Development and validation of TaqMan probe-based real-time polymerase chain reaction for detection of Porcine Circovirus 2

Abstract

Pigs are under constant threat from many infectious diseases and post-weaning multisystemic syndrome (PWMS) is one among them. The syndrome is caused by the Porcine Circovirus 2 (PCV2) which belongs to the Circoviridae family. Because the symptoms of PCV2 infection and other porcine infectious illnesses, especially porcine reproductive and respiratory syndrome (PRRS) overlap, diagnosis of the former based on clinical indicators could be challenging. A study was conducted to develop a TaqMan real-time polymerase chain reaction (PCR) for the detection of PCV2 genotypes prevalent in Kerala. Primers and TaqMan probes based on the ORF2 nucleotide sequences of the PCV2 prevalent in Kerala were designed. On testing, it was observed that the TaqMan real-time PCR was not able to detect the PCV2genotypes prevalent in Kerala. However, the designed primers (but not the probe) were able to detect these genotypes. Hence, another TaqMan assay specific for detection of 2d was designed as that genotype was predominant in Kerala. The detection limit estimated using the cloned template was found to be 310 copies of the viral genome. The assay was more sensitive in detecting the virus compared to conventional PCR.

Keywords

• pcv2
• orf2
• taqman real-time pcr