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Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same

A kind of Vibrio cholerae, the smallest technology, applied in the identification and calculation of Vibrio cholerae and Vibrio the smallest, in the field of detection, can solve the problem of not being able to distinguish between the two

Inactive Publication Date: 2006-03-15
NICHIREI FOODSKK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is not possible to clearly distinguish the two

Method used

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  • Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same
  • Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same
  • Primer and probe for detecting vibrio cholerae or vibrio mimicus and detection method using the same

Examples

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Embodiment 1

[0042]An example of using the gene amplification primers (shown in Table 2) designed and obtained according to the present invention using regions specific to Vibrio cholerae and Vibrio minimal is given below. In addition, the primers described in claim 5(2) and (3) and claim 11(2) and (7) correspond to CMgF, CMgR, CMrF, and CMrR in Table 2, respectively. PCR was performed using chromosomal DNA extracted from the test strain as a template, AmpliTaq Gold (PE Applied Biosystems) and a total of 20 µl of the reaction solution. Regarding the thermal cycle conditions, after heating at 95°C for 10 minutes, cycle 35 times at 94°C for 1 minute, anneal for 1 minute (see the annealing temperature in Table 5), and 72°C for 1 minute, and finally extend the reaction at 72°C for 10 minutes . The sample obtained after the reaction was subjected to 1% agarose gel electrophoresis, and then stained with ethidium bromide. The presence or absence of amplified genes was confirmed under UV irradia...

Embodiment 2

[0044] An example of using the gene amplification primers (shown in Table 3 and Table 4) designed and obtained according to the present invention using a region specific to Vibrio cholerae or Vibrio minimal is given below. Furthermore, the primers described in claims 19(1), (3), (4), and (5) and claims 25(1) and (14) are specific to Vibrio cholerae. These primers are described in Table 3 as CF1, CF2, CR2, CR1, CrF1, and CrR1. The primers described in claims 33(1), (3), (4), and (5) and in claims 39(7) and (13) are specific for Vibrio minimum. These primers correspond to MF1, MF2, MR2, MR1, MrF1, and MrR1 in Table 4, respectively.

[0045] In a similar manner to the example, PCR was performed using chromosomal DNA extracted from the test strain as a template (see Table 5 for annealing temperatures). In all cases, only amplification products derived from Vibrio cholerae were detected using V. cholerae-specific primers, and only amplification products derived from Vibrio minima...

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Abstract

The invention produces high-performance specific gene amplification primers for detecting, quantifying, or identifying Vibrio cholerae and Vibrio mimicus, having low risk of misidentification and practically sufficient amplification efficiency and amplification specificity. We have determined partial nucleotide sequences of rpoD and gyrB genes of Vibrio cholerae, Vibrio mimicus, and closely related species, revealed their phylogenetic relationship, and then identified nucleotides characteristic of Vibrio cholerae and Vibrio mimicus, respectively. Thus, we have made it possible to design probes having high specificity and gene amplification primers having high specificity and excellent amplification efficiency, both of which contain the characteristic nucleotides.

Description

technical field [0001] The present invention relates to methods for detecting, identifying and enumerating Vibrio cholerae and Vibrio mimicus in food inspection, epidemiological environment inspection, and clinical inspection. Background technique [0002] Vibrio cholerae produces cholera toxin and induces severe diarrhea and vomiting following oral infection. In some cases, infectious pathogens can cause death from extreme dehydration in infected patients. The only Vibrio cholerae considered to be toxic to humans is O1 cholera, which can be agglutinated by using an anti-O1 antibody. Vibrios, which differ from bacteria, are also classified as NAG (O1-nonagglutinable)-vibrios which are not toxic to humans. However, in Bengal, India, in 1995, a new strain of Vibrio cholerae was isolated without the O1 antigen, which caused symptoms similar to those caused by traditional cholera, revealing that the new strain of Vibrio cholerae had a characteristic called O139 new O-antigen....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12Q1/68C07K14/28
CPCC12Q2600/156C07K14/28C12Q1/689Y02A50/30
Inventor 小泉雄史西山叶子山本敏福山正文古畑胜则大仲贤二
Owner NICHIREI FOODSKK
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