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Kit, primer, and probe for detecting Japanese encephalitis virus via real-time fluorescence isothermal amplification

A Japanese encephalitis virus, detection kit technology, applied in biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc., can solve the problem of time-consuming, low sensitivity, unsuitable for rapid detection and clinical emergency Disease treatment and other issues to achieve the effect of improving detection sensitivity and ensuring specificity

Active Publication Date: 2015-09-09
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus isolation and culture method is cumbersome and time-consuming, and is not suitable for rapid detection and clinical emergency treatment; enzyme-linked immunoassay technology is limited by itself, and its sensitivity is relatively low, which is not conducive to early diagnosis of the disease; RT-PCR, fluorescent RT -PCR and RT-LAMP are also detection methods based on nucleic acid amplification, and their sensitivity is much higher than the previous two methods. Ten heating and cooling cycles take a long time, and the LAMP method has too high requirements on the primer design area, so it is difficult to achieve good application on RNA viruses with large and frequent mutations

Method used

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  • Kit, primer, and probe for detecting Japanese encephalitis virus via real-time fluorescence isothermal amplification
  • Kit, primer, and probe for detecting Japanese encephalitis virus via real-time fluorescence isothermal amplification
  • Kit, primer, and probe for detecting Japanese encephalitis virus via real-time fluorescence isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the preparation of positive control substance

[0036] The virus particles were purified and harvested from the Japanese Japanese encephalitis virus cell culture medium by ultracentrifugation, and the viral RNA was extracted. The NS1 gene amplification primers N-Primer-1 and N-Primer-2 were designed, and the Japanese encephalitis Japanese encephalitis NS1 gene fragment of about 520bp was amplified by RT-PCR technology. The obtained RT-PCR product was inserted into the pGEM-4z cloning plasmid to construct the recombinant plasmid pGEM-4z-NS1, and its sequence was determined. The sequencing results were compared with 10 randomly selected Japanese encephalitis virus NS1 gene sequences of 5 different genotypes in GenBank to ensure that their homology reached more than 95%. After enrichment of positive clones, plasmid DNA was extracted, and after linearization, the inserted NS1 gene was transcribed in vitro using the T7 promoter on the plasmid to obtain single-...

Embodiment 2

[0040] Example 2, the establishment and optimization of Japanese Japanese encephalitis virus real-time fluorescent NASBA detection system

[0041] The reaction conditions were optimized for the important conditional factors affecting the real-time fluorescence NASBA detection system.

[0042] 1. Various factors and optimization methods

[0043] ① AMV buffer volume: Configure 2×NASBA reaction solution according to the reaction system in Table 1, fix other parameters, adjust the volume of AMV buffer to 2.4 μL, 2.8 μL, 3.2 μL, 3.6 μL, 4.0 μL, 4.4 μL, and use Japanese encephalitis virus In vitro transcribed RNA of NS1 gene was used as a template for real-time fluorescent NASBA amplification. After the real-time end, compare the effects of different AMV buffers on the amplification efficiency and fluorescence curve. The results are as follows: figure 1 . The volume of AMV buffer and the concentration of each component are compared in Table 3.

[0044] Table 3 AMV buffer volume ...

Embodiment 3

[0051] Embodiment 3, Japanese encephalitis virus real-time fluorescent NASBA kit components and detection method

[0052] 1. Composition of the kit (stored at -20°C)

[0053] ① 2× real-time NASBA reaction solution: containing Tris-HCl 100mM, KCl 100mM, MgCl 2 20 mM, DTT 20 mM, DMSO 20%, NTP 4 mM, dNTP 1 mM, upstream primer, downstream primer (Primer-1, Primer-2) 0.125 μM each, and molecular beacon probe (Tag1) 0.25 μM.

[0054] ②4×Enzyme Mixture: Contains AMV Reverse Transcriptase 1.6U / μL, T7 RNA Polymerase 8U / μL, Rnase H 0.04U / μL, Ribonuclease Inhibitor 2U / μL, BSA 2.0μg.

[0055] ③ Positive control: it is the RNA fragment of NS1 gene transcribed in vitro by Japanese encephalitis virus.

[0056] ④ Negative control: It is sterile physiological saline, which is extracted in parallel with the specimen at the same time as the nucleic acid extraction and used as a negative control.

[0057] ⑤ DEPC water: Nuclease-free ultrapure water treated with DEPC, used as a blank control. ...

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Abstract

The invention discloses a kit, a primer, and a probe for detecting Japanese encephalitis virus via real-time fluorescence isothermal amplification. The kit comprises the following components: 100mM of Tris-HCl, 100mM of KCl, 20mM of MgCl2, 20mM of DTT, 20% of DMSO, 4mM of NTP, 1mM of dNTP, 0.125MuM of each of Primer-1 and Primer-2, 0.25MuM of a molecular beacon probe, 1.6U / MuL of an AMV reverse transcriptase, 8U / MuL of T7RNA polymerase, 0.04U / MuL of rnase h, 2U / MuL of ribonuclease inhibitor, and 0.1% of BSA. According to the invention, a specific forward primer, reverse primer, and molecular beacon probe are optimizedly selected via design and via combination with clinical sample screening; three different areas of the encephalitis virus are synchronously recognized; amplification specificity is ensured; the encephalitis virus RNA, which is of single copy number, contained in a sample can be detected; and sensitivity of detecting encephalitis is improved.

Description

technical field [0001] The invention belongs to the technical field of virus nucleic acid detection, and relates to a rapid detection technology of Japanese encephalitis virus based on nucleic acid sequence-based amplification (Nucleic acid sequence-based amplification, NASBA) technology. It specifically relates to a Japanese Japanese encephalitis virus real-time fluorescence isothermal amplification rapid detection kit, and a primer pair and a molecular beacon probe specific to the Japanese encephalitis NS1 gene used in the kit. Background technique [0002] Japanese encephalitis virus (JEV) belongs to the Flaviviridae family and is a serious zoonotic pathogen. The virus is a single-stranded positive-strand RNA virus, and its nucleic acid sequence encodes 3 structural proteins and 7 nonstructural proteins (Nonstructural protein). good candidate genes. Japanese encephalitis B is transmitted by mosquitoes and is more common in summer and autumn. It causes irreversible damag...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 周丹娜田永祥刘学郭锐段正赢杨克礼袁芳艳刘威刘泽文孟丽
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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