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Porcine epidemic diarrhea virus S gene complete sequence amplifying method and application thereof

A porcine epidemic diarrhea and full sequence technology is applied in the field of full sequence amplification of porcine epidemic diarrhea virus S gene, which can solve the problems of inability to amplify target fragments in hypervariable regions and heavy workload, so as to avoid inaccurate sequence information, Improve the success rate and reduce the effect of amplification failure

Active Publication Date: 2020-09-18
LONGYAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, the S gene of porcine epidemic diarrhea virus is divided into multiple overlapping gene segments for primer design and amplification, and the full-length sequence of the S gene is obtained after splicing. Not only is the workload heavy, but there are hypervariable regions that cannot be amplified. Increased risk of obtaining targeted fragments

Method used

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  • Porcine epidemic diarrhea virus S gene complete sequence amplifying method and application thereof
  • Porcine epidemic diarrhea virus S gene complete sequence amplifying method and application thereof
  • Porcine epidemic diarrhea virus S gene complete sequence amplifying method and application thereof

Examples

Experimental program
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Embodiment 1

[0033] A method for amplifying the full sequence of the S gene of porcine epidemic diarrhea virus, including the following steps:

[0034] Step 1. Extract viral RNA: Take an appropriate amount of porcine epidemic diarrhea virus-positive fecal material, add 5 times the volume of sterile PBS, and store at -20°C after homogenization; repeated freezing and thawing 3 times, using Trizol reagent method to extract the virus RNA;

[0035] The specific method of the Trizol reagent method is: a. Take 1 ml of the tissue suspension and centrifuge at 5000 rpm for 10 min at 4°C to obtain the first supernatant; b. Take 300 μL of the first supernatant. Add 500μL of Trizol to the RNase-free sterilized 1.5ml EP tube, mix well, and let stand at room temperature for 10min; c. Add 500μL of chloroform, mix well, let stand at room temperature for 10min, at 4℃, Centrifuge at 12000 rpm for 10 min to obtain the second supernatant; d. Take 500 μL of the second supernatant into a new 1.5 mL EP tube, add 1.0 ...

Embodiment 2

[0044] A method for amplifying the full sequence of the S gene of porcine epidemic diarrhea virus. The disease material used in step 1 is the porcine epidemic diarrhea virus-positive small intestine disease material. The remaining steps are the same as in Example 1. In the gene fragment detection and sequencing steps, the electrophoresis results Such as figure 1 The lane 3 in is shown, and the sequencing result is shown in SEQ ID NO:5. The PEDV strain contained in the disease material sample was named LY-201803.

Embodiment 3

[0046] A method for amplifying the full sequence of the S gene of porcine epidemic diarrhea virus. The disease material used in step 1 is the porcine epidemic diarrhea virus-positive small intestine disease material. The remaining steps are the same as in Example 1. In the gene fragment detection and sequencing steps, the electrophoresis results Such as figure 1 Lane 5 in is shown, and the sequencing result is shown in SEQ ID NO:6. The PEDV strain contained in the disease material sample was named LY-201805.

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Abstract

The invention discloses a porcine epidemic diarrhea virus S gene complete sequence amplifying method which includes the following steps of step one, extracting viral RNA, step two, cDNA synthesis, step three, PCR amplification and step four, gene fragment detection and sequencing. The invention further provides application of the porcine epidemic diarrhea virus S gene complete sequence amplifyingmethod, and the application is used for the detection and analysis of a porcine epidemic diarrhea virus S gene complete sequence. According to the amplifying method, a conventional scheme of adoptingmulti-pair primers for splicing is not needed, by designing one pair of primers, the porcine epidemic diarrhea virus S gene complete sequence can be amplified, the operation is convenient, and the risk that a target fragment cannot be obtained due to amplifying cannot be performed in a hypervariable region can be avoided.

Description

Technical field [0001] The present invention discloses the field of porcine epidemic diarrhea diseases, in particular to a method for amplifying the full sequence of the S gene of porcine epidemic diarrhea virus and its application. Background technique [0002] Porcine epidemic diarrhea is an acute and infectious intestinal disease of pigs caused by Porcine Epidemic Diarrhea Virus (PEDV). Clinically, sick piglets vomiting, watery diarrhea, dehydration and weight loss are typical symptoms. In my country, before 2010, it was mainly distributed. After 2010, the disease broke out in an all-round way, and showed new epidemic characteristics: sick piglets showed more severe symptoms of diarrhea, vomiting and dehydration, and the mortality rate was as high as 80-100%. [0003] PEDV belongs to α-coronavirus and presents a typical coronavirus structure. The full length of the genome is about 28kb, and it is composed of at least 7 open reading frames (ORFs), which encode 4 structural prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12Q1/6806
CPCC12Q1/701C12Q1/686C12Q1/6806C12Q2521/107C12Q2565/125C12Q2537/165C12Q2535/122C12Q2531/113Y02A50/30
Inventor 包银莉黄翠琴范克伟杨小燕董波
Owner LONGYAN UNIV
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