Detection kit and detection method for Cyprinid herpesvirus III

The technology of koi herpes virus and detection kit is applied in the detection field of fish pathogenic virus to achieve the effects of ensuring fish health, simple and easy operation procedures, and improved detection efficiency

Inactive Publication Date: 2017-01-25
INST OF AQUATIC LIFE ACAD SINICA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no research report on quantitative PCR detecti

Method used

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  • Detection kit and detection method for Cyprinid herpesvirus III
  • Detection kit and detection method for Cyprinid herpesvirus III
  • Detection kit and detection method for Cyprinid herpesvirus III

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Primer Design and Screening for Fluorescent Quantitative PCR Detection of Koi Herpesvirus Type III TK Gene

[0046] 1. Bioinformatics method to design primers and conduct primer screening

[0047] Download the full sequence of Koi Herpesvirus Type Ⅲ collected by GenBank (GeneBank: NC_009127.1), and also download the negative control virus genome, Koi Herpesvirus Type Ⅰ, Koi Herpesvirus Type Ⅱ, and Carp Spring Viremia Virus, please See Table 1. After the comparison by Clustal X, select the appropriate region to design primers. This region is specifically expressed in Koi herpesvirus type III, but there is at least 10%-30% difference compared to the negative control. After selecting the region, use ABI PrimerExpress 3.0 real-time fluorescence quantitative PCR primer design software, design synthetic primers. The extracted alternative primers were screened according to the following requirements:

[0048] 1) The primers should be designed in the conserved region...

Embodiment 2

[0114] The optimization of embodiment 2 fluorescence quantitative PCR primer consumption

[0115] 1. The first optimization of the amount of fluorescent quantitative PCR primers

[0116] With diluted L1 (10 -4 ) and L2 (10 -5 ) positive control plasmid was used as a template for optimizing the amount of primers, and gradient optimization was performed on the upstream and downstream amounts of primers respectively. The concentration of the primer working solution was 10 pmol / μL. Polymerase (5U / μL) 0.6 μL, real-time fluorescent PCR buffer (2×) 12.5 μL (2.5 μL of ThermoFisher’s 10× buffer 2.5 μL, 2 μL of 10 mM dNTP and 10000×SYBRgreen I 0.0025 μL and 8 μL of DEPC water mixture), supplemented with sterile water to 25 μL. The PCR reaction conditions are: 95°C for 3min; 95°C for 10sec, 55°C for 20sec, 72°C for 20sec, 75°C for 5sec, and read plate for a total of 45 cycles. The results are shown in Table 3. It can be analyzed from the results that the PCR primers can work normally...

Embodiment 3

[0127] Example 3 Koi Herpesvirus Type III TK Gene Fluorescent Quantitative PCR Detection

[0128] 1. Establishment of standard curve

[0129] The positive control plasmid DNA was used as a template for fluorescent quantitative PCR detection to establish a standard curve. The specific operation is as follows: the plasmid DNA was serially diluted 10 times to I: 1.0×10 6 copies / μL; II: 1.0×10 5 copies / μL; III: 1.0×10 4 Copy / μL; Ⅳ: 1.0×10 3 Copy / μL; V: 1.0×10 2 Copy / μL; VI: 1.0x10 1 Copy / μL; VII: 1.0×10 0 copies / μL. Each dilution was repeated 3 times in parallel. Standard product detection PCR reaction system is: upstream primer 2.0 μL (10 pmol / μL), downstream primer 2.0 μL (10 pmol / μL), plasmid DNA 5 μL, Taq DNA polymerase (5U / μL) 0.6 μL, real-time fluorescent PCR buffer ( 2×) 12.5 μL (prepared with 2.5 μL of 10× buffer purchased from ThermoFisher, 2 μL of 10 mM dNTP, and a mixture of 0.0025 μL of 10000×SYBR green I and 8 μL of DEPC water), supplemented with sterile wate...

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Abstract

The invention discloses a primer pair which is used for amplification of a TK gene sequence of Cyprinid herpesvirus III. The invention also discloses an amplification method for the TK gene sequence of the Cyprinid herpesvirus III. The invention discloses a detection kit for the Cyprinid herpesvirus III. The invention discloses a method for detecting the Cyprinid herpesvirus III by using a fluorescent quantitative detection kit. The detection kit and the method can provide assurance for rapid and accurate detection of the Cyprinid herpesvirus III, and provide guarantee for disease prevention, scientific medication and fish health. The detection method and the kit on the basis of a special primer provided by the invention can be used for a nucleic acid test of a TK gene of main diseased tissues (the liver, the brain and the kidney) of fish.

Description

technical field [0001] The invention relates to the field of detection of fish pathogenic viruses, in particular to a detection kit and detection method of fish pathogenic viruses, in particular to a detection kit and detection method of koi herpes virus type III. Background technique [0002] Koi herpesvirus (Koi herpesvirus), referred to as KHV. It is mainly caused by DNA herpes virus infection, also known as carp nephritis and gill gangrene virus. The virus has an envelope, the nucleocapsid is an icosahedron with a diameter of 170-230nm, and the viral nucleic acid is double-stranded DNA. [0003] Koi Herpes Virus (KHV) was successfully isolated in May 1998 in the Magan Michael area of ​​Israel. In the same year, the pathogenic Koi Herpes Virus (KHV) was successfully isolated. (Cyprinid Herpesvirus) member, also known as Cyprinid Herpes virus type III (CyHV-3). Koi herpes virus disease spread rapidly to all parts of the world. In 2002, it was confirmed that the disease...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/705C12Q2531/113C12Q2563/107
Inventor 邹红熊凡张金李文祥吴山功王桂堂
Owner INST OF AQUATIC LIFE ACAD SINICA
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