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Published on: November 28, 2016
High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal
Manuel Miller1, Julia Zorn1, Markus Brielmeier1
1Research Unit Comparative Medicine, German Research Center for Environmental Health, Neuherberg, Germany.
Abstract:
Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.
Insights
This study introduces a new real-time PCR and high-resolution melting curve analysis (HRM) method for rapid identification of Pasteurellaceae bacterial pathogens in lab mice. This technique ensures accurate health monitoring for experimental animals.
Area of Science:
- Microbiology
- Veterinary Science
- Molecular Diagnostics
Background:
- Pasteurellaceae are common bacterial pathogens in laboratory mice, impacting research integrity.
- Accurate identification and differentiation of these bacteria are crucial for effective health monitoring programs in animal facilities.
Purpose of the Study:
- To develop and evaluate a novel real-time PCR combined with high-resolution melting curve analysis (HRM) assay.
- To enable sensitive, rapid, and reliable identification and differentiation of key Pasteurellaceae species commonly found in laboratory mice.
Main Methods:
- Real-time PCR assay targeting a variable region of the 16S rRNA gene.
- High-resolution melting curve analysis (HRM) for differentiating bacterial species based on unique melting profiles.
- Validation using six reference strains and 25 unknown isolates from an external diagnostic laboratory.
Main Results:
- Distinct melting profiles were observed for the targeted Pasteurellaceae species (Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium) due to sequence variations.
- The real-time PCR/HRM assay demonstrated high concordance with partial 16S rRNA sequencing for identifying unknown isolates.
- The method proved to be sensitive, rapid, and a closed-tube procedure.
Conclusions:
- The developed real-time PCR/HRM assay is a valuable tool for the accurate identification of Pasteurellaceae species.
- This method significantly enhances the health monitoring of laboratory mice, ensuring the reliability of research outcomes.
- The closed-tube nature of the assay minimizes contamination risks and improves laboratory workflow efficiency.
