- Original article
- Open Access
Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae
© The Author(s) 2015
- Received: 4 January 2015
- Accepted: 19 June 2015
- Published: 1 December 2015
Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment]) in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR) assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.
- multiplex real-time PCR
- Streptococcus pneumoniae
- pneumococcal surface adhesin A
Streptococcus pneumoniae is an important bacterial pathogen in humans that is recognised as a major cause of pneumonia, meningitis, sinusitis, otitis media, and as an uncommon cause of a variety of other infectious diseases . Pneumococcal disease is under-reported, as only a small portion of presumptive cases can be confirmed by conventional techniques. Isolation of S. pneumoniae from blood occurs in only 20–30% of adult cases of pneumococcal pneumonia and less than 10% of cases among children [2,3]. Even when present in blood, S. pneumoniae may be missed due to a low density of the pathogen, the fastidious nature of the organism, and previous administration of antibiotics . Serologic assays for both antibody and antigen detection lack specificity and sensitivity [5,6].
Accurate and rapid assays are indispensable for prompt diagnosis and effective therapy. Molecular assays with increased sensitivity and specificity are inherently beneficial for detection of infectious agents and are not abated by non-viable organisms . Various molecular assays, including loop-mediated isothermal amplification method (LAMP) , DNA probe test , and TaqMan® quantitative real-time polymerase chain reaction (qPCR) assay  have been developed and employed to assist investigations.
Studies have revealed that genes encoding pneumolysin (ply) , autolysin (lytA) , pneumococcal surface adhesion A (psaA) , penicillin binding protein , and manganese-dependent superoxide dismutase  have been targeted for identification of S. pneumoniae by PCR-based assays. The ply and lytA genes are among the more common gene targets used for screening S. pneumoniae. Although lytA is present in S. pneumoniae as well as related species such as oral streptococci, the lytA gene has sufficient variability that properly designed primers afford specificity for S. pneumoniae [14,15].
Amongst pneumococcus-like viridans group streptococci, a newly recognised species classified as S. pseudopneumoniae which is positive by AccuProbe assay has been described and characterised . Studies have shown that lytA and psaA gene sequences can reliably distinguish S. pneumoniae from S. pseudopneumoniae . Further, Suzuki et al.  developed a highly specific Spn9802 primer set for S. pneumoniae in order to discriminate S. pneumoniae from pneumococcus-like oral streptococci harbouring the ply and lytA genes; however, these Spn9802 primers may not discriminate S. pseudopneumoniae . For the identification of pneumococci, PCR tests have been applied by various researchers, however, there are reports of ambiguous results . Such ambiguities are perhaps not surprising given the diversity that exists within the organisms that are closely related to pneumococci. It is evident that these organisms can harbour pneumococcal virulence determinants such as ply and lytA genes that are notionally used for diagnosis for pneumococci . So, straightforward amplification of ply by PCR or in combination with lytA is unable to resolve the identification of strains with equivocal reactions for serotype, optochin, or bile solubility [5,18]. The development of a diagnostic test based upon single-target identification is an ambitious hope in a clinical laboratory setting. Given the genetic plasticity of the pneumococcus and its naturally transformable relatives, the combination of 3 or more unique loci is needed to reduce the possibility of false positives .
Accurate detection of pneumococcal infection is of importance to estimate disease burden, tracking changes in the epidemiology of the disease and to assess the effectiveness of currently used vaccines. In the present work, we have standardised and evaluated the quantitative multiplex real-time PCR (qmPCR) system for the accurate and rapid identification of S. pneumoniae.
2.1 Bacterial isolates
S. pneumoniae ATCC 49619 (American Type Culture Collection, USA) was used as a reference strain for optimisation of the assay. Twenty-three characterised and typed S. pneumoniae strains were procured from Statens Serum Institute, Denmark (serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F). Twenty invasive S. pneumoniae strains isolated from whole blood samples of children with pneumonia and 29 negative control organisms were obtained from Central Research Laboratory, Kempegowda Institute of Medical Sciences, Hospital and Research Centre, Bangalore. These organisms were cultured under conditions appropriate for each species .
