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

Abstract

Background: Anatid 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.

Results: The 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 x 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.

Conclusions: The 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.

Figure 1

The amplification curves (Figure 1.a.) and standard curve (Figure 1.b.) of the TaqMan™ FQ-PCR detection. Ten-fold dilutions of standard DNA ranging from 1.0 × 10⁸ to 1.0 × 10¹ copies/reaction were used (1-8), as indicated in the x-axis, whereas the corresponding Ct values are presented on the y-axis. The correlation coefficient and the slope value of the regression curve were calculated and indicated.

Figure 2

The sensitivity of TaqMan™ FQ-PCR detection. Ten-fold serial dilutions of AHV-1 standard template were used (1-6), 1.0 × 10⁵-1.0 × 10⁰ copies/reaction of AHV-1 standard template. As shown in the figure, the detection limit for the assay was 1.0 × 10¹ copies.

Figure 3

The specificity of TaqMan™ FQ-PCR detection. The pcDNA3.1-gC (1), AHV-1 Cha (2), AHV-1 Chv (3), gosling new type viral enteritis virus (4), duck hepatitis virus type1 (5), duck adenovirus (6), goose parvovirus (7), Marek's disease virus (8), Pasteurella multocida (5:A) (9), Escherichia coli (O78) (10), Salmonella enteritidis (No. 50338) (11), the liver DNA of the healthy duck (12) and NTC (13) were tested to evaluate the specificity of the assay by FQ-PCR.