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Analysis of TGEV ORF7 Gene and Establishment of Sybr Green I RT-PCR Detection Assay

Author: GuJiangPing
Tutor: YangZhiBiao
School: Shanghai Jiaotong University
Course: Preventive Veterinary Medicine
Keywords: TGEV ORF7 Sequence analysis SYBR Green RT-PCR
CLC: S858.28
Type: Master's thesis
Year: 2012
Downloads: 92
Quote: 0
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Abstract


Transmissible gastroenteritis (TGE) is a highly contagious disease inpigs that is caused by the transmissible gastroenteritis virus (TGEV), andis characterized by vomiting, watery diarrhea, and dehydration. Thedisease has a high mortality rate in newborns, and piglets that recovermay suffer from dysplasia or growth retardation. Moreover, older animalsthat recover can have impaired immune function, weight loss, and maynot feed efficiently. The disease seriously endangers the pig industry bywasting resources and drugs and causing serious economic lossesworldwide.TGEV was isolated from porcine fecal samples detection whichwere collected from Shanghai farms. The open reading frame 7 (ORF 7)was amplified by the RT-PCR. The amplified PCR product was cloned,sequenced, and compared with the sequences of 24 TGEV referencestrains. The nucleotide and amino acid sequences comparison showedORF 7 is relative conserved. In phylogenetic analysis of the ORF 7 gene,Chinese TGEV strains were clustered into three groups. Chinese TGEVstrains and Asian strains were related to the TGEV strains isolated in theUSA, and alien to the European strains.This study established a SYBR Green I real-time quantitative PCRassay (RT-qPCR) for the detection of the porcine TGEV using specific primers designed to amplify the highly conserved porcine TGEV S genesequence. The threshold cycle (Ct) and the log plasmid copy numbers hada good linear relationship with an efficiency of 1.05 (R2 = 0.999). Theadvantages of utilizing this approach for the rapid detection of TGEVinclude excellent sensitivity, reproducibility, and low cost. Ninety-sixporcine fecal samples were tested in this study, and 7 samples more thanPCR assay were detected by this assay.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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