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

Inactive Publication Date: 2011-06-15
EAST CHINA NORMAL UNIV +1
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  • Abstract
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Problems solved by technology

[0008] One of the technical problems to be solved by the present invention is to overcome the shortcomings of the prior art, such as long time consumption, high missed detection rate, and high requirements for experimental equipment, and provide a detection method with more comprehensive detection effect, high specificity, good sensitivity, and low missed detection rate. Low, convenient and easy-to-operate cholera toxin virulence gene detection kit

Method used

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  • Cholera toxin virulence gene detection kit and detection method thereof
  • Cholera toxin virulence gene detection kit and detection method thereof
  • Cholera toxin virulence gene detection kit and detection method thereof

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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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Abstract

The invention relates to a cholera toxin virulence gene detection kit and a detection method thereof. The kit of the invention contains three pairs of primers which are designed by using vibrio cholera ctxA gene as target gene on the basis of the loop-mediated isothermal amplification technology, namely inner primers FIP/BIP, outer primers F3/B3 and ring primers LF/LB and also contains Bst DNA polymerases, reaction solution, sample pretreatment solution, coloring liquid, stabilizing solution and positive control. The method for detecting the cholera toxin virulence gene comprises the following steps: extracting bacterial DNA, performing the loop-mediated isothermal amplification of the cholera toxin virulence gene and coloring for detection. The kit of the invention has high amplificationefficiency, specificity and sensitivity, low omission ratio and obvious coloring effect and is suitable for the rapid detection of toxigenic vibrio cholera.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnostic reagents, in particular to a cholera toxin virulence gene detection kit and a detection method thereof. Background technique [0002] Cholera is a severe infectious disease with diarrhea as the main symptom. So far, seven world pandemics have occurred. The seventh world pandemic of cholera, caused by Vibrio cholerae Eltor, began in 1961 and affected 140 countries and regions. More than 4 million cases were reported. According to the WHO expert meeting, there are about 5.5 million cases worldwide each year, among which Asia, Africa and Latin America are more serious, causing 100,000 deaths in Asia and 20,000 deaths in Africa. To this day, cholera remains one of the most dangerous infectious diseases. Therefore, early rapid and correct diagnosis is of great significance to the treatment and prevention of the spread of this disease. [0003] At present, Vibrio cholerae has been divided...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王婷张胜萍陆晔田桢干王传贵杜正平
Owner EAST CHINA NORMAL UNIV
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