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GB/T 25165-2010 PDF English

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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
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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 is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GBT25165-2010
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 ......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.


      

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