The isolates were identified as S. pneumoniae by colony morphology, alpha haemolysis, Gram stain, optochin sensitivity, and bile solubility tests by standard methods . The identification was confirmed by automated MicroScan WalkAway-40 (Siemens Healthcare Diagnostics Ltd, UK) system with rapid identification panels .
2.2 Serum specimens
Twenty serum samples from patients who were positive for pneumococcal blood culture, 26 from patients who were negative for pneumococcal blood culture, and 30 from healthy subjects were included in the study. The DNA extracted from the serum samples of healthy subjects was subsequently spiked with 4.29 × 102 copies/µl or 1 pg/µl of S. pneumoniae ATCC 49619 genomic DNA. Serum samples were sourced from the Pneumococcal Research Division, Central Research Laboratory, Kempegowda Institute of Medical Sciences, Hospital and Research Centre, Bangalore.
2.3 DNA extraction/or qmPCR analysis
DNA extraction from isolates and serum specimens was performed using QIAamp DNA Mini Kit with automated DNA extracter, QIAcube (Qiagen, Germany), as per manufacturer’s protocol. Briefly, a loopful of the overnight growth from a blood agar plate was suspended in 180 µl of suspension buffer followed by 20 µl proteinase K and incubated at 56°C for 30 min. For serum, 200 µl of lysis buffer was added to 200 µl of clinical material followed by 20 µl of proteinase K and incubated at 70°C for 10 min. After washing steps, DNA was eluted in 100 µl of elution buffer and stored at −20°C.
2.3.1 Quantification and quality determination of extracted DNA
Quantification and quality of the extracted DNA was determined spectrophotometrically at absorbance 260 nm using Nanodrop 2000 (Thermo Fisher Scientific, USA). The optical density of extracted DNA was measured at the wavelengths of 260 and 280 nm. The DNA purity was estimated by measuring the ratio between the absorbance values.
2.4 Primer and hydrolysis probe oligonucleotide design
Primer and probe sequences for quantitative multiplex real-time PCR
Product size (bp)
GenBank accession no.
5′-CAL Fluor Red 610-CTAGCACATGCTACAAGAATGATTGCAGAAAGAAA-3′-BHQ-2
5′-CAL Fluor Orange 560-ATCAGATTGAAGCTGATAAAACGATAC-3′-BHQ-1 plus
The Rotor-Gene Q (Qiagen) thermocycler has 5 preset channels: Green, Yellow, Orange, Red and Crimson, with the detection range from 450 to 712 nm. Each channel detects reporter dyes that emit light at a particular wavelength. Based on the quenching range of BHQ-1 (480–580 nm), BHQ-2 (559–670 nm), emission wavelength of the reporter dye and detection optics of the instrument, we confirmed that each selected reporter dye is compatible and detected by respective detection channels.
2.5 qmPCR assay
Quantitative multiplex real-time PCR (qmPCR) set-up
2X Rotor-Gene Multiplex PCR Master Mix
20X primer-probe mix *5a
0.5 µM forward primer*5
0.5 µM reverse primer *5
0.2 µM probe *5
DNase/RNase free water
Total reaction volume
2.5.1 Standard curve and lower limit of detection
For assessments of the lower limit of detection (LLD), serial 10-fold dilutions equivalent to from 4.29 × 105 to 0.4 genome copies/µl (1 ng/µl to 1 fg/µl) of purified DNA were prepared and the aliquots were tested by the ply-lytA-, psaA-, and Spn9802-specific primer-probe sets in multiplex reaction [2,7].
2.5.2 Sensitivity and specificity on culture isolates
Sensitivity of the qmPCR assay was determined by testing 10 ng/µl of DNA from 43 confirmed S. pneumoniae isolates. Specificity determination was made by testing 10 ng/µl of DNA extracted from 29 non-pneumococcal isolates in the multiplex reaction. We could not test S. pseudopneumoniae due to non-availability of the strain.
