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SN/T 2051-2008 English PDF (SNT2051-2008)

SN/T 2051-2008 English PDF (SNT2051-2008)

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SN/T 2051-2008: Determination of bovine, ovine, porcine-derived materials in food, cosmetic and feed-Real-time PCR method

This standard specifies the real-time PCR detection methods for bovine, ovine (including sheep and goat), porcine-derived materials in food, cosmetic and feed.
SN/T 2051-2008
SN
Entry-Exit Inspection and Quarantine Standards
of the PEOPLE Republic of China
Determination of bovine, ovine, porcine-derived materials in
food, cosmetic and feed - Real-time PCR method
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
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Terms, definitions and abbreviations ... 4
4 Principle ... 5
5 Materials and reagents ... 6
6 Sampling ... 7
7 Operation methods ... 7
8 Judgment criteria ... 8
9 Low-limit of determination ... 10
Annex A ... 11
Annex B ... 15
Annex C ... 16
Annex D ... 17
Annex E ... 18
Annex F ... 19
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 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. 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 6682-1992, neq ISO 3696.1987)
GB 19489 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
3.1.1
bovine-derived materials
Bovine species specificity DNA fragment.
3.1.2
ovine-derived materials
Ovine species specificity DNA fragment.
3.1.3
porcine-derived materials
Porcine species specificity DNA fragment.
3.1.4
real-time fluorescent PCR
result.
5 Materials and reagents
5.1 Instrumentation and materials
5.1.1 Real-time PCR detection system.
5.1.2 High velocity refrigerated centrifuge (maximum rotation 12 000 r/min above). 5.1.3 Desk centrifuge (maximum rotation 2 000 r/min).
5.1.4 Vibrator.
5.1.5 Refrigerator (2??C ~ 8??C and -20??C or -80??C).
5.1.6 Micro-scale adjustable pipette and matched sucker (10 ??L, 100 ??L, 1 000 ??L). 5.1.7 Real-time PCR reaction pipe.
5.1.8 Centrifuge pipe.
5.1.9 Water bath.
5.1.10 Heater.
5.1.11 Mobile ultraviolet lamp.
5.1.12 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 6682. All the reagents shall be stored in the container which will not pollute DNA enzyme.
5.2.1 The composition, functions and use attentions of genomic DNA extraction kit 1 ) are prescribed in Annex A.
5.2.2 The composition, functions and use attentions of real-time PCR detection kit for bovine-derived materials 1) are prescribed in Annex B.
5.2.3 The composition, functions and use attentions of real-time PCR detection kit for ovine-derived materials 1) are prescribed in Annex C.
5.2.4 The composition, functions and use attentions of real-time PCR detection kit for porcine derived materials 1) are prescribed in Annex D.
5.2.5 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.
Sample DNA (1 ng/??L ~ 100 ng/??L) 1??L
(ddH2O) Up to 25??L
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.
7.3.2 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. 7.3.3 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 7.3.2 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
8.2.1 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.
8.2.2 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.
8.2.3 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 28.0.
8.2.4 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 28.0.
8.3 Judgment criteria and specifications
8.3.1 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. - - +
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 t...

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