CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A.
BACKGROUND
Serotype-specific PCR assays targeting Salmonella enterica serotypes Typhi and Paratyphi A, the causal agents of typhoid and paratyphoid fevers, are required to accelerate formal diagnosis and to overcome the lack of typing sera and, in some situations, the need for culture. However, the sensitivity and specificity of such assays must be demonstrated on large collections of strains representative of the targeted serotypes and all other bacterial populations producing similar clinical symptoms.


METHODS
Using a new family of repeated DNA sequences, CRISPR (clustered regularly interspaced short palindromic repeats), as a serotype-specific target, we developed a conventional multiplex PCR assay for the detection and differentiation of serotypes Typhi and Paratyphi A from cultured isolates. We also developed EvaGreen-based real-time singleplex PCR assays with the same two sets of primers.


RESULTS
We achieved 100% sensitivity and specificity for each protocol after validation of the assays on 188 serotype Typhi and 74 serotype Paratyphi A strains from diverse genetic groups, geographic origins and time periods and on 70 strains of bacteria frequently encountered in bloodstream infections, including 29 other Salmonella serotypes and 42 strains from 38 other bacterial species.


CONCLUSIONS
The performance and convenience of our serotype-specific PCR assays should facilitate the rapid and accurate identification of these two major serotypes in a large range of clinical and public health laboratories with access to PCR technology. These assays were developed for use with DNA from cultured isolates, but with modifications to the assay, the CRISPR targets could be used in the development of assays for use with clinical and other samples.
Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborative Center for Reference and Research on Salmonella, Institut Pasteur, Paris, France.
Laetitia
L
Fabre
Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborative Center for Reference and Research on Salmonella, Institut Pasteur, Paris, France.
Simon
S
Le Hello
Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborative Center for Reference and Research on Salmonella, Institut Pasteur, Paris, France.
Chrystelle
C
Roux
Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborative Center for Reference and Research on Salmonella, Institut Pasteur, Paris, France.
Sylvie
S
Issenhuth-Jeanjean
Unité des Bactéries Pathogènes Entériques, Centre National de Référence des Escherichia coli, Shigella et Salmonella, WHO Collaborative Center for Reference and Research on Salmonella, Institut Pasteur, Paris, France.
François-Xavier
FX
Weill
D001431:Bacteriological Techniques / Q000379:methods; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010284:Paratyphoid Fever / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012482:Salmonella paratyphi A / Q000235:genetics*; D012485:Salmonella typhi / Q000235:genetics*; D012680:Sensitivity and Specificity; D014435:Typhoid Fever / Q000175:diagnosis* / Q000382:microbiology