SN/T 5227.2-2019Rapid detection of pig-derived components in exported foods Recombinase-mediated strand replacement nucleic acid amplification method (RAA method) (English PDF)

3.1 Terms and definitions

The following terms and definitions apply to this document.

3.1.1 Pig Sus

Artiodactyla, Suigoides, Suidae, Suidae are divided into domestic pigs and wild boars. The subspecies include. Central European wild boar, Southeast Asian wild boar and Indian wild boar, it is generally believed that these three subspecies constitute the breeding of domestic pigs.

3.1.2 Real-time RAA real-time RAA

A constant-temperature nucleic acid rapid amplification technology (referred to as RAA technology) that recombinase-mediated strand replacement. Utilize what is obtained from bacteria or fungi Recombinant enzyme, at a constant temperature (usually 37 °C ~ 42 °C), the recombinase can be tightly combined with the primer to form a polymer of the enzyme and the primer.

With the help of the single-stranded DNA binding protein, the double-stranded structure of the template DNA is opened. When the primer is searched on the template DNA, it is completely matched with it.

When matching complementary sequences, under the action of DNA polymerase, new complementary DNA strands are formed, and the amplified products grow exponentially. Use fluorescent with the labeling of the optical probe, as the RAA reaction proceeds, the RAA product and the increase in the fluorescence signal show a corresponding relationship.

3.1.3 Ct value cycle threshold

The number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold.

3.1.4 T value Time threshold

The time required for the fluorescence signal in each reaction tube to reach the set threshold.

3.2 Abbreviations

The following abbreviations apply to this document.

3.2.1 RAA. recombinase-aid amplification, recombinase-mediated amplification.

3.2.2 DNA. deoxyribonuleic acid, deoxyribonucleic acid.

3.2.3 CTAB. cetyltrithylammonium bromide, cetyltrithylammonium bromide.

3.2.4 Tris. tris (Hydroxymethyl) aminomethane.

3.2.5 EDTA. ethylene diaminetetraacetic acid.

3.2.6 dNTPs. deoxyribonuleoside triphosphate, deoxyribonuleoside triphosphate.

3.2.7 SSB. single stranded DNA binding protein, single stranded DNA binding protein.

3.2.8 SC-recA. Streptomyces coelicolor recombinase, Streptomyces coelicolor recombinase.

3.2.9 BS-recA. Bacillus subtilis recombinase, Bacillus subtilis recombinase.

3.2.10 Bsu. Bacillus subtilis, Bacillus subtilis.

3.2.11 Tricine. N-tris [Hydroxymethyl] methylglycine, N-tris hydroxymethyl methyl amino acid.

7.1 DNA extraction

Take 0.2 g of crushed or ground sample into a clean 1.5 mL centrifuge tube (if the sample contains impurities and condiments, add 1 mLdd Wash twice with H2O), add 600 µL CTAB extraction buffer, vortex to mix well, incubate at 70 °C for 15 min, inverted during Heart tube 2~3 times; centrifuge at 12 000r/min for 5 min, take the supernatant into a new clean 1.5 mL centrifuge tube; add 500 µL of phenol. chlorine Imitation. Isoamyl alcohol (25.24.1), turn the centrifuge tube upside down 2 to 3 times, vortex and shake to mix, centrifuge at 12 000 r/min for 5 min;

Transfer the upper aqueous phase to a new 1.5 mL centrifuge tube, add 0.7 times the volume of isopropanol, invert the centrifuge tube up and down 2~3 times, and let stand at 4 °C Centrifuge at 12 000 r/min for 30 min at 4 °C for 3 min. Carefully discard the supernatant; add 700 µL 70% ethanol, resuspend the pellet, and separate at 12 000 r/min.

Discard the supernatant carefully for 1 minute; open the lid of the tube, evaporate the dry liquid at room temperature, and add 50 µL~100 µL TE (pH 8.0) buffer to dissolve it.

Decompose DNA and store at -20 °C for later use.

DNA extraction can also be performed with equivalent DNA extraction kits.

7.2 Determination of DNA concentration and purity

Use a nucleic acid protein analyzer or an ultraviolet spectrophotometer to detect the absorbance values A260 and A280 at 260 nm and 280 nm, respectively. DNA the concentration of is calculated according to formula (1).

7.3 Real-time fluorescent RAA amplification

7.3.1 Real-time fluorescence RAA reaction system

7.3.2 Real-time fluorescent RAA reaction program

One of the following two amplification instruments can be used for detection, and the reaction procedure is as follows.

7.3.2.1 Real-time fluorescent PCR instrument

39°C, 60s, 1 cycle; 39°C, 30s, 40 cycles, collect fluorescence in each cycle.

7.3.2.2 Constant temperature fluorescence detector

39°C, 1min; 39°C, 20min, start to collect fluorescence in the second stage.

7.3.3 Experimental control

In the detection process, a positive control, a negative control, and a blank control are set respectively. Use a sample containing porcine ingredients as a positive control sample the sample without pig-derived ingredients was used as a negative control, and double distilled water of the same volume as the template was used as a blank control.

8.1 Real-time fluorescent PCR instrument

8.1.1 Blank control. there is no logarithmic increase in fluorescence, and the corresponding Ct value is not reported.

8.1.2 Negative control. there is no logarithmic increase in fluorescence, and the corresponding Ct value is not reported.

8.1.3 Positive control. there is a fluorescence logarithmic increase, and a typical amplification curve appears in the fluorescence channel, and the corresponding Ct value is <= 30.0.

8.2 Constant temperature fluorescence detector

8.2.1 Blank control. no fluorescence logarithmic growth, corresponding non-reported T value (time).

8.2.2 Negative control. no fluorescence logarithmic growth, corresponding non-reported T value (time).

8.2.3 Positive control. there is a fluorescence logarithm increase, and a typical amplification curve appears in the fluorescence channel, the corresponding T value (time) <= 15min.

9.1 Judgment of results

9.1.1 Real-time fluorescent PCR instrument

9.1.1.1 The result can be judged to be valid only if it meets the requirements of Clause 8.1.

9.1.1.2 If the Ct value is <= 35.0, the test sample is determined to be positive.

9.1.1.3 If 35.0 Appendix A

(Informative appendix) Gene amplification target reference sequence of pig-derived components Note. The boxed area is the primer binding area, and the shaded area is the probe binding area.