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

Abstract

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 realtime PCR and RFLP provides a rapid, specific method that enables detection and characterization of ILTV directly from field cases.

Molecular epidemiology of infectious laryngotracheitis: a review

“…In North America, target genes sequenced either in their entirety or partially include ICP4, UL47, gB, gG and gM (Ojkic et al, 2006;Callison et al, 2009). In South America the ICP4 gene has been sequenced (Chacon & Ferreira, 2009;Chacon et al, 2010), and in the UK the TK and ICP4 genes are the targets (Creelan et al, 2006). In Europe, larger scale sequencing investigation has been performed using the target genes TK, ICP4, gG, gE, ORFB-TK as well as the gene blocks containing ORFB, ORFC, ORFD, ORFE, gH, TK, and ICP18.5 (Neff et al, 2008;Moreno et al, 2010).…”

“…Differentiation of field isolates and vaccine strains from Brazil was achieved using PCR-RFLP of the TK, ICP4, UL47/gG, and gE genes combined with sequencing of the TK and ICP4 genes (Chacon & Ferreira, 2009;Chacon et al, 2010). Differentiation of field isolates and vaccine strains in the UK combined PCR-RFLP of the TK and ICP4 genes with partial sequencing analysis of the ICP4 gene (Creelan et al, 2006). Differentiation of isolates from Europe generally involved PCR-RFLP of the gE, gG, ICP18.5, ORFB-TK and TK genes in combination with sequencing of the ICP4, TK, gE, gG, ORFB-TK, and ICP18.5 to UL43 genes (Neff et al, 2008;Moreno et al, 2010).…”

“…It is relevant to note that amplification and partial sequencing of either the TK gene or the ICP4 gene had been utilized frequently in the differentiation of GaHV-1 strains from South America, the UK, Africa, Asia and the Middle East. Either one of these two genes have been fundamental in the development of fast, cost-efficient methods to identify and differentiate GaHV-1 isolates (Creelan et al, 2006;Chacon & Ferreira, 2009;Chakma et al, 2010;Sadeghi et al, 2011;Shehata et al, 2013). In addition to PCR-RFLP and PCR-sequencing assays for the differentiation of field isolates and vaccine strains of GaHV-1, development of very sensitive GaHV-1 nucleic acid detection assays have advanced our understanding of the disease epidemiology.…”

“…PCR assays targeting the gC, gE, ICP4, TK and gB genes have been the most widely used method for amplification of GaHV-1 nucleic acid. GaHV-1 PCR assays utilize either nested primers for increased sensitivity (Humberd et al, 2002;Chacon & Ferreira, 2008;Davidson et al, 2009), SYBR Green (Creelan et al, 2006;Callison et al, 2009;Mahmoudian et al, 2011) or Taqman probes (Callison et al, 2007;Ou et al, 2012;Zhao et al, 2013) for realtime detection. Loop-mediated isothermal amplification assays targeting the ICP4 and TK genes have also been utilized in the detection of GaHV-1 nucleic acid (Xie et al, 2010;Ou et al, 2012).…”

Molecular biology of avian infectious laryngotracheitis virus

“…However, more recently protein detection by immunoperoxidase tests, and DNA detection by conventional polymerase chain reaction (PCR) or real-time PCR have become preferred methods of virus diagnosis [1,16,38,54,81,96]. In combination with analyses of restriction fragment polymorphisms and DNA sequencing the PCR technique also facilitates differentiation of virus isolates for epidemiological and phylogenetic studies [16,54].…”