Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled fluorescent probes.
A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical (Cl), variant (V) and very virulent (vv) strains of infectious bursal disease virus (IBDV). The assay was highly sensitive and could detect as little as 3 x 10(2) to 3 x 10(3) copies of viral template. Viral genomic copy number could be accurately assayed over a broad range of 7-8 logs of viral genome. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. A total of 130 field and experimental variant strain isolates were tested using this assay. The classical sequence-specific probe also demonstrated high sensitivity and specificity, and positively detected a total of 87 STC isolates, both field and experimental isolates, while differentiating between isolates that were variant and classical strains. The very virulent sequence-specific probe detected positively the Holland vvIBDV isolate and did not react with classical or variant strains. Rapid identification of viral strain is a primary concern to poultry flock health programs to ensure administered vaccines will protect against current strains of virus circulating in the flock. The ability to quantify virus concurrently is also of assistance in identifying the progression of disease outbreaks within the flock.
Peters
Michelle A
MA
Department of Veterinary Pathobiology, Purdue University, 406 S. University St., West Lafayette, IN 47907-2065, USA.
Lin
Tsang Long
TL
Wu
Ching Ching
CC
D000818:Animals; D001483:Base Sequence; D002060:Bursa of Fabricius / Q000821:virology; D002645:Chickens / Q000821:virology*; D005456:Fluorescent Dyes; D007243:Infectious bursal disease virus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000662:veterinary; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D015678:Viral Structural Proteins / Q000235:genetics
D005456:Fluorescent Dyes; C478245:VP4 protein, infectious bursal disease virus; D015678:Viral Structural Proteins