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...
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ftdoajarticles:oai:doaj.org/article:92c375e7de4c494195f4255dab2290ac 2023-05-15T15:12:17+02:00 CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. Laetitia Fabre Simon Le Hello Chrystelle Roux Sylvie Issenhuth-Jeanjean François-Xavier Weill 2014-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0002671 https://doaj.org/article/92c375e7de4c494195f4255dab2290ac EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC3907412?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0002671 https://doaj.org/article/92c375e7de4c494195f4255dab2290ac PLoS Neglected Tropical Diseases, Vol 8, Iss 1, p e2671 (2014) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2014 ftdoajarticles https://doi.org/10.1371/journal.pntd.0002671 2022-12-31T08:44:01Z 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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 8 1 e2671 |
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Directory of Open Access Journals: DOAJ Articles |
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English |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Laetitia Fabre Simon Le Hello Chrystelle Roux Sylvie Issenhuth-Jeanjean François-Xavier Weill CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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. |
format |
Article in Journal/Newspaper |
author |
Laetitia Fabre Simon Le Hello Chrystelle Roux Sylvie Issenhuth-Jeanjean François-Xavier Weill |
author_facet |
Laetitia Fabre Simon Le Hello Chrystelle Roux Sylvie Issenhuth-Jeanjean François-Xavier Weill |
author_sort |
Laetitia Fabre |
title |
CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. |
title_short |
CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. |
title_full |
CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. |
title_fullStr |
CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. |
title_full_unstemmed |
CRISPR is an optimal target for the design of specific PCR assays for salmonella enterica serotypes Typhi and Paratyphi A. |
title_sort |
crispr is an optimal target for the design of specific pcr assays for salmonella enterica serotypes typhi and paratyphi a. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2014 |
url |
https://doi.org/10.1371/journal.pntd.0002671 https://doaj.org/article/92c375e7de4c494195f4255dab2290ac |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 8, Iss 1, p e2671 (2014) |
op_relation |
http://europepmc.org/articles/PMC3907412?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0002671 https://doaj.org/article/92c375e7de4c494195f4255dab2290ac |
op_doi |
https://doi.org/10.1371/journal.pntd.0002671 |
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PLoS Neglected Tropical Diseases |
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8 |
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1 |
container_start_page |
e2671 |
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1766342995761692672 |