Four-color fluorescent PCR kit for detection of vibrios in water body and detection method

A kit and technology for Vibrio, applied in the field of four-color fluorescent PCR kits for detecting Vibrio in water, can solve the problems of long time and time consuming for detection, reduce detection cost, reduce workload, and improve detection. The effect of the rate

Inactive Publication Date: 2014-01-29
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the traditional detection method of pathogenic Vibrio is still isolated and identified through microbial culture, which is not perfect enough. It needs to go through multiple screening steps, requires a variety of media, and takes a long time; there is also a monoclonal antibody using Vibrio The detection method requires picking a single colony and then using immunological methods to identify it after a large number of bacteria. The specificity is high, but the time required for the detection of the two methods is relatively long

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]A four-color fluorescent PCR kit for detecting pathogenic Vibrio in water, the kit is composed of multiple fluorescent PCR reaction mother solution, bacteria enrichment reagent (containing PEG8000 with a final concentration of 20% by mass percentage, chlorinated sodium), DNA extraction reagent (containing tris hydrochloride at a final concentration of 20mM, potassium chloride at a final concentration of 100mM, ethylenediaminetetraacetic acid at a final concentration of 2.5mM, and NP40 at a final concentration of 1% by mass) , Taq DNA polymerase with a concentration of 5U / ul, wherein the multiplex fluorescent PCR reaction mother solution is by volume ratio: multiplex 10× fluorescent PCR reaction buffer (containing trishydrochloric acid with a final concentration of 100mM, and a final concentration of 500mM Potassium chloride, glycerol with a final concentration of 50% by mass, dATP, dGTP, dTTP and dGTP with a final concentration of 0.2mM, magnesium chloride with a final co...

Embodiment 2

[0036] 1. Select the following pathogenic microorganisms, the experimental group: O1 group Vibrio cholerae, O139 group Vibrio cholerae, Vibrio parahaemolyticus; control group: Vibrio alginolyticus, Vibrio mimicus, Vibrio river, Vibrio vulnificus, Shigella Bacteria and Salmonella, the above pathogenic strains were isolated and cultured from environmental samples: Vibrio cholerae O1 group, Vibrio cholerae O139 group, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio river, Vibrio vulnificus, Chi Shigella and Salmonella were collected, isolated and cultivated in various water bodies in Ningbo around March 2011 by Zhou Donggen and other comrades of our research institute. The contact number is 0574-87169626.

[0037] 2. Sample preparation in the simulated environment: each concentration of 0.5ml was 1×10 5 CFU / ml of O1 group Vibrio cholerae, O139 group Vibrio cholerae, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio river, Vibrio vulnificu...

Embodiment 3

[0044] The 3 samples of drinking water to be tested were tested in the same manner as steps 3, 4, 5, 6, and 7 in Example 2. As a result, the ROX channel of 1 sample of drinking water to be tested showed an S-shaped amplification curve, and the Ct value was between 34 During the period, there was Vibrio parahaemolyticus infection.

[0045] Ningbo Institute of Inspection and Quarantine Science and Technology

[0046] A four-color fluorescent PCR kit and detection method for detecting Vibrio in water

[0047] 12

[0048] PatentIn version 3.5

[0049]

[0050] 1

[0051] 22

[0052] DNA

[0053] Artificial sequence

[0054]

[0055] 1

[0056] CCAGATTGTA AAGCAGGATG GA 22

[0057]

[0058] 2

[0059] 19

[0060] DNA

[0061] Artificial sequence

[0062]

[0063] 2

[0064] GGTCATCTGT AAGTACAAC 19

[0065]

[0066] 3

[0067] 27

[0068] DNA

[0069] Artificial sequence

[0070]

[0071] 3

[0072] CCCGGAGTTT GTAAGCCCAC TACCGGG 27

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PUM

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Abstract

The invention discloses a four-color fluorescent PCR kit for detection of vibrios in a water body and a detection method. According to the method, innovative bacterium enrichment reagent technology, DNA extraction reagent technology, specific fluorescent probe hybridization PCR detection technology and multiplex PCR technology are employed, bacterium enrichment culture of a sample is not needed, false positive interference is minimized as much as possible on the premise that high sensitivity is maintained, and four populations of two kinds of vibrios can be detected at one time. The invention mainly has the following beneficial effects: no need for bacterium enrichment of the sample in advance; high sensitivity and good specificity in detection of bacteria; reduction of the false positive incidence in conventional PCR amplification; and realization of rapid, accurate and specific detection of vibrio cholera and vibrio parahaemolyticus at one time.

Description

technical field [0001] The invention relates to the detection of Vibrio, in particular to a four-color fluorescent PCR kit and a detection method for detecting Vibrio in water. Background technique [0002] At present, the traditional detection method of pathogenic Vibrio is still isolated and identified through microbial culture, which is not perfect enough. It needs to go through multiple screening steps, requires a variety of media, and takes a long time; there is also a monoclonal antibody using Vibrio The detection method requires picking a single bacterial colony and then using immunological methods to identify it. The specificity is high, but the time required for the detection of the two methods is relatively long. In terms of molecular biology testing, the FDA standard is still to design a PCR program for the purpose of detecting a pathogenic Vibrio. Domestic detection reagents design PCR detection method can only detect 3 kinds of Vibrio at the same time, and the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12R1/63
CPCY02A50/30
Inventor 周冬根王燕翟敏刘超群张升倪敏君李红
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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