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

CN104152580AInactive Publication Date: 2014-11-19中国检验认证集团吉林有限公司 +1
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2014-11-19
Estimated Expiration
Not applicable · inactive patent

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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.
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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...

Examples

Embodiment 1

[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), use the PrimerExplorer V4 online software http: / / primerexplorer.jp / elamp4.0.0 / index.html to design and synthesize LAMP primers for the detection of Koi herpes virus: By comprehensively considering factors such as base composition, GC content, secondary structure formation, Tm value, distance between primers, and stability of primer ends, the parameters of the primer design software were set and adjusted, and a total of 10 sets of primers were designed and synthesized. 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, which directly determine the sensitivity and specificity of amplification. The 10 sets of primers synthesized in this example and the nucle...

Embodiment 2

[0091] Example 2 Primer set detects SPH gene

[0092] (1) template:

[0093] Synthesis has the DNA molecule of nucleotide sequence shown in SEQ ID NO:41, and this DNA molecule is the gene sequence corresponding to koi herpes virus DNA polymerase gene (Sph gene) through individual nucleotide replacement, this DNA molecule Labeled as SPH, dilute the concentration of the resulting SPH DNA molecule to 85 ng / μL. Here, the reason for carrying out individual nucleotide substitutions in the Koi herpes virus Sph gene is to make it unable to be translated into an active functional protein to prevent environmental pollution.

[0094] (2) Amplification:

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

Embodiment 3

[0103] Example 3 Primer set detects SPH gene

[0104] (1) template:

[0105] Same as the template in Example 2, that is, a DNA molecule having the nucleotide sequence shown in SEQ ID NO:46.

[0106] (2) Amplification:

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

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

[0109] components

The final concentration in the reaction system

FIP

2.0μmol / L

BIP

2.0μmol / L

...