Primer set for detection of koi herpesvirus Sph gene and application of primer set

A koi herpes virus and primer set technology, applied in the biological field, can solve the problems of low specificity and sensitivity, complicated operation, etc., and achieve the effects of improving specificity and sensitivity, simple operation and convenient identification

Inactive Publication Date: 2014-11-19
中国检验认证集团吉林有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For koi herpes virus, the PCR method provided by the OIE manual involves two genes, the DNA polymerase gene (Sp

Method used

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  • Primer set for detection of koi herpesvirus Sph gene and application of primer set
  • Primer set for detection of koi herpesvirus Sph gene and application of primer set
  • Primer set for detection of koi herpesvirus Sph gene and application of primer set

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0077] Example 1 Screening of specific primer sets

[0078] Primer design:

[0079] According to the Koi herpes virus Sph gene (GenBank accession number AB375381.1), the PrimerExplorer V4 online software http: / / primerexplorer.jp / elamp4.0.0 / index.html was used to design and synthesize the LAMP primers for detecting Koi herpes virus: Through comprehensive consideration of base composition, GC content, formation of secondary structure, Tm value, distance between primers, stability of primer ends and other factors, set and adjust primer design software parameters, design and synthesize 10 sets of primers. Used for the screening of LAMP primers. Among the primers required by the LAMP method, the upstream external primer, downstream external primer, upstream internal primer and downstream internal primer are necessary, and directly determine the sensitivity and specificity of amplification. The 10 sets of primers synthesized in this example and the nucleotide sequence corresponding to ...

Example Embodiment

[0091] Example 2 Detection of SPH gene by primer set

[0092] (1) Template:

[0093] Synthesize a DNA molecule with the nucleotide sequence shown in SEQ ID NO: 41. The DNA molecule is the gene sequence corresponding to the Koi herpes virus DNA polymerase gene (Sph gene) replaced by individual nucleotides, and the DNA molecule Label it as SPH, and dilute the concentration of the resulting SPH DNA molecule to 85ng / μL. The reason why the Koi herpes virus Sph gene is replaced with individual nucleotides is that it cannot be translated into an active functional protein to prevent environmental pollution.

[0094] (2) Amplification:

[0095] The DNA molecule obtained in step (1) is used as a template, and the DNA molecule having the nucleotide sequence shown in SEQ ID NO: 1 is used as the upstream external primer (ie FOP), with the nucleotide sequence shown in SEQ ID NO: 2 The DNA molecule is the downstream external primer (i.e. BOP), and the DNA molecule with the nucleotide sequence show...

Example Embodiment

[0103] Example 3 Detection of SPH gene by primer set

[0104] (1) Template:

[0105] It is the same as the template in Example 2, which is a DNA molecule with the nucleotide sequence shown in SEQ ID NO:46.

[0106] (2) Amplification:

[0107] The DNA molecule obtained in step (1) is used as a template, and the DNA molecule having the nucleotide sequence shown in SEQ ID NO: 1 is used as the upstream external primer (ie FOP), which has the nucleotide sequence shown in SEQ ID NO: 2 The DNA molecule is the downstream external primer (i.e. BOP), and the DNA molecule with the nucleotide sequence shown in SEQ ID NO: 3 is the upstream internal primer (i.e. FIP), and has the nucleotide sequence shown in SEQ ID NO: 4 The DNA molecule of is the downstream internal primer (ie BIP), set up the amplification system, see Table 4.

[0108] Table 4 LAMP reaction system (25μL)

[0109] Component

[0110] KCl

[0111] The experiment is divided into three groups, experimental group 1, experimental g...

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Abstract

The invention belongs to the technical field of biology and discloses a primer set for the detection of a koi herpesvirus Sph gene and the application of the primer set. The premier set for the detection of the koi herpesvirus Sph gene comprises a forward outer primer having the nucleotide sequence shown in SEQ ID No.1, a backward outer primer having the nucleotide sequence shown in SEQ ID No.2, a forward inner primer having the nucleotide sequence shown in SEQ ID No.3 and a backward inner primer having the nucleotide sequence shown in SEQ ID No.4. The primer set provided by the invention is high in specificity and sensitivity, and suitable for amplification of koi herpesvirus Sph gene through an LAMP (Loop-Mediated Isothermal Amplification) method, and can be used for preparation of a reagent for detection of the koi herpesvirus Sph gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a primer set for detecting the Sph gene of koi herpes virus and its application. Background technique [0002] Koi herpesvirus disease (KHVD) is a highly pathogenic disease of koi and carp. It is the main aquatic infectious disease that endangers freshwater fish farming, especially carp farming in recent years. The harm caused by the newly discovered waterborne viral infectious disease has been paid attention to by the whole world. Koi herpes virus disease can lead to mass mortality of carp and koi, causing huge economic losses and severely affecting freshwater fish farming. At the same time, international trade has promoted the spread and spread of the disease, making the disease prevalent all over the world. Because there is no suitable prevention and control method at present, it is difficult to control once it breaks out. In recent years, the International Organization for Anim...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 王伟利孟庆峰魏春艳刘韬姚贵哲
Owner 中国检验认证集团吉林有限公司
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