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Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a...

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Autores principales: Xiong, Dan, Song, Li, Pan, Zhiming, Jiao, Xinan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079294/
https://www.ncbi.nlm.nih.gov/pubmed/30108571
http://dx.doi.org/10.3389/fmicb.2018.01718
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author Xiong, Dan
Song, Li
Pan, Zhiming
Jiao, Xinan
author_facet Xiong, Dan
Song, Li
Pan, Zhiming
Jiao, Xinan
author_sort Xiong, Dan
collection PubMed
description Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/μL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations.
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spelling pubmed-60792942018-08-14 Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay Xiong, Dan Song, Li Pan, Zhiming Jiao, Xinan Front Microbiol Microbiology Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional Salmonella serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/μL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations. Frontiers Media S.A. 2018-07-31 /pmc/articles/PMC6079294/ /pubmed/30108571 http://dx.doi.org/10.3389/fmicb.2018.01718 Text en Copyright © 2018 Xiong, Song, Pan and Jiao. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Xiong, Dan
Song, Li
Pan, Zhiming
Jiao, Xinan
Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
title Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
title_full Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
title_fullStr Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
title_full_unstemmed Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
title_short Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay
title_sort identification and discrimination of salmonella enterica serovar gallinarum biovars pullorum and gallinarum based on a one-step multiplex pcr assay
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079294/
https://www.ncbi.nlm.nih.gov/pubmed/30108571
http://dx.doi.org/10.3389/fmicb.2018.01718
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