Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes

Abstract

We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well-characterized isolates, representing all 90 serotypes, and 268 “unknowns.” The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera.

Previously, we developed a molecular capsule type prediction system for 90 Streptococcus pneumoniae serotypes, based on a combination of partial cpsA-cpsB sequencing and serotype- and/or serogroup-specific PCR (3, 6). While this system is useful, it is too slow, expensive, and labor-intensive for routine use.

Multiplex PCR-based reverse line blot hybridization (mPCR/RLB) is a promising method for simultaneous detection and genotyping of microorganisms, which we have used previously in several applications (5, 9, 10). In the present study we applied it to identification of the 23 S. pneumoniae serotypes represented in the polysaccharide vaccine (Pneumovax 23; Merck & Co, Inc.).

The design of primers and probes was based on recently published full cps gene cluster sequences of all 90 pneumococcal serotypes (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) and others available in GenBank (3, 6; F. Kong, G. L. Gilbert, L. Wang, D. Liu, and J. Tao, 13 April 2004, Australian Patent Office). For the mPCR, we modified the primers we had used previously for serotype-specific PCR for the 23 vaccine serotypes (3, 6; Kong et al., Australian Patent Office) and a published S. pneumoniae-specific primer (7). For the RLB, we designed two probes for each of the 23 serotypes, as well as two S. pneumoniae-specific control probes (Table 1). Probes and primers were designed to have similar physical characteristics so as to allow simultaneous amplification and hybridization in a multiplex reaction system (5); they were synthesized by Sigma-Aldrich (Sydney, Australia). Primers were biotinylated at the 5′ end and probes had a 5′-amine group (5).

TABLE 1.

Oligonucleotide primers and probes for multiplex PCR-based reverse line blot assay used in this study

