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Journal of Clinical Microbiology, February 2003, p. 601-607, Vol. 41, No. 2
0095-1137/03/$08.00+0 DOI: 10.1128/JCM.41.2.601-607.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
David B. Griffiths,1 Siobhán A. Martin,2 Robert C. George,2 and Lucinda M. C. Hall1*
Department of Medical Microbiology, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London E1 2AD,1 Respiratory and Systemic Infection Laboratory, Central Public Health Laboratory, London NW9 5HT, United Kingdom2
Received 22 April 2002/ Returned for modification 25 September 2002/ Accepted 29 October 2002
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ABSTRACT |
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INTRODUCTION |
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New pneumococcal conjugate vaccines, which include up to 11 of the 90 known capsule types, are undergoing extensive trials. A seven-valent conjugate vaccine covering serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F has been shown to be safe, immunogenic, and efficacious in the prevention of invasive pneumococcal disease in American children (4) and is licensed for use in the United States and for use for European infants and children between the ages of 2 months and 2 years. Conjugate vaccines are being recommended for routine use to reduce pneumococcal disease in early childhood (9). With the limited coverage of conjugate vaccines, it is important to maintain regular monitoring of serotype prevalence in case widespread vaccination programs lead to a selective increase in serotypes not covered by the conjugate vaccines (20).
Serotype prevalence is currently monitored by culture of the organism, followed by serology. We have previously demonstrated that evaluation of the polymorphisms within the cpsA and cpsB genes common to all capsule loci can be used as a DNA-based method to predict pneumococcal serotype (14). However, a limitation of both these methods is that the presence of multiple serotypes may not be detected (10). A potential solution is the use of PCR to detect DNA from regions of the capsule locus that directly determine serotype and serogroup. The sequences of the genes responsible for capsule production in several serotypes have been published, and serotype-specific genes have been identified. Unfortunately, this approach requires multiple PCRs for each sample, and manual analysis would barely be feasible in laboratories typing large numbers of isolates.
To make a serotype-specific PCR method amenable for use for routine testing, we have investigated the use of a multiplex PCR procedure coupled with capillary electrophoresis for automated analysis. This was applied to the detection of five commonly occurring serotypes for which capsule locus sequence data are available, namely, serotypes 1, 3, 14, 19F, and 23F, and also to the detection of three serogroups, serogroups 6, 19, and 23. The potential of the system to determine serogroups and serotypes was examined by using pneumococcal cultures submitted to the major reference center in the United Kingdom.
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MATERIALS AND METHODS |
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Other streptococci used to test the specificity of the primers were Streptococcus dysgalactiae (NCTC 4669), S. gordonii (NCTC 7865), S. bovis (NCTC 8133), group G streptococcus (NCTC 9603), group K streptococcus (NCTC 10232), S. anginosus (NCTC 10713), S. mutans (NCTC 10832), S. sanguis (NCTC 11085), S. oralis (NCTC 11427), S. pyogenes (NCTC 12068), S. vestibularis (NCTC 12166), S. mitis (NCTC 12261), S. parasanguis (NCTC 12854), S. agalactiae (NCTC 12906), S. viridans (NCTC 11189), S. intermedius (NCTC 11324), S. constellatus (NCTC 11325), and S. equisimilis (NCTC 11564).
Serotyping of isolates. Preliminary serotyping was performed by slide agglutination (6) with capsular typing sera and factor sera (Statens Serum Institut). Any discrepancies or equivocal results were rechecked by the Quellung reaction.
DNA extraction. S. pneumoniae reference strains and streptococci of other species were cultured overnight in 20 ml of Todd-Hewitt broth (Oxoid, Basingstoke, United Kingdom). After sedimentation the cells were resuspended in 0.1 ml of TE (10 mM Tris HCl, 1 mM EDTA [pH 8.0]). Lysozyme (10 µl at 50 mg/ml) was added, and the cells incubated for 30 min at 37°C, followed by addition of 0.5 ml GES (5 M guanidine thiocyanate, 0.1 M EDTA [pH 8.0], 0.5% Sarkosyl) and incubation at room temperature for 10 min. Ammonium acetate (0.25 ml at 7.5 M) was added, and the mixture was placed on ice for 10 min. Chloroform-isoamyl alcohol (24:1) was added, the contents were mixed and separated by centrifugation, and 0.7 ml of the aqueous phase was recovered. DNA was precipitated with 0.54 volume of isopropanol and then redissolved and reprecipitated in 70% ethanol. The DNA was finally resuspended in 50 to 100 µl of TE.
Pneumococcal DNA from clinical isolates was prepared as a crude extract. A loopful of growth from a fresh plate was suspended in 100 µl of TE, and the mixture was heated to 100°C for 10 min and then placed on ice until it was used.
