Cargando…
Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children
Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-medi...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8803124/ https://www.ncbi.nlm.nih.gov/pubmed/35111144 http://dx.doi.org/10.3389/fmicb.2021.816997 |
_version_ | 1784642803683295232 |
---|---|
author | Du, Shuheng Yan, Chao Du, Bing Zhao, Hanqing Xue, Guanhua Zheng, Ping Feng, Yanling Cui, Jinghua Gan, Lin Feng, Junxia Fan, Zheng Xu, Ziying Fu, Tongtong Fu, Hanyu Zhang, Qun Li, Nannan Zhang, Rui Liu, Shiyu Li, Xiaoran Cui, Xiaohu Zhou, Yao Zhang, Qi Chen, Yaodong Yuan, Jing |
author_facet | Du, Shuheng Yan, Chao Du, Bing Zhao, Hanqing Xue, Guanhua Zheng, Ping Feng, Yanling Cui, Jinghua Gan, Lin Feng, Junxia Fan, Zheng Xu, Ziying Fu, Tongtong Fu, Hanyu Zhang, Qun Li, Nannan Zhang, Rui Liu, Shiyu Li, Xiaoran Cui, Xiaohu Zhou, Yao Zhang, Qi Chen, Yaodong Yuan, Jing |
author_sort | Du, Shuheng |
collection | PubMed |
description | Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4–100%), specificity 99.0% (95% CI 97.8–99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy. |
format | Online Article Text |
id | pubmed-8803124 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-88031242022-02-01 Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children Du, Shuheng Yan, Chao Du, Bing Zhao, Hanqing Xue, Guanhua Zheng, Ping Feng, Yanling Cui, Jinghua Gan, Lin Feng, Junxia Fan, Zheng Xu, Ziying Fu, Tongtong Fu, Hanyu Zhang, Qun Li, Nannan Zhang, Rui Liu, Shiyu Li, Xiaoran Cui, Xiaohu Zhou, Yao Zhang, Qi Chen, Yaodong Yuan, Jing Front Microbiol Microbiology Streptococcus pneumoniae (S. pneumoniae) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4–100%), specificity 99.0% (95% CI 97.8–99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae. This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy. Frontiers Media S.A. 2022-01-17 /pmc/articles/PMC8803124/ /pubmed/35111144 http://dx.doi.org/10.3389/fmicb.2021.816997 Text en Copyright © 2022 Du, Yan, Du, Zhao, Xue, Zheng, Feng, Cui, Gan, Feng, Fan, Xu, Fu, Fu, Zhang, Li, Zhang, Liu, Li, Cui, Zhou, Zhang, Chen and Yuan. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Du, Shuheng Yan, Chao Du, Bing Zhao, Hanqing Xue, Guanhua Zheng, Ping Feng, Yanling Cui, Jinghua Gan, Lin Feng, Junxia Fan, Zheng Xu, Ziying Fu, Tongtong Fu, Hanyu Zhang, Qun Li, Nannan Zhang, Rui Liu, Shiyu Li, Xiaoran Cui, Xiaohu Zhou, Yao Zhang, Qi Chen, Yaodong Yuan, Jing Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children |
title | Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children |
title_full | Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children |
title_fullStr | Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children |
title_full_unstemmed | Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children |
title_short | Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children |
title_sort | development of loop-mediated isothermal amplification assay targeting lyta and psaa genes for rapid and visual diagnosis of streptococcus pneumoniae pneumonia in children |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8803124/ https://www.ncbi.nlm.nih.gov/pubmed/35111144 http://dx.doi.org/10.3389/fmicb.2021.816997 |
work_keys_str_mv | AT dushuheng developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT yanchao developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT dubing developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT zhaohanqing developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT xueguanhua developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT zhengping developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT fengyanling developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT cuijinghua developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT ganlin developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT fengjunxia developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT fanzheng developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT xuziying developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT futongtong developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT fuhanyu developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT zhangqun developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT linannan developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT zhangrui developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT liushiyu developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT lixiaoran developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT cuixiaohu developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT zhouyao developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT zhangqi developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT chenyaodong developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren AT yuanjing developmentofloopmediatedisothermalamplificationassaytargetinglytaandpsaagenesforrapidandvisualdiagnosisofstreptococcuspneumoniaepneumoniainchildren |