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.

Automated summary

The accuracies of PCR and LAMP assay in detecting salmonella in poultry clinical samples were compared with bacterial culture. The LAMP assay showed high sensitivity and specificity, and can be used in the field. HRM curve analysis following PCR can differentiate salmonella isolates based on their melting points.