Method for the detection of Vibrio cholerae by multiple crossover constant temperature amplification combined with gold nanobiosensor
A technology of cross constant temperature amplification and biosensing, applied in the field of detection of Vibrio cholerae, can solve the problems of uncertainty, efficient and specific amplification of MCDA-LFB, restricting the development of non-diagnostic detection of Vibrio cholerae, etc. The effect of fast detection speed and excellent detection sensitivity
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Embodiment 1
[0057] The feasibility of embodiment 1.MCDA-LFB amplification
[0058] Standard MCDA reaction system: the concentration of cross primer CP1* and CP1 is 30pmol, the concentration of cross primer CP2 is 60pmol, the concentration of displacement primers F1 and F2 is 10pmol, the concentration of amplification primers R1, R2, D1 and D2 is 30pmol, The concentration of amplification primers C1* and C2 is 20pmol, 10mM Betain, 6mM MgSO 4 , 1mM dNTP, 12.5 μL of 2×DNA polymerase buffer, 10U of Bst strand-displacing DNA polymerase, 1 μL of template, add deionized water to 25 μL. The whole reaction was kept at 63°C for 1 hour, and the reaction was terminated at 85°C for 5 minutes.
[0059] After MCDA amplification, three detection methods were used for MCDA amplification discrimination. First, a visible dye (such as HNB reagent, naphthalene hydroxyphenol blue visual reagent) was added to the reaction mixture, the color of the positive reaction tube changed from purple to blue, and the ne...
Embodiment 2
[0063] Embodiment 2. measure the optimal reaction temperature of MCDA technology
[0064] Under standard reaction system conditions, the DNA template for Vibrio cholerae and the designed corresponding MCDA primers were added, and the template concentration was 10 pg / μl. The reaction was carried out under constant temperature conditions (60-67°C), and the results were detected by a real-time turbidimeter. Different dynamic curves were obtained at different temperatures, see image 3 . image 3 It represents the temperature dynamic curve of the MCDA primers designed for the ompW gene sequence to detect Vibrio cholerae. 61-64°C ( image 3 B-E) are recommended as the optimal reaction temperature for MCDA primers. Subsequent verification in the present invention selects 63°C as the constant temperature condition for MCDA amplification.
Embodiment 3
[0065] Embodiment 3.MCDA-LFB detects the sensitivity of Vibrio cholerae
[0066] After the standard MCDA amplification reaction was carried out using the serially diluted genomic DNA of Vibrio cholerae, the LFB detection showed that the detection range of MCDA-LFB was 10ng-10fg, and red lines appeared in the TL and CL regions of LFB (Figure 4A1-A3) . When the amount of genomic template in the reaction system was reduced to below 10fg, the LFB only appeared a red line in the CL region, indicating a negative result (Figure 4A4-A5). Figure 4A Use LFB to visually read the MCDA amplification results; Figures 4A1 to A5 indicate that the template amounts of Vibrio cholerae are 10ng, 10pg, 10fg, 1fg and 0.1fg, and Figures 4A6, A7, and A8 indicate Enterococcus faecalis (10pg), Shigella bacteria template (10pg), blank control (1 microliter double distilled water).
[0067] In order to further verify the sensitivity of MCDA-LFB in detecting Vibrio cholerae, three other detection metho...
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