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GB/T 25165-2010 (GBT 25165-2010)

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GB/T 25165-2010English110 Add to Cart 0--3 minutes. Auto immediate delivery. Protocol of identification of bovine, caprine, ovine and porcine derived materials in gelatin -- Real time PCR method GB/T 25165-2010 Valid GBT 25165-2010


   
BASIC DATA
Standard ID GB/T 25165-2010 (GB/T25165-2010)
Description (Translated English) Protocol of identification of bovine, caprine, ovine and porcine derived materials in gelatin. Real time PCR method
Sector / Industry National Standard (Recommended)
Classification of Chinese Standard B43
Classification of International Standard 65.020.30
Word Count Estimation 6,675
Date of Issue 2010-09-26
Date of Implementation 2011-05-01
Quoted Standard GB/T 6682; GB 6783; GB/T 14699.1; GB/T 27403-2008
Drafting Organization Chinese Academy of Inspection and Quarantine
Administrative Organization National Standardization Technical Committee Livestock
Regulation (derived from) National Standard Approval Announcement 2010 No.6 (Total No.161)
Proposing organization People's Republic of China Ministry of Agriculture
Issuing agency(ies) Administration of Quality Supervision, Inspection and Quarantine of People's Republic of China; Standardization Administration of China
Summary This standard specifies the gelatin cattle, sheep, real-time PCR detection method for pig -derived ingredients. This standard applies to gelatin cattle, sheep, qualitative detection of porcine -derived ingredients. Fish out limit of the method was 0. 1%.


GB/T 25165-2010: PDF in English (GBT 25165-2010)
GB/T 25165-2010
NATIONAL STANDARD OF THE
PEOPLE’S 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 12.5μL 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: 0.5μL~10μL, 10μL~100μL, 20μL~200μL, 200μL~1000μL.
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
......