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SN/T 1201-2014: PCR protocol for detection of genetically modified plant ingredients in feed
SN/T 1201-2014
ENTRY and EXIT INSPECTION AND QUARANTINE INDUSTRY
STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA
Replacing SN/T 1201-2003
PCR protocol for detection of genetically modified plant
ingredients in feed
ISSUED ON: NOVEMBER 19, 2014
IMPLEMENTED ON: MAY 01, 2015
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 Terms, definitions, abbreviations ... 4 
4 Principles ... 7 
5 Main equipment and reagents ... 7 
6 Anti-contamination measures ... 13 
7 Sampling and sample preparation ... 13 
8 Inspection steps ... 13 
9 Results judgment and presentation ... 16 
10 Storage of sample ... 17 
PCR protocol for detection of genetically modified plant
ingredients in feed
1 Scope
This standard specifies the qualitative real-time fluorescent PCR detection method, for
transgenic plant components in feed.
This standard is applicable to the qualitative detection of genetically modified plant
components, such as soybean, corn, rice, rape, cotton, lucerne, wheat in feed, as well as
the identification of related lines.
This standard is also applicable to the qualitative detection of the genetically modified
components of distiller dried grains with soluble, as well as the identification of related
lines.
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/T 6682 Water for analytical laboratory use - Specification and test methods
GB/T 19495.2 Detection of genetically modified organism and derived products -
General requirements for laboratories
GB/T 19495.7 Detection of genetically modified organisms and derived products -
Methods for sampling and sample preparation
3 Terms, definitions, abbreviations
3.1 Terms and definitions
The following terms and definitions apply to this document.
3.1.1
Feed
The general term for the food of animals, which are raised by all people. The general
feed, in the narrower sense, mainly refers to the food of animals, which are raised in
agriculture or animal husbandry.
3.1.2
Real-time fluorescent polymerase chain reaction
Fluorescent groups are added to the polymerase chain reaction system, the entire
PCR process is monitored in real time by the accumulation of fluorescent signals,
the intensity of fluorescent signals directly reflects the number of templates.
3.2 Abbreviations
The following abbreviations apply to this document.
Acp1: acid phosphatase 1
BAR: phosphinothricin acetyltransferase gene
BnACCg8: acetyl-CoA carboxylase gene
bp: base pair
Btc: the construct specific DNA sequence of transgenic rice TT51-1 and Bt shanyou
63
CaMV35S: 35S promoter from cauliflower mosaic virus
CP4-EPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene
CryIA(b): The cryIA (b) gene of Bacillus thuringiensis subsp [a synthetic gene
encoded the first 648 amino acids, insecticidal-active truncated product identical to
that of cryIA (b) gene of Bacillus thuringiensis subsp, Kurstaki steam HD-1]
CryIA(c): The cryIA (c) gene of Bacillus thuringiensis [a synthetic gene encoded
the 29 to 613 amino acids, insecticidal-active truncated product identical to that of
cryIA (c) gene of Bacillus thuringiensis]
CryIA (b) /CryIA(c): Fused gene of bacillus thuringiensis insecticidal protein cryIA
(b) and cryIA (c)
Ct value: The number of cycle, that the fluorescent signal in each reaction tube will
go through, when it reaches the set threshold (cycle threshold)
CTAB: cetyltrithylammonium bromide
dATP: deoxyadenosine triphosphate
4 Principles
Real-time fluorescence quantitative PCR technology refers to the method of adding
fluorescent groups to the PCR reaction system, using the accumulation of fluorescent
signals to monitor the entire PCR process in real time, finally quantitatively analyzing
the unknown template through the standard curve.
During PCR amplification, a specific fluorescent probe is added at the same time as a
pair of primers. The probe is an oligonucleotide; the two ends are respectively labeled
with a reporter fluorescent group and a quenching fluorescent group. When the probe
is intact, the fluorescent signal emitted by the reporter gene is absorbed by the
quenching fluorescent group. During PCR amplification, the 5'-3' exonuclease activity
of Taq enzyme cleaves and degrades the probe, making the reporter fluorescent group
be separated from the quenching fluorescent group, so that the fluorescence monitoring
system can receive the fluorescent signal, that is, a fluorescent molecule is formed every
time a DNA chain is amplified; thereby, it achieves the complete synchronization
between the accumulation of the fluorescent signal and the formation of the PCR
product.
5 Main equipment and reagents
5.1 Main equipment
Real-time fluorescent PCR instrument, ice maker, nucleic acid protein analyzer or UV
spectrophotometer, constant temperature water bath, centrifuge, mortar and crushing
device, vortex shaker, micropipette (2.5 μL, 20 μL, 200 μL, 1000 μL).
5.2 Main reagents
Unless otherwise specified, the grades of reagents used in all tests shall be of analytical
pure or biochemical reagents, which do not contain DNA and DNase. The water used
in the test shall meet the specifications of grade 1 water in GB/T 6682; all reagents shall
be packaged in containers, which are contaminated by DNase.
