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
SN/T 2051-2008
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
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’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.
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 μL, 100 μL, 1 000 μL).
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/μL ~ 100 ng/μL) 1μL
(ddH2O) 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.
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.
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