Establishment And Application Of Molecular Typing Methods For Streptococcus Pneumoniae

Objective Establishing a nucleic acid-based in vitro variable-temperature amplification of Streptococcus pneumoniae(including ordinary PCR,multiplex PCR,and fluorescent quantitative PCR)and loop-mediated isothermal amplification of molecular serotyping methods to identify and serotyping of Spn simply and rapidly.For the purpose of molecular serotyping of Spn isolates from China,preliminary understanding of the prevalence of Spn in China and the distribution of different serogroups/types;Multilocus sequence typing(MLST)analysis was performed on the invasive Spn 19 A and 19 F serotypes,and genotypic characteristics of invasive Spn 19 A and 19 F serotypes from China were studied;Using high-throughput whole-genome sequencing techniques,the entire genome sequence of Spn was analyzed,to investigate the genetic relationship between different serogroups/types of Spn.Methods Spn strains were collected for biochemical identification and serotyping.Search for serotype-specific nucleic acid sequences,design and synthesize primers and probes,detect species-specific genes and serogroup/type-specific genes of Spn,optimize reaction conditions and systems for variable-temperature amplification and isothermal amplification,and determine methods for Specificity and sensitivity of the established method for the detection of Spn strains and specimen nucleic acids;For clinically isolated invasive Spn 19 A,19F serotype strains,ST typing was performed using multilocus sequence typing(MLST)and BioNumerics software processed the typing data of invasive Spn 19 A,19F serotype strains from China for cluster analysis;High-throughput whole-genome sequencing of Spn strains and phylogenetic analysis of core genomes to study the genetic relationship between different serotypes of Spn isolates in China.Results All primers and probes of variable temperature amplification can specifically identify Spn and serotype,while isothermal amplification primers can identify Spn species specificly,but only some of the primers can identify the serotypes of Spn;The invasive Spn 19 A,19F serotypes in China all have multiple ST types and each has a major ST type.The 19 A serotype is mainly ST320,the 19 F serotype is mainly ST271,and the 19 A,19F serotype strains have common sequence type ST320;WGS cluster analysis of different serotypes of Spn showed that WGS typing can provide more genetic information among different strains of Spn.Conclusion A nucleic acid-based variant amplification and isothermal amplification of Spn strains and serotype/type typing techniques were initially established to study the prevalence of Spn serogroups/types in the population;Through MLST data analysis of invasive Spn 19 A and 19 F serotypes in China,we have mastered the ST type distribution of Spn in China and analyzed the trends of decidual conversion and serotype replacement of Spn to evaluate the apply effection of pneumococcal vaccine;High-throughput sequencing of genome-wide sequence analysis shows the genetic relationship between Spn strains at the genomic level.