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SN/T 5227.10-2019 PDF English

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SN/T 5227.10-2019: (Rapid detection of mink-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 5227.10-2019	English	209	Add to Cart	3 days [Need to translate]	(Rapid detection of mink-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method))	Valid

Standard similar to SN/T 5227.10-2019

GB 5413.12 | GB 5413.10 | SN/T 1888.4 | SN/T 5227.1 | SN/T 5227.2 | SN/T 5227.11 |

Basic data

Standard ID	SN/T 5227.10-2019 (SN/T5227.10-2019)
Description (Translated English)	(Rapid detection of mink-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	9,944
Date of Issue	2019
Date of Implementation	2020-07-01
Issuing agency(ies)	General Administration of Customs

SN/T 5227.10-2019: (Rapid detection of mink-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.
The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards Rapid detection of marten derived ingredient in food for export- Recombinase-aid amplification (RAA) method 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 expected to be divided into the following 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 exported 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 exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method). This part is part 10 of SN/T 5227-2019. This part is drafted in accordance with the rules given in 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, Chengdu Customs of the People's Republic of China, Gongbei of the People's Republic of China Customs, Hefei Customs of the People’s Republic of China, Chinese Academy of Inspection and Quarantine, Beijing Customs of the People’s Republic of China, People’s Republic of China Heguo Dalian Customs, Xiamen Customs of the People's Republic of China, Hangzhou Zhongce Biotechnology Co., Ltd. The main drafters of this section. Miao Li, Lin Hua, Luo Baozheng, Chen Jing, Zong Kai, Wang Ping, Wang Lin, Zheng Qiuyue, Xu Shufei, Kong Fan Germany, Cheng Qi. Rapid detection of mink-derived components in exported food Recombinase-mediated strand replacement nucleic acid amplification method (RAA method)

1 Scope

This part of SN/T 5227-2019 specifies the RAA detection method for mink-derived components in exported food. This section applies to the qualitative detection of mink-derived ingredients in exported food.

2 Normative references

The minimum detection limit (LOD) of the method specified in this section is 0.1% (W/W). The following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. 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 Martes Carnivora, Schizopod suborder, Mustelidae, Mustela subfamily, Mink genus. The common ones are as follows. American mink, yellow-throated mink (sable mink), stone mink (Beech marten), pine marten (forest marten), Japanese marten, fishing marten, sable (sable). There are four types of stone mink, sable mink, fishing mink and yellow-throated mink produced in China. 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.

4 Method summary

Using the extracted DNA as a template, using mink-specific detection primers and probes for real-time fluorescent RAA amplification, according to the real-time fluorescent The increase in RAA has enabled the detection and identification of mink components 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 (volume ratio). 5.5 TE buffer (pH 8.0). 10 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8.0). 5.6 Buffer A. 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 Buffer B. 280 mol/L 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. the speed is greater than or equal to 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. 2mL, 1.5mL.

7 Detection steps

7.1 DNA extraction Take 0.2 g of crushed or ground sample into a clean 1.5 mL centrifuge tube (6.9) (if the sample contains impurities and condiments, add Wash twice with 1 mLdd H2O), add 600 µL CTAB extraction buffer (pH 8.0) (5.1), vortex and mix well, then warm at 70 ℃ Incubate for 15 minutes, invert the centrifuge tube 2~3 times during the incubation; centrifuge at 12,000 r/min for 5 minutes, and take the supernatant in a new clean 1.5 mL centrifuge tube; Add 500 µL of phenol. chloroform. isoamyl alcohol (25.24.1) (5.2), turn the centrifuge tube upside down 2~3 times, then vortex and mix, 12 000 Centrifuge for 5 min at r/min; transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 0.7 times the volume of isopropanol (5.3), and turn upside down Centrifuge the tube 2~3 times, let stand for 30 min at 4 ℃, centrifuge at 12 000 r/min at 4 ℃ for 3 min, carefully discard the supernatant; add 700 µL 70 % Ethanol (5.4), resuspend the pellet, centrifuge at 12 000 r/min for 1 min, carefully discard the supernatant; open the tube cap, and evaporate the dry liquid at room temperature. Add 50 µL~100 µL TE buffer (pH 8.0) (5.5) to dissolve the DNA, and store at -20 ℃ for later use. DNA extraction can also use equivalent DNA extraction kit was performed. 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 is calculated according to formula (1). c - DNA concentration, in micrograms per microliter (μg/μL); A-- Absorbance at 260 nm; N-nucleic acid dilution factor. When the ratio of A260/A280 is between 1.7 and 1.9, it is suitable for RAA amplification. 7.3 Real-time fluorescent RAA amplification 7.3.1 Real-time fluorescence RAA reaction system See Table 2. 7.3.2 Real-time fluorescent RAA reaction program 7.3.2.1 Real-time fluorescent PCR instrument 39 ℃, 60 s, 1 cycle; 39 ℃, 30 s, 40 cycles, collect fluorescence in each cycle. 7.3.2.2 Constant temperature fluorescence detector 39 ℃, 1 min; 39 ℃, 20 min, 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 mink source components as a positive control sample The sample without mink source components was used as the negative control sample, and the double distilled water of the same volume as the template was used as the blank control sample.

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 logarithmic increase in fluorescence, and a typical amplification curve appears in the fluorescence channel, and the corresponding Ct value is less than or equal to 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 logarithmic increase, and a typical amplification curve appears in the fluorescence channel, the corresponding T value (time) is less than or equal to 15 min.

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 less than or equal to 35.0, the test sample is determined to be positive. 9.1.1.3 If the Ct value is greater than 35.0 and less than 40.0, repeat it once. If the test result again is still Ct value greater than 35.0 and less than 40.0, The test sample is determined to be 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) is less than or equal to 15 minutes, the test sample is determined to be positive. 9.1.2.3 If the T value (time) is greater than 15 min and less than 20 min, repeat it once. If the test result is still the T value (time) If it is more than 15 minutes and less than 20 minutes, the test sample is determined 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 "mink component detected". 9.2.2 The sample is negative, which is expressed as "mink component not 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) Reference sequence for gene amplification target of mink-derived components Note. The boxed area is the primer binding area, and the shaded area is the probe binding area.