High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities

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.

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

“…The rnl real-time PCR-HRM delivered the same results as ITS sequencing (Figure 2 and Table S4). Real-time PCR-HRM assays for species identification have previously been established for bacteria [46,47], protozoans [48], and other fungi [38,[49][50][51][52][53][54][55]. Bialek et al (2005) [56] published an alternative PCR-HRM approach using 18 S rDNA primers and an intercalating dye, similar to our study.…”

Development of a real‐time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues

“…The ability to discriminate between both Photobacterium subspecies was validated on a total of 19 Phdp and 11 Phdd strains isolated from Europe, USA and Japan, proving the assay is able to overcome the documented limitation due to the genetic variability of isolates from diverse hosts and geographical origins (Costas et al., ; Magarinos, Toranzo, Barja, & Romalde, ). The observed difference of at least 0.3°C between the two subspecies has been already proved sufficient/suitable for differentiating virus and bacterial species of medical and veterinary importance (Gelaye et al., ; Miller, Zorn, & Brielmeier, ) as well as for the monitoring of bacterial communities in food (Sardaro et al., ). In fact, both classical melting curve analysis and High Resolution Melting (HRM) analysis have been described as powerful techniques for variant scanning and genotyping, enabling to analyse even single genetic variants in nucleic acid sequences (Wittwer, ).…”

RAPD and ERIC-PCR coupled with HRM for species identification of non-dysenteriae Shigella species isolated from clinical specimens; As a potential of alternative method

“…However, melting pro le analysis by Rotor-Gene 6000 series software package was used for clustering of the isolates and differentiating between the serotypes [19]. Miller et al (2015) developed HRM-based assay for identi cation of Pasteurellaceae species by ampli cation of varied regions in 16 s rRNA sequence and they found this method sensitive and rapid for species identi cation [20]. Chen et al (2019) also employed RAPD-HRM assay for differentiation of pathogenic and non-pathogenic Escherichia coli isolates from each other and they found it a sensitive, speci c, and practical assay.…”