Cholera toxin virulence gene detection kit and detection method thereof
A technology for detecting kits and virulence genes, which is applied to biochemical equipment and methods, and microbial measurement/inspection. It can solve the problems of high requirements for experimental equipment, high missed detection rate, and long time consumption, and achieve simple and accurate identification. The effect of high efficiency and high yield
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Embodiment 1
[0035] The preparation of embodiment 1 cholera toxin virulence gene detection kit
[0036] (1) Synthesize oligodeoxynucleic acid primers by DNA synthesizer according to the following sequence:
[0037] Internal primer FIP: 5'-CCAAAATGAACTCGATACCATCCATAAGAAGTTTCTGCTTTAGGTG-3' (SEQ ID NO 1)
[0038] Internal primer BIP: 5'-GGTGCTTGATGAACAATTACATCGTCTGCTGCTGGAGCAATA-3' (SEQ ID NO 2)
[0039] Outer primer F3: 5'-AGTCCTCATCCAGATGAAC-3' (SEQ ID NO 3)
[0040] Outer primer B3: 5'-CCTGCCAATCCATAACCA-3' (SEQ ID NO 4)
[0041] Loop primer LF: 5'-ATATTTGGGAGTATGGAATCC-3' (SEQ ID NO 5)
[0042] Loop primer LB: 5'-TAATAGGGGCTACAGAGATAGATA-3' (SEQ ID NO 6)
[0043] (2) Purchase DNA polymerase: Bst DNA polymerase is placed in the container;
[0044] (3) Preparation of reaction solution and primers: the reaction solution contains 1.4mmol / L dNTPs, 20mmol / L Tris-HCl, 10mmol / LKCl, 10mmol / L (NH 4 ) 2 SO 4 , 8mmol / L MgSO 4 , 0.1% TritonX-100, 0.8mol / L betaine, inner primer FIP / BIP, loop p...
Embodiment 2
[0056] Example 2 Application of Cholera Toxin Virulence Gene Detection Kit
[0057] 1 Materials and methods
[0058] 1.1 Materials
[0059] 1.1.1 Strains
[0060] There are 14 bacterial strains used in the present invention, mainly from the Chinese Center for Disease Control and Prevention, the Shanghai Center for Disease Control and Prevention, and the Pudong New Area Center for Disease Control and Prevention. See Table 1 for details.
[0061] Table 1 Names and sources of strains
[0062]
[0063] 1.2 Identification of isolated strains
[0064] 1.2.1 Inoculate the specimen on TCBS agar and double-washed plate. Yellow colonies appear on TCBS, and colonies with gray-black center appear on the double-washed plate are suspicious colonies. Under the microscope, the bacterium is a slightly curved bacillus, short and comma-shaped, with a single flagella at one end of the bacterium, and the movement is lively and shuttle-like, and Gram staining is negative. Biochemical react...
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