Standard substance for quantitatively detecting bacterial strains producing geosmin and preparation and detection method

A quantitative detection and standard technology, applied in the field of molecular biology, can solve the problem of not being able to support the establishment of an early warning detection system for geosmin

Pending Publication Date: 2019-10-08
SHANGHAI MUNICIPAL WATER RESOURCES DEV & UTILIZATION NAT ENG CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Commonly used geosmin detection methods, such as sensory analysis, gas chromatography-tandem mas

Method used

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  • Standard substance for quantitatively detecting bacterial strains producing geosmin and preparation and detection method
  • Standard substance for quantitatively detecting bacterial strains producing geosmin and preparation and detection method
  • Standard substance for quantitatively detecting bacterial strains producing geosmin and preparation and detection method

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no. 2 example

[0033] figure 1 It is a flow chart of steps for a preparation method of a standard substance for quantitative detection of geosmin-producing strains according to the second embodiment of the present invention. Such as figure 1 Shown, a kind of preparation method of the standard substance of quantitative detection producing geosmin bacterial strain of the present invention comprises the steps:

[0034] Step 101, designing specific primers for the GSY gene.

[0035] Specifically, the GSY gene sequence was downloaded from GenBank (National Center for Biotechnology Information, NCBI), homology comparative analysis was performed, specific primers were designed and synthesized, and the length of the PCR product was 125 bp. The primer sequences are as follows: the upstream primer is 5'-CTAGACCAATGCGGGTTTTA-3' (SEQ ID NO.2), the downstream primer is 5'-CTCAACTACAAGCACACA-3' (SEQ ID NO.3), and the primers are prepared into 100 μM stock with sterile double distilled water solution, a...

no. 3 example

[0042] figure 2 It is a flowchart of steps of a method for quantitatively detecting geosmin-producing strains according to the third embodiment of the present invention. Such as figure 2 Shown, a kind of method of quantitative detection of the present invention produces geosmin bacterial strain, comprises the steps:

[0043] In step 201, the recombinant plasmid is used as a standard template, and real-time quantitative PCR is used for PCR amplification, and a linear regression curve corresponding to the concentration of the standard and the critical cycle number is established, that is, a standard curve is obtained.

[0044] The real-time quantitative PCR amplification system is composed of a real-time quantitative PCR amplification buffer, a primer pair and a template; the primer pair includes upstream and downstream primers, and the concentrations of the upstream and downstream primers in the reaction system are 0.1 to 0.3 μM / L ( 0.2 μM / L is the best), the upstream prime...

Embodiment 1

[0051] Embodiment 1, Quantitative detection of the preparation of the plasmid standard substance of the strain producing geosmin

[0052] 1. Design specific primers for GSY gene

[0053] Download the GSY gene sequence from GenBank, conduct homology comparison analysis, design and synthesize specific primers, and the length of the PCR product is 125bp. The primer sequences are as follows: the upstream primer is 5'-CTAGACCAATGCGGGTTTTA-3' (SEQ ID NO.2), and the downstream primer is 5'-CTCAACTACAAGCACACA-3' (SEQ ID NO.3). The primers were prepared into a 100 μM storage solution with sterile double-distilled water, aliquoted, and stored at -20°C.

[0054] 2. Extraction of total genomic DNA in the sample

[0055] The total DNA was extracted and purified using a DNA extraction kit to obtain a DNA sample.

[0056] 3. Preparation of plasmid standard

[0057] 1) Preparation of the target fragment

[0058] Use the DNA sample in step 2 as a template for PCR amplification, and the re...

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Abstract

The invention discloses a standard substance for quantitatively detecting bacterial strains producing geosmin and a preparation and detection method. The detection method comprises the following stepsthat step one, a recombinant plasmid is used as a standard substance template, PCR amplification is carried out by real-time quantitative PCR, a standard curve corresponding to concentration of the standard substance and a critical recurring number is established, the recombinant plasmid is a recombinant plasmid comprising a GSY gene or a recombinant cell comprising the GSY gene, and a sequence of the GSY gene is composed of 125 deoxyribonucleotides and is a sequence in a sequence table SEQ ID NO.1; and step two, a sample to be tested is used for replacing the standard substance in the step one, real-time quantitative PC amplification is carried out, according to the critical recurring number and the standard curve of an amplified result, a copy number of the GSY gene in the sample to betested is obtained, and namely, the number of the bacterial strains producing the geosmin in the sample to be tested is obtained. According to the standard substance for quantitatively detecting the bacterial strains producing the geosmin and the preparation and detection method, rapid and quantitative detection of the bacterial strains producing the geosmin in a water environment sample can be realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a standard product for quantitative detection of geosmin-producing strains and a preparation and detection method. Background technique [0002] The eutrophication of the water body leads to a large number of algae in the water body. After the cyanobacteria enters the drinking water treatment process, it affects the normal operation of the water plant process, resulting in frequent recoil of the filter, increasing the self-water consumption of the water plant, and the metabolism of the cyanobacteria. Bring corresponding water quality safety risks. Among them, geosmin occupies a large proportion in drinking water smell and taste problems. Geosmin is a saturated cyclic alcohol organic compound with a very stable structure and is difficult to degrade in nature. They have good water solubility and fat solubility, and can be passed through The gills, skin and food intake of aquatic o...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/06C12N15/70C12N1/21C12R1/19
CPCC12Q1/689C12Q1/6851C12N15/70C12N9/1051C12Y204/01011C12Q2600/166C12Q2531/113C12Q2545/114C12Q2537/16
Inventor 陈蕾姜蕾
Owner SHANGHAI MUNICIPAL WATER RESOURCES DEV & UTILIZATION NAT ENG CENT
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