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.

Comparison of colourimetric loop-mediated isothermal amplification (LAMP), PCR and high-resolution melt curve analysis and culture-based diagnostic assays in the detection of three salmonella serotypes in poultry.

The accuracies of two molecular tests, PCR and loop-mediated isothermal amplification (LAMP) assay were compared with bacterial culture in detection of salmonella in poultry clinical samples. The icIR family transcriptional regulator gene was targeted and, out of 56 clinical specimens, 20 poultry field isolates were found positive for salmonella. Along with human isolates, reference strains of three different serovars, Salmonella Enteritidis (S. Enteritidis), S. Typhimurium and S. Infantis, were also tested. Eight different but genetically closely related bacterial genera (Klebsiella, Pseudomonas, Enterobacter, Campylobacter, Staphylococcus, Streptococcus, Escherichia and Pasteurella) were also used to evaluate the specificity of assay. The LAMP assay showed 80.8% sensitivity (95% CI, 0.66-0.95) and 100% specificity (95% CI, 0.71-1.00) when compared with microbiological culture and PCR, both with 100% sensitivity (95% CI, 0.87-1.00) and 100% specificity (95% CI, 0.71-1.00). High-resolution melt (HRM) curve analysis following PCR was able to differentiate between salmonella isolates based on their melting points, and all specimens were genotyped in three distinct HRM curve profiles. Each normalized melt curve profile represented one salmonella serotype and differences between the three melt profiles were correlated with nucleotide variations in the target gene sequences which demonstrated high discriminatory power of this technique. The colourimetric LAMP assay provided an alternative detection method capable of being used in the field, and showed analytical sensitivity for detection of 1 pg of salmonella DNA per reaction. The advantages and disadvantages of each test in detection of salmonella are discussed.