Rapid detection and characterization from field cases of infectious laryngotracheitis virus by real-time polymerase chain reaction and restriction fragment length polymorphism

A real-time polymerase chain reaction ( PCR) assay was developed to specifically amplify infectious laryngotracheitis virus ( ILTV) DNA from field samples. The 222-base-pair PCR fragment was amplified using primers located in a conserved region of the infected cell protein 4 gene that was demonstrated in this work to encompass a single nucleotide polymorphism. Subsequent restriction fragment length polymorphism ( RFLP) analysis of real-time PCR amplified fragments from a range of ILTV isolates using the restriction endonuclease MspI enabled differentiation between older ILTV isolates that were prevalent in the 1960s prior to the availability of vaccine strains and more recent isolates that predominantly are identical to vaccine strains. The assay, using real-time PCR, RFLP and sequence analysis, was used to characterize two recent field cases of infectious laryngotracheitis from Northern Ireland. One of the field cases was demonstrated to be similar to older "wild-type'' isolates, while the other field case was identified to have a concurrent ILTV infection of both "wild-type'' and vaccinal type origin. The assay described here using real-time PCR and RFLP provides a rapid, specific method that enables detection and characterization of ILTV directly from field cases.