5.2.1 CTAB extract: 20 g/L CTAB, 1.4 mol/L NaCL, 0.1 mol/L Tris-HCL, 0.02 mol/L
Na2-EDTA, pH 8.0.
5.2.2 CTAB precipitation solution: 5 g/L CTAB, 40 mmol/L NaCl.
5.2.3 Proteinase K solution: 20 mg/mL.
5.2.4 RNase A: 100 mg/mL.
5.2.5 NaCl solution: 1.2 mol/L.
There are many kinds of feeds; the ingredients are extremely complex. In addition to
various plant ingredients, it may also contain a variety of premixes, such as protein meal,
bone meal, amino acids, vitamins, antibiotics (or veterinary drugs), minerals, antifungal
agents, insect repellents, colorants, seasonings, binders, fermentation products. These
salts, sugars, pigments and other chemicals, in the feed sample, will affect the
subsequent extraction of genomic DNA; it may even inhibit subsequent PCR reactions,
etc. They shall be removed, by washing and other methods. Take 200 mg of crushed
feed sample; put it into a 2 mL centrifuge tube; add 1.5 mL of double-distilled water;
invert upside down; vortex to mix it; centrifuge it at 12000 g for 5 min; discard the
supernatant; add 1.5 mL of double-distilled water to the precipitate; repeat the above
washing process 3 ~ 5 times. The final sediment is used for the extraction of genomic
DNA.
8.2 DNA extraction
8.2.1 CTAB method
The CTAB extraction method is as follows:
a) Add 1000 μL of CTAB extraction buffer and 2 μL of proteinase K solution, into
the above 200 mg pretreated sample. Incubate it at 65 °C, for 60 min. Invert and
mix 3 ~ 5 times during this period. OR incubate it at 65 °C overnight. Centrifuge
it at 12000 g for 10 min. Transfer the supernatant to another clean 2 mL centrifuge
tube.
b) Add the same volume of chloroform as the supernatant. Invert and mix it.
Centrifuge it at 12000 g for 10 min. Transfer the supernatant to another 2 mL
centrifuge tube.
c) Add 2 times the volume of CTAB precipitation solution. Invert and mix it. Let it
stand at room temperature for 30 minutes. Centrifuge it at 12000 g for 10 minutes.
Discard the supernatant.
d) Add 500 μL of sodium chloride solution to the sediment, to dissolve the sediment.
e) Add 2 μL of RNase A. Incubate it at 37 °C, for 30 min.
f) Add an equal volume of chloroform. Invert and mix it. Centrifuge it at 12000 g for
10 min. Transfer the supernatant to another 1.5 mL centrifuge tube.
g) Add 0.7 times the volume of isopropanol, which was pre-cooled at 4 °C. Invert
and mix it. Let it stand at 4 °C, for 30 min. Centrifuge it at 12000 g for 10 min.
Discard the supernatant.
h) Add 500 μL of 4 °C pre-cooled 70% ethanol. Gently rotate the centrifuge tube 2
~ 3 times. Centrifuge it at 12000 g for 10 min. Carefully remove the supernatant.
Dry it at room temperature or in a vacuum drying system.
i) Add 50 μL of TE buffer, to dissolve the DNA. Store it in a 4 °C refrigerator, for
later use.
8.2.2 Test kit method
When using different kits, such as magnetic bead method and spin column adsorption
method, to extract genomic DNA. Operate according to their operating instructions.
8.3 Determination of DNA concentration and purity
Use the UV spectrophotometer, to measure the absorbance of the sample DNA, at 260
nm and 280 nm. Calculate the DNA purity and concentration, according to formula (1)
and formula (2):
DNA purity = OD260/OD280 …………………….. (1)
DNA concentration = 50 x OD260 ……………….. (2)
The DNA purity ratio shall be between 1.7 and 1.9. The concentration shall not be lower
than 10 ng/μL.
8.4 Selection of primers and probes
There are many primers and TaqMan probes, in Table 1, for real-time PCR detection;
their selection depends on the detection sample and purpose. Step 1: Select primers and
probes for detecting exogenous genes, to determine whether the feed sample contains
genetically modified components. Step 2: Select primers and probes, for detecting
endogenous genes, to determine which plant components the feed contains. Step 3:
Select primers and probes, for line-specific detection, to further identify a specific
transgenic line from which the plant transgenic components are derived.
8.5 Control design
during the detection process, it shall set the nucleic acid extraction blank control, PCR
amplification reagent blank control, PCR amplification negative target DNA control,
and PCR amplification positive target DNA control. The detail method shall be in
accordance with the relevant provisions in GB/T 19495.2.
8.6 Fluorescent PCR reaction system
The real-time fluorescent PCR reaction system is as shown in Table 2. Two parallel
reactions are set, for detection of each extracted DNA sample.