2.5.3 Sensitivity and specificity on serum specimens
DNA extracted from 30 serum samples of healthy subjects spiked with 1 pg/µl of S. pneumoniae ATCC 49619 genomic DNA corresponding to 4.29 × 102 genome copies/µl and 20 S. pneumoniae culture positive serum samples were tested by the qmPCR for sensitivity determination. Twenty-six culture-negative serum samples were examined by the qmPCR for specificity determination. The extracted DNA from each sample (5 µl) was used as a template. GAPDH-specific primer-probe set was used to amplify the endogenous internal control in multiplex reactions.
A no template control (NTC) was included in every run. Positive samples were defined as those which showed amplification for ≥3 target specific sequences and LLD of ≥4 genome copies/µl. Culture and serum specimens were run in duplicates.
2.6 Ethics statement
The study was approved by the Kempegowda Institute of Medical Science independent ethics committee (Approval ID: ECR/216/Inst/Kar/2013). The study was conducted according to the guidelines and recommendations of Good Clinical Practice and the Declaration of Helsinki. Written informed consent was obtained from each participant or legal guardian as applicable.
2.7 Statistical analysis
Sensitivity and specificity of qmPCR on culture and serum specimens were calculated using MedCalc Software bvba version 15.6 (Belgium).
3.1 qmPCR standard curves
The qmPCR assay illustrated LLD equivalent to 4 genome copies/µl or 20 genome copies/reaction for ply-, lytA-, psaA- and Spn9802-specific primer-probe sets. There was an increase in quantification cycle (Cq) values with the decrease in template concentration in subsequent reactions.
3.2 Standardisation of qmPCR on culture isolates
Cross-reactivity panel: negative control organisms
Species or serovar(s)(subtypes)
S. agalactiae, S. mitis, S. equi, S. pyogenes, S. sanguis, S. epidermidis
S. flexneri, S. sonii, S. boydii, S. dysenteriae
S. Typhi-I, S. paratyphi, S. typhimurium
V. Inaba, V. halioticoli, V. ordalii
E. fecalis, E. faecium, E. solitarius
S. aureus, Coagulase negative S. aureus
E. coli H1
Sensitivity and specificity of quantitative multiplex real-time PCR (qmPCR)
No. of samples
Culture positive for pneumococci (%)
Culture negative for pneumococci (%)
qmPCR positivea (%)
qmPCR negativeb (%)
S. pneumoniae-culture positive isolates
Spiked serum samples
Culture-positive serum samples
Culture-negative serum samples
3.3 Standardisation of qmPCR on serum specimens
All 30 spiked DNA extracts were qmPCR positive at the expected concentration (4.29 × 102 genome copies/µl or 1 pg/µl) demonstrating that the DNA extraction eluted from serum samples was free from PCR inhibitors.
Quantification data of the 20 Streptococcus pneumoniae-positive serum samples
Calc Conc (genome copies/µl)
Calc Conc (genome copies/µl)
Calc Cone (genome copies/µl)s
Calc Cone (genome copies/µl)s
Calc Conc (genome copies/µl)
Real-time PCR assays have immense potential to serve as sensitive diagnostic tests for the detection of invasive S. pneumoniae. Currently, there is no simple and dependable method to assess its performance; hence, the diagnostic capability of qPCR should be critically evaluated to acquire reliable results [15,25]. In this study, we evaluated a 4 target qmPCR for accurate detection of S. pneumoniae in culture and serum specimens.
