Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children.
Yinghu Chen, Lingfeng Mao, Dengming Lai, Weize Xu, Yuebai Zhang, Sihao Wu, Di Yang, Shaobo Zhao, Zhicong Liu, Yi Xiao, Yi Tang, Xiaofang Meng, Min Wang, Jueliang Shi, Qixing Chen, Qiang Shu
Author Information
Yinghu Chen: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Lingfeng Mao: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Dengming Lai: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Weize Xu: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Yuebai Zhang: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Sihao Wu: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Di Yang: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Shaobo Zhao: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Zhicong Liu: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Yi Xiao: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Yi Tang: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Xiaofang Meng: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Min Wang: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Jueliang Shi: Joint Research Center for Molecular Diagnosis of Severe Infection in Children, Binjiang Institute of Zhejiang University, Hangzhou, China.
Qixing Chen: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Qiang Shu: The Children's Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Objectives: To develop a rapid and low-cost method for 16S rDNA nanopore sequencing. Methods: This 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. Results: The 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. Conclusions: The nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children.
