A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes
ObjectiveRecently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular ass...
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| Format: | Article |
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Frontiers Media S.A.
2024-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1279186/full |
| _version_ | 1827318957112033280 |
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| author | Xinran Gong Xinran Gong Guang Yang Wei Liu Di Wu Di Wu Di Wu Chunyuan Duan Chunyuan Duan Xinjing Jia Xinjing Jia Zhiqiang Li Zhiqiang Li Zhiqiang Li Xiaocang Zou Renfeng Yu Dayang Zou Yong Wang Yong Wang |
| author_facet | Xinran Gong Xinran Gong Guang Yang Wei Liu Di Wu Di Wu Di Wu Chunyuan Duan Chunyuan Duan Xinjing Jia Xinjing Jia Zhiqiang Li Zhiqiang Li Zhiqiang Li Xiaocang Zou Renfeng Yu Dayang Zou Yong Wang Yong Wang |
| author_sort | Xinran Gong |
| collection | DOAJ |
| description | ObjectiveRecently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting mcr genes in clinical isolates.MethodsThe primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes.ResultsThe standard curves for both the single and multiplex systems showed good linearity (R2 > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 102 copies/μL. The specificity test showed positive amplification results only for strains containing the mcr genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method.ConclusionThe assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research. |
| first_indexed | 2024-04-25T00:10:32Z |
| format | Article |
| id | doaj.art-b9a2b2e28ba64609b0e2eb4684320202 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-25T00:10:32Z |
| publishDate | 2024-03-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj.art-b9a2b2e28ba64609b0e2eb46843202022024-03-13T13:45:00ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2024-03-011510.3389/fmicb.2024.12791861279186A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genesXinran Gong0Xinran Gong1Guang Yang2Wei Liu3Di Wu4Di Wu5Di Wu6Chunyuan Duan7Chunyuan Duan8Xinjing Jia9Xinjing Jia10Zhiqiang Li11Zhiqiang Li12Zhiqiang Li13Xiaocang Zou14Renfeng Yu15Dayang Zou16Yong Wang17Yong Wang18School of Public Health, China Medical University, Shenyang, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaThe 5th Medical Center of General Hospital of Chinese People’s Liberation Army, Beijing, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaSchool of Public Health, China Medical University, Shenyang, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaNational Institute for Communicable Disease Control and Prevention, Beijing, ChinaSchool of Public Health, China Medical University, Shenyang, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaSchool of Public Health, China Medical University, Shenyang, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaSchool of Public Health, China Medical University, Shenyang, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaCentre for Evidence-based Chinese Medicine, Beijing University of Chinese Medicine, Beijing, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaSchool of Public Health, China Medical University, Shenyang, ChinaChinese PLA Center for Disease Control and Prevention, Beijing, ChinaObjectiveRecently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting mcr genes in clinical isolates.MethodsThe primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes.ResultsThe standard curves for both the single and multiplex systems showed good linearity (R2 > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 102 copies/μL. The specificity test showed positive amplification results only for strains containing the mcr genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method.ConclusionThe assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research.https://www.frontiersin.org/articles/10.3389/fmicb.2024.1279186/fullcolistinthe mcr genesmultiplex TaqMan real-time PCRrapid detectionmultidrug-resistant |
| spellingShingle | Xinran Gong Xinran Gong Guang Yang Wei Liu Di Wu Di Wu Di Wu Chunyuan Duan Chunyuan Duan Xinjing Jia Xinjing Jia Zhiqiang Li Zhiqiang Li Zhiqiang Li Xiaocang Zou Renfeng Yu Dayang Zou Yong Wang Yong Wang A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes Frontiers in Microbiology colistin the mcr genes multiplex TaqMan real-time PCR rapid detection multidrug-resistant |
| title | A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes |
| title_full | A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes |
| title_fullStr | A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes |
| title_full_unstemmed | A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes |
| title_short | A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes |
| title_sort | multiplex taqman real time pcr assays for the rapid detection of mobile colistin resistance mcr 1 to mcr 10 genes |
| topic | colistin the mcr genes multiplex TaqMan real-time PCR rapid detection multidrug-resistant |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2024.1279186/full |
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