SN/T 2978-2011 English PDF (SNT2978-2011)

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SN/T 2978-2011: PCR method for the detection of chicken components in animal derived products
SN/T 2978-2011
SN
ENTRY-EXIT INSPECTION AND QUARANTINE INDUSTRY
STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA
PCR method for the detection of chicken components
in animal derived products
ISSUED ON: SEPTEMBER 09, 2011
IMPLEMENTED ON: APRIL 01, 2012
Issued by: General Administration of Quality Supervision, Inspection and
Quarantine of PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Normative references ... 4 
3 Reagents and materials ... 4 
4 Equipment ... 5 
5 Selection and preparation of specimen ... 5 
6 Inspection steps ... 5 
7 Judgment and expression of results ... 6 
8 Measures to prevent cross-contamination ... 7 
Appendix A (Informative) Preparation of reagents ... 8 
Appendix B (Informative) GEENBANK reference sequence of PCR
amplification product of chicken-derived component (1877 bp ~ 2008 bp) ... 9 
PCR method for the detection of chicken components
in animal derived products
1 Scope
This standard specifies the PCR method for the detection of chicken
components in animal derived products.
This standard applies to the qualitative detection of chicken components in
animal derived products.
2 Normative references
The following documents are essential to the application of this document. For
the dated documents, only the versions with the dates indicated are applicable
to this document; for the undated documents, only the latest version (including
all the amendments) is applicable to this standard.
GB 4789.1 National food safety standard - Food microbiological
examination - General guidelines
GB/T 5009.1 Methods of food hygienic analysis - Physical and chemical
section - General principles
GB/T 6682 Water for analytical laboratory use - Specification and test
methods
GB/T 14699.1 Feeding Stuffs - Sampling
SN/T 1193 General requirements for the laboratories for gene detection and
identification
3 Reagents and materials
Unless otherwise specified, the reagents are analytical reagents or biochemical
reagents; the experimental water meets the requirements of GB/T 6682.
3.1 Primer (pair) sequence for detection of chicken-derived components:
Upstream: 5’-ctataatcgataatccacgattca-3’
shake to mix it uniformly from time to time. Centrifuge it at 12000 r/min for 5
min. Transfer the supernatant into a clean centrifuge tube. Add 400 μL of
chloroform/isoamyl alcohol (24:1). Mix well. Centrifuge it at 12000 r/min for 5
min. Take the supernatant. Add 0.8 times the volume of isopropanol. Precipitate
it. Centrifuge it at 12000 r/min for 5 min. Discard the supernatant. Use the 75%
ethanol to rinse it once. Dry it naturally. Add 50 μL of double-distilled water
(ddH2O), to dissolve the precipitate, to make a DNA solution. Prepare for use.
It may also use the equivalent DNA extraction kit, to extract sample DNA.
6.2 PCR amplification
Reaction system: The volume is 25 μL, including 12.5 μL of 2 X PCR buffer, 5
μL of dNTPs (2 mmol/L), 0.5 μL of primer pair (10 nmol/L), 1 μL of DNA
polymerase (1 U/μL), 5 μL of template DNA (100 ng ± 50 ng DNA), 0.5 μL of
double-distilled water.
Reaction conditions: PCR reaction conditions vary slightly with different PCR
instruments. Perform pre-denaturation at 94 °C for 1 min ~ 3 min, denaturation
at 94 °C for 30 s ~ 60 s, annealing at 63 °C for 30 s ~ 60 s, extension at 72 °C
for 30 s ~ 60 s, in a total of 35 cycles; perform extension at 72 °C for 5 min;
store it at 4 °C.
In the detection process, set a positive control, a negative control, a blank
control, respectively.
6.3 Electrophoresis detection of the amplified product of PCR
Weigh 1.5 g of agarose in 100 mL of electrophoresis buffer. Heat it to fully melt.
Add ethidium bromide to a final concentration of 0.5 μg/mL. Prepare a gel. Add
the electrophoresis buffer to the electrophoresis tank, so that the liquid level
has just covered the gel. Mix 8 μL of each amplified product of PCR with 3 μL
of sample-added buffer. Apply the sample. Keep constant voltage at 9 V/cm.
Carry out electrophoresis, until the bromophenol blue indicator migrates to the
middle of the gel. Observe and record the electrophoresis results under the gel
imager.
6.4 Sequencing
When the electrophoresis result is suspiciously positive, the PCR amplification
product is subjected to DNA sequencing.
7 Judgment and expression of results
7.1 Electrophoresis and sequence analysis of PCR amplification product