Comparison of PCR-HRM, colorimetric LAMP and culture based diagnostic assays in the detection of endometritis caused by Streptococcus equi subsp. zooepidemicus in mares

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the causative agents of equine endometritis. In this study, a panel of different bacterial species, and colonies derived from bacteriological cultures of 38 clinical samples, were subjected to Loop-Mediated Isothermal Amplification (LAMP) assay and PCR, followed by high-resolution melt (HRM) curve analysis. All clinical samples were genotyped into three distinct groups based on HRM curve analysis. Differences in melting curve profiles were a reflection of DNA variation in sorD gene which was confirmed by DNA sequencing. A mathematical model based on Genetic Confidence Percentage (GCP) was used in HRM curve analysis and a cut-off point value was established which differentiated S. zooepidemicus isolates without requiring visual interpretation of curve profiles. The accuracy of PCR-HRM and bacterial culture in detection of S. zooepidemicus were identical with 100% sensitivity and specificity, while LAMP assay had similar specificity but a lower sensitivity (89.5%). PCR-HRM and LAMP assay provided an effective detection method with a turn-around time of six hours for PCR-HRM and 120 min for LAMP assay, compared to a minimum three days that was required when routine bacteriological culture method was used. In summary, results indicate that LAMP had the quickest turnaround, and HRM curve analysis could potentially be used for genotyping without DNA sequencing. Any mare suspected of endometritis will benefit from developed rapid diagnostic tests for detection of S. zooepidemicus and proper treatment prior to being bred and will mitigate unnecessary treatment and antibiotic resistance.

Automated summary

In this study, a combination of Loop-Mediated Isothermal Amplification (LAMP) and PCR techniques with high-resolution melt (HRM) curve analysis were used to analyze different bacterial species and clinical samples. HRM curve analysis genotyped all clinical samples into three distinct groups based on differences in melting curve profiles, which reflected DNA variation in the sorD gene. A mathematical model based on Genetic Confidence Percentage (GCP) and a cut-off point value were established for HRM curve analysis, enabling differentiation of S. zooepidemicus isolates without visual interpretation of curve profiles. PCR-HRM and LAMP assay showed high accuracy in detecting S. zooepidemicus, with PCR-HRM having a turnaround time of six hours and LAMP assay requiring 120 minutes compared to the minimum three days for routine bacterial culture method. These rapid diagnostic tests can benefit mares suspected of endometritis by allowing early detection of S. zooepidemicus and proper treatment before breeding, reducing unnecessary treatment and antibiotic resistance.