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Lamp primer composition and application thereof for detecting infectious pancreatic necrosis virus

A pancreas necrosis virus and primer combination technology, which is applied in the field of genetic engineering, can solve the problem of no IPNV detection, etc., and achieve the effects of improving epidemic early warning and prevention and control capabilities, high specificity and sensitivity, and simple detection instruments

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

However, this method has not been applied to the detection of IPNV at present.

Method used

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  • Lamp primer composition and application thereof for detecting infectious pancreatic necrosis virus
  • Lamp primer composition and application thereof for detecting infectious pancreatic necrosis virus
  • Lamp primer composition and application thereof for detecting infectious pancreatic necrosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 Design and synthesis of LAMP primer composition of the present invention

[0022] According to the nucleotide sequence (Genbank NO: AF342728.1) encoding fragment A polyprotein in the Genbank IPNV genome, use PrimerExploreV3 and PrimerPremier5.0 software to target 6 regions (F3c, F2c, F1c at the 3' end and B1 at the 5' end) , B2, B3) design LAMP primers, conduct 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 internal primer FIP and reverse internal primer respectively. The sequences of primer BIP, forward outer primer F3, and reverse outer primer B3 are shown in Table 1. Primers were synthesized by Dalian Bao Biological Engineering Co., Ltd.

[0023] Table 1. IPNVRT-LAMP detection primer set

[0024]

Embodiment 2

[0025] The optimization of embodiment 2RT-LAMP reaction condition

[0026] 1. Preparation of Sample RNA Template

[0027] The infectious pancreatic necrosis virus (IPNV) isolate used in the present invention, by literature (Hu Xiaoli, Li Wei, Zhao Huijun, Wu Bin. Isolation and identification of rainbow trout infectious pancreatic necrosis virus, China Animal Quarantine, 2012 , 29(3): 27-30), and was isolated and stored in the Laboratory of Animal Inspection of the Technology Center of Liaoning Entry-Exit Inspection and Quarantine Bureau. 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.

[0028] 2. RT-LAMP reaction system

[0029] The RT-LAMP amplification reaction uses the ribonucleic acid amplification kit (RT-LAMP), Shenzhen Bo Ruixiang Biotechnology Co., Ltd., and the specific operation steps refer to the kit manual.

[0030] RT-LAMP uses a...

Embodiment 3

[0049] Example 3 Different methods determine the RT-LAMP amplification product that utilizes the LAMP primer composition to carry out

[0050] According to the method of step 2 in Example 2, the sample reaction system (abbreviation: IPNV sample) and the negative control reaction system were prepared by adding the RNA template of the infectious pancreatic necrosis virus isolate, and after 60 minutes of constant temperature reaction at 62 ° C, the reaction was stopped. The method for judging the result of the LAMP reaction described in step 3 in Example 2 judges whether the sample contains IPNV.

[0051] 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 IPNV samples and negative control, respectively. image 3 The results showed that the ladder-like specific band appeared in the swimming lane of the IPNV sample, but no ladd...

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Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer composition for detecting an infectious pancreas necrosis virus and an application of the LAMP primer composition. The LAMP primer composition is composed of a forward inner primer (FIP) with a sequence as shown in SEQ ID No.1, a backward inner primer with a sequence as shown in SEQ ID No.2, a forward outer primer F3 with a sequence as shown in SEQ ID No.3 and a backward outer primer B3 with a sequence as shown in SEQ ID No.4. The LAMP primer composition disclosed by the invention is used for rapidly detecting the infectious pancreas necrosis virus in a sample, favorable in specificity and sensitivity for the infectious pancreas necrosis virus, simple in required detecting instruments, capable of rapidly, efficiently and accurately detecting the infectious pancreas necrosis virus and suitable for being popularized to port check and examination organizations and basic laboratories.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a LAMP primer composition for detecting infectious pancreatic necrosis virus and its application. Background technique [0002] Infectious pancreatic necrosis virus (IPNV) is an economically important fish disease pathogen that can cause extremely severe infections in salmonids, resulting in high mortality. At first, it was only popular in some countries in North America and Europe. In recent years, with the increase of aquatic animal import trade, infectious pancreatic necrosis disease has been introduced into my country and became popular in some areas, causing serious economic losses. At present, the detection methods of the pathogen IPNV in my country mainly include virus isolation and identification methods, reverse transcription-polymerase chain reaction methods, enzyme-linked immunosorbent assay methods, etc. Although these detection methods have achieved accurate diagnosis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70C12Q2531/119
Inventor 吴斌肇慧君张雪蔡晓萍
Owner 吴斌