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Double PCR rapid detection kit and method of Portunus trituberculatus Vibrio natriegens

A technology of Portunus trituratus and detection kits, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of staying pathogen isolation and laboratory identification, lack of rapid detection methods, cumbersome and other problems, reducing the need for Sodium vibrio disease, avoid spreading and spreading, the effect of short whole time

Active Publication Date: 2017-07-11
HUAIHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. The classic physiological and biochemical identification method is cumbersome, time-consuming, and requires operators to have good professionalism;
[0005] 2. Bacteria rapid automatic identification system, this method requires expensive equipment and skilled operators;
[0007] However, the current research on this natrivibrio mainly stays in the isolation and laboratory identification of the pathogen, and there is no rapid detection method reported

Method used

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  • Double PCR rapid detection kit and method of Portunus trituberculatus Vibrio natriegens
  • Double PCR rapid detection kit and method of Portunus trituberculatus Vibrio natriegens

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1, a kind of Portunus trituberculatus pathogenic factor needs double PCR rapid detection kit of Navibrio, and this reagent is combined and is made up of following article:

[0048] 1) Negative control, the composition is 1 mL of sterilized double distilled water;

[0049] 2) Positive control, the reagent component is 100ng of sodium-demanding Vibrio ( Vibrio natriegens ) XA1 strain DNA 1 branch;

[0050] 3) Contains Mg 2+ 1 PCR buffer for dNTP and DNA polymerase;

[0051] 4) Primer pair toxR-F / toxR-R

[0052] toxR-F is: 5'-AAT CCG CTT TCC TCT GTT-3' 1 branch;

[0053] toxR-R is: 5'-GGC GTT AGC ACA GGT ACA-3' 1 branch;

[0054] 5) Primer pair Vhh-F / Vhh-R

[0055] Vhh-F: 5'-AAT GTC ATC CGC CAA CGA-3' 1 piece;

[0056] Vhh-R: 5'-CCG TCA GGC GAA TCA ATG-3' 1 branch;

[0057] 6) 1 bottle of sterilized double distilled water;

[0058] 7) Packaging box, a piece of foam board with the same size as the bottom surface of the packaging box, which is installe...

Embodiment 2

[0059] Example 2, using the kit described in Example 1 to carry out the double PCR detection method of Portunus trituberculatus Navibrio pathogen; before the detection, prepare the bacterial genome extraction kit provided by Beijing Quanshijin Biotechnology Co., Ltd.; the detection steps are as follows:

[0060] (1) Extraction of DNA template: 1 mL of overnight cultured Vibrio sodium ( Vibrio natriegens ) XA1 bacteria solution, centrifuge at 12,000g for 1min, discard the supernatant as much as possible; add 100μL LB11 and 20ul proteinase K, shake until the bacteria are completely suspended; incubate at 55°C for 20min; add 20μL RNaseA, mix and let stand for 2min; add 400μL BB11 and vortex Add all the liquid to the spin column, centrifuge at 12,000g for 30s, discard the effluent; add 500μL CB11, centrifuge at 12,000g for 30s, discard the effluent; add 500μL CB11, centrifuge at 12,000g for 30s, discard the effluent; add 500μL WB11, Centrifuge at 12,000g for 30s, discard the effl...

Embodiment 3

[0075] Example 3, the application experiment of the double-PCR rapid detection kit and detection method of Portunus trituberculatus requiring Navibrio trituberculosis pathogen. Adopt the test kit of embodiment 1 and the detection method described in embodiment 2:

[0076] Experimental content: adopt the primer, experimental material and experimental method that Huaihai Institute of Technology provides, carry out random sampling to the seawater, Portunus trituberculatus and prawn of 10 different shrimp and crab breeding ponds in Ganyu District, Lianyungang City, collect altogether 30 Portunus trituberculatus, Portunus trituberculatus, 30 prawns and 30 water samples were then tested by double PCR, 16S rRNA-PCR and in vitro culture in this study.

[0077] The result of the experiment is that the double PCR results of 18 swimming crabs, 9 prawns and 15 water samples are positive, and the 16S rRNA-PCR results of 22 swimming crabs, 17 prawns and 20 water samples are positive. The r...

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Abstract

The invention discloses a double PCR rapid detection kit of Portunus trituberculatus Vibrio natriegens. The kit comprises a negative control, a Vibrio natriegens XA1 strain DNA as a positive control, a PCR buffer solution containing Mg<2+>, dNTP and DNA polymerase, and a primer pair. The method also discloses a double PCR rapid detection method of Portunus trituberculatus Vibrio natriegens. The method comprises extracting a DNA template, preparing a PCR premix solution, carrying out PCR amplification, after the amplification reaction, carrying out electrophoresis and observing the result through an ultraviolet analyzer, wherein if the bands appear at 308bp and 526bp, the detection result is positive, otherwise, the result is negative. The kit and method have high sensitivity, has the lowest detection limit of 13.2pg of the Vibrio natriegens genome DNA, is easy to operate, is free of specialized technicians, has short whole process time and has low time consumption.

Description

technical field [0001] The invention relates to a detection kit and a detection method for pathogenic bacteria, in particular to a double PCR rapid detection kit and detection method for Portunus trituberculatus pathogenic Natrivibrio netabilis. Background technique [0002] Sodium-demanding Vibrio is a common facultative anaerobic bacterium in marine microorganisms. It is widely found in estuaries, seawater near the coast, seabed sediments and marine animals. It is an opportunistic pathogen that can cause aquaculture animals Sickness and death, the mortality rate is as high as 87.5-100%, which seriously endangers the healthy development of my country's mariculture industry. [0003] Current techniques for identifying Vibrio include: [0004] 1. The classic physiological and biochemical identification method is cumbersome, time-consuming, and requires operators to have good professionalism; [0005] 2. Bacteria rapid automatic identification system, this method requires exp...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q1/689C12Q2537/143
Inventor 毕可然
Owner HUAIHAI INST OF TECH
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