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The development of a rapid SYBR one step real-time RT-PCR for detection of Porcine Reproductive and Respiratory Syndrome Virus.

Tian H, Wu J, Shang Y, Cheng Y, Liu X - Virol. J. (2010)

Bottom Line: Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak.Primers were designed based on the sequence of highly conservative region of PRRSV N gene.Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiologic Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.

ABSTRACT

Background: Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak. In this study, a rapid SYBR-based, one step real-time RT-PCR quantitative reverse transcription PCR (qRT-PCR) has been developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Primers were designed based on the sequence of highly conservative region of PRRSV N gene.

Results: The sensitivity of the real-time qRT-PCR assay was achieved through PRRSV ch-1a RNA for the generation of a standard curve. The detection limit of the assay was found to be 9.6 RNA copies per reaction mixture. This assay had excellent intra- and inter-assay reproducibility as in total 65 field samples were screened for the presence of PRRSV by conventional RT-PCR in parallel with qRT-PCR, and the detection rate increased from 60.0% to 76.9%. Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).

Conclusion: The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PRRSV in field samples.

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Related in: MedlinePlus

The sensitivity of real-time qRT-PCR for detection of the PRRSV. A 10-fold dilution series of total RNA extracted from a field sample ranging from 10-1 to 10-7 dilutions were tested in parallel in the qRT-PCR assay and in the conventional RT-PCR. The detection limit for the real-time qRT-PCR assay was a 10-5 dilution of sample RNA.
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Figure 4: The sensitivity of real-time qRT-PCR for detection of the PRRSV. A 10-fold dilution series of total RNA extracted from a field sample ranging from 10-1 to 10-7 dilutions were tested in parallel in the qRT-PCR assay and in the conventional RT-PCR. The detection limit for the real-time qRT-PCR assay was a 10-5 dilution of sample RNA.

Mentions: Next, the detection limit of the real-time qRT-PCR assay and the conventional RT-PCR assay were compared. The real-time qRT-PCR assay was able to detect a 10-5 diluted sample, with a corresponding Ct value of 38.65 ± 0.49 (Fig. 4). In parallel, the analytical sensitivity of the conventional RT-PCR was found to be a 10-4 dilution (Fig. 5). These results indicate that the sensitivity of the qRT-PCR assay was observed to increase one log unit than that of the conventional RT-PCR assay.


The development of a rapid SYBR one step real-time RT-PCR for detection of Porcine Reproductive and Respiratory Syndrome Virus.

Tian H, Wu J, Shang Y, Cheng Y, Liu X - Virol. J. (2010)

The sensitivity of real-time qRT-PCR for detection of the PRRSV. A 10-fold dilution series of total RNA extracted from a field sample ranging from 10-1 to 10-7 dilutions were tested in parallel in the qRT-PCR assay and in the conventional RT-PCR. The detection limit for the real-time qRT-PCR assay was a 10-5 dilution of sample RNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874540&req=5

Figure 4: The sensitivity of real-time qRT-PCR for detection of the PRRSV. A 10-fold dilution series of total RNA extracted from a field sample ranging from 10-1 to 10-7 dilutions were tested in parallel in the qRT-PCR assay and in the conventional RT-PCR. The detection limit for the real-time qRT-PCR assay was a 10-5 dilution of sample RNA.
Mentions: Next, the detection limit of the real-time qRT-PCR assay and the conventional RT-PCR assay were compared. The real-time qRT-PCR assay was able to detect a 10-5 diluted sample, with a corresponding Ct value of 38.65 ± 0.49 (Fig. 4). In parallel, the analytical sensitivity of the conventional RT-PCR was found to be a 10-4 dilution (Fig. 5). These results indicate that the sensitivity of the qRT-PCR assay was observed to increase one log unit than that of the conventional RT-PCR assay.

Bottom Line: Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak.Primers were designed based on the sequence of highly conservative region of PRRSV N gene.Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiologic Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.

ABSTRACT

Background: Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak. In this study, a rapid SYBR-based, one step real-time RT-PCR quantitative reverse transcription PCR (qRT-PCR) has been developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Primers were designed based on the sequence of highly conservative region of PRRSV N gene.

Results: The sensitivity of the real-time qRT-PCR assay was achieved through PRRSV ch-1a RNA for the generation of a standard curve. The detection limit of the assay was found to be 9.6 RNA copies per reaction mixture. This assay had excellent intra- and inter-assay reproducibility as in total 65 field samples were screened for the presence of PRRSV by conventional RT-PCR in parallel with qRT-PCR, and the detection rate increased from 60.0% to 76.9%. Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).

Conclusion: The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PRRSV in field samples.

Show MeSH
Related in: MedlinePlus