Simultaneous detection of Streptococcus pneumoniae and prevention of carryover contamination using multiple cross displacement amplification with Antarctic thermal sensitive uracil-DNA-glycosylase and a lateral flow biosensor

ABSTRACT Streptococcus pneumoniae is an important clinical pathogenic bacterium that is the primary cause of meningitis, septicemia and community-acquired pneumonia. The mortality rate of pneumococcal disease is high, especially in children younger than 5-years-old. Rapid and accurate detection of S...

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Bibliographic Details
Published in:FEMS Microbiology Letters
Main Authors: Yan, Linlin, Zhao, Fan, Niu, Lina, Cai, Yu, Wu, Lei, Zhu, Xiaoxue, Nong, Jinqing, Hu, Shoukui
Other Authors: Education Department of Hainan Province, National Natural Science Foundation of China, Beijing Natural Science Foundation
Format: Article in Journal/Newspaper
Language:English
Published: Oxford University Press (OUP) 2021
Subjects:
Genetics
Molecular Biology
Microbiology
Antarctic
Antarc*
Online Access:http://dx.doi.org/10.1093/femsle/fnab006
http://academic.oup.com/femsle/advance-article-pdf/doi/10.1093/femsle/fnab006/35924612/fnab006.pdf
http://academic.oup.com/femsle/article-pdf/368/3/fnab006/36248725/fnab006.pdf
Description
Summary:ABSTRACT Streptococcus pneumoniae is an important clinical pathogenic bacterium that is the primary cause of meningitis, septicemia and community-acquired pneumonia. The mortality rate of pneumococcal disease is high, especially in children younger than 5-years-old. Rapid and accurate detection of S.pneumoniae is critical for clinical diagnosis. A ply gene-based multiple cross displacement amplification (MCDA) assay, amplifying DNA under 65°C for 40 min, was established to detect S.pneumoniae. Antarctic thermal sensitive uracil-DNA-glycosylase (AUDG) was applied to prevent carryover contamination. A lateral flow biosensor (LFB) was used to indicate the MCDA results. The ply-MCDA assay could detect as low as 10 fg of S. pneumoniae DNA and 447 colony forming units (CFU)/mL of spiked sputum samples. The analytical sensitivity of the ply-MCDA assay to detect clinical specimens was 100 times higher than that of PCR. The specificity of the ply-MCDA assay was evaluated using 15 S.pneumoniae strains and 25 non-S. pneumoniae strains, which confirmed the high selectivity of the ply-MCDA assay for S.pneumoniae. The AUDG enzyme could effectively eliminate carryover contamination and thus prevented false-positive results. In conclusion, ply-AUDG-MCDA-LFB is a simple, rapid and accurate method to detect S.pneumoniae.

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