Phytophthora vignae LAMP (loop-mediated isothermal amplification) detection primers and phytophthora vignae LAMP detection method
A technology for detection primers and detection methods, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieve reliable results, strong specificity, Sensitive effect
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[0042] Example 1: The specific amplification of LAMP primers against Phytophthora cowpea
[0043] 1. LAMP-specific detection of Phytophthora cowpea
[0044] ①LAMP reaction system: the concentration of F3 and B3 primers in 25μl reaction system are 0.2mmol / L, the concentration of FIP and BIP primers is 1.6mmol / L, 20mMTris-HCl, 10mM(NH 4 ) 2 SO 4 , 10mMKCl, 8mMMgSO 4 , Betaine 0.8mol / L, Bst The polymerase is 8U, dNTPs 1.0mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, TWeen-200.1%, template DNA 50ng, and the insufficient part is made up with sterile double-distilled water; LAMP reaction conditions are at 65 ℃ Incubate for 50 minutes and inactivate at 85°C for 10 minutes.
[0045] ② After the LAMP reaction, the result of color development is that the green fluorescence is judged to be positive, and the orange is judged to be negative. Or take 2μl of amplified product and use 2% agarose gel electrophoresis to detect. If there is a characteristic ladder band of LAMP, it is judg...
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[0048] Example 2: Sensitivity detection of LAMP primers to Phytophthora cowpea
[0049] 1. LAMP Sensitivity Detection of Phytophthora Vigna
[0050] A 10-fold concentration serial dilution method was used to dilute the extracted DNA of Phytophthora indica into 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg / μL, a total of 9 different concentration gradients.
[0051] ①LAMP reaction system: F3 and B3 primer concentration in 25μl reaction system are 0.2mmol / L, FIP and BIP primer concentration are 1.6mmol / L, 20mMTris-HCl, 10mM(NH 4 ) 2 SO 4 , 10mMKCl, 8mMMgSO 4 , Betaine 0.8mol / L, Bst The polymerase is 8U, dNTPs1.0mmol / L, calcein 50μmol / L, manganese chloride 500μmol / L, TWeen-200.1%, template DNA 100ng, and the insufficient part is made up with sterile double-distilled water; the LAMP reaction conditions are at 65℃ Incubate for 60 minutes and inactivate at 85°C for 5 minutes.
[0052] ② After the LAMP reaction, the result of color development is that the green fluorescence is judg...
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