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PCR primers, PCR detection method and PCR detection kit for detecting and identifying atypical porcine pestivirus (APPV)

A swine fever virus, APPV-F technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as no detection and identification of atypical swine fever virus, and achieve convenient clinical detection. , Great application prospect, high specificity effect

Inactive Publication Date: 2017-07-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are still few reports on the research progress of APPV virus in my country, and there is no method or kit for detecting and identifying atypical swine fever virus.

Method used

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  • PCR primers, PCR detection method and PCR detection kit for detecting and identifying atypical porcine pestivirus (APPV)
  • PCR primers, PCR detection method and PCR detection kit for detecting and identifying atypical porcine pestivirus (APPV)
  • PCR primers, PCR detection method and PCR detection kit for detecting and identifying atypical porcine pestivirus (APPV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 The design of atypical swine fever virus PCR primer

[0047] 1. The present invention refers to and compares the genome sequence of atypical swine fever virus in GenBank, selects a conserved nucleotide sequence as the amplification region, and designs a pair of detection atypical swine fever virus gene according to the cap protein of atypical swine fever virus PCR primers; the PCR primers are as follows:

[0048] Upstream primer APPV-F: 5'-CTGCCTTATGGGCGGTAGAAT-3' (SEQ ID NO.1);

[0049] Downstream primer APPV-R: 5'-ATCAGCACCATGTTCTTGGGAT-3' (SEQ ID NO.2).

[0050] 2. After verification, the cDNA of atypical swine fever virus was used as a template for amplification, and the results showed that the primer set could specifically amplify and detect atypical swine fever virus.

Embodiment 2

[0051] Example 2 PCR primers detect atypical swine fever virus samples

[0052] 1. RNA extraction

[0053] (1) Take 30 mg of diseased pig tissue in a centrifuge tube, add 1 mL of TRIZOL reagent, grind the homogenate and let it stand for 5 min, then freeze and centrifuge at 12,000 rpm for 10 min at a high speed at 4 °C, and centrifuge the supernatant Transfer the supernatant into a new 1.5 mL centrifuge tube;

[0054] (2) Add 0.2 mL of chloroform, tightly cap the centrifuge tube, shake the centrifuge tube vigorously for 15 s, incubate at 15-30 °C for 2-3 min, and then centrifuge at 12,000 rpm for 15 min at 4 °C;

[0055] (3) After centrifugation, the mixture was separated into three layers, and the RNA was in the upper colorless water sample layer. Transfer the water sample layer to a clean centrifuge tube, add 0.5 mL of isopropanol to mix well, incubate at 15-30 °C for 10 min, and centrifuge at 12,000 rpm for 10 min at 4 °C;

[0056] (4) Remove the upper layer suspension, a...

Embodiment 3

[0074] Implementation example 3 Atypical swine fever virus PCR primer specificity detection

[0075] With reference to step 1 to step 3 in embodiment example 2, utilize the PCR primer described in embodiment 1 to atypical swine fever virus, porcine epidemic diarrhea virus, porcine senega valley virus, foot-and-mouth disease virus, porcine delta coronavirus, porcine library PCR detection of bruvirus, porcine bocavirus and porcine sapero virus were carried out respectively. Electrophoresis results such as figure 2 As shown, the PCR primers were positive for atypical swine fever virus, but for porcine epidemic diarrhea virus, porcine senega valley virus, foot-and-mouth disease virus, porcine deltacoronavirus, porcine Kubu virus, porcine bocavirus Negative reaction with porcine sapero virus, indicating that the primer has strong specificity for atypical swine fever virus.

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Abstract

The invention discloses PCR primers, a PCR detection method and a PCR detection kit for detecting and identifying atypical porcine pestivirus (APPV). By referring to and comparing the genomic sequence of the atypical porcine pestivirus, based on the conserved sequence of the cap protein of the atypical porcine pestivirus, a pair of PCR primers for detecting the atypical porcine pestivirus is designed, and the sequences of the forward primer and the reverse primer of the PCR primers APPV-F / R are shown as SEQ ID NO.1- SEQ ID NO.2 in sequence; the PCR detection method and the PCR detection kit for detecting the atypical porcine pestivirus are further provided. The primers and the detection method can detect and identify the atypical porcine pestivirus fast, conveniently and efficiently, specificity is good, and sensitivity is high; moreover, operation of the detection method is easy and efficient, and the detection method brings convenience to clinical detection and epidemiological investigation, and has a broad application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically discloses a PCR primer, a detection method and a detection kit for detecting and identifying atypical swine fever virus (APPV). Background technique [0002] Congenital tremors of piglets (CT), also known as "piglet shaking disease", refers to the blockage of myelin in the central nervous system above the level of the spinal cord in newborn piglets. A disorder characterized by generalized or localized spasms. The disease was first reported in Germany in 1854, and has since been reported around the world. So far, there is no effective treatment for the disease, and most of the affected piglets are treated with death or elimination. [0003] In 2016, Paulo Arruda and others identified the causative agent through next-generation gene sequencing technology and found that the causative agent was closer to porcine pestivirus (Porcine Pestivirus), and inoculated the fetus in the middle an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 孙媛马静云蓝天麦凯杰
Owner SOUTH CHINA AGRI UNIV
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