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Journal of Clinical Microbiology, October 2007, p. 3411-3415, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.01076-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Less-Common Streptococcus pneumoniae Serotypes by a Multiplex PCR-Based Reverse Line Blot Hybridization Assay{triangledown}

Fei Zhou,1 Fanrong Kong,1 Zhongsheng Tong,1,2 and Gwendolyn L. Gilbert1*

Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia,1 Research Laboratory for Infectious Skin Diseases, Department of Dermatology, Wuhan First Hospital, Wuhan 430022, People's Republic of China2

Received 26 May 2007/ Accepted 29 July 2007


   ABSTRACT
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We developed a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify 50 uncommon pneumococcal serotypes. In combination with a previously described mPCR/RLB assay (3), all 90 pneumococcal serotypes can be identified individually (32 serotypes) or, because of predictable cross-reactions, to within small groups of two to five related serotypes (58 serotypes), which can be distinguished using serotype-specific antisera.


   TEXT
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Recently, we described a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to identify 40 Streptococcus pneumoniae serotypes, including the 23 represented in the polysaccharide vaccine and 17 others that show reproducible cross-reactions (3). In this study, we designed one pair of specific primers and one probe for each of the remaining 50 serotypes to allow provisional identification of all 90 known serotypes by mPCR/RLB (5). The primer and probe sequences were based on recently published full cps gene cluster sequences of all 90 pneumococcal serotypes (1) and on other sequences available in GenBank (4, 7).

Twenty sets of serotype-specific primer pairs and probes were designed; the remaining 30 serotypes shared identical or very similar wzy sequences with one or two others in the same or closely related serogroups, as follows: 7B/7C/40, 10F/10C, 11B/11C, 15F/15A, 19B/19C, 24F/24A/24B, 25F/25A/38, 28F/28A, 32F/32A, 33B/33D, 35F/47F, 35A/35C/42, and 41F/41A. The primers and probes used in this assay are shown in Table 1.


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  		TABLE 1. Oligonucleotide primers and probes for mPCR/RLB assay used in this study

 
Probes and primers were designed with similar physical characteristics to allow simultaneous amplification and hybridization in a multiplex reaction (5) and were synthesized by Sigma-Aldrich (Sydney, Australia). Primers were biotinylated at the 5' end, and probes had a 5' amine group (5). DNA extraction (6) and RLB hybridization were performed as previously described (5).

mPCR was performed according to the QIAGEN Hotstar Taq polymerase kit instructions (5) under the following conditions: 95°C for 15 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; 72°C for 10 min; and a hold at 22°C. The 34-primer-pair mPCR mixture was prepared to contain the following: 2 µl template DNA, 0.075 µl of each forward (100 pmol µl–1) and reverse (100 pmol µl–1) primer, 2.4 µl deoxynucleoside triphosphates (2.5 mM of each deoxynucleoside triphosphate), 3 µl 10x PCR buffer, 4.2 µl 25 mM MgCl2 (final concentration, 5 mM), 0.2 µl QIAGEN Hotstar Taq polymerase (5 units µl–1), and water to 30 µl.

Preliminary testing of reference strains of all 90 serotypes (Statens Serum Institut, Copenhagen, Denmark) showed that all 50 target serotypes (and none of the other 40) were amplified and hybridized by the mPCR/RLB system; 20 serotypes reacted only with the corresponding primers and probe, and 30 exhibited the predicted cross-reactions (Table 1; Fig. 1).


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  		FIG. 1. mPCR/RLB results for a representative sample of 43 clinical isolates (see Table 1 for descriptions and specificities of the probes listed on the left). Conventional serotype-mPCR/RLB results for the isolates shown, from left to right, are 43, 7B, 9A,* 9L,* 9V,* 19B, 11B, 11C, 12A,* 15F, 16F, 17A, 18F,* 19B, 19C, 21, 24F, 24A, 24B, 11B, 27, 28F, 28A, 32F, 32A, 33A,* 33B, 33B, 33F,* 35C, 36, 37,* 39, 40, 41F, 41A, 44,* 43, 7B, 45, 46,* 47F, and 47A. Isolates marked with asterisks belong to one of 40 serotypes identified by our original mPCR/RLB assay (3) and were not amplified in this assay.

