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RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer group for detecting porcine epidemic diarrhea virus, kit and application

A RT-LAMP, coronavirus technology, applied in the field of microbial detection, can solve the problems of long time, expensive instruments, difficult porcine delta coronavirus, etc., and achieve the effect of simple interpretation method and easy operation.

Pending Publication Date: 2017-12-12
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems such as difficult, time-consuming, and expensive instruments for detecting porcine D-coronavirus at the grassroots level, and to provide a simple, fast and accurate test kit for the detection of porcine D-coronavirus at the grass-roots level. The technical scheme used is: a RT-LAMP primer set for detecting porcine D-coronavirus, said primer set is shown in SEQ ID NO: 1-6

Method used

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  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer group for detecting porcine epidemic diarrhea virus, kit and application
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer group for detecting porcine epidemic diarrhea virus, kit and application
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer group for detecting porcine epidemic diarrhea virus, kit and application

Examples

Experimental program
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Effect test

Embodiment 1

[0071] The specificity result of embodiment 1 RT-LAMP detection method

[0072] Carry out RT-LAMP amplification to 1 strain of porcine D-coronavirus, 7 strains of control virus and water control, the results are as follows figure 1 As shown, the porcine D-coronavirus reaction tube showed a rising curve of turbidity in about 15 minutes, which was a positive result, and the 7-strain control virus reaction tube and the water control reaction tube had no amplification, which was a negative result.

Embodiment 2

[0073] Example 2 Sensitivity results of RT-LAMP detection method

[0074] The initial concentration of porcine D-coronavirus genomic RNA was 2.47×10 1 ng / μL, after 10-fold serial dilution, RT-LAMP and common PCR amplification, the results are as follows figure 2 and image 3 As shown, the result shows that the detection limit of the RT-LAMP method of the present invention is about 2.47×10 -10 ng / μL, while the detection limit of common PCR method is 2.47×10 -8 ng / μL.

Embodiment 3

[0075] Example 3 Fluorescence visualization detection results of RT-LAMP detection method

[0076] According to the optimized conditions monitored by the turbidimeter, fluorescent dyes were added, reacted at 63°C for 60 minutes, and observed under ultraviolet light. Figure 4 For observing the results, the left tube is the reaction of porcine D-coronavirus RNA 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 established RT-LAMP method can be used conveniently at the grassroots level. You only need to use the kit with the RT-LAMP primer designed by this method, add the sample, and use a cheap water bath to keep it at 63°C for 60 minutes, and you can quickly observe As a result, without opening the cap, contamination is avoided.

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Abstract

The invention discloses an RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer group for detecting a porcine epidemic diarrhea virus, a kit and application. The primer group is shown as SEQ ID NO:1 to 6; the kit comprises the RT-LAMP primer group, 2*reaction buffer liquid, EM, ultrapure water and a porcine epidemic diarrhea virus RNA (ribonucleic Acid) template. The method comprises the steps of material preparation, RT-LAMP primer design and synthesis, virus genome RNA extraction, RT-LAMP reaction system building, and specific detection, sensibility detection and real-time quantitative detection by an RT-LAMP detection method. The specific detection and the sensibility detection prove that the RT-LAMP kit provided by the invention can monitor the reaction in real time and can perform quantitative detection on the porcine epidemic diarrhea virus copying number; the detection result can be fast and accurately obtained; convenience is brought for simply, conveniently, fast and reliably detecting the porcine epidemic diarrhea virus.

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 porcine D-coronavirus reverse transcription loop-mediated isothermal amplification kit and its use method. Background technique [0002] Pig coronavirus disease ( porcine epidemic diarrhea, PDCo ) is a porcine coronavirus ( porcine epidemic diarrhea virus, PDCoV ) is a highly contagious intestinal infectious disease of pigs, which mainly infects the entire small intestine, especially the jejunum and ileum, causing severe enteritis accompanied by diarrhea and vomiting in piglets. [0003] The virus PDCoV was first reported in Hong Kong, China from 2009 to 2010. Porcine D-coronavirus infection is similar to the clinical incidence of porcine epidemic diarrhea (PED) and porcine transmissible gastroenteritis (TGE), which can cause diarrhea and vomiting in suckling pigs, and the morbidity and mortality are as hi...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2531/119C12Q2561/113C12Q2545/113Y02A50/30
Inventor 何颖卢冰霞陈忠伟秦毅斌赵武段群棚周英宁李斌梁家幸苏乾莲蒋冬福卢敬专杨思仪
Owner GUANGXI VETERINARY RES INST
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