Our qmPCR targeting ply, lytA, psaA, and Spn9802 sequences had 100% sensitivity for detecting S. pneumoniae in culture isolates. The assay also had 100% specificity against a cross-reaction panel of 29 organisms representing diverse genera, including 6 streptococcal species. The present study shows greater sensitivity and specificity than reported by Falquera et al.  (78% sensitivity, 93% specificity), Toikka et al.  (44% sensitivity, 100% specificity), and Michelow et al.  (92% sensitivity, 95% specificity). However, our results are in concordance with the findings of Carvalho et al. , McAvin et al. , and Messmer et al. , who also reported 100% sensitivity and specificity.
qPCR for pneumococci in serum has been reported to be challenging due to the presence of inhibitors in blood and the low number of genomic copies [4,29]. In the present study, qmPCR testing of 30 spiked DNA extracts and 20 pneumococcal culture-positive serum specimens correlated completely with the culture results, indicating 100% sensitivity. High correlation results were also reported by Bayram et al.  (97.2%) and Stralin et al.  (94%). None of the 26 culture-negative serum samples were positive by qmPCR, suggesting 100% specificity. Similar results were reported by Ismail et al. , Carvalho et al.  and McAvin et al. .
In the present study, all the primer-probe sets showed a lower limit of detection equivalent to 4 genome copies/µl. This is similar to that reported by McAvin et al.  and Rudolph et al. . Scott et al.  and Carvalho et al.  reported 1 genome copy/µl and <10 genome copies/µl, respectively. Even though the PCR could detect as little as one genome copy of target DNA as has been described by Scott et al. , the sensitivity of pneumococcal PCR may be poor in clinical evaluations due to the presence of inhibitors. Taq polymerase is highly sensitive to porphyrin inhibitors that are generated from the breakdown of haemoglobin. Most of the inhibitors can be eliminated by an efficient DNA extraction method. In our study, human GAPDH used as an endogenous internal control amplified in all the reactions, suggesting optimal DNA extraction, qmPCR efficiency, and absence of inhibitors.
The efficiency, accuracy, sensitivity, and dynamic range of the PCR assay is determined by the standard curve which is independent of variables associated with the sample preparation. The standard curves generated in this study showed efficiency of 97% to 100%. Similar findings were reported by Carvalho et al. . The high efficiency of this protocol signifies that the amount of PCR product is doubling during each cycle and there is an absence of PCR inhibitors.
By targeting the ply gene, S. pneumoniae DNA in blood samples has been detected with sensitivity ranging from 35% to 100% [15,33]. A matter of concern for the ply PCR is that it is unable to distinguish S. pneumoniae from other streptococcal species . In view of the low specificity of the ply PCR, the lytA PCR has been used and found to have higher specificity [2,34], and pneumococcal DNA was not detected in the blood of healthy subjects irrespective of carrier status . Sequencing and high resolution DNA typing of S. pneumoniae illustrated conservation of the lytA gene. It has been shown that lytA differentiates S. pneumoniae from genotypically related species . Monoclonal antibody studies suggest that psaA is expressed in all 90 serotypes of S. pneumoniae, and PCR-restriction fragment length polymorphism analysis of the 23 vaccine serotypes demonstrated the conservation of the gene . Recently a new S. pneumoniae specific target, the gene fragment Spn9802, has been reported to discriminate S. pneumoniae from pneumococcus-like strains .
Application of multiplex real-time PCR in serum specimens has emerged as a valuable clinical diagnostic tool that offers an opportunity to readdress the problem of the diagnosis of S. pneumoniae infections. The problems associated with microorganisms that are low in number, difficult to culture, or antigenically similar has been minimised with the advancement of sensitive and specific multiplex PCR assays [30,36]. It has several positive outcomes over the singleplex assay as it reduces test costs, eliminates well-to-well variability, conserves precious samples, increases test throughput, and improves turnaround times .
Multiplex PCR assays are popularly adopted for simultaneous detection of various pathogens in the clinical specimens. In the present protocol, multiple specific primer-probe sets were used for detection of a single pathogen to eliminate the spurious negative and positive results. Our study establishes that—with high sensitivity, specificity, and rapidity—the qmPCR assay is a valid platform for detection of pneumococci from serum specimens in clinical laboratories. Finally, the present methodology has broader applications beyond the scope of the present study that demands further investigation. Future studies with large sample sizes should seek to replicate our findings across different geographical locations, particularly in regions with high prevalence of pneumococcal infections.
