Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection
ObjectiveTo develop plasmid DNA reference materials that can be used for polymyxin antibiotic resistance encoding genes rapid detection.MethodsThe reference sequence of mcr1， mcr3 and mcr5 were retrieved from the National Center for Biotechnology Information to construct recombinant plasmids and...
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| Format: | Article |
| Language: | zho |
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The Editorial Office of Chinese Journal of Food Hygiene
2022-03-01
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| Series: | Zhongguo shipin weisheng zazhi |
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| Online Access: | http://www.zgspws.com/zgspwszz/article/abstract/202202003?st=article_issue |
| _version_ | 1797976883999014912 |
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| author | CHEN Jin SU Xiumin YANG Jing CAO Yanwei TIAN Yuanyuan FENG Chengqian ZHAO Hongyang LU Xing’an CUI Shenghui YANG Baowei |
| author_facet | CHEN Jin SU Xiumin YANG Jing CAO Yanwei TIAN Yuanyuan FENG Chengqian ZHAO Hongyang LU Xing’an CUI Shenghui YANG Baowei |
| author_sort | CHEN Jin |
| collection | DOAJ |
| description | ObjectiveTo develop plasmid DNA reference materials that can be used for polymyxin antibiotic resistance encoding genes rapid detection.MethodsThe reference sequence of mcr1， mcr3 and mcr5 were retrieved from the National Center for Biotechnology Information to construct recombinant plasmids and strains. The recombinant strains were subcultured for 15 generations to detect the genetic stability of the target gene. The recombinant plasmid was extracted and vacuum dried to prepare the standard sample. The limit of detection （LOD） of PCR and RTqPCR was determined after the standard sample was hydrated and continuously diluted with 10fold gradient， respectively. Multitube plasmid DNA standard samples were randomly selected to qualitatively and quantitatively evaluate the uniformity and storage stability at 4 ℃， 37 ℃ and -20 ℃ by PCR and UV spectrophotometer.ResultsFragments of mcr1， mcr3 and mcr5 that encoding polymyxin resistance mechanism were successfully obtained. Target genes in the recombinant strains could stably inherited after subcultured for 15 generations. The LOD of PCR and RTqPCR of mcr1， mcr3 and mcr5 standard samples were 1.67×104 and 1.67 copies/μL， 1.31×104 and 13.1 copies/μL， 1.55×105 and 1.55 copies/μL， respectively. The mass F values of 12 standard samples randomly selected for each gene were all less than the F critical value， each gene could be detected by RTqPCR and indicating that the uniformity of the samples met the requirements. When standard samples were stored at 4 ℃ for 90 d， 37 ℃ for 14 d and -20 ℃ for 360 d， the qualitative and quantitative test result showed it was stable without significant difference.ConclusionIn this study， plasmid DNA standard samples that carrying mcr1， mcr3 and mcr5 were prepared. The target genes could be stably inherited， the standard samples had good uniformity and storage stability， and could be used as a quality control samples for polymyxin antibiotics resistance encoding genes detection and mechanisms prediction. |
| first_indexed | 2024-04-11T04:58:05Z |
| format | Article |
| id | doaj.art-daab25db4ef44233b741f598d79ca73b |
| institution | Directory Open Access Journal |
| issn | 1004-8456 |
| language | zho |
| last_indexed | 2024-04-11T04:58:05Z |
| publishDate | 2022-03-01 |
| publisher | The Editorial Office of Chinese Journal of Food Hygiene |
| record_format | Article |
| series | Zhongguo shipin weisheng zazhi |
