Development and comparison of a Primer-Probe Energy Transfer based assay and a 5′ conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5′ conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2 × 108 to 2 × 102 copies/μl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5′ conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV.

► We compare PriProET and MGB real-time PCR methods for the detection of ILTV. ► Assays are linear to 200 and ultimately sensitive to 20 copies of virus equivalents. ► Both assays are rapid and sensitive. ► Assays do not detect non-ILTV isolates.