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Specific primers, kit and method for detecting porcine epidemic diarrhea virus wild strains

A technology of porcine epidemic diarrhea and wild strains, applied in the field of molecular biology, can solve problems that have not been reported, and achieve good sensitivity, high accuracy and good reproducibility

Pending Publication Date: 2020-01-24
POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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  • Application Information

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Problems solved by technology

At present, there are many reports on the fluorescent quantitative detection method of PEDV, but there is no report on the specific detection of porcine epidemic diarrhea virus field strains by means of dye-based fluorescent quantitative PCR

Method used

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  • Specific primers, kit and method for detecting porcine epidemic diarrhea virus wild strains
  • Specific primers, kit and method for detecting porcine epidemic diarrhea virus wild strains
  • Specific primers, kit and method for detecting porcine epidemic diarrhea virus wild strains

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Effect test

Embodiment 1

[0033] Embodiment 1 detects the specific primer, kit and method of porcine epidemic diarrhea virus wild strain

[0034] 1.1 Primer design and synthesis

[0035] The sequence refers to the sequence of each target gene in the Gene Bank database, and the specific primers that can amplify the wild strain of porcine epidemic diarrhea virus are designed. Co., Ltd., and stored at -20°C for future use.

[0036] Three pairs of primers were designed, and their nucleotide sequences are shown in Table 1.

[0037] Nucleotide sequence of table 1 primer

[0038]

[0039] 1.2 Viral nucleic acid extraction and cDNA synthesis

[0040] MagaBio plus virus DNA / RNA purification Kit to extract viral RNA, reverse transcription of viral RNA with TaKaRa reverse transcription kit: prepare 10 μL solution according to RNA 8 μL, 5× reverse transcriptase 2 μL, 37 ° C, 30 min; 85 ° C , 5s; 4 ℃, save. The obtained sample is the target cDNA.

[0041]1.3 Optimization of fluorescent quantitative PCR rea...

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Abstract

The present invention discloses specific primers for detecting porcine epidemic diarrhea virus wild strains. The specific primers comprise PEDV-delta-ORF3-F1 and PEDV-delta-ORF3-R1. Nucleotide sequences of the specific primers are shown in SEQ ID NO.1 and 2. The kit for detecting the porcine epidemic diarrhea virus wild strains comprises the specific primers. The method for detecting the porcine epidemic diarrhea virus wild strains comprises the following steps: extracting viral RNA in tissues of samples to be detected, conducting reverse transcription into cDNA, and using the above specific primers or kit to perform PCR amplification on the cDNA; and result determination is as follows: if amplification products exist, the results are positive; and if the amplification products do not exist, the results are negative. The specific primers, kit and detection method have high specificity and specificity, and are also high in amplification efficiency, good in sensitivity, high in accuracy,good in reproducibility, short in detection cycle, and very high in feasibility and application prospects.

Description

technical field [0001] The invention relates to a specific primer, a kit and a method for detecting wild strains of porcine epidemic diarrhea virus, belonging to the technical field of molecular biology. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is an acute, highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV). Pigs of different ages can be infected, and the disease is the most harmful to suckling piglets, and the mortality rate of piglets within 7 days of age can be as high as 100%. In recent years, the re-emergence of PED caused by mutant PEDV strains has caused huge economic losses to the world's pig industry. The wild PEDV strain ORF3 that has not been attenuated by passaging contains 675 nucleotides and encodes a polypeptide containing 224 amino acid residues, while the ORF3 gene of the attenuated vaccine strain will have partial deletions...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 郭效珍于克戬黄兵吴家强胡峰刘存霞于可响马秀丽李玉峰亓丽红宋玲玲
Owner POULTRY INST SHANDONG ACADEMY OF AGRI SCI
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