PCR. Standard PCR was performed with 50 µl of 50 mM KCl, 15 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, each deoxynucleoside triphosphate at a concentration of 0.2 M, 20 pmol of each primer (Table 1), 1 µl of template DNA (purified or crude extract), and 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, Warrington, United Kingdom). The conditions for PCR were 1 cycle at 94°C for 10 min and then 32 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 60 s in a Perkin-Elmer GeneAmp 2400 instrument (Applied Biosystems). Alternative conditions used during optimization are described in Results. PCRs were set up in a UV-treated cabinet (Template Tamer; Oncor Appligene, Chester-le-Street, United Kingdom) and performed and analyzed in separate rooms.
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A green flash capillary (47 cm by 50 µm [inner diameter]) cut down to 41 cm was used with 0.6x 310 genetic analyzer buffer (containing EDTA) and 2% 310 GeneScan polymer made up in 0.6x 310 genetic analyzer buffer containing EDTA. The separation conditions were 15 kV for 7 min at 60°C with filter set D. The samples were injected at 15 kV for 7 s.
The electropherograms were analyzed with the Genotyper program (version 2.1; Applied Biosystems). Peaks representing the specific PCR products were automatically identified according to their size, as described in Results.
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RESULTS |
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Primers were designed for each selected serotype and serogroup on the basis of the criteria of a melting temperature of 57 ± 1.5°C and the production of products of different sizes. A size range of 100 to 500 bp was chosen to minimize the likelihood of amplification of nonspecific products and increase the thermocycling speed. In the case of primers specific for serotype 19F, which has a high degree of similarity to serotype 19A, primers were designed on the basis of the cps19fI sequence, with the forward and reverse primers positioned so that the last two bases at the 3' terminus were specific for serotype 19F. The genes and product sizes chosen are summarized in Table 1.
Reference laboratories require robust PCRs that are specific and efficient and that can be performed under standardized conditions, necessitating empirical optimization of the reaction conditions. For each serotype-specific primer pair the annealing temperature was tested between 58 and 64°C in 2°C steps on a Perkin-Elmer GeneAmp 2400 instrument. The best compromise among the optimum annealing temperatures for the various primer pairs was identified as 61°C, and that temperature was adopted in all subsequent PCRs. Similarly, for each pair a series of PCRs containing 1 to 3.5 mM MgCl2 in 0.5 nM increments was performed, and 2.5 mM was selected as the optimal concentration. Amplifications were also optimized on alternative thermocyclers, the Perkin-Elmer GeneAmp 9700 instrument and the Hybaid Omni Gene instrument. The Omni Gene instrument was found to have a slow ramping time, so shorter annealing and elongation times of 15 and 60 s, respectively, could be used. Conversely, the 9700 instrument has a very fast ramping time. The difference in ramping between the 9700 instrument and the 2400 instrument was timed, and the ramping rate for the 9700 instrument was adjusted accordingly to 80% for the denaturation to annealing step. These adjustments allowed comparable results to be obtained between the three machines (Table 2).
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To identify PCRs that are falsely negative due to inhibitors or human error, imperative in a clinical situation, an internal positive control (IPC) that could be used in all PCRs was required (3, 7). This was adapted from a 500-bp bacteriophage lambda internal positive control from the GeneAmp Gold PCR kit (Applied Biosystems), but it amplified from bacteriophage lambda DNA a product of 75 bp, well below the size range of the pneumococcal products. The concentrations of the bacteriophage lambda DNA and primers were limited so that amplification of serotype- and serogroup-specific and pneumococcus control products would not be impaired. A consequence of limiting the IPC and IPC primers, as described by Ke et al. (12), is that in the presence of target DNA the IPC is not amplified due to competitive inhibition. This provides a visual advantage during interpretation of the results (Fig. 1).
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The serotype and serogroup specificities of the primers were evaluated with purified genomic DNA from type strains of the 23 most prevalent serotypes (5), corresponding to those contained in current polysaccharide vaccines: serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9A, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The primers for serotypes 19F and 23F and serogroups 6, 19, and 23 were also tested with all subtypes of the same serogroups. No serotype-specific primer pair yielded a product with DNA from any of the other 23 serotypes tested; the primers specific for serotypes 19F and 23F were also negative in assays with isolates of serotypes 19A, 19B, and 19C and serotypes 23A and 23B, respectively. The serogroup-specific primers yielded products from all subtypes of their respective serogroups and were negative with isolates of other serogroups and serotypes.
To ensure that the primers would detect all pneumococcal isolates of the intended serogroup or serotype, each primer pair was tested with crude colony preparations of 9 to 10 recent United Kingdom clinical isolates of the respective serogroup or serotype. Each primer pair yielded products for all isolates of the target serogroup or serotype.
