Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars
Abstract Background While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the ide...
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| Format: | Article |
| Language: | English |
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BMC
2019-12-01
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| Series: | Annals of Clinical Microbiology and Antimicrobials |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12941-019-0338-5 |
| _version_ | 1818384366508703744 |
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| author | Le Zuo Min Jiang Yixiang Jiang Xiaolu Shi Yinghui Li Yiman Lin Yaqun Qiu Yinhua Deng Minxu Li Zeren Lin Yiqun Liao Jianbin Xie Qingge Li Qinghua Hu |
| author_facet | Le Zuo Min Jiang Yixiang Jiang Xiaolu Shi Yinghui Li Yiman Lin Yaqun Qiu Yinhua Deng Minxu Li Zeren Lin Yiqun Liao Jianbin Xie Qingge Li Qinghua Hu |
| author_sort | Le Zuo |
| collection | DOAJ |
| description | Abstract Background While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars. Methods Serovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella. Results All gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/μL to 1.56 ng/μL of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 °C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated. Conclusions With an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis. |
| first_indexed | 2024-12-14T03:21:07Z |
| format | Article |
| id | doaj.art-adf91a09db4e47108040922080bffb66 |
| institution | Directory Open Access Journal |
| issn | 1476-0711 |
| language | English |
| last_indexed | 2024-12-14T03:21:07Z |
| publishDate | 2019-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Annals of Clinical Microbiology and Antimicrobials |
| spelling | doaj.art-adf91a09db4e47108040922080bffb662022-12-21T23:19:02ZengBMCAnnals of Clinical Microbiology and Antimicrobials1476-07112019-12-0118111010.1186/s12941-019-0338-5Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovarsLe Zuo0Min Jiang1Yixiang Jiang2Xiaolu Shi3Yinghui Li4Yiman Lin5Yaqun Qiu6Yinhua Deng7Minxu Li8Zeren Lin9Yiqun Liao10Jianbin Xie11Qingge Li12Qinghua Hu13Shenzhen Center for Disease Control and PreventionShenzhen Center for Disease Control and PreventionShenzhen Center for Disease Control and PreventionShenzhen Center for Disease Control and PreventionShenzhen Center for Disease Control and PreventionShenzhen Center for Disease Control and PreventionShenzhen Center for Disease Control and PreventionCollege of Life Science, Sichuan UniversityCollege of Life Sciences and Oceanography, Shenzhen UniversityCollege of Life Sciences and Oceanography, Shenzhen UniversitySchool of Life Sciences, Xiamen UniversityShenzhen Center for Disease Control and PreventionSchool of Life Sciences, Xiamen UniversityShenzhen Center for Disease Control and PreventionAbstract Background While Salmonella serotyping is of paramount importance for the disease intervention of salmonellosis, a fast and easy-to-operate molecular serotyping solution remains elusive. We have developed a multiplex ligation reaction based on probe melting curve analysis (MLMA) for the identification of 30 common Salmonella serovars. Methods Serovar-specific primers and probes were designed based on a comparison of gene targets (wzx and wzy encoding for somatic antigen biosynthesis; fliC and fljB for flagellar antigens) from 5868 Salmonella genomes. The ssaR gene, a type III secretion system component, was included for the confirmation of Salmonella. Results All gene targets were detected and gave expected Tm values during assay evaluation. Cross reactions were not demonstrated between the 30 serovars (n = 211), or with an additional 120 serovars (n = 120) and other Enterobacteriaceae (n = 3). The limit of identification for all targets ranged from using 1.2 ng/μL to 1.56 ng/μL of DNA. The intra- and inter-assay standard deviations and the coefficients of variation were no more than 0.5 °C and less than 1% respectively, indicating high reproducibility. From consecutive outpatient stool samples (n = 3590) collected over a 10-month period at 11 sentinel hospitals in Shenzhen, China, we conducted a multicenter study using the traditional Salmonella identification workflow and the MLMA assay workflow in parallel. From Salmonella isolates (n = 496, 13.8%) derived by both workflows, total agreement (kappa = 1.0) between the MLMA assay and conventional serotyping was demonstrated. Conclusions With an assay time of 2.5 h, this simple assay has shown promising potential to provide rapid and high-throughput identification of Salmonella serovars for clinical and public health laboratories to facilitate timely surveillance of salmonellosis.https://doi.org/10.1186/s12941-019-0338-5SalmonellaSerovar identificationMultiplex ligation reaction based on probe melting curve analysisFoodborne illness |
| spellingShingle | Le Zuo Min Jiang Yixiang Jiang Xiaolu Shi Yinghui Li Yiman Lin Yaqun Qiu Yinhua Deng Minxu Li Zeren Lin Yiqun Liao Jianbin Xie Qingge Li Qinghua Hu Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars Annals of Clinical Microbiology and Antimicrobials Salmonella Serovar identification Multiplex ligation reaction based on probe melting curve analysis Foodborne illness |
| title | Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars |
| title_full | Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars |
| title_fullStr | Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars |
| title_full_unstemmed | Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars |
| title_short | Multiplex ligation reaction based on probe melting curve analysis: a pragmatic approach for the identification of 30 common Salmonella serovars |
| title_sort | multiplex ligation reaction based on probe melting curve analysis a pragmatic approach for the identification of 30 common salmonella serovars |
| topic | Salmonella Serovar identification Multiplex ligation reaction based on probe melting curve analysis Foodborne illness |
| url | https://doi.org/10.1186/s12941-019-0338-5 |
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