Primera	Target	Specificityb	Tm (°C)c	GenBank accession no.	Sequence (5′ to 3′)d
SpIaSbe	Pneumolysin	S. pneumoniae	61.43	M17717	384ATT TCT GTA ACA GCT ACC AAC GA406
SpSp	Pneumolysin	S. pneumoniae	65.49	M17717	531AGC GAT AGC TTT CTC CAA GTG G552
SpAp	Pneumolysin	S. pneumoniae	75.10	M17717	556ACC CCA GCA ATT CAA GTG TTC GCG579
SpIbAbe	Pneumolysin	S. pneumoniae	61.69	M17717	730GAA TTC CCT GTC TTT TCA AAG TC708
1Sb	wzy	Serotype 1	59.03	Z83335	10307GGG ACT TTA ATT TTA TGC AGT G10328
1Ap	wzy	Serotype 1	59.22	Z83335	10402AAA TTT CAC AAT TAT CAT TGC C10381
1Sp	wzy	Serotype 1	62.00	Z83335	10529CTG GCT TTG GCA ACT TTG10546
1Ab	wzy	Serotype 1	60.67	Z83335	10576CAC AAT GGC TTT AGA AGG TAG AG10554
2Sb	wzy	Serotype 2	59.86	AF026471	9730CGG CAT TGT ATT CTT TAT ATC G9751
2Ap	wzy	Serotype 2	60.40	AF026471	9849CCA ATA AAT CTT GTG TGA ATA TAA CTG9823
2Sp	wzy	Serotype 2	60.76	AF026471	9989GCA ACA TTT CAA TCT TAT GGT G10010
2Ab	wzy	Serotype 2	63.05	AF026471	10049CGT TTG TAT CCA TTT AAC TGC ATC10026
3Sb	wze	Serotype 3	59.32	Z47210	5807TTG ATA TTC CCC TTG ACA ATA G5828
3Sp	wze	Serotype 3	59.62	Z47210	5910TTT ACT ACA GTC CTC TTT CTC TGC5887
3Ap	wze	Serotype 3	58.63	Z47210	6056GCC TCA TCC TTA ATT GGA G6038
3Ab	wze	Serotype 3	61.69	Z47210	6102GGA GGC TTC AAG ATT CAA CTC6082
4Sb	wzy	Serotype 4	61.09	AF316639	9619CCG TCT ATA TTT ATA TGG GTT TGC9642
4Ap	wzy	Serotype 4	60.08	AF316639	9774TTG AAA CCC CAT ATA CTC ATT G9753
4Sp	wzy	Serotype 4	60.49	AF316639	9848GGA TTT TGT TTT GTT ATT CTG TAG G9872
4Ab	wzy	Serotype 4	59.48	AF316639	9934CCT GAT AAT TTT GTA CTT CTG AAT G9910
5Sb	wzy	Serotype 5	61.23	AY336008	6052GTT TTC CCA ATA GTA GTT TGC G6073
5Ap	wzy	Serotype 5	61.86	AY336008	6118TGC GAT AAA ATA GAT AAG GCA ATC6095
5Sp	wzy	Serotype 5	62.20	AY336008	6275TGC TTT GTT AGA TAT AGT TAC GGG AG6300
5Ab	wzy	Serotype 5	62.04	AY336008	6349CCC ACA GCC AAA TAG AGT TG6330
6B6ASb	wzy	Serogroup 6 (6A, 6B)	58.77	AF316640	9263TCA ACC TGC AGT AAT TTT AAC A9284
6B6AAp	wzy	Serogroup 6 (6A, 6B)	60.95	AF316640	9322TTA ACT AGA GCA CTT GCA ATC G9301
6B6Asp	wzy	Serogroup 6 (6A, 6B)	59.01	AF316640	9461GAA AGA AAT AAA TCC TTC AAA GAT AAT9487
6B6AAb	wzy	Serogroup 6 (6A, 6B)	60.19	AF316640	9555CTA CTT TCT GAA TTT CAC GGA TAT AAA G9528
7F7ASb	wzy	Serotypes 7F, 7A	61.48	CR931643	14476GCA AGT GTT TCA ATG GGA GTA14496
7F7AAp	wzy	Serotypes 7F, 7A	58.71	CR931643	14537AAA TTC CAC AAT ATT GGT AAT ACT G14513
7F7Asp	wzy	Serotypes 7F, 7A	58.58	CR931643	14776TTT TTG TAT GAT TTA AAT ATT GCG14799
7F7AAb	wzy	Serotypes 7F, 7A	61.51	CR931643	14820ACG GAG GGA CCA TAC AAT AAG14800
8Sb	wzy	Serotype 8	60.50	AF316641	10827GAT GTT AGT TTC TTC GAT TCC AG10849
8Ap	wzy	Serotype 8	59.15	AF316641	10870GAG GAA ACC CAC AAA GTC AAA AAA C10849
8Sp	wzy	Serotype 8	59.38	AF316641	11012AAA ATT ATG TTT ACT TTA CGA GTT GG11037
8Ab	wzy	Serotype 8	61.32	AF316641	11066TCA TAA TGA ATC GTA CCA ATC AAC11043
9N9LSb	wzy	Serotypes 9N, 9L	62.46	CR931647	9818TCA ATG GCG ACT TTA TTT GC9837
9N9LAp	wzy	Serotypes 9N, 9L	58.53	CR931647	9893GAA CTT TGG GAA TAT AAT CAA AAG9870
9N9LSp	wzy	Serotypes 9N, 9L	62.15	CR931647	10139GTC GGT TTC GAT TCT TTG C10157
9N9LAb	wzy	Serotypes 9N, 9L	60.41	CR931647	10179AGT CTA TTA TCT CCT GTA GGG TGC10156
9V9ASb	wzy	Serotypes 9V, 9A	58.70	AF402095	8546AAC TAT ATT TAC CCT ACT CTC CAC AG8571
9V9AAp	wzy	Serotypes 9V, 9A	58.76	AF402095	8639AAT CAG CGT TAC CTT ATA CTG TG8617
9V9ASp	wzy	Serotypes 9V, 9A	59.62	AF402095	8797GCC TCT TTT TAA CCT TTA TCT TGT8820
9V9AAb	wzy	Serotypes 9V, 9A	61.56	AF402095	8860ACC GGA AAA AGC AAT TGA G8842
10A10BSb	wzy	Serotypes 10A, 10B	61.86	CR931649	7172TGA GCT ATT TAA GGA CCT GGG7192
10A10BAp	wzy	Serotypes 10A, 10B	62.30	CR931649	7206GTT TAG AAA CCT TGC CCA GG7187
10A10BSp	wzy	Serotypes 10A, 10B	62.25	CR931649	7353ACC ATA TGG TAT TTG TTG CCT G7374
10A10BAb	wzy	Serotypes 10A, 10B	61.76	CR931649	7450GCA AGC GTC ACT TTC TTG A7432
11A11DSb	wzy	Serotypes 11A 11D	59.48	CR931653	11368GAA ATA TCG CCA TTC ATC AG11387
