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SN/T 5227.2-2019 PDF English

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SN/T 5227.2-2019: (Rapid detection of pig-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method))
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Std ID	Contents [version]	USD	STEP2	[PDF] delivered in	Standard Title (Description)	Status
SN/T 5227.2-2019	English	209	Add to Cart	3 days [Need to translate]	(Rapid detection of pig-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method))	Valid

Standard similar to SN/T 5227.2-2019

GB 5413.12 | GB 5413.10 | SN/T 1888.4 | SN/T 5227.11 | SN/T 5227.1 | SN/T 5227.10 |

Basic data

Standard ID	SN/T 5227.2-2019 (SN/T5227.2-2019)
Description (Translated English)	(Rapid detection of pig-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method))
Sector / Industry	Commodity Inspection Standard (Recommended)
Classification of Chinese Standard	C53
Classification of International Standard	67.050
Word Count Estimation	9,948
Date of Issue	2019
Date of Implementation	2020-07-01
Issuing agency(ies)	General Administration of Customs

SN/T 5227.2-2019: (Rapid detection of pig-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method))

---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Rapid detection of porcine derived ingredient in food for export Recombinase-aid amplification (RAA) method The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards 2019-12-27 release 2020-07-01 Implementation Issued by the General Administration of Customs of the People's Republic of China

Foreword

SN/T 5227-2019 "Rapid Detection of Animal-derived Components in Exported Foods Recombinase Mediated Strand Replacement Nucleic Acid Amplification Method (RAA) Law)" is divided into 11 parts. Part 1.Rapid detection of chicken-derived components in exported foods, recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 2.Rapid detection of pig-derived components in export foods, recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 3.Rapid detection of sheep-derived components in export foods, recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 4.Rapid detection of duck-derived components in export foods recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 5.Rapid detection of bovine-derived components in exported foods, recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 6.Rapid detection of buffalo-derived components in export foods recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 7.Rapid detection of horse-derived components in export foods, recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 8.Rapid detection of donkey-derived components in export foods recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 9.Rapid detection of fox-derived components in export foods recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 10.Rapid detection of mink-derived components in exported foods, recombinase-mediated strand replacement nucleic acid amplification method (RAA method); Part 11.Rapid detection of rat-derived components in export foods Recombinase-mediated amplification strand replacement nucleic acid method (RAA method). This part is part 2 of SN/T 5227-2019. This standard is compiled in accordance with the rules of GB/T 1.1-2009. This standard was proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this standard. Zhengzhou Customs of the People's Republic of China, Shijiazhuang Customs of the People's Republic of China, China Inspection and Quarantine Science Research Institute, Zhejiang Institute of Food and Drug Inspection, Xiamen Customs of the People's Republic of China, Dalian Customs of the People's Republic of China, Chinese Beijing Customs of the People’s Republic of China, Gongbei Customs of the People’s Republic of China, Hangzhou Customs of the People’s Republic of China, and Chengdu Customs of the People’s Republic of China. The main drafters of this standard. Miao Li, Wang Jianchang, Wang Xiangjun, Han Jianxun, Shen Hong, Xu Shufei, Zheng Qiuyue, Wang Lin, Luo Baozheng, Wu Shan, Lin Hua, Kong Fande. Rapid detection of pig-derived ingredients in export food Recombinase-mediated strand replacement nucleic acid amplification method (RAA method)

1 Scope

This section specifies the RAA detection method for pig-derived ingredients in exported food. This section applies to the qualitative detection of pig-derived ingredients in exported food. The minimum detection limit (LOD) of the method specified in this standard is 0.01% (W/W).

