Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes

• Published in: Clinical microbiology and infection Vol. 17; no. 9; pp. 1426 - 1434
• Main Authors: Richardson, L.J., Tong, S.Y.C., Towers, R.J., Huygens, F., McGregor, K., Fagan, P.K., Currie, B.J., Carapetis, J.R., Giffard, P.M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier Ltd 01.09.2011; Blackwell Publishing Ltd; Wiley-Blackwell; Elsevier Limited
• Subjects: Base Composition; Biological and medical sciences; Clinical isolates; Computational Biology; Databases, Genetic; DNA, Bacterial - chemistry; Genotype; High-resolution melting; Humans; Infectious diseases; Medical sciences; Melting; multilocus sequence typing; Multilocus Sequence Typing - methods; Multilocus Sequence Typing - standards; Polymorphism, Single Nucleotide; Population structure; Reproducibility of Results; Simpsons index of diversity; Single-nucleotide polymorphism; Streptococcal Infections - microbiology; Streptococcus pyogenes; Streptococcus pyogenes - classification; Streptococcus pyogenes - genetics; Typing; typing; multilocus sequence typing; Simpsons index of diversity; Streptococcus pyogenes; High-resolution melting; single nucleotide polymorphism; High resolution; Melting; Typing; Method; Infection; Streptococcaceae; Microbiological investigation; Bacteria; Micrococcales; Single nucleotide polymorphism
• ISSN: 1198-743X; 1469-0691; 1469-0691
• DOI: 10.1111/j.1469-0691.2010.03433.x

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson's Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59–119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.