Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus.
Chicken anemia virus (CAV) is a ubiquitous pathogen of poultry. A CAV specific TaqMan-based PCR and RT-PCR assay for real-time quantitation of viral load and relative quantitation of virus-specific transcript levels was developed. Detection of viral DNA copy number from infected MDCC-CU147 cells was determined by extrapolation from a CAV plasmid-based standard curve. Viral load increased proportionally with increasing cell number harvested, increasing from 4x10(2) copies in 250 cells with 38% virus positive cells in an indirect immunofluorescence assay to 8x10(5) copies in 250,000 cells with 64% infected cells. The estimated average viral copy number per infected cell ranged from 5 to 14. Strain-specific primers were developed to distinguish between the Cux-1 and CIA-1 strains of CAV. These primers exhibited a 3 to 4 log differential in amplification comparing homologous versus heterologous virus-primer combinations. The sensitivity of the real-time assay was found to be comparable to a nested PCR assay using DNA samples from a SPF poultry flock exposed to the SH-1 strain of CAV. The real-time PCR detected from 1.7 to 4.2 target molecules in three out of four samples that were positive by nested PCR using 50% of the DNA used in the nested PCR. Relative viral transcript levels for Cux-1 and CIA-1 infected cell cultures increased proportionally with increasing cell numbers harvested for RNA extraction. This assay will be important for both diagnosis and in understanding the complex pathogenesis of CAV infection.
Markowski-Grimsrud
Carrie J
CJ
Department of Microbiology and Immunology, Unit of Avian Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Miller
Myrna M
MM
Schat
Karel A
KA
D000818:Animals; D002460:Cell Line; D017727:Chicken anemia virus / Q000235:genetics / Q000302:isolation & purification*; D002645:Chickens / Q000821:virology*; D018173:Circoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D004279:DNA, Viral / Q000032:analysis / Q000097:blood; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D011201:Poultry Diseases / Q000175:diagnosis*; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D019914:Taq Polymerase / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D019562:Viral Load
D004279:DNA, Viral; D012367:RNA, Viral; D019914:Taq Polymerase