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Detection primer set, detection kit and detection method for Vibrio parahaemolyticus, Photobacterium damsela and Nocardia seriolea

A technology of Nocardia japonica and a detection kit, which is applied in the field of detection of pathogenic bacteria in aquaculture animals, can solve problems such as cross-infection, omission, and hidden dangers of infection between shrimp and fish, and achieve rapid pathogen detection, low technical quality requirements, and application wide range of effects

Active Publication Date: 2015-12-23
深圳市富炜城投资有限公司 +2
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, this method does not essentially determine whether the prawns are suffering from AHPNS disease. The probability of reducing the spread of pathogens is not high with this method, and some diseased prawns may still be missed in the water without being cleaned by fish. Then there are still hidden dangers of infectivity to other healthy prawns, and there is a probability of cross-infection between prawns and fish in this method, so there are some shortcomings

Method used

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  • Detection primer set, detection kit and detection method for Vibrio parahaemolyticus, Photobacterium damsela and Nocardia seriolea
  • Detection primer set, detection kit and detection method for Vibrio parahaemolyticus, Photobacterium damsela and Nocardia seriolea
  • Detection primer set, detection kit and detection method for Vibrio parahaemolyticus, Photobacterium damsela and Nocardia seriolea

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Experimental program
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Effect test

Embodiment 1

[0033] The extraction of embodiment 1 bacterial genome DNA

[0034] Vibrio parahaemolyticus, Photobacterium mermaidi, and Nocardia aerevisiae (self-preserved in the Fishery Biological Disease Research Laboratory of South China Sea Fisheries Research Institute) were collected respectively, and the DNA extraction was carried out by the following steps:

[0035] (1) Resuspend the bacteria with 500-550 μL of TE buffer;

[0036] (2) Add 30 μL of SDS solution with a mass concentration of 10% and 15 μL of proteinase K (20 mg / mL) to the solution resuspended in step (1), mix well and incubate at 37° C. for 1 h;

[0037] (3) Add 100 μL of 5mol / L NaCl solution to the solution incubated in step (2), mix well and then add 80 μL of NaCl solution of LCTAB (dissolve CTAB in 0.5mol / L of NaCl solution, CTAB and all The mass volume ratio of the above NaCl solution is 1:20), and incubated at 65°C for 20min;

[0038] (4) Add phenol-chloroform-isoamyl alcohol (the volume ratio of phenol, chlorofo...

Embodiment 2

[0042] Example 2 Design and effectiveness detection of triple PCR detection primer sets for Vibrio parahaemolyticus, Photobacterium mermaid and Nocardia aerevisiae

[0043] 1. Select the specific genes (GI: 6714616, GI: 2988378, GI: 696553489) of Vibrio parahaemolyticus, Photobacterium mermaidi and Nocardia aerevisiae respectively, and use Primer Premier 5.0 software to analyze and design corresponding primer pairs. The primer pairs can specifically identify the specificity of the above-mentioned bacteria respectively, and the primer sequences are as follows:

[0044]

[0045]

[0046] 2. Effectiveness testing:

[0047] (1) Synthesize the primer set of the above-mentioned design, carry out single PCR verification to the DNA of Vibrio parahaemolyticus, Photobacterium mermaidus and Nocardia aerevisiae respectively with the primer of primer set, wherein single PCR reaction system is as follows:

[0048]

[0049] (2) Simultaneously perform triple PCR verification on Vibr...

Embodiment 4

[0062] Embodiment 4 detection kit

[0063] The detection kit of this embodiment can be used for rapid triple PCR diagnosis of Vibrio parahaemolyticus, Nocardiella aerevisiae and Photobacterium mermaidi in samples Bacillus detection primer set, proteinase K, SDS solution, phenol-chloroform-isoamyl alcohol mixed solution, isopropanol, ethanol with a volume concentration of 70%, TE buffer, CTAB NaCl solution, positive control substance and PCRDsMix ,in:

[0064] (1) Primer set for detection of Vibrio parahaemolyticus, Photobacterium mermaidi and Nocardia amberii: 1 tube contains Vibrio parahaemolyticus primers V.pa-F and V.pa-R, the nucleotide sequences of which are respectively shown in SEQ ID NO .1 and shown in SEQIDNO.2; 2 tubes are equipped with photobacterium mermaid primers N.se-F and N.se-R, and their nucleotide sequences are shown in SEQIDNO.3 and SEQIDNO.4 respectively; 3 tubes are equipped with amberjack The nucleotide sequences of the primers P.da-F and P.da-R for Ka...

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Abstract

The invention discloses a detection primer set, detection kit and detection method for Vibrio parahaemolyticus, Photobacterium damsela and Nocardia seriolea. The primer set comprises a primer pair V.pa, a primer pair N.se and a primer pair P.da, and the nucleotide sequences of the primer pairs are disclosed as SEQ ID NO.1-6. The detection kit comprises the detection primer set. The detection method uses the detection kit and comprises the following steps: carrying out pretreatment on a sample, extracting genome DNA (deoxyribonucleic acid) as a sample set DNA, and detecting the sample by a PCR (polymerase chain reaction) process. The triplex PCR detection method established by the invention can be used for tracing detection on Vibrio parahaemolyticus, Photobacterium damsela and Nocardia seriolea in various stages, avoids transmission and prevalence of pathogens, and enhances the scientific management efficiency, thereby having very high practical value.

Description

technical field [0001] The invention belongs to the field of detection of pathogenic bacteria in aquaculture animals, and in particular relates to a detection primer set, a detection kit and a detection method for Vibrio parahaemolyticus, Photobacterium mermaida and Nocardia aerevisiae. Background technique [0002] Vibrio parahaemolyticus (Vibrio Parahaemolyticus), Photobacterium damsel (Photobacterium damsel) and Nocardia seriolae (Nocardiaseriolae) are three common aquatic pathogens, of which Vibrio parahaemolyticus and Photobacterium damsel belong to Gram-negative bacteria, Karstella belongs to Gram-positive bacteria. These three bacteria are widely distributed in the marine environment and aquaculture environment, and can cause a large number of deaths of a variety of aquaculture animals and cause serious economic losses. [0003] Vibrio parahaemolyticus is halophilic and can be transmitted by the fecal-oral route. Eating raw or unprocessed seafood, especially oysters,...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCY02A50/30
Inventor 许海东郭志勋陈日和冯娟苏友禄郑维谦
Owner 深圳市富炜城投资有限公司
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