Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses

A primer set and multiplex technology, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of EHV false positives, etc., and achieve the effect of strong specificity and easy operation.

Active Publication Date: 2017-09-22
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

However, the existing PCR detection is based on the design of primers based on the DNA polymerase gene sequence of EHV. Because the DNA pol

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  • Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses
  • Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses
  • Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses

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Embodiment Construction

[0034] In order to describe the technical content, structural features, achieved goals and effects of the present invention in detail, the following will be described in detail in conjunction with the embodiments and accompanying drawings.

[0035] (1) Design and optimization of primers

[0036] Eight strains of EHV were isolated and identified, the open reading frame sequences of 6 genes including EHV ORF8 and ORF95 were cloned, their homology was analyzed, 6 pairs of primers were designed in the conserved regions, and the sizes of the amplified products were all set at about 250bp. Using the EHV DNA isolated and cultured from the cells as a template, PCR amplification is carried out by a conventional PCR method, and the amplified product is analyzed by gel electrophoresis. The primer set 1 with high amplification efficiency was screened out.

[0037] Cell separation and identification of 3 JEECV-positive disease samples, the open reading frame sequence of JEECV LTLG were clo...

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Abstract

The invention provides a multiplex PCR primer set, a kit and a method for simultaneous detection of EHV (Eel herpes virus), JEECV (Japanese eel endothelial cells-infecting virus) and AmPV (Anguilla marmorata polyoma-like virus). According to obtained nucleic acid sequences, specific primers for amplification of EHV, JEECV and AmPV are designed to avoid formation of stable primer dimmers between primers, specificity of primer amplified sequences is analyzed to obtain the primer set that is composed of 6 nucleic acid sequences including SEQ ID NO:1-6 and has high amplification sensitivity and specificity on EHV, JEECV and AmPV simultaneously. The multiplex PCR primer set, kit and method provided by the invention can simultaneously detect and identify EHV, JEECV and AmPV in one PCR reaction system, have the advantages of simple operation and strong specificity, etc., and can directly determine the result according to different amplification lengths, thus being more efficient and practical.

Description

technical field [0001] The invention belongs to the technical field of detection of aquatic disease microorganisms, in particular to a method capable of simultaneously detecting eel herpesvirus (Eel herpesvirus, EHV), Japanese eel epidermal cell necrosis virus (Japanese eel endothelial cells-infecting virus, JEECV) and flower eel polyomavirus ( Anguilla marmorata polyoma-likevirus (AmPV) multiplex PCR primer set, kit and detection method for three DNA viruses. Background technique [0002] my country is one of the main eel breeding countries in the world, and its output value occupies an important position in my country's aquatic product export trade. The species cultivated are mainly Japanese eel (A.japonica), European eel (A. anguilla), American eel (A.rostrata), and flower eel (A.Marmorata). Due to the large-scale intensive breeding, the outbreak of major viral diseases has become increasingly frequent, causing huge economic losses. Identifying the causative agent of a ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/705C12Q1/708C12Q2600/16C12Q2537/143
Inventor 葛均青杨金先柯翎
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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