Development of PCRSeqTyping—a novel molecular assay for typing of Streptococcus pneumoniae
Nagaraj et al. Pneumonia
Development of PCRSeqTyping-a novel molecular assay for typing of Streptococcus pneumoniae
Geetha Nagaraj 0
Feroze Ganaie 0
Vandana Govindan 0
Kadahalli Lingegowda Ravikumar 0
0 Central Research Laboratory, KIMS Hospital and Research Centre , KR Road, VV Purum, Bangalore, Karnataka 560 004 , India
Background: Precise 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. Methods: Polymerase 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). Results: A 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. Conclusion: This 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.
Molecular serotyping; PCRSeqTyping; Streptococcus pneumoniae; cpsB sequencing
Streptococcus pneumoniae, found in the upper
respiratory tract of healthy children and adults, causes a
range of infections including meningitis, septicemia,
pneumonia, sinusitis, and otitis media. Children < 2 years
of age and adults aged ≥65 years of age are particularly
susceptible . According to the Morbidity and
Mortality Weekly Report, April 26 2013 , an estimated 14.5
million cases of serious pneumococcal disease (including
pneumonia, meningitis, and sepsis) occur each year in
children aged <5 years worldwide, which has resulted in
approximately 500,000 deaths, mostly in low- and
middle-income developing countries.
The high morbidity and mortality caused by
pneumococci are not clearly understood. The pathogenicity of
pneumococci has been linked to various virulence
factors such as capsule, cell wall and its component
polysaccharides, pneumolysin, PspA, complement factor
H-binding component, autolysin, neuraminidase, peptide
permeases, hydrogen peroxide, and IgA1 protease [3–5].
Capsular polysaccharide (CPS) is the primary virulence
factor, and is also used to categorize, S. pneumoniaeinto
more than 90 different serotypes [6–8]. Capsule is
important for the survival of bacteria at infection site as
it provides resistance to phagocytosis .
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Pneumococcal CPS is generally synthesized by the
Wzx/Wzy-dependent pathway, except for types 3 and
37, which are produced by the synthase pathway [10,
11]. Most genes required for synthesis of capsule are
within the capsule polysaccharide synthesis (cps) operon,
which ranges from 10 kb (serotype 3) to 30 kb (serotype
38). Cps operon is flanked by dexB in 5′ end and aliA at
3′ end. Neither of these participates in capsule synthesis.
The 5′-end of the CPS loci starts with regulatory and
processing genes wzg, wzh, wzd, and wze (also known as
cpsABCD), which are conserved with high sequence
identity in all serotypes, followed by the central region
consisting of serotype specific genes [12, 13].
Pneumococcal serotyping is necessary for
epidemiological and vaccine impact studies. It also aids in
understanding the pathogenicity of the organism and
closely monitors for the emergence of non-vaccine
strains, replacement serotypes, and new serovars [14, 15].
Widespread use of pneumococcal vaccines has led to
replacement with serotypes that are not included in the
vaccines. Continuous monitoring of serotypes is therefore
essential for epidemiological surveillance and long-term
vaccine impact studies [16–20].
Several phenotypic and genotypic methods are
currently used to identify pneumococcal group and type.
The phenotypic serotyping methods of capsular swelling
reaction, latex agglutination and coagglutination tests
are costly, require skilled personnel, and cannot detect
all serotypes. Genotypic typing methods that assess
genome variation include sequential multiplex
polymerase chain reaction (PCR), sequential real-time PCR,
restriction fragment length polymorphism (RFLP),
microarray, sequetyping, and matrix-assisted lazer
desorption ionization-time of flight (MALDI-TOF)
analysis. In addition to general applicability and a high
discriminatory power, these genotypic assays are
economical, detect pneumococci directly from the
clinical specimen, and detect emerging serovars,
replacement strains, and vaccine escape recombinants .
However, many of these methods are multistep, intricate,
and do not discriminate all serotypes [22–26].
It is crucial to develop a robust, simple method with
complete serotype coverage for serotype detection and
pneumococcal serogroup/serotype surveillance .
Herein, the authors describe an innovative serotyping
approach that relies on sequencing of assembly genes
located in the capsular operon to identify all
There were 91 reference serotype strains of S.
pneumoniae obtained from Staten Serum Institute, Copenhagen,
Denmark (Table 1).
