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GB/T 25165-2010 English PDF (GBT25165-2010)

GB/T 25165-2010 English PDF (GBT25165-2010)

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GB/T 25165-2010: Protocol of identification of bovine, caprine, ovine and porcine derived materials in gelatin -- Real time PCR method

This Standard specifies the real time PCR detection method of bovine, ovine and porcine derived components in gelatin. This Standard applies to the qualitative identification of bovine, ovine and porcine derived components in gelatin. The detection limit of this method is 0.1%.
GB/T 25165-2010
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
ICS 65.020.30
B 43
Protocol of identification of bovine, caprine, ovine and
porcine derived materials in gelatin - Real time PCR method
ISSUED ON: SEPTEMBER 26, 2010
IMPLEMENTED ON: MAY 01, 2011
Issued by: General Administration of Quality Supervision, Inspection and Quarantine;
Standardization Administration of the People's Republic of China.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Terms and definitions... 4
4 Principle ... 5
5 Primers and probes for detection ... 5
6 Reagents ... 5
7 Solution preparation ... 6
8 Instruments ... 6
9 Sample collection ... 6
10 Inspection steps ... 6
11 Result determination ... 8
12 Measures to prevent cross-contamination during detection ... 8
Protocol of identification of bovine, caprine, ovine and
porcine derived materials in gelatin - Real time PCR method
1 Scope
This Standard specifies the real time PCR detection method of bovine, ovine and porcine derived components in gelatin.
This Standard applies to the qualitative identification of bovine, ovine and porcine derived components in gelatin.
The detection limit of this method is 0.1%.
2 Normative references
The provisions in following documents become the provisions of this Standard through reference in this Standard. For dated references, the subsequent amendments (excluding corrigendum) or revisions do not apply to this Standard, however, parties who reach an agreement based on this Standard are encouraged to study if the latest versions of these documents are applicable. For undated references, the latest edition of the referenced document applies.
GB/T 6682, Water for analytical laboratory use - Specification and test methods GB 6783, National Food Safety Standard Food additive - Gelatine
GB/T 14699.1, Feeding Stuffs - Sampling
GB/T 27403-2008, Criterion on quality control of laboratories - Molecular biological testing of food
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply. 3.1 real time PCR
add fluorescent groups to the PCR reaction system, and use the accumulation of fluorescent signals to monitor the entire PCR amplification process in real time 3.2 cycle threshold
meets the requirements of secondary water in GB/T 6682.
7 Solution preparation
7.1 Lysate
Ingredients include: 2% (mass concentration) CTAB (cetyltrithylammonium bromide, cetyltrimethylammonium bromide), 100mmol/L Tris (tris hydroxymethyl
aminomethane, trishydroxymethyl aminomethane), 1.4 mol/L NaCl, 20mmol/L EDTA (ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid). Use HCl to adjust pH to 8.0.
7.2 Real time PCR reaction mix
The 12.5??L reaction system includes: 1U~2U (Unit, enzymatic unit) of Taq enzyme, 1??PCRbuffer, 2.5mmol/L~4.0mmol/L Mg2+, 0.2U~1U UNG enzyme, 0.2mmol/L d
(A,C,G) TPs, 0.2mmol/L~0.4mmol/L dUTP, 400nmol/L ROX dye (some fluorescent PCR instruments do not require ROX correction).
8 Instruments
8.1 Real time PCR instrument.
8.2 Constant temperature water bath.
8.3 Centrifuge: the centrifugal force is not less than 3000g.
8.4 Micropipette: 0.5??L~10??L, 10??L~100??L, 20??L~200??L, 200??L~1000??L.
8.5 Nucleic acid protein analyzer or UV spectrophotometer.
8.6 pH meter.
8.7 Balance: the resolution is 0.01g.
9 Sample collection
Conduct sampling of edible gelatin according to GB 6783. Sample feed gelatin according to GB/T 14699.1. Pulverize the experimental sample. Mix well and set aside. 10 Inspection steps
10.1 DNA extraction

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