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SN/T 5227.1-2019 PDF English

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SN/T 5227.1-2019: (Rapid detection of chicken-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 5227.1-2019	English	209	Add to Cart	3 days [Need to translate]	(Rapid detection of chicken-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method))	Valid

Standard similar to SN/T 5227.1-2019

GB 5413.12 | GB 5413.10 | SN/T 1888.4 | SN/T 5227.2 | SN/T 5227.11 | SN/T 5227.10 |

Basic data

Standard ID	SN/T 5227.1-2019 (SN/T5227.1-2019)
Description (Translated English)	(Rapid detection of chicken-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	9,928
Date of Issue	2019
Date of Implementation	2020-07-01
Issuing agency(ies)	General Administration of Customs

SN/T 5227.1-2019: (Rapid detection of chicken-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 chicken 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 strand replacement nucleic acid amplification method (RAA method). This part is part 1 of SN/T 5227-2019. This part is compiled in accordance with the rules of GB/T 1.1-2009. This part is proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this section. Zhengzhou Customs of the People’s Republic of China, Chinese Academy of Inspection and Quarantine Sciences, Shijia of the People’s Republic of China Zhuang Customs, Hefei Customs of the People's Republic of China, Beijing Customs of the People's Republic of China, Gongbei Customs of the People's Republic of China, People of China Xiamen Customs of the Republic of China, Chengdu Customs of the People’s Republic of China, Dalian Customs of the People’s Republic of China, Hangzhou Customs of the People’s Republic of China. The main drafters of this section. Miao Li, Wang Ping, Wang Jianchang, Zong Kai, Deng Tingting, Wang Lin, Luo Baozheng, Kong Fande, Lin Hua, Zheng Qiuyue, Wu Shan. Rapid detection of chicken-derived ingredients in exported food Recombinase-mediated strand replacement nucleic acid amplification method (RAA method)

1 Scope

This section specifies the RAA detection method for chicken-derived ingredients in exported food. This section applies to the qualitative detection of chicken-derived ingredients in exported food. The minimum detection limit (LOD) of the method specified in this standard is 0.1% (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 Chicken Gallus gallus domesticus Galliformes, Phasianidae, Pheasant tribe, Archaeopteryx genus, Red original chicken species, domestic chicken. Originated from wild wild fowl. There are turkey, black-bone chicken, pheasant and so on. 3.1.2 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 chicken-specific detection primers and probes for real-time fluorescent RAA amplification. The increase of RAA enables the detection and identification of chicken 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. Note. See Appendix A for the target gene sequence. F is the upstream primer, R is the downstream primer, P is the probe, FAM-dT, THF, BQH-dT and C3spacer All are probe modification groups. 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, 250 ng/μL creatine kinase. An equivalent commercial kit can also be used instead. 5.8 B Buffer. 280 mM 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 Micro pipette. 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 0.2g 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 and mix well, incubate at 70 ℃ for 15 min, invert during Heart tube 2 to 3 times; centrifuge at 12 000 r/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 to 3 times, statically at 4 ℃ Leave it for 30 min at 4 ℃ Centrifuge at 12 000 r/min for 3 min, carefully discard the supernatant; add 700 μL of 70% ethanol, resuspend the pellet, and centrifuge at 12 000 r/min 1 min, carefully discard the supernatant; open the cap, evaporate the dry liquid at room temperature, add 50 μL ~100 μL TE (pH 8.0) buffer to dissolve DNA should be stored at -20°C 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 chicken ingredients as a positive control sample The sample without chicken ingredients was used as the negative control, and the double distilled water of the same volume as the template was used as the 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 "chicken ingredients detected". 9.2.2 The sample is negative, which is expressed as "no chicken 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) Chicken-derived component gene amplification target reference sequence Note. The boxed area is the primer binding area, and the shaded area is the probe binding area.