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Method for detecting porcine sapeloviruses based on reverse transcription loop-mediated isothermal amplification technology

A ring-mediated isothermal and technical detection technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as time-consuming, delayed disease control, and inability to achieve detection, achieving high detection sensitivity and detection Convenient, fast, and easy-to-identify effects

Inactive Publication Date: 2013-06-26
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation and enzyme-linked immunosorbent assay are the most basic detection methods, but they are also the most time-consuming detection methods. For the early diagnosis of sudden epidemic diseases, early detection cannot be achieved, which may delay the progress of the epidemic. control
The detection methods of molecular biology have been paid more and more attention, such as RT-PCR has become a routine detection method, but these methods require high-precision instruments and skilled techniques
For testing facilities and animal clinics in remote areas, these methods cannot play their role

Method used

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  • Method for detecting porcine sapeloviruses based on reverse transcription loop-mediated isothermal amplification technology
  • Method for detecting porcine sapeloviruses based on reverse transcription loop-mediated isothermal amplification technology
  • Method for detecting porcine sapeloviruses based on reverse transcription loop-mediated isothermal amplification technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: RT-LAMP detects the optimal reaction temperature of porcine Sapero virus

[0040] Obtained the whole genome of China's first porcine Sapero virus (accession number is HQ875059) on GeneBank, and screened out the conserved sequence region 5'UTR of the genome according to the principle of LAMP primer design, targeting six specific sequence regions of this sequence Four primers were designed and synthesized. The RNA template of Sapero virus was obtained by using the RNA\DNA extraction kit of Tiangen, and extracting the virus pure liquid according to the instructions. The reaction system was configured according to the operation manual of the RT-LAMP reaction kit purchased by Beijing Lanpu Biotechnology Co., Ltd., with a total volume of 25 μL. The final concentration of outer primers F3 and B3 was 0.2 μM, and that of inner primers FIP and BIP was 1.6 μM.

[0041] Configure the reaction solution according to the above reaction system, and design three reaction te...

Embodiment 2

[0042] Embodiment 2: RT-LAMP detects the specificity of porcine sapero virus

[0043] Porcine Jieshen virus (gifted by PTV\Harbin Veterinary Research Institute), porcine enterovirus 9 (PEV9\laboratory preservation), porcine foot-and-mouth disease virus (FMDV\purchased from China China Animal Husbandry Co., Ltd.) that do not contain Sapero virus were selected. , porcine enterovirus 9 (PEV9\laboratory preservation), encephalomyocarditis virus (EMCV\Lanzhou Veterinary Research Institute gift), porcine transmissible gastroenteritis (TGEV\laboratory preservation), porcine epidemic diarrhea (PEDV laboratory preservation ), porcine blue ear virus (PRRSV\laboratory storage) nucleic acid, configure the reaction solution according to the above reaction system, and place two parallel reaction tubes in the real-time monitoring turbidimeter and water bath respectively, set the temperature at 63 °C, and store 70min, 5min at 80°C to end the reaction. Real-time monitoring of the test results...

Embodiment 3

[0044] Example 3: Sensitivity of RT-LAMP to detect porcine Sapero virus

[0045] The RNA of the extracted porcine Sapero virus was diluted with RNase-free water in a 10-fold gradient, and the concentration of the RNA mother solution was: 2.97×10 9 copies / µl. serially diluted to 2.97×10 8 copy / microliter, 2.97×10 7 copy / microliter, 2.97×10 6 copy / microliter, 2.97×10 5 copy / microliter, 2.97×10 4 copy / microliter, 2.97×10 3 Copy, 2.97×10 2 copies / μl and 2.97 x 10 1 copies / µl. Configure the reaction solution according to the above reaction system. Two sets of parallel reaction tubes are placed in the real-time monitoring turbidimeter and the water bath respectively. The temperature is set at 63°C, stored for 60 minutes, and the reaction is completed at 80°C for 5 minutes. The result analysis method is the same as above, and the experimental results finally show that the minimum detection limit of RT-LAMP of porcine Sapero virus is 2.97×10 2 copies / microliter, but the best...

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Abstract

The invention relates to the application field of biotechnology, and provides a method for detecting porcine sapeloviruses based on a reverse transcription loop-mediated isothermal amplification technology. The method comprises the following steps: screening a conserved sequence block 5' UTR of the porcine sapeloviruses and designing four primers according to the conserved sequence block; preparing a sample to be detected; preparing a reaction liquid and carrying out a reverse transcription loop-mediated isothermal amplification reaction (RT-LAMP); and identifying a reaction result according to the real-time detection of an amplification curve output by a turbidity meter, the color changing conditions of the calcein of a fluorescent dye or the direct observation of the turbidity change of the reaction liquid. For relevant detection departments or companies, the method which is low in price, rapid in speed, high in efficiency and high in specificity is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting porcine sapero virus. Background technique [0002] Porcine Sapelovirus (Porcine Sapelovirus) is a porcine virus belonging to the family Sapelovirus. It can cause a combination of nervous disorders, reproductive failure, respiratory failure, pneumonia and diarrhea in pigs. The virus can be co-infected with other pig viruses, which brings a very huge economic threat to the pig industry. Therefore, the detection of this virus is an important measure for early prevention, early control and elimination of mutual infection among pig herds. This experiment is aimed at the first strain of Sapero virus (PSV-Csh) in China (gene number: HQ875059). The full length of the virus genome is 7502 base pairs, with only one open reading frame, encoding a polyprotein front body, at the 5' and 3' ends of the genome are the 5'UTR and 3'UTR of the untranslated region of the genom...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 崔立王春艳郁达义华修国刘雨潇汤赛冬
Owner SHANGHAI JIAO TONG UNIV
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