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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 specificity and sensitivity of one step SYBR green real-time RT-PCR. Plot of the amplification of a 10-fold serial dilution of ch-1a RNA to calculate the detection limit and sensitivity of real-time RT-PCR by analyzing the fluorescence curve of the 228 bp DNA amplification product. NTC is the negative control; ND is the non-diluted sample (9.6 × 105); CSFV is classical swine fever virus; SVDV is swine vesicular disease virus; VESV is vesicular exanthema of swine virus; ch-1a dilutions are 10-2 to 10-7 with copies from 9.6 × 105 down to 9.6 per reaction mixture.
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Figure 2: The specificity and sensitivity of one step SYBR green real-time RT-PCR. Plot of the amplification of a 10-fold serial dilution of ch-1a RNA to calculate the detection limit and sensitivity of real-time RT-PCR by analyzing the fluorescence curve of the 228 bp DNA amplification product. NTC is the negative control; ND is the non-diluted sample (9.6 × 105); CSFV is classical swine fever virus; SVDV is swine vesicular disease virus; VESV is vesicular exanthema of swine virus; ch-1a dilutions are 10-2 to 10-7 with copies from 9.6 × 105 down to 9.6 per reaction mixture.

Mentions: PRRSV, CSFV, SVDV and VESV strains from widely different geographical regions were selected for amplification in the qRT-PCR assay. We found that specific signal was found in PRRSV but not in CSFV, SVDV and VESV. To determine the sensitivity of the one step SYBR green real-time RT-PCR method, ch-1a RNA were diluted 10-fold serially from undiluted to10-6. The diluted ch-1a was still positive at 10-5 dilution (Ct = 38.65), the sensitivity of this method was 9.6 copy numbers per reaction mixture. No primer-dimers or non-specific product were found in negative control (Fig. 2 and Fig. 3).


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 specificity and sensitivity of one step SYBR green real-time RT-PCR. Plot of the amplification of a 10-fold serial dilution of ch-1a RNA to calculate the detection limit and sensitivity of real-time RT-PCR by analyzing the fluorescence curve of the 228 bp DNA amplification product. NTC is the negative control; ND is the non-diluted sample (9.6 × 105); CSFV is classical swine fever virus; SVDV is swine vesicular disease virus; VESV is vesicular exanthema of swine virus; ch-1a dilutions are 10-2 to 10-7 with copies from 9.6 × 105 down to 9.6 per reaction mixture.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The specificity and sensitivity of one step SYBR green real-time RT-PCR. Plot of the amplification of a 10-fold serial dilution of ch-1a RNA to calculate the detection limit and sensitivity of real-time RT-PCR by analyzing the fluorescence curve of the 228 bp DNA amplification product. NTC is the negative control; ND is the non-diluted sample (9.6 × 105); CSFV is classical swine fever virus; SVDV is swine vesicular disease virus; VESV is vesicular exanthema of swine virus; ch-1a dilutions are 10-2 to 10-7 with copies from 9.6 × 105 down to 9.6 per reaction mixture.
Mentions: PRRSV, CSFV, SVDV and VESV strains from widely different geographical regions were selected for amplification in the qRT-PCR assay. We found that specific signal was found in PRRSV but not in CSFV, SVDV and VESV. To determine the sensitivity of the one step SYBR green real-time RT-PCR method, ch-1a RNA were diluted 10-fold serially from undiluted to10-6. The diluted ch-1a was still positive at 10-5 dilution (Ct = 38.65), the sensitivity of this method was 9.6 copy numbers per reaction mixture. No primer-dimers or non-specific product were found in negative control (Fig. 2 and Fig. 3).

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