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

Zou Q, Sun K, Cheng A, Wang M, Xu C, Zhu D, Jia R, Luo Q, Zhou Y, Chen Z, Chen X - Virol. J. (2010)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China.

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.

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Related in: MedlinePlus

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.
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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.

Mentions: The specificity test showed that pcDNA3.1-gC, AHV-1 attenuated vaccine (AHV-1 Cha) strain virus and AHV-1 virulent (AHV-1 Chv) strain virus were found positive for AHV-1 by the established FQ-PCR assay, while the bacteria, remaining viruses including negative control (liver sample of the healthy duck) were negative (Figure 3.). The results were confirmed by gel electrophoresis, there was a band of the expected size (78 bp) observed exclusively from samples of pcDNA3.1-gC, AHV-1 Cha and AHV-1 Chv. It indicated that the established FQ-PCR assay was highly specific.


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

Zou Q, Sun K, Cheng A, Wang M, Xu C, Zhu D, Jia R, Luo Q, Zhou Y, Chen Z, Chen X - Virol. J. (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2837632&req=5

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.
Mentions: The specificity test showed that pcDNA3.1-gC, AHV-1 attenuated vaccine (AHV-1 Cha) strain virus and AHV-1 virulent (AHV-1 Chv) strain virus were found positive for AHV-1 by the established FQ-PCR assay, while the bacteria, remaining viruses including negative control (liver sample of the healthy duck) were negative (Figure 3.). The results were confirmed by gel electrophoresis, there was a band of the expected size (78 bp) observed exclusively from samples of pcDNA3.1-gC, AHV-1 Cha and AHV-1 Chv. It indicated that the established FQ-PCR assay was highly specific.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Avian Disease Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China.

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.

Show MeSH
Related in: MedlinePlus