SN/T 5227.5-2019Rapid detection of cattle-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 Bovine

Artiodactyla, ruminant suborder, Bovidae, Bovis subfamily, Bovis, Boss, there are 6 species under the genus Buffalo (Indian bison), Yak, Kuwa bison (white rump bison), Cambodian bison (Khmer cow), primitive cow, bison (white-legged bison).

3.1.2 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 37~42 °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 DNA-binding protein, the double-stranded structure of the template DNA is opened, and when the primer is searched on the template DNA, it matches exactly with it Under the action of DNA polymerase, a new complementary DNA strand is formed, and the amplified product grows exponentially. Use fluorescence with the labeling of the probe, as the RAA reaction progresses, the RAA product shows a corresponding relationship with the increase of the fluorescence signal.

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 to 3 times; centrifuge at 12 000 r/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 to 3 times, 4 °C Let stand for 30 min, centrifuge at 12 000 r/min for 3 min at 4 °C, carefully discard the supernatant; add 700 µL of 70% ethanol, resuspend the pellet, Centrifuge at 12 000 r/min for 1 min. Carefully discard the supernatant; open the tube cap, evaporate the dry liquid at room temperature, and add 50 µL ~100 µL TE (PH8.0) Dissolve DNA in buffer 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, 60 s, 1 cycle; 39 °C, 30 s, 40 cycles, collect fluorescence in each cycle.

7.3.2.2 Constant temperature fluorescence detector

39 °C, 1 min; 39 °C, 20 min, 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 bovine ingredients as a positive control sample the sample without bovine 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) Bovine-derived component gene amplification target reference sequence Note. The boxed area is the primer binding area, and the shaded area is the probe binding area.