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Lamp primer composition and application thereof for detecting viral hemorrhagic sepsis virus

A technology of viral hemorrhage and primer composition, applied in the field of genetic engineering, can solve problems such as inability to quickly and accurately detect viruses, high requirements for instruments and equipment, and complicated detection steps, so as to improve epidemic early warning and prevention and control capabilities, and detection time The effect of short, simple detection equipment

Active Publication Date: 2016-07-20
吴斌 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, domestic and foreign detection methods for the pathogen VHSV mainly include virus isolation and culture methods, reverse transcription-polymerase chain reaction methods, indirect fluorescent antibody test methods, enzyme-linked immunosorbent assay, etc. Although these detection methods have reached Accurate diagnosis, but time-consuming, high cost, high equipment requirements, and complicated detection steps cannot meet the needs of rapid and accurate detection of the virus

Method used

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  • Lamp primer composition and application thereof for detecting viral hemorrhagic sepsis virus
  • Lamp primer composition and application thereof for detecting viral hemorrhagic sepsis virus
  • Lamp primer composition and application thereof for detecting viral hemorrhagic sepsis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 Design and synthesis of LAMP primer composition of the present invention

[0023] According to the nucleotide sequence of the N gene in the GenbankVHSV genome (GenbankNo: AB672617.1), use PrimerExploreV3 and PrimerPremier5.0 software for 6 regions (F3c, F2c, F1c at the 3' end and B1, B2, B3 at the 5' end) ) to design LAMP primers, carry out LAMP amplification experiments and detection analysis on different primer sets, and finally screen out a set of LAMP primers with high amplification rate and good specificity, which are forward inner primer FIP, reverse inner primer BIP, forward inner primer The sequences of the outward primer F3 and the reverse outer primer B3 are shown in Table 1. Primers were synthesized by Dalian Bao Biological Engineering Co., Ltd.

[0024] Table 1. VHSVRT-LAMP detection primer set

[0025]

Embodiment 2

[0026] The optimization of embodiment 2RT-LAMP reaction condition

[0027] 1. Preparation of Sample RNA Template

[0028] The viral hemorrhagic septicemia virus isolate (VHSV) used in the present invention is isolated and preserved by the Liaoning Entry-Exit Inspection and Quarantine Bureau Technical Center Animal Inspection Laboratory. Use the TakaraMiniBESTViralRNA / DNAExtractionKitVer.4.0 kit to extract the sample RNA from the virus cell culture, and refer to the kit instructions for the specific operation steps.

[0029] 2. RT-LAMP reaction system

[0030] The RT-LAMP amplification reaction used a ribonucleic acid amplification kit (RT-LAMP), which was purchased from Rongyan Biotechnology (China) Co., Ltd., and the specific operation steps refer to the kit instruction manual.

[0031] RT-LAMP uses a 25 μL reaction system, and the dosage of the components added is as follows:

[0032]

[0033]

[0034] When preparing the reaction system, replace the RNA template wit...

Embodiment 3

[0050] Example 3 Different methods determine the RT-LAMP amplification product that utilizes the LAMP primer composition to carry out

[0051] According to the method of step 2 in Example 2, prepare the sample reaction system (abbreviation: VHSV sample) and the negative control reaction system by adding the RNA template of the viral hemorrhagic sepsis virus isolate, and stop the reaction after reacting at a constant temperature of 62 ° C for 60 min. The RT-LAMP reaction result judging method described in step 3 in Example 2 judges whether VHSV is contained in the sample.

[0052] Method 1: Take 8 μL of the amplified product from the completed reaction tube for gel electrophoresis. The electrophoresis results are as follows: image 3 A, where lane M is DL2000Marker, lanes 1 and 2 are VHSV samples and negative control, respectively. image 3 The results showed that the ladder-like specific bands appeared in the swimming lane of the VHSV sample, but no ladder-like specific bands...

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Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) primer composition used for detecting viral haemorrhagic septicaemia viruses and an application of the primer composition. The LAMP primer composition consists of a forward inner primer FIP with a sequence as show in SEQ ID No.1, a reverse inner primer BIP with the sequence as shown in SEQ ID No.2, a forward outer primer F3 with the sequence as shown in SEQ ID No.3, and a reverse outer primer B3 with the sequence as shown in SEQ ID No.4. The LAMP primer composition disclosed by the invention is used for quickly detecting viral haemorrhagic septicaemia viruses in a sample, and has good specificity and sensitivity for the viral haemorrhagic septicaemia viruses; a needed detecting apparatus can quickly, efficiently and accurately detect the viral haemorrhagic septicaemia viruses, and therefore, the LAMP primer composition is suitable for being popularized at a port inspection and quarantine institution and base-level laboratory.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a LAMP primer composition for detecting viral hemorrhagic sepsis virus and its application. Background technique [0002] Viral hemorrhagic sepsis (Viralhaemorrhagic septicaemia, VHS) is a severe infectious disease caused by rhabdoviruses in more than ten kinds of fish such as salmon, trout, pike and turbot, which can lead to high mortality. The pathogen is Viral hemorrhagic septicaemia virus (VHSV). Viral hemorrhagic septicaemia virus (VHSV) belongs to Rhabdoviridae extragranular Rhabdovirus genus, is a serious infectious disease that can infect salmon and trout, with a fatality rate of 90% to 100%. The disease was first discovered in continental Europe and North America, and spread to Asia in recent years, and is prevalent in some parts of my country. The disease is a disease that must be declared by the World Organization for Animal Health (OIE), and it is listed as a disease...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 吴斌肇慧君胡强王刚
Owner 吴斌
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