Biosensing detection method for salmon trout seed identification

A technology of biological sensing and detection methods, applied in the field of aquatic organisms, can solve the problems of long detection period, high requirements for detection equipment and operators, and achieve the effect of short response time

Inactive Publication Date: 2020-09-29
CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned method has a long detection period, and virus isolation requires virus culture in a specific cell line, which requires high detection equipment and operators, and is not easy to promote in production and operation places such as seedling farms and breeding farms.

Method used

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  • Biosensing detection method for salmon trout seed identification
  • Biosensing detection method for salmon trout seed identification
  • Biosensing detection method for salmon trout seed identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1 identifies whether rainbow trout fry is infected with IHNV

[0064] 1. Solution preparation

[0065] Custom-synthesized primers with base sequences as shown in SEQ ID NO: 1-6 (Shanghai Shenggong), each primer has a ferrocene modification at the 5' end, dissolved in nuclease-free sterilized deionized water, and prepared to contain 1 μM each of primers SEQ ID NO: 1 and SEQ ID NO: 2, 8 μM each of primers SEQ ID NO: 3 and SEQ ID NO: 4, and 2 μM each of primers SEQ ID NO: 5 and SEQ ID NO: 6. Primers SEQ ID NO: 3 and SEQ ID NO: 4 have a ferrocene tag upstream.

[0066] Use nuclease-free sterilized deionized water to prepare a dNTP use solution, which contains 10 mM each of dATP, dCTP, and dGTP, 8 mM of dTTP, and 2 mM of biotin-16-dUTP.

[0067] 2. Total RNA Extraction

[0068] The body length of the rainbow trout fry to be tested is about 2-3cm. Dissect 5 tails, take the viscera and mix them, grind them with liquid nitrogen, use the RNAsimple Total RNA Extract...

Embodiment 2

[0084] Embodiment 2 Identify whether golden trout eggs carry IHNV

[0085] 1. Solution preparation

[0086] Custom-synthesized primers with base sequences as shown in SEQ ID NO: 1-6 (Shanghai Sangong), the 5' ends of primers SEQ ID NO: 1-4 are all modified with ferrocene, dissolved in nuclease-free sterilized Deionized water, formulated to contain 1 μM each of primers SEQ ID NO: 1 and SEQ ID NO: 2, 8 μM each of primers SEQ ID NO: 3 and SEQ ID NO: 4, 2 μM each of primers SEQ ID NO: 5 and SEQ ID NO: 6 The primer using solution, wherein primers SEQ ID NO: 3 and SEQ ID NO: 4 are labeled with ferrocene upstream.

[0087] Use nuclease-free sterilized deionized water to prepare a dNTP use solution, which contains 10 mM each of dATP, dCTP, and dGTP, 8 mM of dTTP, and 2 mM of biotin-16-dUTP.

[0088] 2. Total RNA Extraction

[0089] Take about 20 pieces of golden trout eggs to be tested, mix them, grind them with liquid nitrogen, use the RNAsimple Total RNA Extraction Kit (Tiangen D...

Embodiment 3I

[0102] The quantitative detection of embodiment 3IHNV

[0103] 1. Solution preparation

[0104] With embodiment 1.

[0105] 2. IHNV control plasmid extraction

[0106] According to the whole genome sequence (KJ421216.1) of IHNV Chinese strain Ch20101008 in NCBI database, the synthetic matrix (matrix, M) gene and its 10 bp sequences (2245-2852) at both ends of the flanks, a total of 608 bp, were connected to the pGM-T vector, An IHNV control plasmid with a molecular weight of 3639bp was constructed, transformed into DH5αEscherichia coli, and expanded for culture. Use the endotoxin-free plasmid medium extraction kit (Tiangen DP108) to extract the plasmid, dilute it in a 10-fold concentration gradient, and prepare it to contain 1-10 13 A solution of plasmid copies.

[0107] 3. Nucleic Acid Amplification

[0108] The amplification reaction system is as follows:

[0109]

[0110] Each reaction tube was placed in a 60 °C water bath for 30 min.

[0111] 4. Electrode Surface...

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Abstract

The invention discloses a biosensing detection method for identification of trout fries. The biosensing detection method is characterized in that on the basis of conservative analysis results of IHNV (infectious hematopoietic necrosis virus) gene sequence from different plant sources in a public database, six specific primers using IHNV virus matrix protein (M) encoding genes as amplification targets are designed, the constant-temperature reverse transcription amplification is performed on the to-be-measured total RNA template of the fish egg or fry sample, and the amplification time is only 15 to 30min; all or part of primers have the electrochemical activity markers in the amplification reaction, one part of dNTP has an anchor marker, the successfully amplified virus M gene sequence simultaneously has the electrochemical activity marker and the anchor marker, the amplified product can be captured onto the electrode surface by the anchor marker, and the carrying of IHNV in the sample can be accurately judged through detecting the electrochemical signal at the surface of the electrode. The biosensing detection method has the advantages that the operation is simple, the reaction is quick, the requirement on equipment precision is low, and the method is suitable for the site quick detection of production and business workplaces of fry farms, culture farms, and the like.

Description

technical field [0001] The invention belongs to the technical field of aquatic organisms, and relates to a biosensing detection method for identification of salmon trout fry, and relates to a method for identifying whether salmon trout eggs and fry carry infectious hematopoietic necrosis virus (IHNV) detection technology. Background technique [0002] Salmon trout is the general name of salmon family (Salmonidae). As an important cold-water famous aquatic species, it has been artificially cultured in 26 provinces and municipalities in my country. The scale of breeding has been steadily expanding year by year. tons. Infectious hematopoietic necrosis (IHN) is an explosive disease that seriously threatens the development of the salmon and trout farming industry. ) etc., the mortality rate after infection is as high as more than 90%, and there is a lack of effective therapeutic drugs. At present, in the salmon and trout farming industry, the more effective way to prevent and c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/607
Inventor 赵紫霞许建江炎亮张瀚元
Owner CHINESE ACAD OF FISHERY SCI
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