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

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 °C 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 °C 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 °C 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 °C 2 min, 95 °C denaturation 15 s, 60 °C annealing/extension 30 s, 40 cycles. Annealing/extension in each cycle Read the fluorescence signal at the end of the extension step at 60 °C; after the end of the total extension, add the melting curve analysis step (60 °C, 2 min; at 0.1 °C/s the rate of opening the temperature; up to 95 °C; 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 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...