High-resolution melting curve analysis for infectious bronchitis virus strain differentiation

Area of Science:

• Veterinary Virology
• Molecular Diagnostics

Background:

• Avian infectious bronchitis virus (IBV), a member of the Coronaviridae family, causes significant diseases in poultry.
• Current IBV classification relies on S1 gene sequencing, a time-consuming and expensive method.
• There is a need for rapid, cost-effective diagnostic tools for IBV strain identification.

Purpose of the Study:

• To develop and evaluate a novel quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) assay for IBV strain classification.
• To assess the cost-effectiveness and speed of the qRT-PCR/HRM assay compared to traditional sequencing methods.

Main Methods:

• A qRT-PCR assay was developed targeting a partial fragment of the IBV S1 gene.
• High-resolution melting (HRM) curve analysis was performed on 23 IBV-positive samples from Jordan.
• The assay utilized common IBV vaccine strains (Mass and 4/91) as references.
• Sequencing of the S1 gene was conducted to validate the qRT-PCR/HRM results.

Main Results:

• The qRT-PCR/HRM assay successfully classified IBV samples into four distinct clusters based on melting point similarities.
• Cluster 1 showed similarity to the 4/91 vaccine strain; Cluster 2 to the Mass vaccine strain.
• Cluster 3 contained novel IBV strains, including a variant II-like (IS-1494-06) field strain, not matching reference vaccines.
• Cluster 4 identified a unique Q1 IBV strain.

Conclusions:

• The developed qRT-PCR/HRM assay is a rapid and cost-effective tool for IBV strain detection and classification.
• This method accurately identifies both vaccine-related and novel IBV strains, as confirmed by S1 gene sequencing.
• The assay offers a valuable alternative to traditional sequencing for routine IBV diagnostics in poultry.