There were 28 clinical isolates of S. pneumoniae selected
from isolates submitted to Central Research Laboratory,
KIMS Hospital, Bangalore (Table 2). They were isolated
from blood (n = 23), cerebrospinal fluid (CSF) (n =3) and
pleural fluid (n = 2).
Media and culture conditions
Strains were stored in skim milk, tryptone, glucose, and
glycerol (STGG) media at −80 °C. They were cultured
on 5% sheep blood agar (Chromogen, Hyderabad) for
18–24 hrs at37°C with 5% CO2. The isolates were
characterized as S. pneumoniae by colony morphology, alpha
hemolysis, bile solubility, and optochin susceptibility.
Quellung reaction was performed using Pneumotest kit
and type-specific antisera (SSI, Denmark), as
recommended by the manufacturer.
PCRSeqTyping assay was performed in two steps. Step I
involved PCR amplification and sequencing of the cpsB
gene from genomic DNA. There were 91 serotypes that
were divided into non-homologous group (Group I, 59
serotypes) and homologous group (Group II, 32
serotypes) based on the cpsB sequence data. The
homologous group was further subdivided into 10
subgroups based on the sequence homology. The second
step involved PCR and sequencing of each homology
group by using specific primers in order to identify the
Nucleic acid extraction
Genomic DNA was extracted from bacterial strains
using QIAamp DNA mini kit (Qiagen, Germany), as per
the manufacturer’s protocol.
PCR reaction was performed using the primers designed
by Leung et al.  with modifications. Primers used in
the study were cps1-FP (5′-GCAATGCCAGACAGT
AACCTCTAT-3)′, cps2-RP (5′-CCTGCCTGCAAGTCT
TGATT-3′) and cps-2538-RP (5′-CTTTACCAACCTT
TGTAATCCAT-3′). The reaction mixture was modified
to contain 50–100 ng of genomic DNA, 0.75 units XT-5
polymerase (3 U/μl, Merck, which is a mixture of
thermo stable enzymes Taq DNA polymerase and
proofreading [PR] polymerase), 1X XT5A-Assay buffer, 1 μl
deoxynucleoside triphosphates (dNTPs, 2.5 mM each
[Fermentas, United States]), 1 μl forward primer
(100 ng/μl), 1 μl of reverse primer mix (100 ng/μl). The
final reaction volume was made up to 25 μl with DNase/
RNase-free distilled water (Gibco, United States). Thermal
Table 1 PCRseqtyping results for 91 SSI strains
Sl. NO Serogroup Serotype
NCBI ACCESSION NO 1 2 3
Table 1 PCRseqtyping results for 91 SSI strains (Continued)
44 19 19 F
48 20 20
49 21 21
50 22 22 F
52 23 23 F
55 24 24 F
58 25 25 F
60 27 27
61 28 28 F
63 29 29
64 31 31
65 32 32 F
67 33 33 F
72 34 34
73 35 35 F
77 36 36
78 37 37
79 38 38
80 39 39
81 40 40
82 41 41 F
84 42 42
85 43 43
86 44 44
Table 1 PCRseqtyping results for 91 SSI strains (Continued)
cycling was performed in GeneAmp PCR system 9700
(Applied Biosystems, United States) under the following
conditions: 94 °C for 5 min, followed by 35 amplification
cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min
and final extension at 72 °C for 5 min. The PCR products
were separated by electrophoresis on 1.2% agarose gel for
45 min at 80 V in 1X Tris-acetate EDTA buffer. Ethidium
bromide-stained DNA products were visualized under
ultraviolet (UV) illumination and size of the DNA
products was determined by using a 1–kb DNA molecular size
Sequencing and data analysis
PCR products were purified using QIA quick PCR
purification kit (Qiagen, Germany) following
manufacturer’s protocol. Purified PCR products were subjected
Table 2 Serotype distribution of the clinical isolates of Streptococcus pneumoniae from Central Research Laboratory, KIMS Hospital,
Sl.No Sample ID SEX AGE YRS SOURCE PCRSeq Typing data Quellung data
7 F 19A 3
to sequencing, employing the Big Dye Sequence
Terminator kit V3.1 (Applied Biosystems) and analyzed on ABI
3730 XL Genetic Analyzer (Applied Biosystems).
Sequencing was performed in one direction using
forward primer (cps1), 5′-GCA ATG CCA GAC AGT
AAC CTC TAT-3′ and Long Seq Module (ABI). DNA
sequences that were obtained were analyzed for
sequence similarity using GenBank database (http://
www.ncbi.nlm.nih.gov/blast) and then assigned to
serotype . Serotype of the cpsB nucleotide sequence was
determined from GenBank with the highest BLAST bit
score of > 99% sequence identity with the query
‘amplicon nucleotide sequence’.