2 Normative references

The following documents are indispensable for the application of this document. For dated reference documents, only the dated version is applicable to this file. 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/T 27403-2008 Laboratory Quality Control Specification Food Molecular Biology Testing 3 Terms, definitions and abbreviations 3.1 Terms and definitions The following terms and definitions apply to this document. 3.1.1 Pig Sus Artiodactyla, Suigoides, Suidae, Suidae are divided into domestic pigs and wild boars. The subspecies include. Central European wild boar, Southeast Asian wild boar and Indian wild boar, it is generally believed that these three subspecies constitute the breeding of domestic pigs. 3.1.2 Real-time RAA real-time RAA A constant-temperature nucleic acid rapid amplification technology (referred to as RAA technology) that recombinase-mediated strand replacement. Utilize what is obtained from bacteria or fungi Recombinant enzyme, at a constant temperature (usually 37 ℃ ~ 42 ℃), the recombinase can be tightly combined with the primer to form a polymer of the enzyme and the primer. With the help of the single-stranded DNA binding protein, the double-stranded structure of the template DNA is opened. When the primer is searched on the template DNA, it is completely matched with it. When matching complementary sequences, under the action of DNA polymerase, new complementary DNA strands are formed, and the amplified products grow exponentially. Use fluorescent With the labeling of the optical probe, as the RAA reaction proceeds, the RAA product and the increase in the fluorescence signal show a corresponding relationship. 3.1.3 Ct value cycle threshold The number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold. 3.1.4 T value Time threshold The time required for the fluorescence signal in each reaction tube to reach the set threshold. 3.2 Abbreviations The following abbreviations apply to this document. 3.2.1 RAA. recombinase-aid amplification, recombinase-mediated amplification. 3.2.2 DNA. deoxyribonuleic acid, deoxyribonucleic acid. 3.2.3 CTAB. cetyltrithylammonium bromide, cetyltrithylammonium bromide. 3.2.4 Tris. tris (Hydroxymethyl) aminomethane. 3.2.5 EDTA. ethylene diaminetetraacetic acid. 3.2.6 dNTPs. deoxyribonuleoside triphosphate, deoxyribonuleoside triphosphate. 3.2.7 SSB. single stranded DNA binding protein, single stranded DNA binding protein. 3.2.8 SC-recA. Streptomyces coelicolor recombinase, Streptomyces coelicolor recombinase. 3.2.9 BS-recA. Bacillus subtilis recombinase, Bacillus subtilis recombinase. 3.2.10 Bsu. Bacillus subtilis, Bacillus subtilis. 3.2.11 Tricine. N-tris [Hydroxymethyl] methylglycine, N-tris hydroxymethyl methyl amino acid.

4 Method summary

Take the extracted DNA as a template, and use pig-specific detection primers and probes for real-time fluorescent RAA amplification. The increase in RAA has enabled the detection and identification of pig ingredients in food and feed.

5 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. 5.1 CTAB extraction buffer (pH8.0). 10 g/L CTAB, 0.7 mol/L NaCl, 0.05 mol/L Tris-HCl, 0.01 mol/L Na2EDTA. 5.2 Phenol. Chloroform. Isoamyl alcohol=25.24.1. 5.3 Isopropanol. 5.4 70% ethanol (V/V). 5.5 TE buffer (pH 8.0). 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8.0). 5.6 A Buffer. 20% polyethylene glycol. 5.7 Real-time fluorescent RAA amplification system. 2.5 mmol/L dNTPs, 225 ng/µL SSB, 300 ng/µL recA recombinase protein (SC- recA/BS-recA), 75 ng/µL Bsu DNA polymerase, 75 ng/µL Exo exonuclease, 250 mmol/L Tricine, 12.5 mmol/ L dithiothreitol, 250ng/µL creatine kinase. An equivalent commercial kit can also be used instead. 5.8 B Buffer. 280mM magnesium acetate.

6 Equipment

6.1 Real-time fluorescent PCR machine. 6.2 Constant temperature fluorescence detector. 6.3 Nucleic acid protein analyzer or ultraviolet spectrophotometer. 6.4 Constant temperature water bath. 6.5 Centrifuge. speed ≥ 12 000 r/min. 6.6 Micropipette. measuring range 0.5 µL~10 µL, 10 µL~100 µL, 20 µL~200 µL,.200 µL~1000 µL. 6.7 Mortar and crushing device. 6.8 Vortex oscillator. 6.9 Centrifuge tube. 2 mL, 1.5 mL.

