Quantitative competitive polymerase chain reaction for detection and quantification of infectious bursal disease virus cDNA and RNA.
A polymerase chain reaction (PCR)-based method to measure complementary DNA (cDNA) and RNA levels of infectious bursal disease virus (IBDV) was developed. Quantification was achieved by quantitative competitive PCR (QC-PCR) amplification. A competitor, a deletion mutant of the wild type IBDV cDNA, was 10-fold serially diluted and co-amplified with IBDV cDNA after being reversely transcribed from the viral RNA. After agarose gel electrophoresis, staining, and densitometric scanning, the bands on the digitized images were analyzed and quantified by computer-assisted image analysis. Complementary DNA of standard, as well as variant strains, of serotype 1 IBDV was detected and quantified using the same QC-PCR procedures. The assay could measure IBDV cDNA levels ranging from 1 microgram to 45 fg and RNA levels ranging from 9 micrograms to 45 fg. The results indicated that QC-PCR is sensitive, easy to perform, and suitable for routine quantitation of IBDV cDNA or RNA levels.
Wu
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CC
Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907-1175, USA.
Lin
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TL
Akin
A
A
D000818:Animals; D002645:Chickens; D018076:DNA, Complementary; D004279:DNA, Viral / Q000032:analysis*; D007243:Infectious bursal disease virus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011201:Poultry Diseases / Q000821:virology; D012367:RNA, Viral / Q000032:analysis*
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral