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Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof

A coronavirus and primer set technology, applied in the field of virus detection, can solve the problem of inability to meet the increasing number of import and export animals

Inactive Publication Date: 2016-08-17
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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Problems solved by technology

[0005] In order to improve the detection efficiency of animal diseases, save time, and solve the problem that traditional detection methods cannot satisfy the growing situation of imported and exported animals, the present invention has finally explored a method for detecting porcine delta coronavirus by using the ring-mediated isothermal amplification method through experimental testing. This method has the characteristics of rapid detection, convenience, low cost, and is suitable for application in field sites and grassroots departments.

Method used

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  • Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof
  • Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof
  • Primer combination used for rapid isothermal detection of porcine deltacoronavirus, and method thereof

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preparation example Construction

[0039] 1.3 RNA extraction and cDNA preparation

[0040] Take the EP tube treated with DEPC water, add 1mL TRIzol and 200μL virus solution, mix well and let stand at room temperature for 5min; add 200μL chloroform, shake vigorously for 30s, let stand at room temperature for 3min, and centrifuge at 4°C for 15min to obtain a layered solution; take the upper layer Transfer to a clean EP tube, add 500 μL of isopropanol, let stand at -20°C for 30 minutes, and centrifuge at 4°C for 15 minutes; remove the supernatant; add 1 mL of 75% DEPC ethanol, vortex, and centrifuge at 4°C for 10 minutes; remove the supernatant , dried in air for 5 min; add 50 μL DEPC water to the test tube. Reverse transcription was performed according to the M-MuLV reverse transcriptase process to obtain cDNA, which was stored at -20°C for later use.

[0041] 1.4 Construction of positive recombinant plasmids

[0042]Using specific primers for the N-segment gene of porcine delta coronavirus, the cDNA was amplif...

Embodiment 1

[0052] Amplification of N segment gene of porcine deltacoronavirus

[0053] Take the sample, extract the virus genome, and carry out reverse transcription according to the M-MuLV reverse transcriptase process to obtain cDNA, use specific primers to amplify the N segment gene of porcine delta coronavirus, and obtain the S segment gene after amplification 1029bp, consistent with the theoretical amplification value.

Embodiment 2

[0055] Detection of porcine delta coronavirus by LAMP method

[0056] 1.1 Observation of gel electrophoresis results

[0057] After exploring and optimizing the reaction conditions, a LAMP detection method for the N-segment gene of porcine deltacoronavirus was established. The detection results are as follows: figure 1 As shown, the nucleic acid electrophoresis bands in the amplification results of the N-segment gene region were distributed in a ladder shape, which was consistent with the theoretical results, and no specific products appeared in the negative control.

[0058] 1.2 Enzyme digestion and identification of amplified products

[0059] Recover the positive amplification product of the N-segment gene, and use Dpn II endonuclease for enzyme digestion identification. The result is as figure 2 As shown, the electrophoresis results of the N gene after enzyme digestion were 164bp and 45bp bands, which were consistent with the theoretical results, and the positive ampli...

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Abstract

The invention discloses a primer combination used for rapid isothermal detection of porcine deltacoronavirus, and a method thereof. The method using a loop-mediated isothermal amplification (LAMP) technology to detect the porcine deltacoronavirus, disclosed in the invention, is used to detect the porcine deltacoronavirus in an environment sample, a medical sample and the sanitation and epidemic prevention field. The method comprises the following steps: designing specific primers by adopting an N segment encoding region gene sequence in a porcine deltacoronavirus genome as a target sequence, optimizing a reaction system, and carrying out target gene specific amplification. The method only needs a constant temperature device, does not need thermotropy and long-time temperature circulation of a template, allows a result to be directly observed, and has the characteristics of low cost, high efficiency and simple operation. The porcine deltacoronaviru nucleic acid LAMP detection method established in the invention also has the characteristics of strong specificity, high sensitivity, high convenience and fastness, can be implemented in a base laboratory and a miniature experiment station, and provides a new technology and method for detection of the porcine deltacoronaviru.

Description

technical field [0001] The invention relates to a virus detection technology, in particular to using a loop-mediated isothermal amplification method to detect porcine delta coronavirus. Background technique [0002] Piglet diarrhea is a common multiple disease that affects pig production. Porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), porcine rotavirus (Porcine rotovirus (PRoV) is the main pathogen of epidemic viral piglet diarrhea in my country, and TGEV and PEDV are both coronaviruses. In 2012, scholars at the University of Hong Kong detected a new type of porcine coronavirus - porcine deltacoronavirus (porcine deltacoronavirus, PDCoV), and gene sequence analysis showed that it was a deltacoronavirus. In 2012, Patrick et al. tested 169 porcine diarrhea samples, showing that the positive rate of porcine delta coronavirus was 10%; Marthaler et al. tested samples from pig farms in some parts of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/70
Inventor 王乃福陈小金吴冬雪黄晨陈本龙董志珍赵祥平王玉玲王建华
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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