7 Detection steps

7.1 DNA extraction Take 0.2 g of crushed or ground sample into a clean 1.5 mL centrifuge tube (if the sample contains impurities and condiments, add 1 mLdd Wash twice with H2O), add 600 µL CTAB extraction buffer, vortex to mix well, incubate at 70 ℃ for 15 min, inverted during Heart tube 2~3 times; centrifuge at 12 000r/min for 5 min, take the supernatant into a new clean 1.5 mL centrifuge tube; add 500 µL of phenol. chlorine Imitation. Isoamyl alcohol (25.24.1), turn the centrifuge tube upside down 2 to 3 times, vortex and shake to mix, centrifuge at 12 000 r/min for 5 min; Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 0.7 times the volume of isopropanol, invert the centrifuge tube up and down 2~3 times, and let stand at 4 ℃ Centrifuge at 12 000 r/min for 30 min at 4 ℃ for 3 min. Carefully discard the supernatant; add 700 µL 70% ethanol, resuspend the pellet, and separate at 12 000 r/min. Discard the supernatant carefully for 1 minute; open the lid of the tube, evaporate the dry liquid at room temperature, and add 50 µL~100 µL TE (pH 8.0) buffer to dissolve it. Decompose DNA and store at -20 ℃ for later use. DNA extraction can also be performed with equivalent DNA extraction kits. 7.2 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 of is calculated according to formula (1). 7.3 Real-time fluorescent RAA amplification 7.3.1 Real-time fluorescence RAA reaction system 7.3.2 Real-time fluorescent RAA reaction program One of the following two amplification instruments can be used for detection, and the reaction procedure is as follows. 7.3.2.1 Real-time fluorescent PCR instrument 39°C, 60s, 1 cycle; 39°C, 30s, 40 cycles, collect fluorescence in each cycle. 7.3.2.2 Constant temperature fluorescence detector 39°C, 1min; 39°C, 20min, start to collect fluorescence in the second stage. 7.3.3 Experimental control In the detection process, a positive control, a negative control, and a blank control are set respectively. Use a sample containing porcine ingredients as a positive control sample The sample without pig-derived ingredients was used as a negative control, and double distilled water of the same volume as the template was used as a blank control.

8 Quality control

8.1 Real-time fluorescent PCR instrument 8.1.1 Blank control. there is no logarithmic increase in fluorescence, and the corresponding Ct value is not reported. 8.1.2 Negative control. there is no logarithmic increase in fluorescence, and the corresponding Ct value is not reported. 8.1.3 Positive control. there is a fluorescence logarithmic increase, and a typical amplification curve appears in the fluorescence channel, and the corresponding Ct value is ≤ 30.0. 8.2 Constant temperature fluorescence detector 8.2.1 Blank control. no fluorescence logarithmic growth, corresponding non-reported T value (time). 8.2.2 Negative control. no fluorescence logarithmic growth, corresponding non-reported T value (time). 8.2.3 Positive control. there is a fluorescence logarithm increase, and a typical amplification curve appears in the fluorescence channel, the corresponding T value (time) ≤ 15min.

9 Judgment and expression of results

9.1 Judgment of results 9.1.1 Real-time fluorescent PCR instrument 9.1.1.1 The result can be judged to be valid only if it meets the requirements of Clause 8.1. 9.1.1.2 If the Ct value is ≤ 35.0, the test sample is determined to be positive. 9.1.1.3 If 35.0 < Ct value < 40.0, repeat it once. If the result of re-testing is still 35.0 < Ct value < 40.0, it is judged The test sample is positive. 9.1.1.4 If there is no report of Ct value or no increase in fluorescence logarithm, the test sample is judged to be negative. 9.1.2 Constant temperature fluorescence detector 9.1.2.1 The result can be judged to be valid only if it meets the requirements of Clause 8.2. 9.1.2.2 If the T value (time) ≤ 15min, the test sample is judged to be positive. 9.1.2.3 If 15min < T value (time) < 20min, repeat once. If the result of re-testing is still 15min < T value (time Time) < 20min, the test sample is judged to be positive. 9.1.2.4 If there is no report of T value (time) or no increase in fluorescence logarithm, the test sample is judged to be negative. 9.2 Presentation of results 9.2.1 If the sample is positive, it is expressed as "pig component detected". 9.2.2 The sample is negative, which is expressed as "no pig ingredients detected". 10 Measures to prevent cross contamination during testing 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.

Appendix A

(Informative appendix) Gene amplification target reference sequence of pig-derived components Note. The boxed area is the primer binding area, and the shaded area is the probe binding area.