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Primer combination for TGEV, PEDV and PDCoV triple PCR detection, kit and and application of primer combination and kit

A primer combination and kit technology, applied in the field of biomedicine, can solve the problems of large number of primers and low primer specificity, etc.

Active Publication Date: 2021-08-06
广州博达锦弘生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] ①, the specificity of the primer is not high;
[0023]②, need to use 3 pairs of 6 primers, the number of primers is large

Method used

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  • Primer combination for TGEV, PEDV and PDCoV triple PCR detection, kit and and application of primer combination and kit
  • Primer combination for TGEV, PEDV and PDCoV triple PCR detection, kit and and application of primer combination and kit
  • Primer combination for TGEV, PEDV and PDCoV triple PCR detection, kit and and application of primer combination and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Primer Design

[0074] A common downstream degenerate primer was designed using a homologous region of PDCoV, TGEV and PEDV, which reduced the number of primers by two, and the expected amplified band sizes were 361bp, 528bp and 763bp, respectively.

Embodiment 2

[0075] Construction of embodiment 2 positive plasmid

[0076]There are three kinds of positive plasmids in the present invention: TGEV and PEDV positive plasmids are constructed by PCR method, and the target fragment lengths are 528bp and 763bp respectively; PDCoV positive plasmids (GenBank accession number: KJ601778.1) are synthesized by Shanghai Jierui Bioengineering Co., Ltd The target fragment is 361 bp in length.

[0077] 2.1 Using total RNA of TGEV and PEDV as templates, carry out reverse transcription and PCR amplification respectively, the system and procedure are as follows:

[0078] A. Reverse transcription:

[0079] (1) Configure the following systems:

[0080] ①Total RNA: 2μg

[0081] ②dNTP (2.5μM): 4μL

[0082] ③Random Primer (9mer): 2μL

[0083] ④Add water to 10μL

[0084] 70°C, water bath for 5 minutes, ice bath for 2 minutes.

[0085] (2) Configure the following systems:

[0086] ① Reverse transcriptase: 4 μL

[0087] ② RNase inhibitor: 1 μL

[0088] ...

Embodiment 3

[0120] The determination of embodiment 3 single PCR primer concentration

[0121] In the present invention, the concentration of each primer solution is configured as 5 gradients of 10 μM, 8 μM, 6 μM, 4 μM and 2 μM. When configuring the reaction system, take 1 μL each of the upstream and downstream primers. The reaction system is as follows:

[0122] ①Plasmid: 1 μL

[0123] ② Upstream primer: 1 μL

[0124] ③Downstream primer: 1μL

[0125] ④5×buffer: 5μL

[0126] ⑤dNTP (2.5μM): 2μL

[0127] ⑥FastPfu Fly DNA polymerase: 0.5μL

[0128] ⑦Water: 14.5μL

[0129] The reaction procedure is as follows:

[0130]

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Abstract

The invention relates to the field of biological medicine, in particular to a primer combination for TGEV, PEDV and PDCoV triple PCR detection, a kit and application of the primer combination and the kit. According to the invention, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine deltacoronavirus are taken as detection objects, and a triple PCR detection method is established. Software such as Snapgene, DNAMAN, Primer5 and the like is utilized to find a segment with relatively high homology of the three to design a degenerate primer, and the method has important practical significance and clinical application value.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a primer combination, a kit and an application thereof for triple PCR detection of TGEV, PEDV and PDCoV. Background technique [0002] For a long time, the pig industry has been plagued by three big mountains: reproductive system diseases, digestive system diseases, and respiratory system diseases. Among them, the symptoms of digestive system diseases are "more death". Since outbreaks of epidemic diarrheal viruses in China in 2010 and the United States in 2013, porcine coronaviruses in particular have caused severe economic losses to the swine industry worldwide. Because porcine diarrhea virus often attacks young pigs, especially newborn piglets, it can carry the virus but is not susceptible to adult pigs, resulting in perennial virus infection in pig herds. [0003] 1Coronavirus causing swine diarrhea [0004] Acute infectious diarrhea is one of the main causes of high morbidity and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/686
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143Y02A50/30
Inventor 郝鹏飞
Owner 广州博达锦弘生物科技有限公司
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