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1 Translated English of Chinese Standard: SN/T SN Entry-Exit Inspection and Quarantine Standards of the People s Republic of China Determination of bovine, ovine, porcine-derived materials in food, cosmetic and feed - Real-time PCR method 食品 化妆品和饲料中牛羊猪源性成分检测方法实时 PCR 法 Issued on: April 29, 2008 Implemented on: November 01, 2008 Issued by: General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China Page 1 of 21

2 Table of Contents Foreword Scope Normative references Terms, definitions and abbreviations Principle Materials and reagents Sampling Operation methods Judgment criteria Low-limit of determination Annex A Annex B Annex C Annex D Annex E Annex F Page 2 of 21

3 Foreword Annex F is normative; Annexes A, B, C, D and E are informative. This Standard shall be under the jurisdiction of Certification and Accreditation Administration of the People s Republic of China. Drafting organizations of this standard: Liaoning Entry-Exit Inspection and Quarantine Bureau, Takara Biotechnology (Dalian) Co., Ltd. AND Chinese Academy of Inspection and Quarantine. Main drafters of this standard: Cao Jijuan, Li Jingquan, Zhen Qiuyue, Yu Aili, Chen Yin, Yao Shan and Xie Yan. This is the first-time to publish this entry-exit inspection and quarantine standard. Page 3 of 21

4 Determination of bovine, ovine, porcine-derived materials in food, cosmetic and feed - Real-time PCR method 1 Scope This standard specifies the real-time PCR detection methods for bovine, ovine (including sheep and goat), porcine-derived materials in food, cosmetic and feed. This standard is applicable to the identification detection of bovine, ovine (including sheep and goat), porcine-derived materials in food, cosmetic and feed. 2 Normative references The articles contained in the following documents have become part of this standard when they are quoted herein. For the dated documents so quoted, all the subsequent modifications (including all corrections) or revisions made thereafter do not apply to this standard. However, the parties who reach an agreement according to this standard are encouraged to study whether the latest versions of these documents are applicable. For the undated documents so quoted, the latest versions (including all modification sheets) apply to this document. GB/T 6682 Water for analytical laboratory use - Specification and test methods (GB/T , neq ISO 3696:1987) GB Laboratories - General requirements for biosafety 3 Terms, definitions and abbreviations For the purpose of this standard, the following terms, definitions and abbreviations apply. 3.1 Terms and definitions bovine-derived materials Bovine species specificity DNA fragment ovine-derived materials Ovine species specificity DNA fragment porcine-derived materials Porcine species specificity DNA fragment real-time fluorescent PCR Page 4 of 21

5 result. 5 Materials and reagents 5.1 Instrumentation and materials Real-time PCR detection system High velocity refrigerated centrifuge (maximum rotation r/min above) Desk centrifuge (maximum rotation r/min) Vibrator Refrigerator (2 C ~ 8 C and -20 C or -80 C) Micro-scale adjustable pipette and matched sucker (10 μl, 100 μl, μl) Real-time PCR reaction pipe Centrifuge pipe Water bath Heater Mobile ultraviolet lamp Magnetic shelf. 5.2 Reagents Unless otherwise specified, all the reagents are analytically pure. The water is grade-1 water as prescribed in GB/T All the reagents shall be stored in the container which will not pollute DNA enzyme The composition, functions and use attentions of genomic DNA extraction kit 1) are prescribed in Annex A The composition, functions and use attentions of real-time PCR detection kit for bovine-derived materials 1) are prescribed in Annex B The composition, functions and use attentions of real-time PCR detection kit for ovine-derived materials 1) are prescribed in Annex C The composition, functions and use attentions of real-time PCR detection kit for porcine derived materials 1) are prescribed in Annex D The composition, functions and use attentions of real-time PCR detection kit for bovine and ovine-derived materials 1) are prescribed in Annex E. 1) Provided by the designated supplier. The information given here is for the convenience of the user of this standard but does not represents the approval to the product. If other equivalent products have the same effect, these equivalent products can also be used. Page 6 of 21