 
The method was further evaluated using 173 previously studied (4, 7) clinical isolates from China, Australia, and Canada, which included one to four isolates of all pneumococcal serotypes except for 10C, 11F, 23B, 25A, and 33D (for which clinical isolates were not available). They were tested by mPCR/RLB without knowledge of the serotype. Sixty-eight isolates belonging to 1 of 40 serotypes identified by our original mPCR/RLB assay were not amplified. One nonserotypable isolate was also not amplified by either the previous (3) or new mPCR, although it was confirmed to be S. pneumoniae by species-specific PCR and phenotypic characteristics. This result suggests that this isolate either contains a significant cps deletion or mutation (8) or belongs to a novel serotype (10).

Forty-four isolates were identified as being of individual serotypes corresponding with those identified by conventional serotyping, and 59 were identified as being one of a group of two or three related serotypes which, with one exception, included the serotype identified by conventional serotyping (Table 2).


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  		TABLE 2. mPCR/RLB and serotype identification results for 173 previously studied isolates from China, Australia, and Canada

 
The exception was an isolate from China, which was identified as serotype 33B/33D by mPCR/RLB and as serotype 33C by conventional serotyping. Both methods were repeated and the initial (discrepant) results confirmed. Single PCR was positive with 33B/33D-specific and negative with 33C-specific primers. Sequencing of a portion of wzy of this isolate showed that it was different from that of any known serotype (1, 7) but more closely related to that of 33C than to that of 33B/33D. Although the discrepancy between molecular and immunological serotype identifications remained unresolved, our results suggest that wzy is not the only determinant of antigenic specificity (3). Further investigation of this isolate is in progress.

Finally, the mPCR/RLB assay was used to test 152 clinical isolates collected during 2000-2007 at the Centre for Infectious Diseases and Microbiology and selected as belonging to one of the 50 uncommon serotypes identified by the new mPCR/RLB system.

Thirteen isolates were not initially amplified by mPCR. Of these, 11 were successfully identified after modification of the DNA extraction method (5) by adding 2 µl of proteinase K (20 mg/ml; Sigma, Australia) to tubes before heating them at 100°C for 10 min. Two isolates that still were not amplified had been identified, using antisera, as serotypes 11F and 16A. Conventional serotyping was repeated and showed that they belonged to serotypes 11A and 18C, respectively (not represented in the current mPCR/RLB system). Two isolates, previously identified as serotypes 35A and 29, were identified by mPCR/RLB as 35F and 35B, respectively. Retesting with antisera confirmed the mPCR/RLB results.

Seventy-three isolates were identified by mPCR/RLB as single serotypes corresponding with those of conventional serotyping; 75 isolates gave positive results with probes cross-reacting with two or three related serotypes, which included the serotypes identified by conventional serotyping. The mPCR/RLB-predicted serotypes for these 152 isolates are shown in Table 3.


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  		TABLE 3. mPCR/RLB and conventional serotyping results for 152 clinical isolates from Centre for Infectious Diseases and Microbiology

 
Because serotype-specific targets within cps gene clusters are not always available, this mPCR/RLB assay is less discriminatory—although more objective and reproducible—than conventional serotyping (2, 4). In combination with our previous mPCR/RLB assay (3), it can identify 32 serotypes precisely and 58 as belonging to one of two to five related serotypes. This is the first report of a practical molecular assay that can predict all 90 serotypes without sequencing. Potentially, it can be used to identify multiple serotypes directly in clinical (including culture-negative) specimens. It could be adapted to other platforms, such as "liquid" molecular beacons (9) and solid microarrays (12).

For routine serotyping of pure pneumococcal cultures, our first mPCR/RLB assay (identification of 40 serotypes) would be used initially to identify the most prevalent serotypes (>95% of invasive isolates, based on unpublished data from the NSW Pneumococcal Reference Laboratory) (3). The current assay would then be needed, infrequently, to identify the small proportion of isolates belonging to less-common serotypes.


   ACKNOWLEDGMENTS
 
Fanrong Kong and Fei Zhou had similar contributions to the work and should be seen as co-first authors.

We thank Mitchell Brown and Shahin Oftadeh for conventional serotyping and for selecting appropriate clinical isolates.

This study was funded, in part, by National Health and Medical Research Council project grant 358351 (to G. L. Gilbert).


   FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Darcy Road, Westmead, New South Wales, 2145 Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: lyng{at}icpmr.wsahs.nsw.gov.au Back

{triangledown} Published ahead of print on 8 August 2007. Back


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Journal of Clinical Microbiology, October 2007, p. 3411-3415, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.01076-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




This Article
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