A kind of rpa primer for detecting Lawsonia intracellulare and its detection method
A Lawsonia, purpose technology, applied in the detection of Lawsonia intracellular RPA primers and its detection field, can solve the problems of guidance, unable to provide clinical control of epidemic diseases, time-consuming and other problems, achieve low cost, easy to promote and use at the grassroots level, The effect of high sensitivity
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Embodiment 1
[0036] Example 1: Synthesis of primers for detection of Lawsonia intracellulare
[0037] According to the comparison results of the Lawsonia intracellulare gene sequence published in GenBank, the sequence at the 960bp-988bp position (5'-AAATCCAAAAGTCGAGTATCTAACTGCGG-3') in the nucleotide sequence of the Lawsonia intracellulare genome was selected as the RPA amplification reaction Upstream primer, when synthesizing the upstream primer, label carboxyfluorescein (FAM) at its 5' end; use the reverse complementary sequence of the 1219bp-1251bp position sequence in the Lawsonia intracellulare genome nucleotide sequence as the downstream primer of the RPA amplification reaction, When synthesizing the downstream primer, label the 5' end of the downstream primer with biotin: (5'-Biotin-TAAAAACCCAGAGCAAAATCGTGATACCAGGCG-3').
[0038] Primer synthesis was completed by Sangon Bioengineering (Shanghai) Co., Ltd.
Embodiment 2
[0039] Embodiment 2: the preparation of colloidal gold lateral flow immunochromatography test strip
[0040] (1) Preparation of streptavidin-coated gold nanoparticles: Take 1 ml of gold nanoparticle solution (0.15 pmol / mL), add 200 mM borax solution, and adjust the pH value to 9.5. At the same time, in another test tube, 2 μl of streptavidin (2 mg / ml) was mixed with 398 μl of borax solution (2 mM); The amount is gradually added, stirring while adding. Place the mixture at room temperature for 45 minutes, add 155.6 μl of 2 mM borax solution containing 10% BSA, continue to place the solution at room temperature for 10 minutes, centrifuge at 4500 g for 15 minutes, discard the liquid, and wash with 1 ml of washing solution (containing 10 g / L BSA 2mM borax solution) to resuspend the precipitate, centrifuge at 4500g for 15 minutes, aspirate and discard the liquid, and resuspend the red precipitate in 250 μl buffer solution containing 5% BSA, 137mM NaCl and 0.025% Tween-20. Accordi...
Embodiment 3
[0042] Example 3: DNA Extraction
[0043] The specific operation steps for DNA extraction of Lawsonia intracellulare are as follows: ① Add 350 μl of bacterial stock solution to a 1.5ml eppendorf tube, add an equal amount (350 μl) of preheated tissue lysate, and then add proteinase K (final concentration is 0.2mg / ml), mix well, and put in a water bath at 56°C for 2 hours; ②add 700μl saturated phenol to each tube, mix well, and centrifuge at 12000rpm for 10min; 350 μl of chloroform: isoamyl alcohol (24:1) was mixed evenly, and centrifuged at 12000 rpm for 10 min; ④ Take 700 μl of the upper layer solution and put it into a new 1.5ml eppendorf tube, add 700 μl of chloroform: isoamyl alcohol (24:1) and mix well, Centrifuge at 12000rpm for 10min; ⑤Pipe 700μl of the upper layer solution into a new 1.5ml eppendorf tube, add 700μl of chloroform to mix, and centrifuge at 12000rpm for 10min; / 10 volume (40μl) of 3M NaAC (pH=5.2), mix well, and place at -20°C for 2 hours; ⑦12000rpm, 4°C...
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