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Set of reagents for detection of infectious pancreatic necrosis virus

A pancreas necrosis virus, infectivity technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. time, high sensitivity

Active Publication Date: 2019-01-18
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above methods all need to be completed in the laboratory with professional equipment, and the test conditions are relatively high.

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  • Set of reagents for detection of infectious pancreatic necrosis virus
  • Set of reagents for detection of infectious pancreatic necrosis virus
  • Set of reagents for detection of infectious pancreatic necrosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the preparation of the complete set of reagents that detect infectious pancreatic necrosis virus

[0073] This embodiment provides a set of reagents for the detection of infectious pancreatic necrosis virus by using the loop-mediated constant temperature amplification method. -LooF and B2-looB are composed of six single-stranded DNAs; B2-F3 is the single-stranded DNA shown in SEQ ID No.1 in the sequence listing; B2-B3 is the single-stranded DNA shown in SEQ ID No.2 in the sequence listing ; B2-FIP is the single-stranded DNA shown in SEQ ID No.3 in the sequence listing; B2-BIP is the single-stranded DNA shown in SEQ ID No.4 in the sequence listing; B2-LooF is SEQ ID No.5 in the sequence listing The single-stranded DNA shown; B2-looB is the single-stranded DNA shown in SEQ ID No.6 in the sequence listing.

[0074] In this set of reagents, each single-stranded DNA is packaged independently, and the molar ratio of B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-...

Embodiment 2

[0075] Example 2, Establishment of a method for detecting infectious pancreatic necrosis virus

[0076] The total RNA of IPNV ChRtm213 strain and Cannabis embryonic cells (CHSE-214) were extracted respectively, and the method for detecting infectious pancreatic necrosis virus by using the set of reagents in Example 1 was optimized.

[0077] 1. Mg in the reaction system 2+ content optimization

[0078] Prepare a 25 μL RT-LAMP reaction system. The substances and contents in the reaction system are as follows: the concentrations of B2-F3 and B2-B3 are both 0.4 μM, the concentrations of B2-FIP and B2-BIP are both 1.6 μM, and the concentrations of B2-LooF and The concentration of B2-looB is 0.8μM, the concentration of dNTPs is 1.0mM (dNTPs contain four kinds of dNTPs, and the concentration of each dNTP in the reaction system is 1.0mM), 2.5μL 10×Isothermal Amplification Buffer [contains a final concentration of 2mM MgSO 4 ] (NEB company, product number B0537S), the concentration ...

Embodiment 3

[0091] Embodiment 3, the specificity of the complete set of reagents that detect infectious pancreatic necrosis virus

[0092] The samples to be tested are: IPNV ChRtm213 strain, infectious hematopoietic necrosis virus (Infectioushematopoietic necrosis virus, IHNV) Sn1203 and viral hemorrhagic septicemia virus (Viralhemorrhagic septicemia virus, VHSV) RNA, wherein, IHNV Sn1203 (Xu L M, Zhao J Z, LiuM, et al.High throughput screening of recombinant antibodies against hematopoietic necrosis virus from a combinatorial antibody library[J].Aquaculture,2016,460:32-36.); VHSV is a product of China Center for Type Culture Collection, strain No. VR-1387, from China Type Culture Collection Center, recorded in (Liu Miao, Xu Liming, Lu Tongyan, et al. Establishment and application of RT-LAMP detection method for fish infectious hematopoietic organ necrosis virus[J].China Aquatic Science, 2014, 21(5): 1065-1071.) in the article.

[0093] Each viral RNA was detected according to the optima...

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Abstract

The present invention discloses a set of reagents for detection of infectious pancreatic necrosis virus. The kit for detecting infectious pancreatic necrosis virus disclosed in the invention consistsof six single-stranded DNAs named B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-looB; B2-F3, B2-B3, B2-FIP, B2-BIP, B2-LooF and B2-looB are respectively SEQ ID No. in the sequence listing. Single-stranded DNA as shown in 1-6. The experiment proves that the kit of the present invention has good specificity for detecting infectious pancreatic necrosis virus, and does not cross-react with infectious hematopoietic organ necrosis virus (IHNV) and viral hemorrhagic septicavirus (VHSV); sensitivity is high, sensitivity is 0. .0008fg / 25[mu]L reaction system; detection is simple and time-saving.

Description

technical field [0001] The invention relates to a set of reagents for detecting infectious pancreatic necrosis virus in the field of biotechnology. Background technique [0002] Infectious pancreas necrosis virus (IPNV) is the pathogen that causes infectious pancreas necrosis (IPN) in salmon and trout. The genus (Aquabirnavirus), for susceptible salmon and trout, the fatality rate can be as high as 90%, and the surviving fish are potential sources of infection, carrying the virus for life. The disease has a wide range of susceptibility, high fatality rate, and strong infectivity , causing serious damage to the global fish farming industry. Since entering China in the 1980s, the virus has spread to most rainbow trout farms in my country, causing serious economic losses. [0003] The detection methods for IPNV mainly include enzyme-linked immunosorbent assay (ELISA), RT-PCR, fluorescent quantitative PCR and probe fluorescent quantitative RT-PCR. The above-mentioned methods ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2537/1376C12Q2521/107
Inventor 徐黎明杨瑶赵景壮卢彤岩任广明
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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