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Detection of bacterium by utilizing dnaj gene and use thereof

A technology of bacteria and genes, applied in the determination/inspection of microorganisms, recombinant DNA technology, resistance to vector-borne diseases, etc., can solve the problem of large sequence differences (Non-patent document 8, high polymorphism level at the species level).

Inactive Publication Date: 2009-10-14
GIFU UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, it is reported that the DnaJ gene polymorphism is common among the serotypes of Mycobacterium avium (Non-Patent Document 7) and although there is only a short sequence of 200 bases, the DnaJ gene Very conserved, with large sequence differences at the species level of the same genus (Non-Patent Document 8)
In addition, it has also been reported that in the genus Legionella, the DnaJ gene is conserved between serotypes of L. pneumophila, but the level of polymorphism at the species level is high (Non-Patent Document 9 )

Method used

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  • Detection of bacterium by utilizing dnaj gene and use thereof
  • Detection of bacterium by utilizing dnaj gene and use thereof
  • Detection of bacterium by utilizing dnaj gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0362] Staphylococcus aureus (S.aureus) of the genus Staphylococcus is an important species that causes food poisoning, and it is known that in terms of 16S rDNA, it has a similarity of 99.5 to the closely related bacteria Staphylococcus epidermidis (S. %( Figure 1A ), so it is impossible to design strain-specific primers, but for the DnaJ gene, the similarity of these two strains is only 81.1% ( Figure 1B ). Therefore, primers can be easily designed for the DnaJ gene. Moreover, the primer sequence is relatively conservative among the 11 strains of Staphylococcus aureus (S.aureus) whose sequence has been determined ( figure 2 ). In this example, based on the determined DnaJ sequences of 9 pathogenic bacteria causing food poisoning, primers for these pathogens were prepared. The primer sequences for each pathogen are as follows.

[0363] Primers for Staphylococcus aureus

[0364] SEQ ID NO: 13:5-GGACAAGGATTCAATGGCTCT

[0365] SEQ ID NO: 14:5-TTGCGGTGCATTTGGATCTCT

[0...

Embodiment 2

[0410] Instead of DNA of Staphylococcus aureus (S. aureus) used in Example 1, DNA of the following strains was used, and a mixed primer set was used under the same conditions. DNA amplified with a Light Cycler (Roche) capillary was monitored by an increase in CYBR green fluorescence intensity. The results are shown in Figure 4 .

[0411] [Strains used]

[0412] 1. Campylobacter jejuni GTC 0259 strain

[0413] 2. Vibrio cholerae (Vibrio cholerae) GTC 0037 01 E1 Tol type

[0414] 3. Vibrio parahaemolyticus GTC 0056 (=CDC A3308)

[0415] 4. Escherichia coli (Escherichia coli) GTC 1061 0157 (VT1 and VT2 positive)

[0416] 5. Listeria monocytogenes (Listeria monocytogenes) GTC0149 (=ATCC15313) type strain

[0417] 6. Salmonella enterica var. Typhimurium GlFU 11566 ST-1

[0418] 7. Yersinia enterocolitica (Yersinia enterocolitica) GTC 0127 strain

[0419] 8. Clostridium perfringens GTC 0785 (=ATCC13124)

[0420] Such as Figure 4 As shown, it was confirmed that the DNA of...

Embodiment 3

[0422] (Amplification of the DnaJ gene of Vibrio bacteria)

[0423] A primer solution obtained by mixing a universal forward primer (SEQ ID NO: 43) for bacteria belonging to the genus Vibrio (SEQ ID NO: 43) and a species-specific reverse primer within the same genus other than those described below was prepared in the following manner.

[0424] Vibrio universal primer (SEQ ID NO: 43) 2 μM

[0425] Vibrio cholerae (SEQ ID NO: 44) 1 μM

[0426] Vibrio mimicus (SEQ ID NO: 47) 1 μM

[0427] Vibrio vulnificus (SEQ ID NO: 53) 1 μM

[0428] Vibrio fluvialis (SEQ ID NO: 134) 1 μM

[0429] Vibrio parahaemolyticus (SEQ ID NO: 50) 1 μM

[0430] Vibrio alginolyticus (SEQ ID NO: 58) 1 μM

[0431] In addition, this primer solution was used to prepare a PCR reaction solution in the following manner.

[0432] Primer mixed solution (forward μM, reverse each 1μM): 2μl

[0433] 2×CYBER premixed Hotstart Ex Taq (Takara): 10μl

[0434] DNA of each Vibrio (single): 5ng

[0435] Water was a...

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PUM

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Abstract

Disclosed is a method for detecting the species of a bacterium in a simple manner. Specifically disclosed is a method for detecting the species of a bacterium, which can detect at least one bacterial species selected from Staphylococcus species, Streptococcus species, Klebsiella species, Escherichia species, Mycobacterium species, Legionella species, Vibrio species, Bacillus species, Neisseria species, Campylobacter species, Chlamydia species, Chlamydophila species, Mycoplasma species, Listeria species, Salmonella species and Yersinia species, and which comprises the steps of: a) contacting a sample suspected to contain a nucleic acid derived from the bacterium species to be detected with a nucleic acid molecule capable of hybridizing with at least a part of a DnaJ gene derived from the bacterial species; and b) detecting the presence or absence of the hybridization between the nucleic acid molecule and the nucleic acid contained in the sample.

Description

technical field [0001] The present invention relates to the detection of bacteria using the DnaJ gene and its utilization. In detail, it relates to a method for detecting bacteria using the DnaJ gene, a nucleic acid molecule used in the method, and applications of the method and the nucleic acid molecule in diagnosis and identification . Background technique [0002] For a long time, the detection and identification of various bacteria such as pathogenic bacteria requires the use of special reagents and skilled techniques, and the use of special reagents and skilled techniques for the cultivation of target bacteria. The growth of pathogenic bacteria is accompanied by a certain degree of danger. In addition, when considering pathogenic bacteria with various possibilities, it is necessary to cultivate them under various conditions. Furthermore, in order to identify bacterial species, it is necessary to examine in detail, for example, biochemical characteristics. Therefore, t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/09C12Q1/04
CPCY02A50/30
Inventor 江崎孝行大楠清文吉田滋
Owner GIFU UNIVERSITY
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