Quantitative PCR detecting kit and method for detecting vibrio parahaemolyticus thereof
A detection kit, a technology for Vibrio hemolyticus, applied in the determination/inspection of microorganisms, biochemical equipment and methods, resistance to vector-borne diseases, etc., can solve the problem of easy contamination, no molecular beacon probe, false positive and other problems, to achieve the effect of overcoming contamination, overcoming false positives, and good specificity
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example 1
[0034] The quantitative PCR detection kit consists of the following 7 tubes:
[0035] (1) The reaction solution in the PCR reaction tube consists of the following components: 5 μl of 10×Buffer buffer, 25mM MgCl 2 3 μl of solution, 8 μl of 1.25 mM dNTP solution, 0.5 μl of 50 μM upstream primer, 0.5 μl of 50 μM downstream primer, 0.5 μl of 50 μM circular molecular beacon probe and 27 μl of deionized water;
[0036] (2) Sample treatment solution tube The treatment solution in the tube is composed of the following components: 0.5mol / LEDTA (PH8.0) 0.01ml, 1% SDS 0.25ml, 20mg / ml proteinase K 5μl and distilled water 0.235ml;
[0037] (3) 100 μl of recombinant positive plasmid DNA containing the following insert sequence in the positive control tube;
[0038] (4) Negative control tube containing 100 μl of recombinant negative plasmid DNA;
[0039] (5) 3 tubes of standard quality control, each containing 10 7 、10 6 、10 5 copy / ml recombinant positive plasmid DNA 100μl;
[0040] (6) ...
example 2
[0073] The components contained in the tubes of the positive control tube, negative control tube, standard product tube, polymerase tube and deionized water tube in the quantitative PCR detection kit are the same as those in Example 1, and the content of each tube depends on the specifications of the kit. It depends, that is, how many servings to make. The quantitative PCR detection kit described in Example 1 is designed for 20 people, and this implementation and the following examples can be deduced by analogy (common knowledge that those of ordinary skill in the art should know). The reaction solution in the tube of the PCR reaction tube is composed of the following components: 4 μl of 10×Buffer buffer solution, 25mM MgCl 2 2 μl of solution, 4 μl of 1.25 mM dNTP solution, 0.2 μl of 50 μM upstream primer, 0.2 μl of 50 μM downstream primer, 0.2 μl of 50 μM circular molecular beacon probe and 34.4 μl of deionized water; Composition: 0.5mol / LEDTA (PH8.0) 0.008ml, 1% SDS 0.20ml,...
example 3
[0077] The components and contents in the positive control tube, negative control tube, standard product tube, polymerase tube and deionized water tube in the quantitative PCR detection kit are the same as those in Example 1. The reaction solution in the tube of the PCR reaction tube is composed of the following components: 6 μl of 10×Buffer buffer solution, 25mM MgCl 2 4 μl of solution, 8 μl of 1.25 mM dNTP solution, 1.0 μl of 50 μM upstream primer, 1.0 μl of 50 μM downstream primer, 1.0 μl of 50 μM circular molecular beacon probe and 24 μl of deionized water; Composition: 0.5mol / LEDTA (PH8.0) 0.012ml, 1% SDS 0.30ml, 20mg / ml protease K 8μl and distilled water 0.250ml;
[0078] The preparation of the quantitative PCR detection kit and the method for detecting Vibrio parahaemolyticus can be implemented with reference to Example 1.
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