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 

TITLE=Development of Loop-Mediated Isothermal Amplification Assay Targeting lytA and psaA Genes for Rapid and Visual Diagnosis of Streptococcus pneumoniae Pneumonia in Children

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.816997

DOI=10.3389/fmicb.2021.816997

ISSN=1664-302X

ABSTRACT=<p><italic>Streptococcus pneumoniae</italic> (<italic>S. pneumoniae</italic>) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify <italic>S. pneumoniae</italic> form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect <italic>S. pneumoniae</italic> pneumonia in children, we designed specific LAMP primers targeting <italic>lytA</italic> and <italic>psaA</italic> 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 <italic>S. pneumoniae</italic> 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 <italic>Mycoplasma pneumoniae</italic>. This LAMP assay detected <italic>S. pneumoniae</italic> in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for <italic>S. pneumoniae</italic> early diagnosis and effective antibiotic therapy.</p>