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LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof

A technology for hematopoietic organ necrosis and infectivity, applied in recombinant DNA technology, microbiological measurement/inspection, biochemical equipment and methods, etc., can solve problems such as economic losses in salmon and trout farming, and achieve low cost and good specificity , high sensitivity effect

Inactive Publication Date: 2014-01-22
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

IHNV can cause nearly 100% mortality in salmon and trout fry, causing huge economic losses to the world's salmon and trout farming industry

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof
  • LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof
  • LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Preparation and use of the loop-mediated isothermal amplification kit for detecting infectious hematopoietic necrosis virus

[0040] 1. Preparation of loop-mediated isothermal amplification primer set for detection of infectious hematopoietic necrosis virus

[0041] The loop-mediated isothermal amplification primer set used in this embodiment for detecting infectious hematopoietic necrosis virus consists of primer 1, primer 2, primer 3, primer 4, primer 5 and primer 6. These six primers were prepared according to the following method: according to the multiple gene sequences of the IHNV polymerase protein included in GenBank, compare them, select the specific conserved segment of the polymerase gene, and use the online software PrimerExplorer V4 (https: / / primerexplorer.jp / elamp4.0.0 / ) Designed 3 pairs of RT-LAMP primers for the conserved region of the L gene. The sequences are shown in Table 1. The primers were synthesized by Harbin Boshi Biological Company....

Embodiment 2

[0065] Example 2. Specificity Analysis of the Loop-Mediated Isothermal Amplification Kit for Detection of Infectious Hematopoietic Necrosis Virus

[0066] respectively containing 10 5 Genomic RNA of pfu's IHNV-WRAC (ATCC: VR-1392), IPNV (ATCC: VR-890) and VHSV (ATCC: VR-1387) was used as a template, and was detected using the kit in Example 1 and the established RT-LAMP IHNV method, specificity comparison experiment. The specific operation was carried out according to the method of using the kit described in Step 3 of Example 1.

[0067] After the reaction, take 5 μl from the amplification product, add 2 μl of 100-fold diluted SYBR Green I to the remaining amplification product, shake gently, and directly observe the color change of the reaction solution to judge the result. At the same time, 5 μl of the product was taken out for 2% agarose gel electrophoresis, and the results were observed. Result judgment standard refers to step 3 of embodiment 1.

[0068] The results sh...

Embodiment 3

[0069] Example 3. Sensitivity Analysis of the Loop-Mediated Isothermal Amplification Kit for Detection of Infectious Hematopoietic Necrosis Virus

[0070] to contain 10 5 Genomic RNA of pfu IHNV-WRAC (ATCC: VR-1392) was used as a standard template, and it was serially diluted according to a certain ratio, and diluted to contain 10 4 、10 3 、10 2 , 50, 25, 10, and 5 pfu of IHNV RNA, using the IHNV RNA of each dilution as a template, the sensitivity analysis was performed on the kit of Example 1 and the established RT-LAMP method for detecting IHNV. The specific operation was carried out according to the method of using the kit described in Step 3 of Example 1.

[0071] After the reaction, take 5 μl from the amplification product, add 2 μl of 100-fold diluted SYBR Green I to the remaining amplification product, shake gently, and directly observe the color change of the reaction solution to judge the result. At the same time, 5 μl of the product was taken out for 2% agarose ge...

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Abstract

The invention discloses an LAMP (Loop-Mediated Isothermal Amplification) primer used for detecting infectious haematopoietic necrosis virus and application thereof. The primer disclosed by the invention is a primer group, wherein the primer group consists of a primer 1, a primer 2, a primer 3, a primer 4, a primer 5 and a primer 6; or the primer group consists of the primer 1, the primer 2, the primer 3 and the primer 4; nucleotide sequences of the primer 1, the primer 2, the primer 3, the primer 4, the primer 5 and the primer 6 are respectively expressed as a sequence 1, a sequence 2, a sequence 3, a sequence 4, a sequence 5 and a sequence 6 in a sequence table. Due to the adoption of the primers, an IHNV RT-LAMP (Infectious Haematopoietic Necrosis Virus Reverse Transcription Loop-Mediated Isothermal Amplification) visual detection method is established by adding SYBR Green I fluorescent dye into a reaction solution; meanwhile, according to the method, the sensitivity is high, the specificity is good, the cost is low and field high-flux fast detection can be realized without depending on any special instrument equipment, so that an important significance isi provided for introduction of trout fries, breeding of fishes without special pathogen type and pollution-free cultivation of trout.

Description

technical field [0001] The invention relates to a LAMP primer for detecting infectious hematopoietic organ necrosis virus and its application. Background technique [0002] Infectious haematopoietic necrosis (IHN) is caused by infectious haematopoietic necrosis virus (IHNV), a common acute viral infectious disease that harms salmon and trout. Since the first outbreak in the United States in the 1950s, IHNV has spread to more than ten countries including Europe, Australia, and Asia. IHNV can cause nearly 100% mortality of salmon and trout fry, and has caused huge economic losses to the salmon and trout farming industry in the world. Therefore, the establishment of a practical, rapid, sensitive, and accurate IHNV detection technology is of great significance in the early diagnosis of IHN, the supervision of aquatic product hygiene quality, and epidemic monitoring. Currently, several diagnostic methods for IHN recommended by the World Organization for Animal Health include ce...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/70C12Q1/6844C12Q2531/119
Inventor 徐黎明卢彤岩刘淼
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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