Establishment and application of real-time fluorescence-based quantitative PCR for detection of infectious laryngotracheitis virus using SYBR Green I.

Abstract

In this study, a pair of primers were designed and synthesized for the gB gene of infectious laryngotracheitis virus (ILTV) (GenBank accession number: EU104985). The recombinant plasmid was constructed as the positive reference material, and a real-time fluorescence-based quantitative PCR (RFQ-PCR) method was established to detect ILTV using synergy brands (SYBR) Green I. This method could detect 3.34 × 103 copies/μL viral nucleic acid in the initial template, the sensitivity of this method was higher than that of the conventional PCR, and the coefficient of variation (CV) in the repeatability test by this method was 3.35%. At the same time, the method was used to detect 14 suspected pathological samples for clinical analysis, and the results showed that 10 positive samples were detected, and the standard S-shaped curve was amplified. It was concluded that the RFQ-PCR method established in this study was highly sensitive, specific and repeatable, it was suitable for early clinical detection and epidemiological investigation of ILTV, and was significant in effectively controlling the occurrence and transmission of ILTV.