Homology group assignment and PCRSeqTyping
Amplifiable serotypes that shared identical interceding
sequences (e.g. sequences for serotypes 2 and 41A, 7B, and
40) were grouped into 10 different groups based on their
homology by in silico analysis of cpsB region. Individual
primer sets were designed for each subgroup. Sequetyping
data obtained in Step I was used to assign the homologous
strains into subgroups (Fig. 1). Serotypes were
considered homologous when the highest bit score was
shared between two or more serotypes (i.e. the same
amount of nucleotide variation between query and
database sequences), and then assigned to one of the
10 groups (Table 3).
For homologous strains, a second round of PCR was
performed using group specific primers as specified in
Table 3. PCR products were subjected to sequencing
reaction. The nucleotide sequence data was used to
assign the serotype.
PCRSeqTyping results for reference strains
The 91 pneumococcal serotype reference strains
(sourced from SSI) were tested with PCRSeqTyping
protocol. All 91 strains were amplified using the
modified method. In Step I of amplification and sequencing,
59 strains of the non-homologous group (Group I) were
correctly assigned to their respective serotype. There
were 32 strains (Group II) identified along with their
homologous type. The homologous types were correctly
assigned to their respective type in Step II by performing
a second round of amplification using group specific
primers and sequencing. Quellung reaction performed
using Pneumotest kit (SSI), in parallel with
PCRSeqTyping, showed 100% concordant results (Table 1).
The results were further evaluated by blinded testing
of PCRSeqtyping. Samples were evaluated randomly by
assigning codes. Quellung reaction data showed no
discrepancies between serotypes assigned by Quellung
and PCRSeqTyping for all reference strains.
PCRSeqTyping results for clinical isolates
Twenty eight pneumococcal isolates tested in the study
were from children <5 years with invasive pneumococcal
disease. The predominant serotypes were 1, 6B, 19A,
19 F, 14 and 7 F (Table 2). PCRSeqTyping results and
serotyping results by Quellung reaction were in
concordance, without any discrepancies. Among 28 isolates,
Fig. 1 Homology group assignment for 91 pneumococcal serotypes
TGTCCAATGAAGAGCAAGACTTGAC CAGAAAAAGTAGCCTTATTTCTTAAGA ATGAAGCTATTCAAAGTTTGTTAGC TGAATCCTCTAATCCTTGCATGA
25 isolates were assigned to their serotype with the first
step of PCRSeqTyping. Three isolates belonging to the
homologous group were subsequently identified with the
second step of PCRSeqTyping.
There is a renewed interest in pneumococcal capsular
typing techniques, as a result of an increased complexity in the
management of pneumococcal disease and the widespread
use of pneumococcal vaccines . The ability to
differentiate pneumococcal strains efficiently is essential to track the
emerging serovars, and for epidemiological investigations.
The limitations of the Quellung serotyping method, many
DNA-based typing protocols, PCR, restriction fragment
length polymorphisms, hybridization assays, microarrays
and sequencing for S. pneumoniae are well known.
Different PCR strategies, namely multiplex PCR,
sequential PCR, serotype-specific PCR, and real time
multiplex PCR [25, 28–36] targeting serotype-specific
regions of cps could detect only 22 serotypes uniquely, and
48 serotypes along with their homologous types [37, 38].
Despite the fact these methods cover imited serotypes,
PCR is a widely used technique, which avoids the use of
serological reagents and requires specific expertise to
Methods using multiple restriction enzymes and long
cps fragments [39, 40] for PCR make the amplification
difficult and inconsistent. Another protocol based on
sequencing of regulatory region of cps [30, 31] shows
poor resolution with cross reactivity of serotypes. An
approach targeting serotype-specific glycosyl transferase
genes  was only tested for serogroup 6 and serotype
19 F. The cross reactivity of serotypes, along with the
requirement for a higher number of primers, and poor
resolution limits their wide usage.
