Development of Quantitative Real-Time Polymerase Chain Reaction for Duck Enteritis Virus DNA

Yang, Falong; Jia, Wenxiang; Yue, Hua; Luo, Wei; Chen, Xi; Xie, Yonghui; Zen, Wei; Yang, W.

doi:10.1637/7338-020305r.1

Cited by 23 publications

(12 citation statements)

References 15 publications

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“…Execution of the above-mentioned methods are complex and the results are only semi-quantitative. The development of fluorescence qPCR technology provides a quicker and easier method for detecting the copy number in different samples; hence, it has been widely used in many research fields (Higgins et al, 2001;Yang et al, 2005). In the current study, we established a method for detecting the FVII copy number by using SYBR GreenI in CHO-K1 cells.…”

Section: Discussionmentioning

confidence: 99%

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Xiao¹,

Li²,

Xiao³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 μg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 μg/ mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

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Section: Discussionmentioning

confidence: 99%

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Xiao¹,

Li²,

Xiao³

et al. 2013

Genet. Mol. Res.

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“…Beside diagnostics, the rapidity, sensitivity and specificity of these assays could provide a suitable tool towards understanding the epizootology of DP (Hansen et al 1999;Pritchard et al 1999;Hansen et al 2000;Sandhu & Shawky 2003;Cheng et al 2004). DEV-specific DNA segments can be amplified from infected cell culture supernatant and tissues from esophagus, liver, kidney and spleen (Plummer et al 1998;Thayer & Beard 1998;Hansen et al 1999Hansen et al , 2000Pritchard et al 1999;Yang et al 2005;Xuefeng et al 2008;Liu et al 2009;Qi, Yang, Cheng, Wang, Guo, and Jia 2009;Wu et al 2011b;Lin et al 2013). Plummer et al (1998) diagnosed the DEV by targeting the highly conserved domain of the UL6 gene.…”

Section: Molecular Diagnosis Of Devmentioning

confidence: 99%

Duck virus enteritis (duck plague) – a comprehensive update

Dhama

Kumar

Saminathan

et al. 2017

Veterinary Quarterly

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Duck virus enteritis (DVE), also called duck plague, is one of the major contagious and fatal diseases of ducks, geese and swan. It is caused by duck enteritis virus (DEV)/Anatid herpesvirus-1 of the genus Mardivirus, family Herpesviridae, and subfamily Alpha-herpesvirinae. Of note, DVE has worldwide distribution, wherein migratory waterfowl plays a crucial role in its transmission within and between continents. Furthermore, horizontal and/ or vertical transmission plays a significant role in disease spread through oral-fecal discharges. Either of sexes from varying age groups of ducks is vulnerable to DVE. The disease is characterized by sudden death, vascular damage and subsequent internal hemorrhage, lesions in lymphoid organs, digestive mucosal eruptions, severe diarrhea and degenerative lesions in parenchymatous organs. Huge economic losses are connected with acute nature of the disease, increased morbidity and mortality (5%-100%), condemnations of carcasses, decreased egg production and hatchability. Although clinical manifestations and histopathology can provide preliminary diagnosis, the confirmatory diagnosis involves virus isolation and detection using serological and molecular tests. For prophylaxis, both live-attenuated and killed vaccines are being used in broiler and breeder ducks above 2 weeks of age. Since DEV is capable of becoming latent as well as shed intermittently, recombinant subunit and DNA vaccines either alone or in combination (polyvalent) are being targeted for its benign prevention. This review describes DEV, epidemiology, transmission, the disease (DVE), pathogenesis, and advances in diagnosis, vaccination and antiviral agents/therapies along with appropriate prevention and control strategies.

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“…The disease is caused by duck enteritis virus (DEV), which was epidemic in many countries and regions and has produced significant economic losses to the commercial duck industry because of the consequent high mortality and low egg production rates [19][20][21][22][23][24][25][26]. Currently, the DEV genome and molecular biology research is very rare，most previous research has focused on the epidemiology [27], diagnosis [28] and prevention of DVE and the structure and morphogenesis of DEV [29,30]. However, a DEV genomic library was constructed successfully in our laboratory [31], and the DEV UL18 gene was first discovered by selection, sequence determination and bioinformatics analysis.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Codon usage bias in the newly identified DEV UL18 gene

et al. 2011

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In this study, Codon usage bias (CUB) of DEV UL18 gene was analyzed, the results showed that codon usage bias in the DEV UL18 gene was strong bias towards the synonymous codons with A and T at the third codon position. Phylogenetic tree based on the amino acid sequences of the DEV UL18 gene and the 27 other herpesviruses revealed that UL18 gene of the DEV CHv strain and some fowl herpesviruses such as MeHV-1, GaHV-2 and GaHV-3 were clustered within a monophyletic clade and grouped within alphaherpesvirinae. The ENC-GC3S plot indicated that codon usage bias has strong species-specificity between DEV and 27 reference herpesviruses, and suggests that factors other than gene composition, such as translational selection leading to the codon usage variation among genes in different organisms, contribute to the codon usage among the different herpesviruses. Comparison of codon preferences of DEV UL18 gene with those of E. coli , yeast and humans showed that there were 20 codons showing distinct usage differences between DEV UL18 and yeast, 22 between DEV UL18 and humans, 23 between DEV UL18 and E.coli, which indicated the codon usage bias pattern in the DEV UL18 gene was similar to that of yeast. It is infered that the yeast expression system may be more suitable for the DEV UL18 expression.

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Development of Quantitative Real-Time Polymerase Chain Reaction for Duck Enteritis Virus DNA

Cited by 23 publications

References 15 publications

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells

Duck virus enteritis (duck plague) – a comprehensive update

Characterization of Codon usage bias in the newly identified DEV UL18 gene

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