LUX real-time PCR assay for the detection of porcine circovirus type 2.
Light Upon eXtension real-time PCR (LUX real-time PCR) assay was developed for the detection of porcine circovirus type 2 (PCV2). The primers flanking a 114 bp fragment were selected from ORF1. The optimized assay could detect 20 viral copies of pBluescript SK+ plasmid containing inserted PCV2 DNA. The dynamic range of quantitative analysis covered a 7-order interval ranging from 20 to 2 x 10(8) genome equivalents per assay with the best results in the range from 2 x 10(2) to 2 x 10(7) viral copies. The LUX real-time PCR assay had a high specificity since it detected PCV2 but not PCV1, CSFV, PRRSV or negative samples. There was good agreement between the LUX real-time PCR and the conventional PCR when lymph nodes from PCV2 infected animals were tested. A comparison of the LUX real-time PCR with the TaqMan PCR and SYBR Green PCR indicated that the amount of viral copies determined using linear calibration curve differed from assay to assay but not more than an order. LUX real-time PCR, similar to the TaqMan PCR, was more specific for generation of fluorogenic signal than SYBR Green PCR.
University of Veterinary Medicine and Pharmacy, Dept. of Infectious Diseases and Parasitology, SK-041 81 Kosice, Slovakia. vilcek@uvm.sk
Stefan
S
Vilcek
Michaela
M
Vlasakova
Anna
A
Jackova
D000818:Animals; D018173:Circoviridae Infections / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D017925:Circovirus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D008198:Lymph Nodes / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology
D017931:DNA Primers; D004279:DNA, Viral