SN/T 5227.8-2019Rapid detection of donkey-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

Donkey Equus asinus

Perissodactyla, Equatiformes, Equidae, Equidae, Equus, and horses belong to the same genus Equus.

3.1.2

Real-time fluorescence 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.

3.2 Abbreviations

The following abbreviations apply to this document.

7.1 DNA extraction

Take 0.2 g of the crushed or ground sample into a clean 1.5mL centrifuge tube (6.9) (if the sample contains impurities and condiments, add Wash twice with 1 mLdd H2O), add 600 µL CTAB extraction buffer (pH 8.0) (5.1), vortex and mix well, then warm at 70 °C Incubate for 15 minutes, invert the centrifuge tube 2~3 times during this period; centrifuge at 12 000 r/min for 5 minutes, take the supernatant and centrifuge in a fresh 1.5 mL in the tube; add 500 µL of phenol. chloroform. isoamyl alcohol (25.24.1) (5.2), turn the centrifuge tube upside down 2 to 3 times and then vortex.

Mix well, 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 (5.3), Invert the centrifuge tube up and down 2~3 times, let stand for 30 min at 4 °C, centrifuge at 12 000 r/min at 4 °C for 3 min, carefully discard the supernatant; add 700 µL 70% ethanol (5.4), resuspend the pellet, centrifuge at 1.2000 r/min for 1 min, carefully discard the supernatant; open the tube lid, and evaporate at room temperature Dry liquid, add 50 µL ~100 µL TE buffer (pH 8.0) (5.5) to dissolve DNA, and store at -20 °C for later use. DNA extraction is also available Use equivalent DNA extraction kit.

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 is calculated according to formula (1).

c - DNA concentration, in micrograms per microliter (µg/µL);

A-- Absorbance at 260 nm;

N-nucleic acid dilution factor.

When the ratio of A260/A280 is between 1.7 and 1.9, it is suitable for RAA amplification.

7.3 Real-time fluorescent RAA amplification

7.3.1 Real-time fluorescence RAA reaction system

See Table 2.

7.3.2 Real-time fluorescent RAA reaction program

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 donkey ingredients as a positive control sample the sample without the donkey source component is used as the negative control sample, and the double distilled water of the same volume as the template is used as the blank control sample.

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 logarithmic increase in fluorescence, and a typical amplification curve appears in the fluorescence channel, and the corresponding Ct value is less than or equal to 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 logarithmic increase, and a typical amplification curve appears in the fluorescence channel, the corresponding T value (time) is less than or equal to 15 min.

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 less than or equal to 35.0, the test sample is determined to be positive.

9.1.1.3 If the Ct value is greater than 35.0 and less than 40.0, repeat it once. If the result of the second test is still greater than 35.0 and less than 40.0, it is judged the tested sample is positive.

9.1.1.4 If there is no report of Ct value or no increase in fluorescence logarithm, the test sample is judged to be negative.

9.1.2 Constant temperature fluorescence detector

9.1.2.1 The result can be judged to be valid only if it meets the requirements of Clause 8.2.

9.1.2.2 If the T value (time) is less than or equal to 15 minutes, the test sample is determined to be positive.

9.1.2.3 If it is more than 15 min and less than 20 min, repeat it once. If the test result is still greater than 15 min and less than 20 min, the test sample is determined to be positive.

9.1.2.4 If there is no report of T value (time) or no increase in fluorescence logarithm, the test sample is judged to be negative.

9.2 Presentation of results

9.2.1 If the sample is positive, it is expressed as "donkey component detected".

9.2.2 The sample is negative, which is expressed as "donkey components not detected".