Check patentability & draft patents in minutes with Patsnap Eureka AI!

Primer and method for detecting variable adenylate site usage of ostc gene mrna

A variable adenosine, gene technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low virus content, short viremia time, high detection cost, and achieve specificity. The effect of good performance, convenient and fast detection method and high sensitivity

Inactive Publication Date: 2020-05-29
SUN YAT SEN UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation and identification are not only valuable for clinical diagnosis, but also crucial for epidemiological and etiological research. Big
Serological methods such as ELISA are mainly used to detect samples such as feather follicle epithelium, cytolyzed infected lymphocyte tissue and cell culture. Low content, resulting in low detection rate

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer and method for detecting variable adenylate site usage of ostc gene mrna
  • Primer and method for detecting variable adenylate site usage of ostc gene mrna
  • Primer and method for detecting variable adenylate site usage of ostc gene mrna

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Use 1×10 5 pfu Md5 (Marek's disease virus Md5, MDV Md5) infected a single layer of CEF cells (chicken embryo fibroblasts), using uninfected CEF cells as a control, collected cells after 18h, 36h, 54h, and 72h, and extracted total RNA; After digesting DNA, detect OD260 / 280, and the ratio is 2.0-2.1 as qualified. Qualified total RNA was constructed for SAPAS (sequence of alternative polyadenylation site, SAPAS) library, and the constructed library was subjected to high-throughput sequencing on the Hi-Seq-2500 platform, and the sequencing results were analyzed by APA (alternative polyadenylation, APA) .

[0027] The result is as figure 2 As shown, comparing the APA analysis results of Md5-infected and non-Md5-infected CEF cells, it was found that the use of OSTC gene polyadenylation sites has changed significantly, and the LMAN2 gene of Md5-infected CEF cells uses distal APA sites. The increased ratio of mRNA can be used as a biomarker of MDV infection.

Embodiment 2

[0029] The primers used to detect the use of variable adenylate sites in OSTC gene mRNA in this example are shown in Table 1. The primer was designed with primer premier 5 based on the OSTC gene (gene bank Accession: NM_001006442.1). The first pair of primers is designed according to the common region and used to amplify the Proximal fragment of the common region; the second pair of primers is designed according to the Extended region and used to amplify the Distal fragment of the Extended region. Two pairs of primers were synthesized by Invitrogen Company.

[0030] Table 1. Primer sequence list

[0031] Primer name sequence OSTC-short-F (SEQ ID NO: 1) tgtcttcagc tctgccttga OSTC-short-R (SEQ ID NO: 2) cagacacgtt gcatttggac OSTC-long-F (SEQ ID NO: 3) actgtatgaa tgtgcttttg OSTC-long-R (SEQ ID NO: 4) gaaaccaact acccaactat

[0032] A method for detecting the use of variable adenine acid sites in OSTC gene mRNA, comprising the following...

Embodiment 3

[0043] Embodiment 3, animal experiments

[0044] Infect ten one-day-old chickens with 2000pfu Md5, collect the whole blood of chickens infected with Md5 30 days later, and collect the whole blood of ten chickens not infected with MDV at the same time, separate lymphocytes from the blood as samples, and carry out the detection of OSTC gene mRNA The usage of variable adenosine acid sites is detected, and the detection method is the same as that in Example 2. The CT value of control group in this embodiment dis / CT value pro The average value is 0.6, the CT value of the virus-infected group dis / CT value pro The average value is 0.71, and the result is consistent with Example 2. Therefore, the polyadenylation site usage of OSTC gene can be used as a biomarker for MDV infection in chickens.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses primers and a method for detecting use conditions of variable adenosine loci of OSTC (oligosaccharyltransferase complex subunit) gene mRNA (messenger ribonucleic acid). The primers comprise a first primer pair and a second primer pair, and sequences of the primers are the same as two pairs of sequences or complementary sequences thereof in the 3'untranslated region of an OSTC gene. The method comprises steps as follows: total RNA of a sample is extracted, and cDNA (complementary deoxyribonucleic acid) is obtained through reverse transcription; cDNA is taken as a template, Proximal fragments of a common region are subjected to qPCR (quantitative polymerase chain reaction) amplification by the aid of the first primer pair, and a CT value pro is obtained; cDNA is taken as the template, Distal fragments of an Extended region are subjected to amplification by the aid of the second primer pair, and a CT value dis is obtained; a ratio of the CT value dis to the CT value pro is calculated. The method for detecting the use conditions of the variable adenosine loci of the OSTC gene mRNA is convenient and fast and is suitable for being further popularized in simple laboratories and developing countries.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a primer and a method for detecting the use of variable adenine acid sites of OSTC gene mRNA. Background technique [0002] Marek's disease (Marek's disease, MD) is a highly contagious, contagious, lymphoproliferative neoplastic disease of poultry caused by Marek's disease virus (MDV), which mainly harms chickens, crane quail, and pheasant Also have morbidity.This disease can cause the nerve enlargement of the lymphocytoma of visceral organ, muscle and peripheral nerve.Clinically, MD diseased flock shows symptoms such as growth unevenness, extreme emaciation, death, and the most common pathological changes are nerve damage and Lymphatic tumors of various solid organs, liver and spleen tumors are the most common, asymmetric progressive paralysis caused by nerve damage, and eventually can cause complete paralysis of one or more limbs. [0003] MDV belongs to the herpesviru...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156C12Q2600/158
Inventor 曹永长薛春宜李杰何亮亮
Owner SUN YAT SEN UNIV
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com