Authors : Jiming Gao, Ning Kong, Yihong Xiao, Angke Zhang and En-Min Zhou
Abstract: The objective of this study was to develop an RNA-dependent real-time reverse-transcriptase PCR (real-time RT-PCR) Method for the detection of relative levels of CD163 and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) N genes in Marc-145 cells. Primers were designed based on the sequence of highly conservative region of PRRSV N, CD163 and β-actin gene as the reference gene. The specificity and sensitivity of real-time RT-PCR Method were determined with the amplification signals Tm peaks in positive controls and the standard curve generated from PRRSV N gene positive plasmid and total RNA dilution end-point standard curve from Marc-145, respectively. The minimum detection levels for PRRSV N gene, CD163 and β-actin gene were 10 copies, 0.25 and 0.025 ng total RNA per reaction mixture, respectively. The R2 and efficiency of standard curves were 0.983 and 102.166% for CD163, 1 and 101.453% for PRRSV N and 0.996 and 90.969% for β-actin genes. The developed real time RT-PCR Method described in this report is more rapid, specific and sensitive than the conventional RT-PCR for the detection of relative levels of PRRSV N and CD163 gene in Marc-145 cell line.
Jiming Gao, Ning Kong, Yihong Xiao, Angke Zhang and En-Min Zhou, 2012. Development of Real-Time PCR Methods for the Detection of CD163 and Porcine Reproductive and Respiratory Syndrome Virus N Genes in Marc-145 Cell. Journal of Animal and Veterinary Advances, 11: 4489-4493.