6 Sample DNA (1 ng/μl ~ 100 ng/μl) (ddh2o) 1μL Up to 25μL Note 1: For blank control experiment, ddh2o replaces sample DNA. Note 2: For negative control experiment, non-target-derived constituent replaces sample DNA. Note 3: For positive control experiment, the corresponding bovine or ovine or porcine DNA mixture replaces sample DNA Sample addition The addition of sample shall be carried out in the sampling area. Add the prepared template DNA solution to each setup PCR reaction pipe. Enclose the pipe and centrifuge it for 5s ~10s Real-time PCR detection The real-time PCR detection shall be carried out in the detection area. Put the real-time PCR reaction pipe which has been centrifuged in accordance with in the real-time PCR detection system and record the placement sequence of sample. For the setup of cycling conditions, 95 C/10 s, one cycle; 95 C/5 s, 60 C/20s (24s, 30s, 31s, 34s) 2), 40 cycles and fluorescence collected after the annealing of each cycle. At the end of detection, the results can be judged from amplification curve and Ct value. 8 Judgment criteria 8.1 Setup of result analysis conditions The detection results can be directly read. The principle for the setup of valve values shall be adjusted based on the noise conditions of instrumentation. 8.2 Quality control criteria In the case of negative control experiment for bovine, ovine, porcine-derived materials, if HEX fluorescence signal is detected and the typical amplification curve appears, Ct value shall be less than 28.0 but no FAM fluorescence signal is detected In the case of negative control experiment for bovine and ovine-derived materials, if HEX fluorescence signal is detected and the typical amplification curve appears, Ct value shall be less than 28.0 but no FAM or ROX fluorescence signal is detected In the case of positive control experiment for bovine, ovine, porcine-derived materials, if FAM and HEX fluorescence signals are detected and the typical amplification curve appears, Ct value shall be less than In the case of positive control experiment for bovine and ovine-derived materials, if FAM, ROX and HEX fluorescence signals are detected and the typical amplification curve appears, Ct value shall be less than Judgment criteria and specifications Valid principles Ct value less than or equal to 35 is regarded as valid value and Ct value greater than 35 is regarded as invalid value. 2) For Real Time PCR amplifier of Applied Biosystem Ltd., the time setup shall be based on the model of instrument: 7700/7900HT, 30s; 7000/7300, 31s; 7500, 34s; 500 Fast, 24s. Page 8 of 21

7 In the case of actual detection when the results of positive control experiments conducted at the same time are acceptable, if HEX fluorescence signal is detected and no FAW fluorescence is detected, it is judged not containing bovine-derived materials; if no ROX fluorescence is detected, it is judged not containing ovine-derived materials. PCR reaction fails. The following aspects shall be noted if another reaction is conducted:: a) If the results of positive control experiments are acceptable, it is the problem of the preparation of sample DNA, e.g. materials to restrain PCR reaction contained in the sample; b) If the results of positive control experiments are not accepted, it is the problem of operation failure or reagent inactivation Result description The result is described as bovine, ovine, porcine-derived materials detected or bovine, ovine, porcine-derived materials not detected. 9 Low-limit of determination Under the conditions as described above, the low-limit of the determination by this method is 0.1%. Page 10 of 21

8 A Centrifuge the centrifugation pipe filled with the lysis buffer of 0.9mL for 30s at r/min. A Add the sample of 100μL ~ 150μL to the centrifugation pile filled with this lysis buffer and turn it upside down for 2 or 3 times. A When vibrating the silica suspension in the vibrator, quickly add the silica suspension of 50μL in each pipe filled with the lysis buffer as prescribed in A Vibrate it for well mixture. A Put the centrifugation pipe at the room temperature for 10min (no more mixture). Centrifuge all the centrifugation pipes for 2min at r/min. A Mount the centrifugation pipe on the magnetic shelf to make silica form into globule. Remove the supernatant carefully (when absorbing the supernatant, please do not take away the centrifugation pipe and mount it on the magnetic shelf to prevent stirring globule). Add the washing buffer 1 of 400μL to each centrifugation pipe. A Dismantle the centrifugation pipe from the shelf and turn the centrifugation pipe upside down gently for many times until the globule fully suspended. A Repeat the washing procedure as prescribed in A to A.2.4.6, where, washing buffer 1 of 400μL to wash for 1 time, washing buffer 2 of 500μL to wash for 2 times and washing buffer 3 of 500μL to wash for 1 time. Note: do not let the sample in the washing buffer 3 for long-time stay or otherwise the extraction of nucleic acid will be reduced. A In the last washing step, the pipette of 100μL shall be used to remove the residual washing buffer 3. A Add the washing buffer of 50μL to each centrifugation pipe. A Dismantle the centrifugation pipe from the shelf and carefully knock at the centrifugation pipe until the globule fully suspended. A Put the centrifugation pipe of silica for re-suspension at 60 C for 5 min to elute nucleic acid. A Mount the pipe on the magnetic shelf to make the silica form into globule. A Transfer the extracted nucleic acid to a clean centrifugation pipe (ensure the silica will not be transferred). This is the template DNA solution. The template DNA solution shall be stored on the ice. In the case of long-term storage, it shall be placed in the refrigerator at -20 C or -80 C. A.2.5 Use attentions A Wear gloves during test. A The direct contact with skin or clothes shall be avoided. A The operations shall be carried out in the well-ventilated chemical hood to protect human from breathing in harmful gases. Page 14 of 21