Design of multiplex reactions. Primers were tested in combination, since multiplexing of the reactions would reduce labor, time, and cost. Three multiplex reactions were developed: one for serotypes 3, 14, and 23F, one for serotypes 1 and 19F, and one for serogroups 6, 19, and 23. The concentrations of the primers and the reaction conditions were adjusted to allow similar levels of amplification for each product when a mixture of DNA from all target serotypes and serogroups was tested.
Capillary electrophoresis and automated detection of products. Capillary electrophoresis was chosen for automated detection of PCR products. This allows detection on the basis of both the product size and the color of the fluorescent label, but it is less cumbersome than gel-based methods (15). The system used was an ABI Prism 310 genetic analyzer and GeneScan analysis software (the software assigns size labels to peaks). Analysis (serotype assignment and detection of control products) was automated by using ABI Prism Genotyper software.
For fluorescence detection of the PCR products, one primer from each pair was labeled at its 5' terminus with 6-carboxyfluorescein (6-FAM) or hexachlorofluorescein (HEX) during synthesis. When they are excited by the argon laser (488 and 514.5 nm), these dyes fluoresce blue and green, respectively. Serotype- and serogroup-specific primers were labeled with 6-FAM, while the pneumococcus control and IPC primers were labeled with HEX; thus, the products were easily distinguished. The denatured PCR products were coinjected with a GeneScan 500 size standard (35 to 500 bp) labeled with 6-carboxy-‘x'-rhodamine, which fluoresces red, to enable accurate sizing. Peak height thresholds were set at 40, 500, and 200 fluorescent units for red, blue, and green, respectively, to prevent background noise and nonspecific peaks from being detected.
Initially, the manufacturer's recommended conditions of a 47-cm capillary, 4% polymer (POP4), and the GS STR POP4 (1 ml) D module were used. The module contains the voltage and time setting used during a run. Although the products were correctly detected and sized, each run was 24 min long. Thus, analysis of one isolate involving four multiplex reactions would be completed every 96 min. The protocol was therefore modified to reduce the run time to 7 min, and the products were still found to be correctly sized within the 5-bp resolution of the method. The detection times of the size standards and the sizes of PCR products varied slightly between runs, depending on the temperature of the room.
For analysis of the results, a macro was programmed to scan the electropherograms for peaks of the expected sizes and colors, as defined in Table 3, and to apply a name to the corresponding peak. The results are presented in a tabular format, with each PCR generating two table rows, one for blue peaks (multiplexed serotype- and serogroup-specific PCRs) and one for green peaks (pneumococcus control and IPC). An example of the output for one isolate is given in Table 4. PCR products could also be separated by agarose gel electrophoresis for comparison with the result obtained with the genetic analyzer (Fig. 1). Interpretation of the results was always consistent between the two methods, although the small IPC product was only weakly stained with ethidium bromide.
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The results of the prediction of pneumococcal species and serotypes by the multiplex PCR assay are summarized in Table 5. All 93 isolates were correctly predicted to be pneumococci. Thirty-two isolates with serotypes among the five vaccine types covered by the PCR were correctly assigned. Of these, the nine serotype 19F isolates and the three serotype 23F isolates were also correctly assigned to serogroups 19 and 23, respectively. Thirteen serotype 6B isolates and six serotype 6A isolates were correctly predicted to be serogroup 6, while one serotype 9V isolate was incorrectly predicted to be serogroup 6. Forty-one isolates with serotypes not covered by the PCRs were correctly predicted to have other serotypes. The IPC was present and detected in all PCRs with negative results. Overall, these results demonstrate that the multiplex PCR has a sensitivity of 100% and a specificity of 99% for the detection of the serotypes and serogroups covered.
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DISCUSSION |
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It is envisaged that the present method, like conventional pneumococcal serotyping, would be performed largely in reference laboratories in the first instance. With an appropriate infrastructure in place, the multiplex PCR described here is estimated to have running costs similar to those of conventional serotyping, although a lower proportion of isolates would be assigned a type at present.
We have previously described a method for serotype prediction in which a fragment from cpsA-cpsB common to the capsule loci of all serotypes was amplified and restriction polymorphisms were detected (14). Although there was a strong linkage between the cpsA-cpsB polymorphism and serotype, this was not absolute. The method required only one PCR for each isolate, and a correlation to the serotype did not necessitate knowledge of the capsule locus sequence for each serotype. In comparison to the earlier method, the method described here involves a larger number of PCRs, but interpretation of the results is simpler and the procedure is readily amenable to further automation. Perhaps most significant is the fact that the present method will allow the detection of more than one serotype in a single sample.
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ACKNOWLEDGMENTS |
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E.R.L. was supported by a studentship from the Medical Research Council. The work was supported by the Special Trustees of the Royal London Hospital and by the Public Health Laboratory Service Central Public Health Laboratory.
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FOOTNOTES |
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Present address: DNA Research Innovations Ltd., Sittingbourne Research Centre, Kent ME9 8PX, United Kingdom.
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REFERENCES |
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