Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction.

Jie Che; Bo Han Chen; Li XU; Yuan Gao; Meng Meng Yue; Zi Man Chen; Mao Jun Zhang; Zhu Jun Shao

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Establishment and Modification of Ninety-seven Pneumococcal Serotyping Assays Based on Quantitative Real-time Polymerase Chain Reaction.

Author: Jie CHE ¹ ; Bo Han CHEN ¹ ; Li XU ¹ ; Yuan GAO ¹ ; Meng Meng YUE ^{2
,

3} ; Zi Man CHEN ¹ ; Mao Jun ZHANG ¹ ; Zhu Jun SHAO ^{2
,

3}
Author Information

1. National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
2. National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
3. School of Public Health, Nanjing Medical University, Nanjing 210029, Jiangsu China.
Publication Type:Journal Article
Keywords: Quantitative real-time PCR (qPCR); Serotyping; Streptococcus pneumoniae; cps loci
MeSH: Real-Time Polymerase Chain Reaction; Serotyping; Streptococcus pneumoniae/genetics*; Serogroup
From: Biomedical and Environmental Sciences 2023;36(9):787-799
CountryChina
Language:English
Abstract: OBJECTIVE:To establish and modify quantitative real-time polymerase chain reaction (qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.
METHODS:A database of capsular polysaccharide ( cps) loci sequences was generated, covering 97 pneumococcal serotypes. Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs. A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR, while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay. A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.
RESULTS:A total of 97 pneumococcal serotyping assays based on qPCR were established and modified, which included 64 serotypes previously reported as well as an additional 33 serotypes. Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system. A total of 97 pneumococcal serotypes, which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups, could not be identified as individual serotypes. The sensitivity of qPCR assays based on 27 target sequences was 1-100 copies/µL. The specificity of the qPCR assays was 100%, which were tested by a panel of 90 serotypes of the pneumococcal reference strains.
CONCLUSION:A total of 27 novel qPCR assays were established and modified to analyze 97 pneumococcal serotypes.