, Then it can be determined that the sample is suspected to contain pangolin-derived components, and then sequence the amplified Cytb sequence; if the sample 35 ≤ Ct value < 40, repeat the experiment, continue to appear 35 ≤ Ct value < 40, and the sample dissolution curve peak is single and the same as the positive control amplification product Tm value Like, it can be determined that the sample is suspected of containing pangolin-derived components, otherwise the sample is determined to be negative for pangolin-derived components; if there is no target band, The pangolin-derived component of the sample was determined to be negative. 5.5.3 Determination of pangolin species The sequence obtained by sequencing and the Chinese pangolin, Malay pangolin, South African pangolin, Indian pangolin, giant pangolin, long tail pangolin A and tree pangolin Cytb reference sequence (see Appendix E) for multiple sequence alignment, if the sequence with the closest homology to the obtained sequence belongs to When one of the 7 types of pangolins, including Chinese pangolin and Malay pangolin, and the homology reaches 98% or more, it can be determined that the corresponding experiment is The result was positive, and the Chinese pangolin, Malay pangolin, South African pangolin, Indian pangolin, giant pangolin, and long-tailed pangolin were detected. Mangolin or tree pangolin-derived component.

Appendix A

(Normative) Overview of the Morphological Characteristics of Seven Pangolins A.1 Overview of the morphological characteristics of Chinese pangolin The head and body length are 40 cm to 55 cm, the tail length is 27 cm to 33 cm, and the adult weight is 3.1 kg to 5.2 kg. The percentage of tail length to head body length is about Is 65%. The hind legs are 64 cm long, and the ears are 1.96 cm long. The skull is stubby, the head is large, the limbs are sturdy, and the front and rear feet each have 5 toes. The cheeks, eyes and throat are not covered with scales, but with yellowish white or reddish brown hair. The whole body is covered with hard keratin that is arranged in a tile-like shape and resembles the scales of a fish Thick scales, between the scales where the scales are covered, there are yellow-brown bristles, with 5-6 clusters extending out of the scales. The chest and abdomen are flat and reach the base of the tail The lower part and the inner side of the limbs are not covered with scales but covered with thin yellow-white or red-brown hairs, with very few hairs. The scales are semicircular in length and are in line with the body axis Parallel, there are about 560-688 slices in the whole body. The scales are dark-brown or gray-brown, the edges of the elderly individuals are yellow-brown or orange-brown, and the young ones have not yet The keratinized scales are yellow. The back is raised, the scales are diamond-shaped and each row of scales is about 15-16 pieces, and the ventral side has about 3 rows of ridged scales, the ridge The scales are blunt and rounder than Malay pangolins. The scales of the forelimbs are arranged obliquely behind the back from the distal end to the proximal end, while the hind limbs are arranged from the proximal end covering. tail It is flat, with about 18 scales on the dorsal tail of the tail, and there are 9 to 10 longitudinal scales on the tail. As shown in Figure A.1 a). A.2 Morphological characteristics of Malay pangolin The head and body length is about 65 cm, the tail length is 56 cm, and the adult weight is 2.7 kg to 4.7 kg. The percentage of the tail length to the head and body length is about 90%. male Individuals are larger than females. The front and rear feet each have 5 toes, and the claws are thick and strong. The skull is longer than that of the Chinese pangolin. The ears are small. kiss Slender and narrow, without teeth, the tongue is long and sticky. The body is covered by rows of scales and hairs. There are 16 scales on the back Column, every... ...