11A11DAp	wzy	Serotypes 11A, 11D	59.34	CR931653	11407TGT AAG ATG AAA AAA GCA TGC11387
11A11DSp	wzy	Serotypes 11A, 11D	59.42	CR931653	11676GGA TTT CTC GTA TCA GCA TAT TAC11699
11A11DAb	wzy	Serotypes 11A, 11D	62.68	CR931653	11744CAA CAG CAA CTG TGC CAC T11726
124446Sb	wzy	Serotypes 12F, 12A, 12B, 44, 46	59.41	CR931660	8914TGA ATA TGG ACG GTG GAG8931
124446Ap	wzy	Serotypes 12F, 12A, 12B, 44, 46	58.48	CR931660	8951GAA GAA GTT CAA CAA TCG CT8932
124446Sp	wzy	Serotypes 12F, 12A, 12B, 44, 46	59.37	CR931660	9104GCA TGA TAT GGG AAG TTT TG9123
124446Ab	wzy	Serotypes 12F, 12A, 12B, 44, 46	59.69	CR931660	9155AGC AAA GAA AGC CGA AAG9138
14Sb	wzy	Serotype 14	60.41	X85787	7376CCT ACT TCC AAA ACA GTT TAT GC7398
14Ap	wzy	Serotype 14	59.77	X85787	7494CCA TAC AAA AAG ACG GTG TAT C7473
14Sp	wzy	Serotype 14	59.30	X85787	7587GCA TTT AAT TGG CTA ATA GCA G7608
14Ab	wzy	Serotype 14	59.69	X85787	7652GTC AAT ATT GAT TGG CAT TTT C7631
15B15CSb	wzy	Serotypes 15B, 15C	62.63	CR931664	7797TAA TAA GCG GAT GAT TGT AGC G7818
15B15CAp	wzy	Serotypes 15B, 15C	58.19	CR931664	7837GAG GTA TAG TTG GAT AAA ACG C7816
15B15CSp	wzy	Serotypes 15B, 15C	62.54	CR931664	7976GAG CAG GAA TCA GAA CAC AAT C7997
15B15CAb	wzy	Serotypes 15B, 15C	60.91	CR931664	8148TAT ACT GAT TAA CTT TCC AGA TGG G8124
17FSb	wzy	Serotype 17F	59.84	CR931670	13988AGA GGG ATT GTT GAA GGT ATT C14009
17FAp	wzy	Serotype 17F	60.89	CR931670	14035GGA AGT GAA CGT CAA ATC TTT T14014
17FSp	wzy	Serotype 17F	61.75	CR931670	14250CAA TGC TAT GTC GCA AAT ATT G14271
17FAb	wzy	Serotype 17F	61.34	CR931670	14295AGT AGT CTC GCA TTT CTA TCA TCC14272
18Sb	wzy	Serogroup 18 (18F, 18A, 18B, 18C)	60.45	AF316642	12208AAT TGT TCT TTT CCT GTA CTC AGT C12232
18Ap	wzy	Serogroup 18 (18F, 18A, 18B, 18C)	58.72	AF316642	12260ATT CAA C/TTG GGT TCA TTA CG12241
18Sp	wzy	Serogroup 18 (18F, 18A, 18B, 18C)	58.74	AF316642	12425GGA GGA CTT AGT CAA TTT ATC TTG12448
18Ab	wzy	Serogroup 18 (18F, 18A, 18B, 18C)	62.13	AF316642	12478CGA ACC ATT GAA ACT ATC ATC TG12456
19ASb	wzy	Serotype 19A	61.40	AF094575	9260TGT ATT TGC CCT TAT TAA TGT GC9282
19AAp	wzy	Serotype 19A	61.12	AF094575	9336TGC CAC TAA TAA TCA AAA GAT AAG C9312
19ASp	wzy	Serotype 19A	60.81	AF094575	9422CCA ATT CTG GAA AAT AGC TCT TAC9445
19AAb	wzy	Serotype 19A	60.63	AF094575	9506AAG TGC AAG ATT ATG AAT CTC TCT C9482
19FSb	wzy	Serotype 19F	59.55	U09239	7693TCA GTA TTT GCA CTG GTT AAT TC7715
19FAp	wzy	Serotype 19F	61.85	U09239	7800TGC CAT TAA AGG AAT CGA AA7781
19FSp	wzy	Serotype 19F	60.35	U09239	7858CAA TTT TGG AAA ATT GCT CTA AC7880
19FAb	wzy	Serotype 19F	59.39	U09239	7941AAG AAC AAG GTT GTA TAT TTC CTT C7917
20Sb	wzy	Serotype 20	61.18	CR931679	7711CTT TAT CAG GAA TAC GCC AAT C7732
20Ap	wzy	Serotype 20	60.16	CR931679	7759GCA TAA AAA ACA ATC GCT GTA G7738
20Sp	wzy	Serotype 20	62.31	CR931679	7933CTG GGT CTG AAT TTG TAT CTC G7954
20Ab	wzy	Serotype 20	59.11	CR931679	8010CTG TAT AAT AAC GAG AAC CAA CG7988
22F22ASb	wzy	Serotypes 22F, 22A	61.86	CR931682	13190AGG ATG CAG TAG ATA CCA GTG G13211
22F22AAp	wzy	Serotypes 22F, 22A	59.68	CR931682	13263TAA TAC CAT GGC ACT AGG AAT AAC13240
22F22ASp	wzy	Serotypes 22F, 22A	58.30	CR931682	13503ATG GCT ATC AAC TTT ATC TAG GAC13526
22F22AAb	wzy	Serotypes 22F, 22A	60.25	CR931682	13543TAT AAA CGG AGG TTG TTG TCC13523
23FSb	wzy	Serotype 23F	60.39	AF057294	8583TGA TAG TGA ACT TGG GAT TGT C8604
23FAp	wzy	Serotype 23F	61.24	AF057294	8694CTA TTT GCA AAC ACG TTG AGA G8673
23FSp	wzy	Serotype 23F	58.36	AF057294	8735GGG ATT AAT TTA CAA AAT CTT CC8757
23FAb	wzy	Serotype 23F	58.19	AF057294	8827CTT TAT CGG TAA GGT GGA TAA G8806
33F33A37Sb	wzy	Serotypes 33F, 33A, 37	61.77	AJ006986	11362TCA ACT AGT CAA GGA TTT GAT GG11384
33F33A37Ap	wzy	Serotypes 33F, 33A, 37	60.62	AJ006986	11421TGA TAC CAC AAG TAA CAG AGT CG11399
33F33A37Sp	wzy	Serotypes 33F, 33A, 37	59.52	AJ006986	11589GAT GAT TTT GCA GAT GTA CTA TGA11612
33F33A37Ab	wzy	Serotypes 33F, 33A, 37	59.67	AJ006986	11639CGT ATC AGA TTT GCG ATT TC11620

