Reverse restriction fragment length polymorphism (RRFLP): A novel technique for genotyping infectious laryngotracheitis virus (ILTV) live attenuated vaccines.
A novel technique, the reverse restriction fragment length polymorphism (RRFLP) assay, was developed as a means of detecting specific informative polymorphic sites in the infectious laryngotracheitis virus (ILTV) genome. During the RRFLP procedure, DNA is digested with restriction enzymes targeting an informative polymorphic site and then used as template in a real-time polymerase chain reaction (PCR) with primers flanking the informative region. The analysis of the DeltaC(t) values obtained from digested and undigested template DNA provides the genotype of the DNA. In this study, the RRFLP assay was applied as a method to differentiate between the two types of infectious laryngotracheitis virus attenuated live vaccines. Sequence analysis of ILTV vaccines revealed an informative polymorphic site in the 5'-non-coding region of the infected cell protein (ICP4) gene. Unique AvaI and AlwI restriction enzyme sites were identified in the tissue culture origin and chicken embryo origin attenuated vaccines, respectively. These two informative polymorphic sites were used in a RRFLP assay to genotype rapidly and reproducibly ILTV attenuated live vaccines.
GTCAllison, LLC, Mocksville, NC 27028, United States.
Scott A
SA
Callison
Sylva M
SM
Riblet
Andres
A
Rodríguez-Avila
Maricarmen
M
García
D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D002645:Chickens; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D001352:Herpesvirus 1, Gallid / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D014613:Vaccines, Attenuated; D014765:Viral Vaccines
D004279:DNA, Viral; D014613:Vaccines, Attenuated; D014765:Viral Vaccines; C028678:CYCGRG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C044346:GGATC-specific type II deoxyribonucleases