The qmPCR assay targeting ply, lytA, and psaA genes and Spn9802 DNA fragment is a sensitive and specific assay for the rapid identification of S. pneumoniae. This technology should offer an added advantage when it is used in conjunction with other assays for pneumococcal disease diagnosis. The efficacy of the DNA extraction procedure, degree of sensitivity, and specificity supports the use of this procedure for the direct detection of S. pneumoniae in patient specimens.
The authors would like to thank Dr. Anjana Gopi, Kempegowda Institute of Medical Sciences, Bangalore, for providing the non-pneumococcal isolates.
Funding source: The authors have no funding to report.
Competing interests: All authors have no competing interests to declare
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Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
- Musher DM. Streptococcus pneumoniae. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases. 5th ed. Philadelphia: Churchill Livingstone; 2000: 2128–47.Google Scholar
- Carvalho MG, Tondella ML, McCaustland K, Weidlich L, McGee L, Mayer LW et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol 2007;45:2460–6. https://doi.org/www.dx.doi.org/10.1128/JCM.02498-06 PMID:17537936View ArticleGoogle Scholar
- Korppi M, Koskela M, Jalonen E, Leinonen M. Serologically indicated pneumococcal respiratory infection in children. Scand J Infect Dis 1992;24:437–43. https://doi.org/www.dx.doi.org/10.3109/00365549209052629 PMID:1411309View ArticleGoogle Scholar
- Dagan R, Shriker O, Hazan I, Leibovitz E, Greenberg D, Schlaeffer F et al. Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR. J Clin Microbiol 1998;36:669–73. PMID:9508293PubMedPubMed CentralGoogle Scholar
- Kaijalainen T, Rintamäki S, Herva E, Leinonen M. Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae. J Microbiol Methods 2002;51:111–8. https://doi.org/www.dx.doi.org/10.1016/S0167-7012(02)00061-1 PMID:12069896View ArticleGoogle Scholar
- Sørensen UB, Henrichsen J. Cross-reactions between pneumococci and other streptococci due to C polysaccharide and F antigen. J Clin Microbiol 1987;25:1854–9. PMID:3499450PubMedPubMed CentralGoogle Scholar
- McAvin JC, Reilly PA, Roudabush RM, Barnes WJ, Salmen A, Jackson GW et al. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol 2001;39:3446–51. https://doi.org/www.dx.doi.org/10.1128/JCM.39.10.3446-3451.2001 PMID:11574554View ArticleGoogle Scholar
- Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M. Loop-mediated isothermal amplification method targeting the lytA gene for detection of Streptococcus pneumoniae. J Clin Microbiol 2005;43:1581–6. https://doi.org/www.dx.doi.org/10.1128/JCM.43.4.1581-1586.2005 PMID:15814969View ArticleGoogle Scholar
- Davis TE, Fuller DD. Direct identification of bacterial isolates in blood cultures by using a DNA probe. J Clin Microbiol 1991;29:2193–6. PMID:1939570PubMedPubMed CentralGoogle Scholar
- Corless CE, Guiver M, Borrow R, Edwards-Jones V, Fox AJ, Kaczmarski EB. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J Clin Microbiol 2001;39:1553–8. https://doi.org/www.dx.doi.org/10.1128/JCM.39.4.1553-1558.2001 PMID:11283086View ArticleGoogle Scholar
- Morrison KE, Lake D, Crook J, Carlone GM, Ades E, Facklam R et al. Confirmation of psaA in all 90 serotypes of Streptococcus pneumoniae by PCR and potential of this assay for identification and diagnosis. J Clin Microbiol 2000;38:434–7. PMID:10618136PubMedPubMed CentralGoogle Scholar
