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Loop-mediated isothermal amplification method for detection of dengue virus

A dengue virus and dengue virus technology, applied in the field of warm amplification dengue virus detection method and its kit, can solve the problems of limiting the application of PCR-related technologies, and achieve stable and reliable results, less sample volume, and simple operation

Active Publication Date: 2021-09-21
云南省寄生虫病防治所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR-based methods require thermal cyclers and other equipment, and there are also requirements for the detection site, and quantitative PCR requires special training, which limits the application of PCR-related technologies in rapid detection

Method used

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  • Loop-mediated isothermal amplification method for detection of dengue virus
  • Loop-mediated isothermal amplification method for detection of dengue virus
  • Loop-mediated isothermal amplification method for detection of dengue virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Primer design and screening

[0147] Obtain the whole genome sequence of all dengue viruses from GenBank, perform multiple sequence alignment and sequence analysis by subtype, and find conserved regions for different subtypes. The 10359bp-10578bp segment of dengue virus type 1 is highly conserved and suitable for primer design; the 3343bp-3553bp segment of dengue virus type 2 is highly conserved and suitable for primer design; dengue virus 3 The 3311bp-3509bp segment of type 4 has high conservation and is suitable as a primer design region; the 2316bp-2565bp segment of dengue virus type 4 is highly conserved and is suitable as a primer design region. The above regions were extracted from the comparison results, and primers were designed after re-alignment.

[0148] Screen the designed primers with the established RT-LAMP detection system to obtain primers that meet the requirements, and obtain 6 primers as shown in Table 2, and make these 6 primers into A, B, and C as ...

Embodiment 2

[0157] RNA in vitro transcription

[0158] In the embodiment of in vitro transcription, four subtypes of dengue virus laboratory cultured strains were selected, and specific primers were used to amplify the four subtypes of dengue virus nucleic acid extracts respectively, and the amplified products were amplified with 1% Perform electrophoresis analysis on agarose gel, and after the correct size is determined, the target band is cut and recovered, and the purified DNA product can be used as a template for in vitro transcription.

[0159] Promega's "RiboMAX Large Scale RNA Production System-T7" kit was used for in vitro transcription, and the reaction process used the method provided by the reagent manufacturer. The concentration and purity of RNA products transcribed in vitro were measured using Nanodrop, and the unit was converted into copies / μL (copy / μL). To avoid repeated freezing and thawing of RNA, take an appropriate amount and dilute to 1×10 4 copies / μL were aliquoted...

Embodiment 3

[0161] System establishment and optimization

[0162] With the dengue type 1 virus RNA fragment as a template, the system in Table 1 is used as the basic system, and Tween (0%, 10%, 30%), dNTP (1.0, 1.4, 1.6mM), betaine in the optimized reaction system (0,0.4,0.8M) and the final concentration of magnesium ions (6,8,10mM), four factors set three levels of memory orthogonal experiment, designed 9 condition combinations as shown in Table 4. The real-time fluorescence quantitative detection system uses SYTO9 as the dye, and the channel for collecting fluorescence is set as SYBR Green I.

[0163] The reaction method and steps are as follows:

[0164] 62℃, 120s, 1 cycle,

[0165] 62°C, 60s, 60 cycles, collect fluorescence

[0166] The channel for collecting fluorescence was set to SYBR Green I, using SYTO 9 dye.

[0167] Table 4 Dengue virus system optimization condition combination

[0168]

[0169] The change process of the fluorescence amplification curve during the react...

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Abstract

The invention provides a loop-mediated isothermal amplification (LAMP) primer and a related kit and a method for detecting dengue virus (Denguevirus, DV) using the primer. Using the primers can specifically loop-mediated isothermal amplification of dengue virus nucleic acid. The method of the invention has good specificity, high sensitivity, good repeatability, simple operation, convenience and quickness, can be well applied to identify dengue virus, and is suitable for clinical and laboratory detection.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and nucleic acid detection, and in particular relates to a dengue virus detection method based on a loop-mediated isothermal amplification and a kit thereof. Background technique [0002] Dengue fever is a group of insect-borne infectious diseases caused by dengue virus (Denguevirus, DV), and the main vector is Aedes aegypti. Dengue fever, dengue hemorrhagic fever, and dengue shock syndrome can occur after virus infection. Infections caused by DV are widely prevalent in tropical and subtropical regions of the world, especially in more than 100 countries and regions in Southeast Asia, the Western Pacific, Central and South America, and Africa. The World Health Organization estimates that 2.5 billion people in the world are threatened by dengue fever. Every year, about 5 to 10 million people are infected with dengue fever, 250,000 to 500,000 people suffer from dengue hemorrhagic fever, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119Y02A50/30
Inventor 周红宁周毅姜进勇
Owner 云南省寄生虫病防治所
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