9 Annex C (Informative) Composition of Real-time PCR Detection Kit for Ovine-derived Materials C.1 Composition of kit Take the real-time PCR detection kit for ovine-derived determination produced by Takara Bio (Dalian) Co., Ltd. (Art. No. D319) for example. Each kit can handle 50 reactions (25μL reaction) including the constituents in Table C.1. Composition of reagent Table C.1 Volume 2 pre-mixture for ovine-derived determination 650μL Primer mixture for ovine-derived determination Probe mixture for ovine-derived determination ddh2o Positive template for ovine-derived determination (10 ng/μl) 50μL 50μL 1mL 15μL C.2 Specifications C.2.1 All the constituents above shall be stored at -20 C. C.2.2 The 2X pre-mixture for ovine-derived determination shall include dntp mixture, buffer and enzyme etc. required for reaction. C.2.3 The primer mixture for ovine-derived determination shall include the primer and internal control which can amplify the ovine-genomic DNA (including sheep and goat) and internal control. C.2.4 The probe mixture for ovine-derived determination shall include the probe for the detection of ovine-genomic DNA (including sheep and goat) and the probe for the detection of internal control. The probe mixture shall be stored avoiding direct sunlight. C.3 Functions This kit can be used for the determination of ovine-derived materials (including sheep and goat) in food, cosmetic and feed. C.4 Use attentions C.4.1 The cross contamination among different samples shall be avoided during the detection process. C.4.2 Check whether there are bubbles in the reaction pipe and the air-tightness of reaction pipe before operation to prevent the detection of fluorescence signal or the leakage of fluorescent substance contaminating the instrument. Page 16 of 21

10 Annex E (Informative) Composition of Real-time PCR Detection Kit for Bovine and Ovine-derived Materials E.1 Composition of kit Take the real-time PCR detection kit for bovine and ovine-derived determination produced by Takara Bio (Dalian) Co., Ltd. (Art. No. D321) for example. Each kit can handle 50 reactions (25μL reaction) including the constituents in Table E.1. Table E.1 Composition of reagent 2 pre-mixture for bovine and ovine-derived determination Primer mixture for bovine and ovine-derived determination Probe mixture for bovine and ovine-derived determination ddh2o Positive template for bovine and ovine-derived determination (1 ng/μl for each) Volume 650μL 50μL 50μL 1mL 15μL E.2 Specifications E.2.1 All the constituents above shall be stored at -20 C. E.2.2 The 2X pre-mixture for bovine and ovine-derived determination shall include dntp mixture, buffer and enzyme etc. required for reaction. E.2.3 The primer mixture for bovine and ovine-derived determination shall include the primer and internal control which can amplify the bovine and ovine-genomic DNA (including sheep and goat) and internal control. E.2.4 The probe mixture for bovine and ovine-derived determination shall include the probe for the detection of bovine and ovine-genomic DNA (including sheep and goat) and the probe for the detection of internal control. The probe mixture shall be stored avoiding direct sunlight. E.3 Functions This kit can be used for the determination of bovine and ovine-derived materials (including sheep and goat) in food, cosmetic and feed. E.4 Use attentions E.4.1 The cross contamination of different samples shall be avoided during the detection process. E.4.2 Check whether there are bubbles in the reaction pipe and the air-tightness of reaction pipe before operation to prevent the detection of fluorescence signal or the leakage of fluorescent substance contaminating the instrument. Page 18 of 21

11 Heater (Heating Block); Micro-scale sample injector (0.5μL~10μL, 5μL~20μL, 20μL~200μL, 200μL~1 000μL); Mobile ultraviolet lamp. F.2.2 Reaction mixture preparation area The reaction mixture preparation area shall be configured with the following instrumentation: 2 C ~ 8 C refrigerator; 20 C refrigerator; Desk centrifuge (2 000 r/min); Vibrator; Micro-scale sample injector (0.5μL~10μL, 5μL~20μL, 20μL~200μL, 200μL~1 000μL); Mobile ultraviolet lamp. F.2.3 Sample area The sample area shall be configured with the following instrumentation: 2 C ~ 8 C refrigerator; 20 C refrigerator; Desk centrifuge (2 000 r/min); Micro-scale sample injector (0.5μL~10μL, 5μL~20μL, 20μL~200μL, 200μL~1 000μL); Mobile ultraviolet lamp. F.2.4 Detection area The detection area shall be configured with the following instrumentation: Real-time PCR detection system; Mobile ultraviolet lamp; Printer. F.3 Functions and attentions for each working area F.3.1 Sample preparation area The functions and attentions for sample preparation area are as follows: The sample storage, nucleic acid extraction, storage and addition to the reaction pipe shall be carried out in the sample preparation area; To avoid the cross contamination in the sample room, enclose the reaction pipe containing reaction mixture after the addition of nucleic acid to be tested; Page 20 of 21

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