- Ubukata K, Asahi Y, Yamane A, Konno M. Combinational detection of autolysin and penicillin-binding protein 2B genes of Streptococcus pneumoniae by PCR. J Clin Microbiol 1996;34:592–6. PMID:8904421PubMedPubMed CentralGoogle Scholar
- Kawamura Y, Whiley RA, Shu SE, Ezaki T, Hardie JM. Genetic approaches to the identification of the mitis group within the genus Streptococcus. Microbiology 1999;145:2605–13. PMID:10517614View ArticleGoogle Scholar
- Ramadan IAE, El-sharkawy AA, Elsherbini MT, Yehia WE, Mohammed SA. Molecular diagnostic value of pneumococcal pneumonia among Egyptian children. International Journal of Development Research 2014;4:355–8.Google Scholar
- Song JY, Eun BW, Nahm MH. Diagnosis of pneumococcal pneumonia: current pitfalls and the way forward. Infect Chemother 2013;45:351–66. https://doi.org/www.dx.doi.org/10.3947/ic.2013.45.4.351 PMID:24475349View ArticleGoogle Scholar
- Messmer TO, Sampson JS, Stinson A, Wong B, Carlone GM, Facklam RR. Comparison of four polymerase chain reaction assays for specificity in the identification of Streptococcus pneumoniae. Diagn Microbiol Infect Dis 2004;49:249–54. https://doi.org/www.dx.doi.org/10.1016/j.diagmicrobio.2004.04.013 PMID:15313529View ArticleGoogle Scholar
- Suzuki N, Seki M, Nakano Y, Kiyoura Y, Maeno M, Yamashita Y. Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization. J Clin Microbiol 2005;43:4528–34. https://doi.org/www.dx.doi.org/10.1128/JCM.43.9.4528-4534.2005 PMID:16145102View ArticleGoogle Scholar
- Whatmore AM, Efstratiou A, Pickerill AP, Broughton K, Woodard G, Sturgeon D et al. Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of “Atypical” pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 2000;68:1374–82. https://doi.org/www.dx.doi.org/10.1128/IAI.68.3.1374-1382.2000 PMID:10678950View ArticleGoogle Scholar
- Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.Google Scholar
- Ruoff KL, Whiley RA, Beighton D. Streptococcus. In: Murray PR, Barron EJ, Jorgensen JH, Pfaller MA, Yolken RH, eds Manual of clinical microbiology. 8th ed. Washington, DC: American Society for Microbiology; 2003.Google Scholar
- Barman P, Sengupta S, Singh S. Study of a novel method to assist in early reporting of sepsis from the microbiology laboratory. J Infect Dev Ctries 2010;4:822–7. https://doi.org/www.dx.doi.org/10.3855/jidc.978 PMID:21252463View ArticleGoogle Scholar
- Wong SC, Chan JK, Lee KC, Lo ES, Tsang DN. Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens. J Clin Pathol 2005;58:276–80. https://doi.org/www.dx.doi.org/10.1136/jcp.2004.016592 PMID:15735160View ArticleGoogle Scholar
- Strålin K, Herrmann B, Abdeldaim G, Olcén P, Holmberg H, Mölling P. Comparison of sputum and nasopharyngeal aspirate samples and of the PCR gene targets lytA and Spn9802 for quantitative PCR for rapid detection of pneumococcal pneumonia. J Clin Microbiol 2014;52:83–9. https://doi.org/www.dx.doi.org/10.1128/JCM.01742-13 PMID:24153121View ArticleGoogle Scholar
- Adams PS, Seed B. Data analysis and reporting. In: Dorak, MT, ed. Real-time PCR (BIOS advanced methods). Oxford: Taylor & Francis Ltd; 2006: 39–62.Google Scholar
- Wu HM, Cordeiro SM, Harcourt BH, Carvalho M, Azevedo J, Oliveira TQ et al. Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis. BMC Infect Dis 2013;13:26. https://doi.org/www.dx.doi.org/10.1186/1471-2334-13-26 PMID:23339355View ArticleGoogle Scholar
