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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 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.
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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.

Mentions: The amplification curves (Figure 1.a.) and standard curve (Figure 1.b.) of the TaqMan™ FQ-PCR were generated by using the 10-fold dilutions of pcDNA3.1-gC, which has already known its copies to undertake FQ-PCR reaction under optimum conditions with the iCycler IQ Detection System. The curve covered a dynamic range of eight log units of concentration and displayed a clear linear relationship with a correlation coefficient of 1.000 and high amplification efficiency (100%). By using the following formula, we were able to quantify the amount of unknown samples: Y = -3.321X + 45.822 (Y = threshold cycle, X = log starting quantity).


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

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

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.
Mentions: The amplification curves (Figure 1.a.) and standard curve (Figure 1.b.) of the TaqMan™ FQ-PCR were generated by using the 10-fold dilutions of pcDNA3.1-gC, which has already known its copies to undertake FQ-PCR reaction under optimum conditions with the iCycler IQ Detection System. The curve covered a dynamic range of eight log units of concentration and displayed a clear linear relationship with a correlation coefficient of 1.000 and high amplification efficiency (100%). By using the following formula, we were able to quantify the amount of unknown samples: Y = -3.321X + 45.822 (Y = threshold cycle, X = log starting quantity).

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