RT-LAMP method for identifying Japanese encephalitis virus gene types I and III

A RT-LAMP, Japanese encephalitis virus technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of long time, complicated operation, high cost, and achieve easy identification, visualization of results, and timeliness. The effect of sexual dominance

Inactive Publication Date: 2015-03-04
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the genotyping of JEV is mainly through the analysis of the sequence after cloning and sequencing, which requires a long time, high cost and complicated operation. Therefore, a method for quickly identifying JEV genotypes Ⅰ and Ⅲ is needed

Method used

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  • RT-LAMP method for identifying Japanese encephalitis virus gene types I and III
  • RT-LAMP method for identifying Japanese encephalitis virus gene types I and III
  • RT-LAMP method for identifying Japanese encephalitis virus gene types I and III

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] 1. Primer Design

[0076] Sequence comparison: The Japanese Japanese encephalitis virus genome sequence was retrieved from GenBank, and according to the method established by Chen et al. (1990), the sequence was analyzed for phylogenetic evolution using Mega 5.0 to determine the virus genotype, and the genotype I was selected according to the analysis results Genome sequences of type III strains were compared and analyzed using the software DNAman 6.0 and DNAstar 7.1, and the SNPs loci were determined and analyzed.

[0077] Primer design: According to the results of sequence comparison analysis, LAMP primers were designed through the online software http: / / primerexplorer.jp / e / (Primer Explorer V4), and Primer premier 5.0 was used to edit and modify the primers, and two sets of specificity were designed respectively The primers were used to specifically amplify gene type Ⅰ (GI) and type Ⅲ (GⅢ) JEV, and each set of primers included 1 outer primer F3 / B3 and 1 inner primer ...

Embodiment 2

[0120] 1. Effect of reaction temperature on RT-LAMP reaction

[0121] In the present invention, different temperatures are set for the two sets of primers. Under the condition that other conditions are the same, the two sets of primers are respectively subjected to RT- For LAMP reaction, observe the results of each reaction and determine the optimal temperature of the reaction through 1.5-2% agarose gel electrophoresis. The results showed that the reaction could occur between 59-65°C, and finally determined that the optimal temperature for JEV G Ⅰ and GⅢ specific RT-LAMP reactions was 62°C ( Figure 5 , Figure 6 ).

[0122] 2. Effect of reaction time on RT-LAMP reaction

[0123] In the case of other conditions being the same, according to the optimized temperature, adjust the reaction time to 15, 30, 45, 60 and 75 minutes respectively, and explore the minimum reaction time of this method. The results showed that after 30 minutes of reaction, the electrophoresis of JEV G Ⅰ...

Embodiment 3

[0125] 1. Specificity test of RT-LAMP

[0126] The present invention uses the JEV G Ⅰ specific primer set to detect JEV SCYA201201 (type Ⅰ), JEV SA14-14-2 (type Ⅲ), CSFV, PRRSV, PRV, PPV and PCV2 respectively. As a result, only JEV SCYA201201 is positive, and the others are all positive. Negative. At the same time, the present invention uses the JEV GⅢ specific primer set to detect JEV SCYA201201 (type I), JEV SA14-14-2 (type III), CSFV, PRRSV, PRV, PPV and PCV2, and only JEV SA14-14-2 is positive , all others are negative. Both detection methods showed good specificity, that is, JEV G Ⅰ and JEV G Ⅲ specific RT-LAMP detection methods could only detect the corresponding genotypes of JEV ( Figure 9 , Figure 10 ).

[0127] 2. Sensitivity test of RT-LAMP

[0128] Quantitative results of cRNA: G Ⅰ-cRNA A260=0.96, A280=0.52, A260 / A280=1.85, RNA concentration=768ng / μL, copy number: about 1.2×10 12 copies / μL; GⅢ-cRNAA260=0.73, A280=0.38, A260 / A280=1.92, RNA concentration=584ng...

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Abstract

The invention discloses an RT-LAMP method for identifying Japanese encephalitis virus gene types I and III. Through the comparative analysis of Japanese encephalitis virus gene nucleotide sequences of gene types I and III, four specific primers for specific amplification of Japanese encephalitis virus nucleic acids of gene types I and III are designed for six regions on a target gene, the reaction result can be determined according to the color change of liquid in a reaction tube when a reaction is finished after calcein fluorochrome is added to a reaction system, and the reaction result can be determined according to whether a precipitate exists in the reaction tube or not after the reaction is finished or whether a ladder band appears or not during electrophoresis. According to the method, under the constant temperature condition, reverse transcription and nucleic acid amplification are realized within 1h, and the Japanese encephalitis virus gene types I and III can be quickly detected and identified within 1h.

Description

technical field [0001] The invention belongs to an RT-LAMP method for identifying Japanese Japanese encephalitis virus genotype I and genotype III, specifically, using reverse transcription-loop-mediated isothermal amplification technology to identify genotype I and genotype III Japan Japanese encephalitis virus can be used to distinguish between disease diagnosis purposes and non-disease diagnosis purposes. Background technique [0002] Japanese encephalitis B (JE), also known as epidemic encephalitis (epidemic encephalitis B), is a mosquito-borne infectious disease caused by Japanese encephalitis virus (JEV) , JEV is the main causative agent of viral encephalitis in Asia, which is a flavivirus borne by mosquitoes. The population of the endemic area is as high as 3 billion, and at least 50,000 people are reported to be infected with Japanese encephalitis virus every year. Japanese encephalitis virus is widespread from Asia to northern Australia. With the application of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/6844C12Q1/70C12Q2531/119C12Q2563/107
Inventor 曹三杰文心田张亮袁磊刘瀚扬伍锐黄小波文翼平石双艳周玉鹏田耕
Owner SICHUAN AGRI UNIV
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