Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children
ObjectivesTo develop a rapid and low-cost method for 16S rDNA nanopore sequencing.MethodsThis was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the...
| Main Authors: | , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2022.1001607/full |
| _version_ | 1797957681770659840 |
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| author | Yinghu Chen Yinghu Chen Yinghu Chen Lingfeng Mao Dengming Lai Dengming Lai Dengming Lai Weize Xu Weize Xu Weize Xu Yuebai Zhang Yuebai Zhang Sihao Wu Di Yang Shaobo Zhao Shaobo Zhao Zhicong Liu Zhicong Liu Yi Xiao Yi Xiao Yi Tang Xiaofang Meng Min Wang Jueliang Shi Qixing Chen Qixing Chen Qixing Chen Qiang Shu Qiang Shu Qiang Shu |
| author_facet | Yinghu Chen Yinghu Chen Yinghu Chen Lingfeng Mao Dengming Lai Dengming Lai Dengming Lai Weize Xu Weize Xu Weize Xu Yuebai Zhang Yuebai Zhang Sihao Wu Di Yang Shaobo Zhao Shaobo Zhao Zhicong Liu Zhicong Liu Yi Xiao Yi Xiao Yi Tang Xiaofang Meng Min Wang Jueliang Shi Qixing Chen Qixing Chen Qixing Chen Qiang Shu Qiang Shu Qiang Shu |
| author_sort | Yinghu Chen |
| collection | DOAJ |
| description | ObjectivesTo develop a rapid and low-cost method for 16S rDNA nanopore sequencing.MethodsThis was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the experimental procedure. Twenty-one common pulmonary bacteria (7 reference strains, 14 clinical isolates) and 94 samples of bronchoalveolar lavage fluid from children with severe pneumonia were tested. Results indicating low-abundance pathogenic bacteria were verified with the polymerase chain reaction (PCR). Further, the results were compared with those of culture or PCR.ResultsThe turnaround time was shortened to 6~8 hours and the reagent cost of DNA preparation was reduced by employing a single reaction adding barcodes to the 16S primer in advance. The accuracy rate for the 21 common pulmonary pathogens with an abundance ≥ 99% was 100%. Applying the culture or PCR results as the gold standard, 71 (75.5%) of the 94 patients were positive, including 25 positive cultures (26.6%) and 52 positive quantitative PCRs (55.3%). The median abundance in the positive culture and qPCR samples were 29.9% and 6.7%, respectively. With an abundance threshold increase of 1%, 5%, 10%, 15% and 20%, the test sensitivity decreased gradually to 98.6%, 84.9%, 72.6%, 67.1% and 64.4%, respectively, and the test specificity increased gradually to 33.3%, 71.4%, 81.0%, 90.5% and 100.0%, respectively.ConclusionsThe nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children. |
| first_indexed | 2024-04-11T00:08:41Z |
| format | Article |
| id | doaj.art-559b022bd0b244569264f7b66ed0ddfe |
| institution | Directory Open Access Journal |
| issn | 2235-2988 |
| language | English |
| last_indexed | 2024-04-11T00:08:41Z |
| publishDate | 2023-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj.art-559b022bd0b244569264f7b66ed0ddfe2023-01-09T08:26:39ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882023-01-011210.3389/fcimb.2022.10016071001607Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in childrenYinghu Chen0Yinghu Chen1Yinghu Chen2Lingfeng Mao3Dengming Lai4Dengming Lai5Dengming Lai6Weize Xu7Weize Xu8Weize Xu9Yuebai Zhang10Yuebai Zhang11Sihao Wu12Di Yang13Shaobo Zhao14Shaobo Zhao15Zhicong Liu16Zhicong Liu17Yi Xiao18Yi Xiao19Yi Tang20Xiaofang Meng21Min Wang22Jueliang Shi23Qixing Chen24Qixing Chen25Qixing Chen26Qiang Shu27Qiang Shu28Qiang Shu29The Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaThe Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, ChinaNational Clinical Research Center for Child Health, Hangzhou, ChinaJoint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, ChinaObjectivesTo develop a rapid and low-cost method for 16S rDNA nanopore sequencing.MethodsThis was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the experimental procedure. Twenty-one common pulmonary bacteria (7 reference strains, 14 clinical isolates) and 94 samples of bronchoalveolar lavage fluid from children with severe pneumonia were tested. Results indicating low-abundance pathogenic bacteria were verified with the polymerase chain reaction (PCR). Further, the results were compared with those of culture or PCR.ResultsThe turnaround time was shortened to 6~8 hours and the reagent cost of DNA preparation was reduced by employing a single reaction adding barcodes to the 16S primer in advance. The accuracy rate for the 21 common pulmonary pathogens with an abundance ≥ 99% was 100%. Applying the culture or PCR results as the gold standard, 71 (75.5%) of the 94 patients were positive, including 25 positive cultures (26.6%) and 52 positive quantitative PCRs (55.3%). The median abundance in the positive culture and qPCR samples were 29.9% and 6.7%, respectively. With an abundance threshold increase of 1%, 5%, 10%, 15% and 20%, the test sensitivity decreased gradually to 98.6%, 84.9%, 72.6%, 67.1% and 64.4%, respectively, and the test specificity increased gradually to 33.3%, 71.4%, 81.0%, 90.5% and 100.0%, respectively.ConclusionsThe nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children.https://www.frontiersin.org/articles/10.3389/fcimb.2022.1001607/fullnanopore sequencing16S rDNAbacterial pneumoniaBALFprospective study |
| spellingShingle | Yinghu Chen Yinghu Chen Yinghu Chen Lingfeng Mao Dengming Lai Dengming Lai Dengming Lai Weize Xu Weize Xu Weize Xu Yuebai Zhang Yuebai Zhang Sihao Wu Di Yang Shaobo Zhao Shaobo Zhao Zhicong Liu Zhicong Liu Yi Xiao Yi Xiao Yi Tang Xiaofang Meng Min Wang Jueliang Shi Qixing Chen Qixing Chen Qixing Chen Qiang Shu Qiang Shu Qiang Shu Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children Frontiers in Cellular and Infection Microbiology nanopore sequencing 16S rDNA bacterial pneumonia BALF prospective study |
| title | Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children |
| title_full | Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children |
| title_fullStr | Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children |
| title_full_unstemmed | Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children |
| title_short | Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children |
| title_sort | improved targeting of the 16s rdna nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children |
| topic | nanopore sequencing 16S rDNA bacterial pneumonia BALF prospective study |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2022.1001607/full |
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