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Rapid detection primers and detection method of Flavobacterium columnar

A technology of Flavobacterium columnar and detection primers, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of poor accuracy, high cost, festering fish, etc., and achieve low detection cost and easy operation , high accuracy effect

Inactive Publication Date: 2016-08-17
天津市水产技术推广站
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Problems solved by technology

The pathogen of the disease is Flavobacterium columnar, which is a Gram-negative long rod-shaped bacterium. economic loss is huge
At present, the pathogenic mechanism of Flavobacterium columnar is not completely clear. For the prevention and control of Flavobacterium columnar, early detection and early detection of pathogens are very important, and timely effective measures are taken
At present, for the detection of Flavobacterium columnar, the traditional bacterial isolation and cultivation technology is mostly used, which is time-consuming and costly; the production is also judged according to the symptoms of diseased fish and the results of microscope inspection, the accuracy is not good, and professional technicians are required and instruments and equipment, there is an urgent need for a rapid and effective detection technology for Flavobacterium columnar that can be used in the field of breeding in aquaculture production

Method used

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  • Rapid detection primers and detection method of Flavobacterium columnar
  • Rapid detection primers and detection method of Flavobacterium columnar
  • Rapid detection primers and detection method of Flavobacterium columnar

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Design and synthesis of primers

[0039] Blast software was used to analyze the gene sequence of Flavobacterium columnar, and the conserved nucleotide sequence of the 16S rDNA gene sequence of Flavobacterium columnar (GenBank accession number: AB078047) was screened out. According to the primer design principle of LAMP technology, LAMP primers were designed and synthesized for this fragment.

[0040] F3: 5'-GAGTGGCTAAGCGAAAGTGA-3' (SEQ ID NO.1, outer primer upstream primer)

[0041] B3: 5'-GCTGACGACAACCATGCA-3' (SEQ ID NO.2, outer primer downstream primer)

[0042] FIP: 5'-CCACATGCTCCTCCGCTTGTGttttGTATCCCACCTGGGGAGTA-3' (SEQ ID NO.3, internal primer upstream primer)

[0043] BIP: 5'-ACGCGAGGAACCTTACCAAGGggatccGCACCTTGAAAAACGTCCGA-3' (SEQ ID NO.4, internal primer downstream primer)

Embodiment 2

[0045] A rapid detection method for Flavobacterium columnar comprises the following steps:

[0046] (1) Extract the DNA of the sample to be tested;

[0047] DNA extraction kit (Tiangen's rapid DNA extraction and detection kit KG203) was used to extract the genomic DNA of the purely cultured Flavobacterium columnar liquid;

[0048] (2) Add 4 μl of primer mixture and 1 μl of Bst DNA polymerase to two 200 μl PCR reaction tubes containing 19 μl of LAMP reaction base solution respectively, add 1 μl of Flavobacterium columnar sample DNA as a sample to one, and add to the other Add 1 μl of DEPC water as a negative control to make a 25 μl LAMP reaction system; place it at 63°C for 50 minutes, then place it at 85°C for 15 minutes to obtain the test solution;

[0049] The composition of 19μl LAMP reaction base solution: 1.5μl dNTP, 2.5μl 10×Thermopol Roaction Buffer, 1μl calcein and 14μl DEPC water;

[0050] 4 μl of primer mixture consists of the following components: 1 μl concentrati...

Embodiment 3

[0053] Embodiment 3. a kind of Flavobacterium columnar rapid detection method comprises the steps:

[0054] (1) Take 0.1 g of diseased fish gill tissue, add 100 μl of sample pretreatment solution (TE buffer solution, the following examples are TE buffer solution), and grind to slurry;

[0055] (2) Add 4 μl of primer mixture, 1 μl of BstDNA polymerase, and 1 μl of the supernatant obtained in step (1) to a 200 μl PCR reaction tube containing 19 μl of LAMP reaction base solution to form a 25 μl LAMP reaction system; Place it for 60 minutes, then place it at 85°C for 15 minutes to obtain the test solution;

[0056] The composition of the 19 μl LAMP reaction base solution is the same as in Example 2;

[0057] The primer mixture is the same as in Example 2;

[0058] (3) Observe the color reaction with the naked eye, and if it is green, it is positive, indicating that the gill tissue of the diseased fish to be tested contains Flavobacterium columnar.

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Abstract

The invention discloses a flavobacterium columnare rapid detection primer and a detection method. The flavobacterium columnare rapid detection primer is composed of an outer primer body and an inner primer body. The outer primer body is composed of an outer primer upstream primer shown in SEQ ID NO.1 and an outer primer downstream primer shown in SEQ ID NO.2, and the inner primer body is composed of an inner primer upstream primer shown in SEQ ID NO.3 and an inner primer downstream primer shown in SEQ ID NO.4. The flavobacterium columnare rapid detection primer and the detection method have the advantages that the detection period is short, cost is low, and the flavobacterium columnare rapid detection primer is not dependent on professional equipment or technicians and capable of being applied to field detection, and the detection operation is simple and fast; accuracy is high, and the sensitivity of the flavobacterium columnare rapid detection primer is higher than that of a conventional PCR by 1-2 orders of magnitude; the detection cost is low, special instrument devices such as PCR instruments are not needed, only one water bath kettle or thermostat metal bath which controls temperature accurately is needed, operation is easy and convenient, and the flavobacterium columnare rapid detection primer is suitable for field detection.

Description

technical field [0001] The invention relates to a rapid detection primer and detection method of Flavobacterium columnar, belonging to the field of rapid detection of pathogenic bacteria in cultured fish. Background technique [0002] Bacterial tail and gill rot in fish is a disease that occurs worldwide and endangers almost all freshwater cultured fish. The pathogen of the disease is Flavobacterium columnar, which is a Gram-negative long rod-shaped bacterium. The economic loss is huge. At present, the pathogenic mechanism of Flavobacterium columnar is not completely clear. For the prevention and control of Flavobacterium columnar, early detection and early detection of pathogens are very important, and timely effective measures are taken. At present, for the detection of Flavobacterium columnar, the traditional bacterial isolation and culture technology is mostly used, which is time-consuming and costly; the production is also judged according to the symptoms of diseased ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2531/119C12Q2561/101
Inventor 徐晓丽姚学良李贺密钟文慧杨超敬宋昀鹏
Owner 天津市水产技术推广站
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