- Falguera M, López A, Nogués A, Porcel JM, Rubio-Caballero M. Evaluation of the polymerase chain reaction method for detection of Streptococcus pneumoniae DNA in pleural fluid samples. Chest 2002;122:2212–6. https://doi.org/www.dx.doi.org/10.1378/chest.122.6.2212 PMID:12475865View ArticleGoogle Scholar
- Toikka P, Nikkari S, Ruuskanen O, Leinonen M, Mertsola J. Pneumolysin PCR-based diagnosis of invasive pneumococcal infection in children. J Clin Microbiol 1999;37:633–7. PMID:9986825PubMedPubMed CentralGoogle Scholar
- Michelow IC, Lozano J, Olsen K, Goto C, Rollins NK, Ghaffar F et al. Diagnosis of Streptococcus pneumoniae lower respiratory infection in hospitalized children by culture, polymerase chain reaction, serological testing, and urinary antigen detection. Clin Infect Dis 2002;34:E1–11. https://doi.org/www.dx.doi.org/10.1086/324358 PMID:11731965View ArticleGoogle Scholar
- van Haeften R, Palladino S, Kay I, Keil T, Heath C, Waterer GW. A quantitative LightCycler PCR to detect Streptococcus pneumoniae in blood and CSF. Diagn Microbiol Infect Dis 2003;47:407–14. https://doi.org/www.dx.doi.org/10.1016/S0732-8893(03)00129-9 PMID:14522514View ArticleGoogle Scholar
- Bayram A, Kocoglu E, Balci I, Filiz A, Eksi F. Real-time polymerase chain reaction assay for detection of Streptococcus pneumoniae in sputum samples from patients with community-acquired pneumonia. J Microbiol Immunol Infect 2006;39:452–7. PMID:17164946PubMedGoogle Scholar
- Rudolph KM, Parkinson AJ, Black CM, Mayer LW. Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia. J Clin Microbiol 1993;31:2661–6. PMID:8253962PubMedPubMed CentralGoogle Scholar
- Scott JA, Marston EL, Hall AJ, Marsh K. Diagnosis of pneumococcal pneumonia by psaA PCR analysis of lung aspirates from adult patients in Kenya. J Clin Microbiol 2003;41:2554–9. https://doi.org/www.dx.doi.org/10.1128/JCM.41.6.2554-2559.2003 PMID:12791880View ArticleGoogle Scholar
- Lorente ML, Falguera M, Nogués A, González AR, Merino MT, Caballero MR. Diagnosis of pneumococcal pneumonia by polymerase chain reaction (PCR) in whole blood: a prospective clinical study. Thorax 2000;55:133–7. https://doi.org/www.dx.doi.org/10.1136/thorax.55.2.133 PMID:10639531View ArticleGoogle Scholar
- Abdeldaim G, Herrmann B, Mölling P, Holmberg H, Blomberg J, Olcén P et al. Usefulness of real-time PCR for lytA, ply, and Spn9802 on plasma samples for the diagnosis of pneumococcal pneumonia. Clin Microbiol Infect 2010;16:1135–41. https://doi.org/www.dx.doi.org/10.1111/j.1469-0691.2009.03069.x PMID:19832718View ArticleGoogle Scholar
- Azzari C, Cortimiglia M, Moriondo M, Canessa C, Lippi F, Ghiori F et al. Pneumococcal DNA is not detectable in the blood of healthy carrier children by real-time PCR targeting the lytA gene. J Med Microbiol 2011;60:710–4. https://doi.org/www.dx.doi.org/10.1099/jmm.0.028357-0 PMID:21349984View ArticleGoogle Scholar
- Stephen AB. The PCR revolution: basic technologies and applications. New York: Cambridge University Press; 2010. ISBN-13 978-0-511-65839-6Google Scholar
- Wittwer CT, Herrmann MG, Gundry CN, Elenitoba-Johnson KS. Real-time multiplex PCR assays. Methods 2001;25:430–42. https://doi.org/www.dx.doi.org/10.1006/meth.2001.1265 PMID:11846612View ArticleGoogle Scholar