^aS, sense; A, antisense; b, biotin-labeled primer (primers were biotin labeled at the 5′ end); p, probe (probes were 5′ end C6 amine labeled).

^bBased on published sequence data for the whole cps gene cluster, it was not possible to design primers/probes that could distinguish some individual serotypes from one or four closely related ones (shown in boldface), usually (but not always) belonging to the same serogroup.

^cT_m values were provided by the primer synthesizer (Sigma-Aldrich).

^dNumbers represent the base positions at which primer/probe sequences start and finish (starting at “1” of the corresponding GenBank sequence).

^eTwo primers published previously (8) were used as species-specific probes.

The DNA extraction method (4), the mPCR system, and the thermal profiles used were as previously described (5), except that we included 24 (rather than 10) primer pairs (Table 1) in a 25-μl mPCR system and used 1 U (rather than 0.5) of QIAGEN Hotstart Taq polymerase. For serotype 23F, 25 pM of each primer were used, whereas 12.5 pM of each primer was used for all other serotypes.

RLB hybridization was based on previously published methods (http://www.nioo.knaw.nl/cl/me/) (8, 11) with the following modifications: the hybridization temperature was 60°C, and 1.25 pM of each probe, in 150 μl of 500 mM NaHCO₃ (pH 8.4), was used in each slot to label the membrane (Fig. 1).

FIG. 1.

mPCR/RLB results for a representative sample of 43 clinical isolates. See Table 1 for descriptions and specificities of probes listed at left. The choice of probes for this membrane, in addition to two pairs of S. pneumoniae-specific probes, was based on two for each of the serotypes represented in the 11-valent conjugate vaccine (serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F [shown in the top half of the membrane]) and others that are identified relatively commonly (serotypes 8, 9N, 19A, 20, and 33F) and single probes for each of the less common serotypes among those represented in the 23-valent vaccine (serotypes 2, 10A, 11A, 12, 15B, 17F, and 22F). The conventional serotypes of the 43 isolates shown on this membrane are (from left to right): 14, NT, 19F, NT, 19F, 22F, 9V, 6A, 19F, 18C, NSP, 19F, 16F, 11A, 4, 19F, 19F, 19F, 19F, 6B, 10A, 19F, 23F, 19F, 23F, 19A, 23F, 6A, 6B, 9V, 3, 14, 19F, 6B, 11A, 6A, 14, 23F, 19F, 46, 47F, 47A, and 48. NT, nontypeable. NSP, not S. pneumoniae.

