Resources and Protocols
Resources related to identifying Streptococcus pneumoniae and its serotypes or serogroups (small sets of highly related serotypes, for example 9A/9V) are below.
Alternative MLST primers are located about 40 bases upstream of other primers documented for pneumococcal MLST. You can use these primers for obtaining the first few bases of the target sequence.
S. pneumoniae alternative MLST primers:
You can achieve PCR detection of S. pneumoniae by amplifying the lytA gene target. Details for singleplex and multiplex real-time PCR are below. For additional information, contact StrepLab@cdc.gov.
- Oligonucleotides used in the Singleplex Real-time PCR assays for identification of Streptococcal species (Updated June 2021)
- Oligonucleotides used in the triplex real-time PCR assays for identification of S. pneumoniae, N. meningitidis and H. influenzae (Updated June 2021)
PCR based methods for identification of S. pneumoniae serotypes/serogroups
Accurate serotyping is essential for epidemiologic study of S. pneumoniae. CDC devised simple multiplex PCR schemes to reliably deduce specific pneumococcal serotypes from isolate sets and sterile site clinical specimens. This PCR approach is highly reliable and has the potential to reduce reliance upon conventional phenotypic serotyping. This system gives serotype-determining potential to any facility that has equipment necessary for DNA amplification and electrophoresis; you do not need typing sera or other reagents.
Caution should be taken when using PCR-based approaches for serotyping from upper respiratory specimens, in particular oropharyngeal swabs. The recovery of capsular polysaccharide biosynthetic operon (cps) sequences, thought to be solely associated with pneumococcal serotypes, within related nonpneumococcal commensal strains such as S. mitis and S. oralis, may result in some inaccuracy depending upon specimen and carriage population1-6.
Below are protocols detailing methods for extraction of DNA from bacterial isolates and clinical specimens for any type of PCR testing:
- Fast Extraction of DNA for Streptococcal Culture Isolates [1 page]
- Blood and Body Fluid DNA Extraction for Streptococci [2 pages]
I. Real-time PCR Deduction of Pneumococcal Serotypes or Serogroups
Quadriplex real-time PCR identification of pneumococcal serotypes/serogroups
The previous triplex scheme has been updated to 48 real-time PCR assays in 12 quadriplex reactions for detection of 64 serotypes as individual serotypes or small serogroups. The list of primers/probes used in the assays are provided below.
For additional information on the sequential multiplex serotyping schemes and protocols, contact StrepLab@cdc.gov.
II. Conventional PCR Deduction of 40 Pneumococcal Serotypes or Serogroups
a. Conventional PCR Serotype Deduction Protocols
CDC validates and refines primer sets thoroughly through diverse isolate sets representing individual serotypes. Serospecificities are added and primer sets updated to regularly improve specificity. A current updated primer list (41 serospecificities) is available below.
There are 3 important points for using this PCR serotyping scheme:
- Band sizes must exactly match positive controls before assigning a serotype. Non-specific bands have been occasionally detected when performing multiplex PCR testing on clinical specimens.
- A negative cpsA control does not necessarily equate to a non-serotypeable isolate or a pneumococcus-negative clinical specimen. The positive pneumococcal control band for cpsA is negative in 1-2% of PCR-serotypeable isolates that have been encountered. This result most often occurs in serotypes 25 and 38, but has been rarely encountered in serotypes 14 and 35A.
- Using this PCR assay within carriage specimens may result in some inaccuracy depending upon specimen and carriage population1-6. High amounts of amplification of some presumed pneumococcal-specific targets for serotyping from lytA-negative specimens (i.e. presumed not to contain pneumococci) have been found. For example, the six 192 bp sequences directly below correspond to pneumococcal serogroup 10F/10C amplification products (following subtraction of primers).
>Seq1 [organism =Streptococcus infantis] [strain SS1641] wzx gene, partial CDS
>Seq2 [organism=Streptococcus gordonii] [strain SS1245] wzx gene, partial CDS
>Seq3 [organism=Streptococcus salivarius] [strain SS1061] wzx gene partial CDS
>Seq4 [organism=unknown] [human upper respiratory tract specimen 49] wzx gene, partial CDS
>Seq5 [organism=unknown] [human upper respiratory tract specimen 248] wzx gene, partial CDS
>Seq6 [organism=unknown] [human upper respiratory tract specimen 300] wzx gene, partial CDS
These sequences were found in non-pneumococcal species and/or lytA-negative carriage specimens. Additional data from upper respiratory tract specimens have demonstrated non-pneumococcal homologs of additional serogroups and serotypes.1,2
b. Multiplex Conventional PCR Schemes for Pneumococcal Serotype Deduction
The set of conventional PCR serotyping assays can be used in several sequential PCR schemes based on geographic serotype distribution. For additional information on the sequential multiplex serotyping schemes and protocols, contact StrepLab@cdc.gov.
Multiplex real-time PCR identification of antimicrobial resistance (AMR) determinants and Pili genes
The PCR assays provided below can be used to identify key antibiotic resistance determinants [including pbp2B (penicillin susceptibility), tetM (tetracycline resistance), ermB (erythromycin and clindamycin resistance), mef (erythromycin resistance), and cat (chloramphenicol resistance)] and two virulence factor genes (Pilus 1 and 2).
For additional information contact StrepLab@cdc.gov.
Detailed protocols for specimen collection, processing, and storage of nasopharyngeal swabs for pneumococcal carriage studies are provided below.
- Identification and Serotyping of Pneumococci from Carriage [1 page]
- Streptococcus pneumoniae Carriage Study Protocol ‐ Nasopharyngeal (NP) Swab Processing [3 pages]
1Carvalho MG, Bigogo GM, Junghae M, et al. Potential non-pneumococcal confounding of PCR-based determination of serotype in carriage. J Clin Microbiol. 2012;50(9):3146–7.
2Carvalho Mda G, Pimenta FC, et al. Non-pneumococcal mitis-group streptococci confound detection of pneumococcal capsular serotype-specific loci in upper respiratory tract. PeerJ. 2013;1:e97.
3Lessa FC, Milucky J, Rouphael NG, et al. Streptococcus mitis Expressing Pneumococcal Serotype 1 Capsule. Sci Rep. 2018; 8(1):17959.
4Pimenta F, Gertz RE Jr, Park SH, et al. Streptococcus infantis, Streptococcus mitis, and Streptococcus oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to Serotype 5 Pneumococci. Front Microbiol. 2019; 8;9:3199.
5Beall B. Potential Epidemiologic and Historical Implications of Capsular Serotypes Shared by Pneumococci and Their Nonpneumococcal Relatives. J Infect Dis. 2020;222(3):343-346.
6Gertz RE Jr, Pimenta FC, Chochua S, et al. Nonpneumococcal Strains Recently Recovered from Carriage Specimens and Expressing Capsular Serotypes Highly Related or Identical to Pneumococcal Serotypes 2, 4, 9A, 13, and 23A. mBio . 2021 12(3):e01037-21.