Method for detecting blue algae capable of producing 2-MIB by PCR qualification and TaqMan fluorescent quantitative PCR quantity

A technology of fluorescence quantification and detection method, which is applied in the fields of microbial harmful metabolites, water environment monitoring, and aquatic ecology. It can solve problems such as the inability to quantitatively produce 2-MIB cyanobacteria concentrations, and achieve high sensitivity, accuracy, and high application value. , the effect of cumbersome steps

Inactive Publication Date: 2012-03-28
INST OF AQUATIC LIFE ACAD SINICA
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Problems solved by technology

Ordinary PCR detection technology can qualitatively detect whether cyanobacteria produce 2-MIB and whether there are 2-

Method used

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  • Method for detecting blue algae capable of producing 2-MIB by PCR qualification and TaqMan fluorescent quantitative PCR quantity
  • Method for detecting blue algae capable of producing 2-MIB by PCR qualification and TaqMan fluorescent quantitative PCR quantity

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Embodiment 1

[0023] A detection method for qualitative PCR and TaqMan fluorescence quantitative PCR for producing 2-MIB cyanobacteria, the steps of which are:

[0024] 1. Sample collection and DNA extraction:

[0025] Water samples from Lushui Reservoir (Chibi, Hubei) were collected in July and September 2010 for quantification of 2-MIB-producing cyanobacteria, with 6 sampling points.

[0026] 300 mL of water sample was filtered through 0.45 μm cellulose acetate membrane, and the total DNA was extracted and purified on the filter membrane.

[0027] 2. Qualitative PCR:

[0028] Use primers CITf and CITr to carry out qualitative analysis of cyanobacteria producing 2-MIB at each sample point. The reaction system and program are: 10 μl of 2×PCR reaction system (Mix), 1 μl of CITf and CITr, 1 μl of DNA to be detected, and double Distilled water was corrected to a volume of 20 μl; pre-denaturation at 94°C for 3 minutes, followed by 40 cycles, each cycle of 94°C for 30s, 58°C for 30s and 72°C f...

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Abstract

The invention discloses a method for detecting blue algae capable of producing 2-MIB by PCR qualification and TaqMan fluorescent quantitative PCR quantity. The method comprises the following steps: water sample to be measured is collected, filtered through a cellulose acetate film, extracted and purified total genome DNA on the filtered algae; B, a qualitative PCR reaction system comprises: corresponded monoterpene cyclase gene is synthesized, 2-MIB blue algae is taken as positive control and double distilled water is taken as negative control; C, a qualitative PCR reaction progress comprises: denaturation is carried out at the temperature of 94 DEG C for 3 minutes, circulated and finally extended; D, qualitative PCR products and positive control, negative control; E, the fluorescent quantitative PCR reaction system, aseptic double distilled water is added in negative template control; F, the fluorescent quantitative PCR reaction progress; G, stand curve making; H, the concentration of blue algae capable of producing 2-MIB in the water sample is calculated according to the Ct value and the standard curve of the sample to be measured. The method of the invention has the advantages of easy process, simple operation, high sensitivity and accuracy, so that the concentration of blue algae capable of producing 2-MIB in the water sample can be rapidly and accurately quantified.

Description

technical field [0001] The present invention relates to the fields of water environment monitoring, water ecology, microbial harmful metabolites, etc., and more specifically relates to a method for detecting 2-MIB-producing cyanobacteria in the environment by using PCR (polymerase chain reaction) and fluorescent quantitative PCR. It is suitable for early warning, rapid qualitative and quantitative analysis of algae-derived 2-MIB in drinking water sources such as reservoirs, lakes and rivers, and aquatic ecosystems. Background technique [0002] In recent years, with the increasing eutrophication of water bodies, cyanobacterial blooms have broken out in large areas all over the world, especially in my country. The water bloom problem has become a major problem in the field of environmental protection in our country. The hazards of cyanobacterial blooms are manifested in many aspects, among which algal toxins, which are a direct threat to human health and drinking water, have...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 王中杰宋高飞虞功亮李仁辉
Owner INST OF AQUATIC LIFE ACAD SINICA
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