SN/T 1196-2012 English PDF (SNT1196-2012)

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SN/T 1196-2012: Detection of genetically modified components - Maize test methods
SN/T 1196-2012
SN
Industry Standard
of the People’s Republic of China
Replacing SN/T 1196-2003
Detection of genetically modified components –
Maize test methods
ISSUE ON. DECEMBER 12, 2012
IMPLEMENTED ON. JULY 1, 2013
Issued by. State Administration of Quality Supervision and 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 ... 5
4 Anti-pollution measures ... 6
5 Sampling and sample preparation ... 6
6 Common PCR method ... 6
7 Real-time fluorescence PCR method ... 11
8 Recording of results ... 16
9 Expression of results ... 16
Appendix A ... 17
Appendix B ... 21
References ... 22
Foreword
This Standard was drafted in accordance with rules given in GB/T 1.1-2009.
This Standard replaces SN/T 1196-2003 "GMO maize Qualitative PCR detection methods".
Compared with SN/T 1196-2003, the main technical changes of this Standard are as
follows.
- MIDOFY the Chinese and English name of the standard;
- ADD the scope of identifying maize lines, AMEND qualitative detection of genetically
modified lines of MON863, NK603, TC1507, MON88017, 59122, MIR604, MTR162,
DBT418, MON89034, LY038, ES3272, Bt10, and DP98140.
- ADD 2nd method - real-time PCR method.
This Standard references PCR detection method of genetically modified maize in ISO
standards and the EU Joint Research Centre - Community reference laboratory for GM
food and feed Biotechnology and GMOs Unit. In the 1st method – common PCR method, ADD
qualitative detection of genetically modified lines of MON863 NK603, TC1507, and Bt10.
ADD 2nd method - real-time PCR method.
This Standard is proposed and managed by the National Certification and Accreditation
Administration Committee.
Drafting organizations of this Standard. Liaoning Entry-Exit Inspection and Quarantine
Bureau, Chinese Academy of Inspection and Quarantine, Shandong Entry-Exit Inspection
and Quarantine Bureau, Fujian Entry-Exit Inspection and Quarantine Bureau, and
Shenzhen Entry-Exit Inspection and Quarantine Bureau.
Main drafters of this Standard. Cao Jijuan, Xu Junyi, Zhao Xin, Huang Xin, Gao Hongwei,
Chen Wenbing, Yan Pingping, Ling Xingyuan, Shao Biying, and Zhu Shuifang.
The previous standard replaced by this Standard is.
- SN/T 1196-2003.
Detection of genetically modified components –
Maize test methods
1 Scope
This Standard specifies the PCR method and real-time fluorescence PCR method of
detecting genetically modified components in maize and maize-processed products.
The screening detection of this Standard applies to qualitative detection of genetically
modified components in maize and maize-processed products.
Identification detection of this Standard applies to qualitative detection of genetically
modified components of maize lines MON810, Bt11, Bt176, T14/T25, CBH351, GA21,
MON863, NK603, TC1507, MON88017, 59122, MIR604, MIR162, DBT418, MON89034,
LY038, ES3272, Bt10, and DP98140.
2 Normative references
The following documents for the application of this document are essential. Only those
dated documents are applicable to this document. For undated references, the latest
edition (including any amendments) applies to this document.
GB/T 6682 Analytical laboratory use - Specification and test methods
GB/T 19495.1 Detection of genetically modified organisms and derived products.
General requirements and definitions
GB/T 19495.2 Detection of genetically modified organism and derived products.
General requirements for laboratories
GB/T 19495.3 Detection of genetically modified organisms and derived products.
Nucleic acid extraction
GB/T 19495.7 Detection of genetically modified organisms and derived products.
Gene-chip detection
6.2.15 10 X loading buffer. 0.25% bromophenol blue, 40% sucrose.
6.2.16 RNA enzyme (10μg/mL).
6.2.17 UNG enzyme (Uracil N-glycosylase).
6.3 Instruments
Solid grinder and mortar; high-speed refrigerated centrifuge; desktop mini centrifuge, mini
individual centrifuge; bath incubator, incubator, constant temperature incubator; balance.
sensitivity of 0.001 g; autoclave; temperature oven; distilled water maker or double water
heater; refrigerator, freezers; ice maker; vortex oscillator; microwave oven; Cycler;
electrophoresis; PCR clean bench; nucleic acid protein analyzer; micropipette (0.1μL - 2μL,
0.5μL - 10μL, 2μL - 20μL, 10μL - 100μL, 20μL - 200μL, 200μL - 1000μL); gel imaging
system; centrifuge tube. 1.5 mL - 5 mL; PCR reaction tube. two specifications of 200μL,
500μL.
6.4 Detection steps
6.4.1 Settings of contrast
Contrast shall be set in accordance with provisions in GB/T 19495.2 during the detection
procedure.
DNA contrast of negative target. It shall not contain DNA fragment of exogenous target
nucleic acid sequence.
DNA contrast of positive target. It can be the reference DNA, or the DNA extracted from
traceable reference material, or the DNA extracted from positive sample (or organism)
which contains known sequences.
Contrast of amplification reagent. All reaction reagents except the DNA template of test
samples. USE the same volume of water (excluding the nucleic acid) to substitute template
DNA in the PCR reaction system.
6.4.2 Extraction of template DNA
WEIGH 1 g of powder sample into 10 mL centrifuge tube; ADD 5 mL of CTAB lysis buffer
(including appropriate amount of RNA enzymes); MIX well; at 60ºC water bath SHAKE for 1
7.4.7.2 Relation between Ct value and DNA concentration
When Ct value is greater than or equal to 40, there is no amplification of the target DNA in
PCR procedure; if Ct value is between 36 - 40, and difference between each value of
parallel samples is very large, it indicates that there is little amount of target DNA in the
PCR reaction system; it shall appropriately increase the template amount.
7.5 Quality Control of real-time fluorescence PCR detection
Amplification Reagent Contrast. Detected Ct value of exogenous gene is greater than or
equal to 40; detected Ct value of endogenous gene is greater than or equal to 40.
Negative target DNA contrast. Detected Ct value of exogenous gene is greater than or
equal to 40.
Positive target DNA contrast. Detected Ct value of exogenous gene is smaller than or equal
to 34.
If there is one item of above indicators not compliant, it shall repeat the real-time
fluorescence PCR amplification.
7.6 Selection of screening test and identification test
For detection of genetically modified component in maize sample, it may be performed
according to the contents of Appendix B. Firstly SCREEN and TEST CaMV 35S, NOS,
NPTII, PAT, BAR and CryIA(b) genes, if the screening test result is negative, then directly
REPORT the test result.
If the screening test result is positive, then it needs further identification detection of line
specificity gene of MON810, Bt11, Bt176, T14/T25, CBH351, GA21, MON863, NK603,
TC1507, MON88017, 59122, MIR604, MIR162, DBT418, MON89034, LY038, ES3272,
Bt10 and DP98140, so as to determine the line of genetically modified maize.
7.7 Judgment of results
If the detection value Ct of exogenous gene of test sample is greater than or equal to 40,
the detection value Ct of endogenous gene of test sample is smaller than or equal to 24,
and the setting contrast result is normal, then it can be judged that XXX gene is not
References
[1] Protocol for event-specific quantitation of Bt11 in mai...