GB/T 19495.4-2018Detection of genetically modified organisms and derived products—Qualitative real-time polymerase chain reaction (PCR) methods (English PDF)

3.1.1 Transgene

The technology that makes the DNA sequences that the species have or from other species, through biological engineering technology, transcribe or express it in the species, so that the species can obtain new varieties characteristics.

3.1.2 Real-time polymerase chain reaction

Real-time fluorescent polymerase chain reaction. A method of adding fluorescent groups to the polymerase chain reaction system, using the accumulation of fluorescent signals to monitor the entire PCR process in real time, and quantitatively analyzing the unknown template through the standard curve.

NOTE: The intensity of the fluorescent signal directly reflects the number of templates.

3.1.3 Endogenous gene

A gene that is constant in copy number in the tested species and does not exhibit allelic changes.

NOTE: This gene can be used to determine species specificity.

3.1.4 Exogenous gene

Other biological genes that are transferred by bioengineering technology.

NOTE: After the exogenous gene is transferred, the biological specie shows new biological characteristics.

3.1.5 Cycle threshold

The number of cycles through which the fluorescent signal in each reaction tube reaches the set threshold.

3.2 Abbreviations

The following abbreviations apply to this Document.

Alfalfa-Acc: Alfalfa Acetyl-CoA carboxylase.

BAR: phosphinothricin acetyltransferase gene.

bp: base pair.

PSSuAra: the small subunit promoter of Arabidopsis.

pTA29: developmentally regulated promoter from anther-specific TA29 gene from Nicotiana tabacum.

pUbi: promoter of maize Ubiquitin.

SAH7: sinapis Arabidopsis homolog 7 gene.

SDS: sodium dodecylsulfate.

SPS: sucrose phosphate synthase gene.

Taq: Taq DNA polymerase.

TE: Tris-HCL, EDTA buffer.

tE9: terminator derived from pea ribulose diphosphate carboxylase gene

Tris: [tris(hydroxymethyl) aminomethane].

tg7: transglutaminase 7 gene.

tNOS: terminator of nopaline synthase gene from Agrobacterium tumefaciens.

tOCS: terminator of octopine synthase.

t35S: Cauliflower mosaic virus 35 terminator.

UDG: uracil DNA glycosylase.

UGPase: UDP-glucose pyrophosphorylase gene from Solanum tuberosum.

UNG: uracil-N-glycosylase.

Wx012: waxy wheat genes.

zSS II b: [the endosperm-specific SS (starch synthase) II b].

18S rRNA: 18S ribosomal RNA.

5.1 Apparatus

5.1.1 Real-time fluorescent PCR instrument.

5.1.2 Sample crusher or grinder.

5.1.3 Balance: with sensitivity of 0.01g.

5.1.4 Water bath or constant temperature incubator.

5.1.5 Refrigerated centrifuges.

5.1.6 Autoclave.

5.1.7 Vortex shaker.

5.1.8 Biological safety cabinet.

5.1.9 pH meter.

5.1.10 Nucleic acid protein analyzer or UV spectrophotometer.

5.1.11 Micropipettes (2µL, 10µL, 100µL, 200µL, 1000µL)

5.2 Major reagents

Unless otherwise specified, all reagents are of analytically pure or biochemical reagents; and the experimental water shall satisfy the specifications of Grade-1 water in GB/T 6682.

5.2.1 Real-time PCR master mix

A solution prepared by mixing Taq DNA polymerase (5U/µL), PCR reaction buffer, MgCl2 (3 mmol/L~7 mmol/L), dNTPs (containing dATP, dUTP, dCTP, dGTP), UNG enzyme, etc.

5.2.2 ROX

Fluorescence calibration reagent (50×, diluted to 1× when used).

5.2.3 Primers and probes

5.2.3.1 Screening and detecting primer and probes

7.2 Judgment of result

If the Cycle threshold of exogenous gene detection in the test sample is >=40, and the Cycle threshold of endogenous gene detection is <=30, it can be determined that the sample does not contain the tested gene or line.

If the Cycle threshold of exogenous gene detection in the test sample is <=35, and the Cycle threshold of endogenous gene detection is <=30, it can be determined that the sample contains the tested gene or line.

If the Cycle threshold of the exogenous gene in the test sample is 35~40, the template concentration shall be adjusted and the real-time PCR shall be redone. If the Cycle threshold of the exogenous gene after re-amplification is still 35~40, it can be determined that the sample contains the tested gene or line. If the Cycle threshold of the exogenous gene after re-amplification is >=40, it can be determined that the sample does not contain the tested gene or line.