Influenza virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit and application thereof

A loop-mediated isothermal, influenza virus technology, applied in the field of microbial detection, can solve problems such as unfavorable rapid diagnosis of influenza, laboratory contamination, false positive results, etc., and achieve the effect of simple interpretation of results, rapid detection results, and rapid acquisition.

Inactive Publication Date: 2017-05-10
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional method is the isolation and identification of viruses, and the results are accurate and reliable. However, the conventional identification method has the disadvantages of complex operation, complex equipment and long time, which is not conducive to the rapid diagnosis of influenza.
The method of molecular biology is to detect the specific gene

Method used

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  • Influenza virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit and application thereof
  • Influenza virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit and application thereof
  • Influenza virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The specificity result of embodiment 1 RT-LAMP detection method

[0076] The result is as figure 1 As shown, the influenza virus reaction tube showed a rising curve of turbidity in about 20 minutes, which was a positive result, and the curves of the 6 control strain reaction tubes and the negative control reaction tube showed no amplification, which was a negative result.

Embodiment 2

[0077] Example 2 Sensitivity results of RT-LAMP detection method

[0078] The initial concentration of the original RNA of influenza virus was 6.8ng / μL. After 10-fold serial dilution, RT-LAMP and RT-PCR were amplified. The results were as follows: figure 2 with image 3 As shown, the result shows that the detection limit of the RT-LAMP method of the present invention is about 6.8×10 -3 ng / μL, while the detection limit of common RT-PCR method is 6.8×10 -2 ng / μL.

Embodiment 3

[0079] Example 3 Fluorescence visualization detection results of RT-LAMP detection method

[0080] According to the optimized conditions monitored by the turbidimeter, a fluorescent dye was added before the reaction, and after 60 minutes of reaction at 63°C, it was observed under an ultraviolet light. Figure 4 To observe the results, the left tube is the reaction with influenza virus as a template, which is a positive result, and the right tube is a negative control, which is a negative result. The test results show that the RT-LAMP method established by the present invention is convenient for grass-roots use. You only need to use the kit with the RT-LAMP primer designed by this method. Observe the results without opening the cap, avoiding contamination.

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Abstract

The invention discloses an influenza virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit and application thereof. The kit comprises a RT-LAMP primer, 2* reaction buffer solution, enzyme mixture EM, a fluorescence visual detection reagent, ultrapure water and an influenza virus RNA template, wherein the RT-LAMP primer comprises outer primers F3 and B3, and inner primers FIP and BIP. Specificity detection, sensitivity detection and fluorescence visible detection prove that the RT-LAMP detection method can specifically detect influenza virus, can be used for monitoring reactions in real time and quantitatively detecting the number of influenza virus copies so as to quickly and correctly acquire the detection result, and brings convenience to simple, quick and reliable influenza virus detection.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a rapid, visualized and real-time quantitative detection of influenza virus reverse transcription loop-mediated isothermal amplification kit and its application. Background technique [0002] Influenza viruses can cause infection and disease in many animals such as humans, poultry, pigs, horses, and bats, and are the pathogens of human and animal diseases such as human influenza, avian influenza, swine influenza, and equine influenza. Influenza virus is not strong against the outside world. Animal influenza viruses usually do not infect humans, and human influenza viruses generally do not infect animals, but pigs are an exception. Pigs can be infected with both human influenza viruses and avian influenza viruses, but they are mainly infected with swine influenza viruses. After a small number of animal influenza viruses adapt to humans, they can cause human influenza ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 彭昊李军冯世文潘艳陈泽祥胡帅杨威钟舒红马春霞陶立谢永平许力干韦志锋兰美益
Owner GUANGXI VETERINARY RES INST
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