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RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses

A technology of PCR-HRM and dengue virus, which is applied in the field of virus genotyping, can solve the problems of time-consuming, high cost and limited application

Active Publication Date: 2016-07-20
GUANGDONG LAB ANIMALS MONITORING INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the following ten years, there have been many reports on the application of PCR technology to detect dengue virus in Chinese and foreign literature, from multi-tube two-step method to single-tube one-step method, from four pairs of type-specific primers respectively to single-tube multiplex. reaction, but there are still problems such as the need for multiple pairs of primers and the easy contamination of the electrophoresis detection process, resulting in false positives.
Although fluorescent PCR is not easy to contaminate, it requires multiple pairs of primer probes, which is expensive and cannot be widely used
[0006] None of the above methods can overcome the shortcomings of complex operation, time-consuming, high cost, and poor reliability, and their application in clinical practice is limited.

Method used

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  • RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses
  • RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses
  • RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Rapidly distinguish base sites and gene fragments of different serotypes of dengue viruses

[0099] (1) After a lot of experimental research, the present invention found that the following base sites can be used to quickly distinguish different serotypes of dengue viruses: base 10648 G in the genome of the DV1 standard strain with Genbank number EU848545.1, base 10671 Base T, 10672nd base A, 10675th base G;

[0100] The 10638th base A, the 10661st base C, the 10662nd base T, and the 10665th base G in the genome of the DV2 standard strain with Genbank number AF038403.1;

[0101] The 10610th base G, the 10633rd base C, the 10634th base T, and the 10637th base G in the genome of the DV3 standard strain with Genbank number M93130.1;

[0102] The 10570th base A, the 10593rd base T, the 10594th base G, and the 10597th base A in the genome of the DV4 standard strain with Genbank number AY947539.1.

[0103] (2) After further experimental research, the present inven...

Embodiment 2

[0116] Embodiment 2 distinguishes RT-PCR-HRM or PCR-HRM primers of four kinds of serotype dengue viruses rapidly

[0117] The present invention finds RT-PCR-HRM or PCR-HRM primers that can be used to quickly distinguish different serotypes of dengue viruses after experimental screening of a large number of primers designed, and the target fragment amplified by the primers contains the following bases Base point:

[0118] In the DV1 standard strain genome numbered EU848545.1 in Genbank, the 10648th base G, the 10671st base T, the 10672nd base A, and the 10675th base G;

[0119] The 10638th base A, the 10661st base C, the 10662nd base T, and the 10665th base G in the genome of the DV2 standard strain with Genbank number AF038403.1;

[0120] The 10610th base G, the 10633rd base C, the 10634th base T, and the 10637th base G in the genome of the DV3 standard strain with Genbank number M93130.1;

[0121] The 10570th base A, the 10593rd base T, the 10594th base G, and the 10597th b...

Embodiment 3

[0126] Embodiment 3: A kind of method of RT-PCR-HRM that distinguishes four kinds of serotype dengue virus rapidly

[0127] 1) Extraction of RNA from DV:

[0128] The supernatant of dengue virus cell culture fluid isolated clinically was provided by Guangzhou Eighth People's Hospital. Using Tiangen virus DNA / RNA extraction kit (OSR-M202), extract the dengue virus RNA in the supernatant of dengue virus cell culture fluid of four serotypes isolated clinically on the TGuideM16 automatic nucleic acid extractor, as Template for subsequent one-step RT-PCR.

[0129] 2) One-step RT-PCR:

[0130] Use TakaraOneStepPrimeScript? RT-PCRKit (PerfectRealTime) kit, which contains TaKaRaExTaqHS, PrimeScriptRTenzymeMixII, 2XOneStepRT-PCRBufferIII, etc. 20μl system: RNA template is added according to the actual concentration within 5μl, usually 2μl. Add 0.4 μl of TaKaRaExTaqHS, add 0.4 μl of upstream and downstream primers, add 0.4 μl of PrimeScriptRTenzymeMixII, add 10 μl of 2XOneStepRT-PCR...

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Abstract

The invention discloses an RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses. According to the genomic sequence alignment result of the four kinds of serotype dengue viruses, three primers are designed, RNA is adopted as a template, an LC green dye is added for carrying out a one-step RT-PCR, or a built plasmid containing a dengue virus 280bp basic group is adopted as a template and the LC green dye is added for carrying out PCR amplification, the amplification product is subjected to HRM analysis, and the four kinds of serotype dengue viruses can be obviously distinguished through an HRM standard curve and a peak type figure. The method is easy to operate, the reagent is low in cost, high in detecting sensitivity and reliable in parting result, and the method is an innovative method for detecting and parting the four kinds of serotype dengue viruses.

Description

technical field [0001] The invention relates to a virus genotyping method, in particular to a RT-PCR-HRM or PCR-HRM primer and method for rapidly distinguishing four serotype dengue viruses. Background technique [0002] Dengue virus (denguevirus, DV) is an enveloped single-stranded positive-sense RNA virus, which belongs to the family Flaviviridae and is a mosquito-borne virus. Its main vectors are Aedes aegypti and Aedes albopictus. Aedes mosquito. It is the main pathogen that causes human dengue fever (denguefever, DF), dengue hemorrhagic fever (denguehemorrhagicfever, DHF) and dengue shock syndrome (dengueshock syndrome, DSS). In 1779, in Jakarta, Indonesia, Bylon first described this disease called joint fever, which was named dengue fever by the Royal College of Physicians in London in 1869. Dengue fever and dengue hemorrhagic fever are widely prevalent in more than 60 countries and regions in tropical and subtropical regions of the world, especially in Southeast Asi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701C12Q2600/156C12Q2527/107C12Q2521/107Y02A50/30
Inventor 颜丙峰郭鹏举黄韧张复春赵令斋
Owner GUANGDONG LAB ANIMALS MONITORING INST
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