With the characterization of the cps locus of 92
serotypes , Leung et al.  developed sequetyping
protocol using single primer pair, which binds in all
pneumococcal serotypes. Recently, several research
groups [27, 41–43] have published their results using
sequetyping assay. Limitations of the sequetyping
protocol were as follows: (i) only 84 serotypes out of 92 were
predicted to be amplified by in silico analysis; (ii)
crossreacting serotypes (30/84) belonging to homologous
groups could not be uniquely identified; and (iii)
considering the central 732 bp region of the cpsB amplicon
which could be sequenced, only 46 of 54 serotypes could
In the first step of this study’s modified approach,
successful amplification of all 91 serotypes was achieved
with the addition of a new reverse primer to amplify 25A,
25 F and 38 serotypes specifically. Additionally, XT-5
polymerase used in the PCR amplification reactions contains
Taq DNA polymerase and Pfu enzyme. This enzyme blend
utilizes the powerful 5′-3′ polymerase activity of Taq
DNA polymerase and the 3′-5′ exonuclease-mediated
proof-reading activity of PR polymerase, resulting in high
fidelity PCR products . PCR annealing temperature of
50 °C and extension time of 1 min were found to be
optimal for amplification of cpsB gene of all 91 strains.
The serotypes were grouped into homologous (32) and
non-homologous (59) based on cpsB sequence.
Nonhomologous types were identified uniquely. The 32
homologous strains were further subdivided into 10
groups (HG 1–10) based on their sequence similarity.
Homology group-specific primers were designed and
evaluated for their ability to differentiate between strains.
HG primers were designed to be able to assign the
serotype accurately with second step of PCR and sequencing.
The limitation of using 732 bp region of cpsB
amplicon in sequetyping assay, resulting in prediction of 46 of
54 serotypes, was overcome with the use of Long Seq
module. Approximately 1.0 kb quality reads in a single
sequencing reaction were obtained with modification.
This resulted in providing good quality reads up to the
end of the PCR template, identifying cross-reacting
serotypes (15B/15C, 7 F/7A, 18B/18C, 9 L/9 N, 15B/C, 17 F/
33C, 18B/C, 7A/F, 12A/46, 6C/6D) which have a single
SNP in the cpsB region.
A 100% concordance of serotype results of
PCRSeqTyping and Quellung testing was seen for the 28 clinical
isolates. Moving forward, the study will be extended for
serotyping a larger number of clinical isolates and
clinical samples. The limitation of the protocol will be in
quantification and serotype identification in multiple
carriage; however, studies are underway to address these
issues. For multiple carriage, the PCR amplicon obtained
in the first step will be subcloned into T/A cloning
vector and the individual clones will be sequenced for
assigning the specific serotype. As the corresponding
cpsB gene sequence of the recently discovered serotypes
6E, 6 F, 6G, 6H, 11E, 20A, 20B and 23B1 [45–47] were
unavailable at the time of the study design, they will be
included in future studies.
In the study’s center, the typing cost with Pneumotest
Kit (SSI, Denmark) was US$35/isolate, while
PCRSeqTyping cost was US$10 for Group I (non-homologous
strains) and US$15 for Group II (homologous strains).
With the easy availability of outsourced sequencing
services, the accurate and reliable PCRSeqTyping test
can be adopted in a regular microbiology laboratory,
even without the sequencing facility.
This modified typing method has several advantages
over other reported methods. It involves techniques with
a workflow that many microbiology laboratories can easily
implement. The high throughput PCRSeqTyping method
features good discriminatory power, reproducibility, and
portability, making it suitable for epidemiological studies.
The assay has the flexibility of incorporating additional
primers for the characterization of emerging serotypes.
An added advantage of this method is that raw data from
experiments can be reanalyzed upon the addition of new
entries to the serotyping database.
PCRSeqTyping assay is a cost-effective alternative to
currently available phenotypic and molecular typing
methods. The method is simple to perform, robust, and
economical. It can identify all 91 serotypes specifically
Cps: Capsular polysaccharide; DNA: DeoxyRibo Nucleic Acid;
EDTA: Ethylenediaminetetraacetic acid; MALDI-TOF: Matrix Assisted Laser
Desorption Ionization - Time of Flight; PCR: Polymerase chain reaction;
RFLP: Restriction fragment length polymorphism; SSI: Staten Serum Institute;
STG: Serotype/group; STGG: Skim milk, tryptone, glucose, and glycerol
Availability of data and materials
All data generated or analyzed during this study are included in this
published article [and its supplementary information files].
GN – concept, designing the experiment, executing, data analysis and
writing the manuscript. RKL – Guided the experimentation process and
execution, reviewed the manuscript. FG and VG assisted during
experimentation. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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