PCR system for rapidly detecting Vibrio rotiferianus in culture field
A reaction system and rotifer technology, applied in the direction of microorganism-based methods, microbial measurement/testing, microorganisms, etc., can solve the problem that Vibrio rotiferus has no host specificity, cannot use TCBS medium for qualitative identification, does not ferment sucrose, etc. problem, to achieve good inter-species discrimination, simplify the pre-treatment process, and reduce the effect of condition requirements
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Embodiment 1
[0038] (1) First prepare 8 μL of PCR micro reaction system in a 200 μL PCR reaction tube, including 5 μL of 2×Buffer buffer, 0.5 μL of 5 μM DNA forward sequence primer, 0.5 μL of 5 μM DNA reverse sequence primer, 0.5 μL 5 μL of SYBR GREEN fluorescent dye, 0.5 μL of 5U DNA polymerase, 1 μL of RNAse-free H 2 O, a total of 8 μL;
[0039] (2) Pre-freeze the PCR tube containing the above 8 μL micro-reaction system in an ultra-low temperature refrigerator at -80°C for 1 hour, and then freeze-dry it by freeze-vacuum drying to form a pre-mixed reaction system. Cover the PCR tube and seal it with a seal Membrane packaging is available for use.
[0040] (3) In this embodiment, 23 strains of bacteria were selected for the specific detection of Vibrio rotiferus using the specific primers and methods described in the present invention. The selected strains and their sources are shown in Table 1.
[0041] Table 1 Reference strains for specific detection of Vibrio rotiferi
[0042]
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Embodiment 2
[0050] In the aquarium system of this laboratory, 4 experimental groups were respectively set up, and each experimental group cultured 30 flat scorpionfish with a body weight of 40±5g / tail. After 5 days of temporary breeding, they were marked as experimental groups 1, 2, 3 and the control group, in which each fish in experimental group 1 was injected with 0.05 mL at a concentration of 1.0×10 4 cfu / mL Vibrio rotifer live bacteria solution, each fish in experimental group 2 was injected with 0.05mL concentration of 1.0×10 4 cfu / mL live Vibrio anguillarum solution, each fish in experimental group 3 was injected with 0.05mL concentration of 1.0×10 4 cfu / mL live Edwardsiella tarda solution, each fish in the control group was injected with 0.05 mL of 1.5% sterile NaCl solution. The injection sites were the back muscles of the fish.
[0051] On the third day after the injection, five fish were randomly selected from each group, and 0.2 g of muscle tissue at the injection site was c...
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