A collection of 334 reference strains and well-characterized clinical isolates were used to develop the assay, of which 244 had been used in our previous studies (3, 6) and 90 were serotype reference strains, newly purchased from Statens Serum Institut of Denmark (1, 2). Except for serotypes 10C, 11F, 12B, 25A, 33D, and 44, all serotypes were represented by two or more strains.

All of the putative serotype-specific primers or probes yielded mPCR products and RLB signals from isolates with the corresponding serotypes. However, as demonstrated previously, serotype discrimination based solely on the wzy gene (or even the whole cps gene cluster) is not straightforward (3, 6). Primers and probes designed to identify serotypes 6B, 7F, 9N, 9V, 10A, 11A, 12F, 15B, 18C, 22F, and 33F could not distinguish between the target and one or more closely related serotypes, usually but not always in the corresponding serogroup (Table 1 and Fig. 1) because they share virtually identical wzy sequences. Thus, 17 serotypes, in addition to those in the 23-valent polysaccharide vaccine, were amplified and hybridized by the mPCR/RLB system. These cross-reactions are predictable, and individual serotypes can be identified using a limited number of factor antisera. The remaining 12 primer pairs and probe pairs were truly serotype specific, and there was excellent agreement between paired probes for the same serotypes, indicating that single probes would be adequate for most serotypes.

The method was further evaluated using 268 clinical isolates, the serotypes of which were unknown at the time of mPCR/RLB testing. These isolates included 135 consecutive invasive isolates referred to the NSW Pneumococcal Reference Laboratory for serotyping and 133 colonizing isolates from patients with respiratory infections at the Children's Hospital, Westmead, New South Wales, Australia. Conventional serotyping was performed using the Quellung reaction, as previously described (3). Two isolates were not amplified by mPCR/RLB, and phenotypic retesting showed that they were not S. pneumoniae. Of 266 pneumococcus strains, 12 (4.5%) were nontypeable by mPCR/RLB (amplified by mPCR but hybridized only with the pneumococcal control probe); of these, 10 belonged to serotypes not represented in the current mPCR/RLB assay, namely, 16F (one isolate), 23A (three isolates), 35F (four isolates), 35B (one isolate), and 38 (one isolate), and 2 were not serotypeable. Another 4 of the 266 pneumococcus isolates were not serotypeable but were identified by mPCR/RLB as serotype(s) 4, 11A/11D, 14, and 33F/33A/37. The predicted serotype(s) of the other 250 isolates were as follows: 3 (5 isolates), 4 (13 isolates), 6B/6A (45 isolates), 7F/7A (1 isolate), 9V/9A (15 isolates), 10A/10B (2 isolates), 11A/11D (2 isolates), 14 (51 isolates), 15B/15C (4 isolates), 18 (14 isolates), 19F (63 isolates), 19A (12 isolates), 22F/22A (2 isolates), and 23F (21 isolates). Thus, 181 of 254 (71%) isolates that were typeable by mPCR/RLB were identified exactly, and another 73 (29%) were identified to within one to five serotypes, for which a limited number of antisera were needed to distinguish individual serotypes.

Three isolates identified by mPCR/RLB as serotypes 19A, 9V/9A, and 23F had been serotyped, initially, as 19F, 19A, and 6B, respectively. The discrepancies were resolved by repeating the serotyping, which confirmed that the mPCR/RLB results were correct.

Our molecular serotype prediction mPCR/RLB assay for 23 pneumococcal “vaccine” serotypes is clearly less discriminatory than conventional serotyping (1-3, 6; Kong et al., Australian Patent Office) because individual serotype-specific targets are not available within cps gene clusters of some serotypes. However, there are obvious advantages of the mPCR/RLB. First, it uses reagents and techniques that are available in many microbiology laboratories; an uncomplicated, rapid DNA preparation method (4); and a single mPCR/RLB reaction (5). Second, it provides more consistent and objective results than immunological methods such as the Quellung reaction; cross-reactions are predictable and can be resolved with a small number of antisera. Serotypes that are nontypeable by the mPCR/RLB are isolated uncommonly from clinical specimens (∼5% of invasive isolates in NSW in the past 3 years [data not shown]). Third, the method, potentially, could to be used directly to test clinical specimens and so rapidly identify possible vaccine failure.

Currently, there is no single, ideal technique for pneumococcal serotyping, but this mPCR/RLB format is convenient, rapid, objective, reproducible, and discriminatory when used in conjunction with a limited number of antisera.