Primer set for detecting 1,4-dioxane degrading bacterium, and method for detecting and quantifying 1,4-dioxane degrading bacterium

A detection method and primer set technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of time-consuming and uncertain cultivation

Inactive Publication Date: 2018-03-27
TAISEI CORP +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are the following problems: it takes time to cultivate, and the further formed colonies cannot be determined to be 1,4-dioxane degrading bacteria or other bacteria
In addition, non-patent document 6 reports that bacteria having SDIMO other than THF monooxygenase may also degrade 1,4-dioxane

Method used

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  • Primer set for detecting 1,4-dioxane degrading bacterium, and method for detecting and quantifying 1,4-dioxane degrading bacterium
  • Primer set for detecting 1,4-dioxane degrading bacterium, and method for detecting and quantifying 1,4-dioxane degrading bacterium
  • Primer set for detecting 1,4-dioxane degrading bacterium, and method for detecting and quantifying 1,4-dioxane degrading bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] "Example 1" Identification of the genome involved in 1,4-dioxane degradation

[0036] Predict the open reading frame (ORF) from the draft genome data of the D17 strain and select the annotated ORF through BLAST search (BLASTP), and use the eArray system of Agilent Technology to design the DNA probe, thereby designing 7511 A sequence of probes. The 8×15K format of Agilent Technology was used to make a custom DN Amicroarray slide.

[0037] In an inorganic salt medium supplemented with 500 mg / L of 1,4-dioxane (1g / L K 2 HPO 4 , 1g / L(N H 4 ) 2 SO 4 , 50mg / L NaCl, 200mg / L MgSO 4 ·7H 2 O, 10mg / L FeCl 3 , 50mg / L Ca Cl 2 , PH 7.0) was inoculated with colonies of strain D17, and cultured with rotation and shaking at 28°C and 120 rpm. During the culture period, the concentration of 1,4-dioxane in the culture solution was measured over time. As a result, the concentration of 1,4-dioxane decreased by about 40% after 6 days and decreased by about 60% after 7 days. Therefore, a part of t...

Embodiment 2

[0043] "Example 2" Quantification of bacterial cell mass by real-time PCR method targeting thm genome

[0044] The base sequence of the thm genome was identified from the draft genome data of the D17 strain, using the Primer3 program (T. KORESSAAR, M. REMM: Bioinformatics, 23, 10, 1289-1291 (2007) "Enhanc ements and modifications of primer design program Primer3", A.UNTERGASS ER, I.CUTCUTACHE, T.KORESSAAR, J.YE, BCFAIRCLOTH, M.REMM, SGROZEN: Nucleic Acids Res. 40, 15, e115 (2012) "Primer3-new capabilities and interfaces") selected Candidate primers for real-time PCR. Next, the Primer-Blast program (J.YE, G.COULOURIS, I.ZARETSKAYA, I.CUTCUTACHE, S.ROZEN, TLMADDEN: BMC Bioinformatics, 13, 134 (2012) "Primer-BLAST: Atool to design target-specif ic primers for polymerase chain reaction”) Search for the homology between the sequence of the candidate primer and the GenBank registered sequence to select the primer with high specificity. The selected primers with high specificity were ...

Embodiment 3

[0056] "Example 3"

[0057] For the D17 strain, a primer containing the base sequence shown in SEQ ID NO: 1 (5'-TGATTATGTGGG GCTGGTTATG-3') and a reverse primer containing the base sequence shown in SEQ ID NO: 2 (5'-CGAGGAAAGTTGTGTTCGTGATG) -3') The quantitative value and turbidity (OD 600 The relationship between the measured value) and the quantitative value of the direct counting method (A ODC) using acridine orange was investigated.

[0058] The D17 strain was cultured in MGY medium (malt extract: 10 g / L, glucose: 4 g / L, yeast extract: 4 g / L) for 2 weeks. Afterwards, the bacteria were recovered by centrifugal separation, washed twice with physiological saline and made into different turbidity (OD 600 ) Cell solution, which can be used for AODC and real-time PCR. In addition, the OD of each cell solution 600 The values ​​are 0.00015, 0.0089, 0.1058, 1.01305 and 10.01145.

[0059]

[0060] 2.29 mL of stain preservation solution (NaCl: 1.75%, acridine orange: 0.005%, glutaral dehy...

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Abstract

The present invention addresses the problem of providing primers that enable rapid and highly accurate detection and quantification of a 1,4-dioxane degrading bacterium. Provided as a solution is a primer set comprising a primer including a base sequence set forth in SEQ ID NO: 1 and a primer including a base sequence set forth in SEQ ID NO: 2.

Description

Technical field [0001] The present invention relates to a primer set used for polymerase chain reaction (hereinafter referred to as PCR) and a method for detecting and quantifying 1,4-dioxane degrading bacteria using the primer set. Background technique [0002] 1,4-dioxane is a cyclic ether represented by the following formula (1). 1,4-Dioxane has excellent compatibility with water and organic solvents, and is mainly used as a reaction solvent for organic synthesis. [0003] [0004] The production and import volume of 1,4-dioxane in Japan in 2010 was about 4500t / year, and it is estimated that about 300t / year was discharged into the environment. Since 1,4-dioxane is water-soluble, it will spread widely if it is discharged into the water environment. In addition, it is difficult to remove from water due to its low volatility, solid adsorption, photodegradability, hydrolysis, and biodegradability. Since 1,4-dioxane has acute and chronic toxicity, it has also been pointed out that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N15/09C12Q1/689
CPCC12N9/0073C12Q1/689C12Q2561/113C12Y114/00C12N15/00C12N15/09C12Q1/68C12Q1/6853C12Q1/686C12Q2531/113C12Q2600/16
Inventor 山本哲史斋藤祐二泷宽则池道彦黑田真史清和成井上大介
Owner TAISEI CORP
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