SN/T 1201-2014PCR protocol for detection of genetically modified plant ingredients in feed (English PDF)

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

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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

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.

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