Reverse transcription loop-mediated isothermal amplification test kit of hog cholera virus and application thereof

A ring-mediated isothermal, swine fever virus technology, applied in the field of microbial detection, can solve the problems of false positive results, long time required, complicated operation, etc., and achieve the effect of avoiding pollution and easy operation.

Inactive Publication Date: 2015-05-27
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 the virus, and the result is accurate and reliable, but the conventional identification method has the disadvantages of complex operation, complex equipment and long time, which is not conducive to the rapid diagnosis of swine fever
The method of molecular biology is to detect the specific gene of swine fever virus by reverse transcription polymerase c

Method used

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  • Reverse transcription loop-mediated isothermal amplification test kit of hog cholera virus and application thereof
  • Reverse transcription loop-mediated isothermal amplification test kit of hog cholera virus and application thereof
  • Reverse transcription loop-mediated isothermal amplification test kit of hog cholera virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Specific results of RT-LAMP detection method

[0079] RT-LAMP amplification was performed on 3 strains of swine fever virus, 7 strains of control virus strains, 5 strains of control bacteria and water control, and the results are as follows figure 1 As shown in the figure, the swine fever virus reaction tube showed a rising curve of turbidity at about 20 minutes, which was a positive result. No amplification occurred in the curves of the 7 control strain reaction tubes, 5 control bacterial reaction tubes and the water control reaction tube curves. negative result.

Embodiment 2

[0080] Example 2 Sensitivity results of RT-LAMP detection method

[0081] The initial concentration of the original RNA of swine fever virus was 0.8 ng / μL. After 10-fold serial dilution, RT-LAMP and RT-PCR amplification were performed. The results show that the detection limit of the RT-LAMP method of the present invention is about 0.8× 10 -3 ng / μL, while the detection limit of RT-PCR method (Beijing Century Yuanheng Animal Epidemic Prevention Technology Co., Ltd.) is 0.8×10 -2 ng / μL.

Embodiment 3

[0082] Example 3 Fluorescence visualization detection results of RT-LAMP detection method

[0083] According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added before the reaction, and after 60 minutes of reaction at 63 °C, observed under UV light. Figure 4 In order to observe the results, the left tube is the reaction situation with swine fever 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 can be conveniently used at the grassroots level. It is only necessary to use the kit to cooperate with the RT-LAMP primers designed by this method, and after adding the sample, use an inexpensive water bath to keep 63° C. for 60 minutes. Quickly observe results without opening the cap, avoiding contamination.

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Abstract

The invention discloses a reverse transcription loop-mediated isothermal amplification test kit of a hog cholera virus and an application thereof. The test kit comprises an RT-LAMP primer, a 2*reaction buffer solution, an EM, a fluorescence visual detection reagent, ultrapure water and a hog cholera virus RNA template. The RT-LAMP primer comprises outer primers F3 and B3, inner primers FIP and BIP and a loop primer LF. The specific detection, the sensibility detection and the fluorescence visual detection prove that by adopting the RT-LAMP detecting method, the hog cholera virus can be specifically detected, the reaction can be real-time monitored, the copy number of the hog cholera virus can be quantitatively determined, the detecting result can be rapidly and accurately obtained, so that the simple, rapid and reliable detection of the hog cholera virus are facilitated.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a reverse transcription loop-mediated isothermal amplification kit for rapid, visual and real-time quantitative detection of swine fever virus and its application. Background technique [0002] Swine fever (Hog cholera), also known as Hog Cholera (HC), is commonly known as Intestinal Fever in my country. In order to distinguish it from African swine fever, Europe calls it classical swine fever (Classical swine fever, CSF). The pathogen is Hog cholera virus (HCV or Classical swine fever virus, CSFV), which is a severe contact infectious disease in pigs. Swine fever has been included in the list of Class A diseases by the World Organization for Animal Health (OIE). The disease was first discovered in Ohio in the United States in 1833, and it exists in most swine-raising countries in the world, causing huge economic losses to the swine industry. Although the worl...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q1/701
Inventor 冯世文李军潘艳彭昊杨威胡帅陈泽祥钟舒红谢宇舟禤雄标马春霞柳锋陶立许力干谢永平韦志锋秦若甫兰美益
Owner GUANGXI VETERINARY RES INST
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