MOLECULAR DIFFERENTIATION OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) STRAINS

摘要

THIS INVENTION RELATES TO THE DETECTION AND DISTINGUISHING INFECTIOUS BURSAL DISEASE VIRUS (IBDV) STRAINS BY A FLUORESCENT PROBE BASED REAL-TIME POLYMERASE CHAIN REACTION IN CHICKEN OR OTHER BIRDS. MORE PARTICULARLY, THIS INVENTION RELATES TO DISTINGUISHING DIFFERENT INFECTIOUS BURSAL DISEASE VIRUS (IBDV) STRAINS IN CHICKEN AND OTHER BIRD SAMPLE BY NOVEL SUBTYPE SPECIFIC PRIMERS AND FLUORESCENT PROBE BASED ON ONE-TUBE DUPLEX REAL-TIME POLYMERASE CHAIN REACTION (PCR) METHOD. THE PCR CONDITIONS ARE OPTIMIZED IN ORDER TO OBTAIN OPTIMUM PCR PARAMETERS ON THE INGREDIENTS AND PROFILES USING SAMPLES CONTAINING IBDV RNA IN A TAQMAN PROBE BASED DUPLEX REAL-TIME PCR. HENCE, FOR DIFFERENTIATION OF VERY VIRULENT FROM VACCINE STRAINS OF IBDV BY USING PRIMER FWDC (NUCLEOTIDE POSITION: 2084 TO 2102) AND RVSC (NUCLEOTIDE POSITION: 2178 TO 2197) WERE DESIGNED FROM THE CONSERVED REGION OF VP4 OF BOTH VERY VIRULENT AND CLASSICAL STRAINS, RESPECTIVELY, TO GENERATE A 114 BP AMPLICON. A DUAL-LABELED FLUORESCENT PROBE FAM 5'- TAMRA 3', (NUCLEOTIDE POSITION: 2112 TO 2133), WAS DESIGNED WITH THE SEQUENCE SPECIFIC TO ALIGNED VERY VIRULENT IBDV STRAINS (PROBE 1), AND A SECOND DUAL-LABELED FLUORESCENT PROBE HEX 5'-TAMRA 3', (NUCLEOTIDE POSITION: 2112 TO 2133), WAS DESIGNED WITH THE SEQUENCE SPECIFIC TO ALIGNED CLASSICAL IBDV STRAINS (PROBE 2).