Primer and probe combination for detecting phytophthora cambivora based on RPA-lateral flow chromatography technology and application thereof
A lateral flow chromatography and technical detection technology, applied in the biological field, can solve problems such as no related application reports, and achieve good amplification effect, increased sensitivity and good specificity.
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Embodiment 1
[0032] The genomic DNA of the tested pathogenic bacteria strain was extracted, and the specific extraction process was as follows:
[0033] Take a small amount of mycelium powder, add 900 μL 2% CTAB extract and 90 μL 10% SDS, vortex and mix well, put in a water bath at 60°C for 1 hour, and invert several times every 10 minutes. Centrifuge at 12000rpm for 10min, take the supernatant and add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix by inversion, and centrifuge at 12000rpm for 10min; transfer the supernatant to a new tube, add an equal volume of chloroform, Gently invert to mix and centrifuge at 12000rpm for 5min. Take the supernatant and transfer it to a new tube, add 2 times the volume of absolute ethanol and 1 / 10 volume of 3M NaAc (pH5.2), and precipitate at -20°C (>1h). Centrifuge at 12000 rpm for 10 min, pour off the supernatant, wash the precipitate twice with 70% ethanol, and dry at room temperature. Add an appropriate amount of sterilized ultr...
Embodiment 2
[0035] Using the extracted DNA as a template and using the designed RPA primers, perform the RPA reaction in the following reaction system:
[0036] Sample detection: Add 29.5 μL of buffer buffer, 2.1 μL of 10 μM upstream primer, 2.1 μL of 10 μM downstream primer, and 0.6 μL of probe to a 0.2 mL TwistAmp reaction unit tube (TwistAmp Basickits, Twist) containing lyophilized enzyme powder. Add 2.0 μL of DNA and 2.5 μL of MgAc to the inside of the PCR tube cap, make up to 50 μL with deionized water; mix the RPA amplification system well, centrifuge at 5,000×g for 10 seconds, place it on a metal bath at 39°C for 30 minutes, and incubate for 4 minutes , mix the reaction tube again, centrifuge for 3-5s, and put it into a water bath at 39°C to continue the reaction for 30min.
[0037] Wherein, the length of the RPA primer is generally 30 to 35 nucleotides. Too short primers will seriously affect the activity of the recombinase. Long primers do not necessarily improve the amplificati...
Embodiment 3
[0042] In order to verify the specificity of the RPA lateral flow chromatography test strip detection method, the results of the RPA lateral flow chromatography test strip detection method are as test materials (Table 1) with Phytophthora chestnut and other Phytophthora and pathogenic bacteria The test strip showing Phytophthora chestnutensis has two brown bands, one in the control area (Control line) and one in the detection area (Test line), then the result is positive. Test strips for other Phytophthora and pathogenic bacteria If there is only a brown band in the quality control area, and there is no band in the test line, the result is negative, indicating that the sample does not contain Phytophthora solani.
[0043] Select different species (Pytophthora cedarus; Phytophthora winter; Phytophthora meloni; Phytophthora infestans; Phytophthora victoria, etc.) and different genera (Pythium ultima; Fusarium equiseti; Anthracnose flat head; Verticillium dahliae; Verticillium da...
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