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Rapid and specific detection of porcine parvovirus using real-time PCR and high resolution melting (HRM) analysis.

Yu HQ, Cai XQ, Lin ZX, Li XL, Yue QY, Li R, Zhu XQ - BMC Vet. Res. (2015)

Bottom Line: Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world.The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal.Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods.

View Article: PubMed Central - PubMed

Affiliation: Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province, 510630, PR China. haiqiongyu@139.com.

ABSTRACT

Background: Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world.

Results: We developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the detection of PPV. Primers targeting the VP gene were highly specific, as evidenced by the negative amplification of closely related viruses, such as porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal. Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods. The assay has the accuracy of 100% in reference to labeled real time PCR, when it was tested on 45 clinical samples.

Conclusions: The present study demonstrated that the established assay integrating real-time PCR and HRM is relatively cost-effective and more stable, which provides an alternative tool for rapid, simple, specific and sensitive detection of PPV.

No MeSH data available.


Related in: MedlinePlus

Ten fold diluted genomic DNA of porcine parvovirus (PPV) tested with two kind of real time PCR (100 ng to 0.1 pg). (a) TaqMan real time PCR amplification for 10 fold diluted genomic DNA; (b) Real time PCR with HRM analysis for 10 fold diluted genomic DNA; (c) A linear regression of the data providing a formula of y = -3.1929x + 40.387 (R2 = 0.9972) between Cp value and log concentration (100 ng to 1 pg), when performed with TaqMan real time PCR; (d) A linear regression of the data providing a formula of y = -3.1332x + 38.875 (R2 = 0.9974), between Cp value and log concentration (100 ng to 1 pg) when performed with real time PCR coupled with HRM analysis.
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Fig1: Ten fold diluted genomic DNA of porcine parvovirus (PPV) tested with two kind of real time PCR (100 ng to 0.1 pg). (a) TaqMan real time PCR amplification for 10 fold diluted genomic DNA; (b) Real time PCR with HRM analysis for 10 fold diluted genomic DNA; (c) A linear regression of the data providing a formula of y = -3.1929x + 40.387 (R2 = 0.9972) between Cp value and log concentration (100 ng to 1 pg), when performed with TaqMan real time PCR; (d) A linear regression of the data providing a formula of y = -3.1332x + 38.875 (R2 = 0.9974), between Cp value and log concentration (100 ng to 1 pg) when performed with real time PCR coupled with HRM analysis.

Mentions: In order to evaluate the detection limit and correlation of the real-time PCR coupled with HRM analysis, the sensitivity of the real-time PCR was compared with a TaqMan real time PCR reported previously [7], serial dilutions of plasmid between 107 copies and 101 copies per reaction or 10-fold diluted genomic DNA was tested. Standard curves were generated using the protocol described above. The assay approaches the sensitivity of TaqMan real time PCR (Figure 1).Figure 1


Rapid and specific detection of porcine parvovirus using real-time PCR and high resolution melting (HRM) analysis.

Yu HQ, Cai XQ, Lin ZX, Li XL, Yue QY, Li R, Zhu XQ - BMC Vet. Res. (2015)

Ten fold diluted genomic DNA of porcine parvovirus (PPV) tested with two kind of real time PCR (100 ng to 0.1 pg). (a) TaqMan real time PCR amplification for 10 fold diluted genomic DNA; (b) Real time PCR with HRM analysis for 10 fold diluted genomic DNA; (c) A linear regression of the data providing a formula of y = -3.1929x + 40.387 (R2 = 0.9972) between Cp value and log concentration (100 ng to 1 pg), when performed with TaqMan real time PCR; (d) A linear regression of the data providing a formula of y = -3.1332x + 38.875 (R2 = 0.9974), between Cp value and log concentration (100 ng to 1 pg) when performed with real time PCR coupled with HRM analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4356059&req=5

Fig1: Ten fold diluted genomic DNA of porcine parvovirus (PPV) tested with two kind of real time PCR (100 ng to 0.1 pg). (a) TaqMan real time PCR amplification for 10 fold diluted genomic DNA; (b) Real time PCR with HRM analysis for 10 fold diluted genomic DNA; (c) A linear regression of the data providing a formula of y = -3.1929x + 40.387 (R2 = 0.9972) between Cp value and log concentration (100 ng to 1 pg), when performed with TaqMan real time PCR; (d) A linear regression of the data providing a formula of y = -3.1332x + 38.875 (R2 = 0.9974), between Cp value and log concentration (100 ng to 1 pg) when performed with real time PCR coupled with HRM analysis.
Mentions: In order to evaluate the detection limit and correlation of the real-time PCR coupled with HRM analysis, the sensitivity of the real-time PCR was compared with a TaqMan real time PCR reported previously [7], serial dilutions of plasmid between 107 copies and 101 copies per reaction or 10-fold diluted genomic DNA was tested. Standard curves were generated using the protocol described above. The assay approaches the sensitivity of TaqMan real time PCR (Figure 1).Figure 1

Bottom Line: Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world.The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal.Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods.

View Article: PubMed Central - PubMed

Affiliation: Technical Center, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guangzhou, Guangdong Province, 510630, PR China. haiqiongyu@139.com.

ABSTRACT

Background: Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world.

Results: We developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the detection of PPV. Primers targeting the VP gene were highly specific, as evidenced by the negative amplification of closely related viruses, such as porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal. Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods. The assay has the accuracy of 100% in reference to labeled real time PCR, when it was tested on 45 clinical samples.

Conclusions: The present study demonstrated that the established assay integrating real-time PCR and HRM is relatively cost-effective and more stable, which provides an alternative tool for rapid, simple, specific and sensitive detection of PPV.

No MeSH data available.


Related in: MedlinePlus