Establishment of Real-time (RT-)PCR for Common Swine Respiratory Viral Disease Detection
Date Issued
2010
Date
2010
Author(s)
Hsiao, Chun-Ni
Abstract
Porcine circovirus (PCV2)、porcine reproductive and respiratory syndrome virus (PRRSV)、swine influenza virus (SIV)、Torque teno virus (TTV)、porcine parvovirus (PPV) and classical swine fever virus (CSFV) are the major pathogens of combined infection pigs. The combined infection not only causes serious economic loss but also increases the diagnostic difficulty. Real-time polymerase chain reaction (real-time PCR) is a powerful molecular technology for pathogen detection and quantification via a mathematical and computerizable way. The further development of multiplex real-time PCR can simultaneously analyze several targets in a single reaction, which also reduced sample volume, testing time, and cost. In the present study, we established the SYBR Green real-time (RT-)PCR for each of the above mentioned six important swine viral pathogens. The detection limit of the DNA/RNA assays are 22.7 copies/μL for PCV2, 17.6 copies/μl for PPV, 4.47 copies/μl for TTV1 and 101 TCID50/mL for PRRSV and CSFV. Besides, we also build singleplex TaqMan real-time (RT-)PCR. The detection limit of the DNA/RNA assays are 22 copies/μL for PCV2、15.7 copies/μl for PPV, 4.47 copies/μl for TTV1, 100 TCID50/mL for PRRSV, and 101 TCID50/mL for CSFV. Furthermore, the conditions of duplex TaqMan real-time (RT-)PCR for PCV2/PPV and PRRSV/SIV by decreasing concentrations of probe-primers and the detection limits of triplex TaqMan real-time (RT-)PCR for PCV2/PPV/TTV1 and PRRSV/SIV/CSF without optimization were both evaluated. These assays established may provide a more rapid and sensitive methodologies for swine disease diagnosis in the field.
Subjects
Swine viral disease diagnosis
SYBR Green real-time (RT-)PCR
TaqMan real-time (RT-)PCR
Multiplex
Parallel
Type
thesis
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