| spelling | doaj.art-daab25db4ef44233b741f598d79ca73b2022-12-26T07:33:34ZzhoThe Editorial Office of Chinese Journal of Food HygieneZhongguo shipin weisheng zazhi1004-84562022-03-0134220321010.13590/j.cjfh.2022.02.0031004-8456（2022）02-0203-08Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detectionCHEN Jin0SU Xiumin1YANG Jing2CAO Yanwei3TIAN Yuanyuan4FENG Chengqian5ZHAO Hongyang6LU Xing’an7CUI Shenghui8YANG Baowei9College of Food Science and Engineering， Northwest A&F University， Shaanxi Yangling 712100， ChinaCollege of Food Science and Engineering， Northwest A&F University， Shaanxi Yangling 712100， ChinaYangling Quality and Technical Inspection Institute， Shaanxi Yangling 712100， ChinaHebei Forest， Grass and Flower Quality Inspection and Testing Center， Hebei Shijiazhuang 050081， ChinaCollege of Food Science and Engineering， Northwest A&F University， Shaanxi Yangling 712100， ChinaMarket Supervision and Administration Bureau of Hantai District of Hanzhong， Shaanxi Hanzhong 723000， ChinaChinese Academy of Inspection and Quarantine， Beijing 100176， ChinaChinese Academy of Inspection and Quarantine， Beijing 100176， ChinaNational Institutes for Food and Drug Control， Beijing 100500， ChinaCollege of Food Science and Engineering， Northwest A&F University， Shaanxi Yangling 712100， ChinaObjectiveTo develop plasmid DNA reference materials that can be used for polymyxin antibiotic resistance encoding genes rapid detection.MethodsThe reference sequence of mcr1， mcr3 and mcr5 were retrieved from the National Center for Biotechnology Information to construct recombinant plasmids and strains. The recombinant strains were subcultured for 15 generations to detect the genetic stability of the target gene. The recombinant plasmid was extracted and vacuum dried to prepare the standard sample. The limit of detection （LOD） of PCR and RTqPCR was determined after the standard sample was hydrated and continuously diluted with 10fold gradient， respectively. Multitube plasmid DNA standard samples were randomly selected to qualitatively and quantitatively evaluate the uniformity and storage stability at 4 ℃， 37 ℃ and -20 ℃ by PCR and UV spectrophotometer.ResultsFragments of mcr1， mcr3 and mcr5 that encoding polymyxin resistance mechanism were successfully obtained. Target genes in the recombinant strains could stably inherited after subcultured for 15 generations. The LOD of PCR and RTqPCR of mcr1， mcr3 and mcr5 standard samples were 1.67×104 and 1.67 copies/μL， 1.31×104 and 13.1 copies/μL， 1.55×105 and 1.55 copies/μL， respectively. The mass F values of 12 standard samples randomly selected for each gene were all less than the F critical value， each gene could be detected by RTqPCR and indicating that the uniformity of the samples met the requirements. When standard samples were stored at 4 ℃ for 90 d， 37 ℃ for 14 d and -20 ℃ for 360 d， the qualitative and quantitative test result showed it was stable without significant difference.ConclusionIn this study， plasmid DNA standard samples that carrying mcr1， mcr3 and mcr5 were prepared. The target genes could be stably inherited， the standard samples had good uniformity and storage stability， and could be used as a quality control samples for polymyxin antibiotics resistance encoding genes detection and mechanisms prediction.http://www.zgspws.com/zgspwszz/article/abstract/202202003?st=article_issueantibiotic resistance geneplasmid dna reference materialuniformitystability |
| spellingShingle | CHEN Jin SU Xiumin YANG Jing CAO Yanwei TIAN Yuanyuan FENG Chengqian ZHAO Hongyang LU Xing’an CUI Shenghui YANG Baowei Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection Zhongguo shipin weisheng zazhi antibiotic resistance gene plasmid dna reference material uniformity stability |
| title | Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection |
| title_full | Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection |
| title_fullStr | Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection |
| title_full_unstemmed | Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection |
| title_short | Preparation of plasmid DNA reference material for polymyxin antibiotics resistance encoding gene detection |
| title_sort | preparation of plasmid dna reference material for polymyxin antibiotics resistance encoding gene detection |
| topic | antibiotic resistance gene plasmid dna reference material uniformity stability |
| url | http://www.zgspws.com/zgspwszz/article/abstract/202202003?st=article_issue |
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