Development of PCRSeqTyping—a novel molecular assay for typing of Streptococcus pneumoniae

Abstract

BackgroundPrecise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need.MethodsPolymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark).ResultsA 100% correlation of PCRSeqTyping results was observed with Pneumotest results. Fifty-nine reference strains were uniquely identified in the first step of PCRSeqTyping. The remaining 32 homologous strains out of 91 were also uniquely identified in the second step.ConclusionThis study describes a PCRSeqTyping assay that is accurate and rapid, with high reproducibility. This assay is amenable for clinical testing and does not require culturing of the samples. It is a significant improvement over other methods because it covers all pneumococcal serotypes, and it has the potential for use in diagnostic laboratories and surveillance studies.

Comparison of sequential multiplex PCR, sequetyping and whole genome sequencing for serotyping of Streptococcus pneumoniae

“…However, the low identity of the best HSP (96%) could help to discriminate this isolate. A recent method based on sequetyping including a second analysis step for homologous strains allowed to obtain more accurate results for these strains [ 35 ]. Such protocol could putatively help to obtain better results and make sequetyping more attractive.…”

Bacteremic Community-Acquired Pneumonia in Ethiopian Children: Etiology, Antibiotic Resistance, Risk Factors, and Clinical Outcome

“…Primers used to amplify 1061 bp of the cps B gene (encoding for phosphotyrosine phosphatase) were forward primer cps1-FP (5’-GCAATGCCAGACAGTAACCTCTAT-3’) and 2 reverse primers, cps2-RP (5’-CCTGCCTGCAAGTCTTGATT-3’) and cps-2538-RP (5’-CTTTACCAACCTT TGTAATCCAT-3’), and the PCR reaction was performed as described previously [18]. Amplification of a 101-bp fragment of the lyt A gene was carried out as previously described [19], using the primers lyt A F (5’-ACGCAATCTAGCAGATGAAGCA-3’) and lyt A R (5’-TCGTGCGTTTTAATTCCAGCT-3’).…”

Etiology, antibiotic susceptibility and prognostic factors of pediatric community-acquired sepsis in Addis Ababa, Ethiopia

“…PCRSeqTyping and serotyping DNA extraction from S. pneumoniae isolates was carried out by alkaline lysis as described previously [21]. Amplification and sequencing of the 1061 bp of the cpsB-gene (encoding for phosphotyrosine phosphatase) were done using PCRSeqTyping as previously described [22]. Twenty μl of the amplicons were sent for sequencing to GATC Biotech (Constance, Germany) and sequencing was performed using the Sanger sequencing technique.…”