Detection of anatid herpesvirus 1 gC gene by TaqMan™ fluorescent quantitative real-time PCR with specific primers and probe

Abstract

BackgroundAnatid herpesvirus 1 (AHV-1) is known for the difficulty of monitoring and controlling, because it has a long period of asymptomatic carrier state in waterfowls. Furthermore, as a significant essential agent for viral attachment, release, stability and virulence, gC (UL44) gene and its protein product (glycoprotein C) may play a key role in the epidemiological screening. The objectives of this study were to rapidly, sensitively, quantitatively detect gC gene of AHV-1 and provide the underlying basis for further investigating pcDNA3.1-gC DNA vaccine in infected ducks by TaqMan™ fluorescent quantitative real-time PCR assay (FQ-PCR) with pcDNA3.1-gC plasmid.ResultsThe repeatable and reproducible quantitative assay was established by the standard curve with a wide dynamic range (eight logarithmic units of concentration) and very good correlation values (1.000). This protocol was able to detect as little as 1.0 × 101 DNA copies per reaction and it was highly specific to AHV-1. The TaqMan™ FQ-PCR assay successfully detected the gC gene in tissue samples from pcDNA3.1-gC and AHV-1 attenuated vaccine (AHV-1 Cha) strain inoculated ducks respectively.ConclusionsThe assay offers an attractive method for the detection of AHV-1, the investigation of distribution pattern of AHV-1 in vivo and molecular epidemiological screening. Meanwhile, this method could expedite related AHV-1 and gC DNA vaccine research.

Duck virus enteritis (duck plague) – a comprehensive update

“…In duck plague virus (DPV) infected ducklings, antiviral immunity comprised of noticeable presence of pattern recognition receptors (PRRs) significantly appears along with observable typical pathological lesions and symptoms. Study based on realtime quantitative PCR and TaqMan TM fluorescent quantitative real-time PCR with specific primers and probes also confirmed hike in the protective innate immune response and is helpful in finding the break points during pathogenesis to lower the establishment of infection of DPV/DEV in ducks (Zou et al 2010;Li, Hong, et al 2016). Even though differences in virulence have been observed among DEV strains, antigenic nature remains identical for most of the isolates (Kisary & Zsak 1983;Akter et al 2004).…”

Distribution characteristics of DNA vaccine encoded with glycoprotein C from Anatid herpesvirus 1 with chitosan and liposome as deliver carrier in ducks

“…Probe-based qPCR technology is recommended as the reference technique for monitoring gene transfer biodistribution [38]. Therefore, in our previous study, we established a qPCR protocol based on the use of a TaqMan™ probe [39]. In contrast to previous studies, equivalent amounts of DNA (100 μg) were injected in the form of pcDNA3.1-gC, DNA-lipid complexes and chitosan-DNA nanoparticles and 200 ng of gDNA were used in each PCR reaction by preparation of the appropriate dilution ratios.…”

“…The levels of gC in negative and positive control DNA (from non-injected or pcDNA3.1-gC), and test sample DNA were quantified by a qPCR assay as described previously [39]. The primers used for PCR amplification were forward primer (FP), 5 ′ -GAAGGACGGAATGGTGGAAG-3 ′ , and reverse primer (RP), 5 ′ -AGCGGGTAACGAGATCTAATATTGA-3 ′ , which amplify a 78 bp fragment of the AnHV-1 gC gene (GenBank; Accession No.…”

Detection and isolation of Duck Plague virus from field outbreaks in Assam, India

“…The expected size of PCR amplicon was 983 bp for UL44.5 (Fig 3) and 446 bp for DNA polymerase (Fig 4) gene. Thus, the standardized PCR test was found to be useful for detection of DPV DNA (Pritchard et al, 1999).Our targeted DNA polymerase gene usually encodes UL30 protein according to the reports of other workers (Pritchard et al, 1999;Hansen et al, 2000;Zou et al, 2010;Wu et al, 2011;Wu et al, 2012).Various workers had targeted the gene because of the highly conserved nature of the gene (Mondal et al, 2010;Xuefeng et al, 2008). The targeted gene is nonstructural gene which have open reading frame (ORF) of UL-30 which play many roles like DNA replication, DNA replication-recombination and DNA cleavage-encapsidation.…”