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Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization

¹Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia ²Department of Dermatology, Beijing Children's Hospital, Affiliate of Capital University of Medical Sciences, Beijing, PR China

Correspondence Gwendolyn L. Gilbert lyng{at}icpmr.wsahs.nsw.gov.au

Received 14 July 2005
Accepted 12 August 2005

Streptococcus agalactiae (group B streptococcus, GBS) is an important cause of sepsis in neonates and their mothers, and the elderly and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution is needed to guide the development and assess the feasibility of GBS conjugate vaccines. The authors previously developed a molecular serotype identification method based on serotype-specific PCR and partial sequencing of cps genes. In this study, a novel 10-primer pair multiplex PCR and reverse line blot (mPCR/RLB) hybridization assay was developed for simultaneous detection and serotype identification of all nine GBS serotypes. For all 316 GBS isolates tested the mPCR/RLB results corresponded with those of conventional serotyping and individual serotype-specific PCR, and the method was more convenient and practical than either alternative.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Streptococcus agalactiae (group B streptococcus, GBS) is the most common cause of neonatal (Ekelund & Konradsen, 2004) and obstetric sepsis, and is an increasingly important cause of septicaemia in the elderly and in immunocompromised patients (Schuchat et al., 2002; Tyrrell et al., 2000). There has been considerable progress in the development of conjugate polysaccharide GBS vaccines (Paoletti & Madoff, 2002). Serotyping of GBS is essential for epidemiological studies and future vaccine surveillance (Paoletti & Madoff, 2002).

Nine GBS capsular polysaccharide serotypes and corresponding capsular polysaccharide synthesis (cps) gene clusters are recognized (Cieslewicz et al., 2005). Their distribution varies according to geographic location and study population (Schuchat, 1998). Conventional serotyping (CS) requires high-titre serotype-specific antisera (Elliott et al., 2004), results are somewhat subjective and a significant proportion of isolates are nontypable (Borchardt et al., 2004). Molecular serotype (MS) identification methods are attractive because of their high discriminatory power (Edwards et al., 2005) and reproducibility (Borchardt et al., 2004). PCR/sequencing and DNA dot blot hybridization-based assays have been used to identify GBS serotypes (Kong et al., 2002; Borchardt et al., 2004), but further simplification, without loss of sensitivity and specificity, is needed to make them more practicable for routine use.

Reverse line blot (RLB) hybridization is a promising method for simultaneous detection, identification and typing of micro-organisms (Gold, 2003) that has been successfully used in our laboratory (Wang et al., 2004). Based on single PCR amplification, with or without nesting, RLB has been used successfully to detect and identify 10 mollicute species (Wang et al., 2004) and to identify 14 serotypes of Chlamydia trachomatis (Molano et al., 2004). In this study we used multiplex PCR (Markoulatos et al., 2002) and RLB to simultaneously detect and identify nine GBS serotypes (Goguet de la Salmoniere et al., 2004).

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
GBS reference strains and clinical isolates.

We used 27 GBS reference strains in this study, as shown in Table 1. In addition, we tested 83 isolates provided by Dr Nicola Jones, Oxford, UK, that had been used previously in a study of multilocus sequence typing (MLST) (Jones et al., 2003) and 260 clinical isolates that have been used in our previous studies (Table 2) (Kong et al., 2002, 2003). Results of conventional serotyping (CS) were available for all 316 isolates and all had been tested by serotype-specific PCR and/or partial cps sequencing [molecular serotyping (MS)] as previously described (Kong et al., 2002; Borchardt et al., 2004). All isolates were identified as GBS by the following criteria: ß-haemolysis on a 5 % horse blood agar, Gram stain showing Gram-positive cocci in pairs or short chains, negative reaction with catalase reagent and Lancefield grouping with type B antisera. For the purpose of this study the identity of isolates was also confirmed by GBS-specific PCR (Ke & Bergeron, 2001).

Multiplex PCR and reverse line blot (mPCR/RLB) hybridization

Oligonucleotide design. We designed two serotype-specific PCR primers and two serotype-specific probes for each of the nine GBS serotypes (Ia, Ib, II–VIII) using published GenBank sequences for cps gene clusters (Cieslewicz et al., 2005) and recently published sequence data for serotypes II, VII and VIII (Borchardt et al., 2004). The cpsH genes were used as primer and probe targets for serotypes Ia, Ib, III, IV, V and VI, for which we had previously developed serotype-specific PCRs (Kong et al., 2002). To compare with previously published primer pairs for serotype II-, VII- and VIII-specific PCRs (Borchardt et al., 2004; Cieslewicz et al., 2005) we used cpsIIK, cpsVIIM and cpsVIIIJ, respectively, rather than cpsH as the targets for primers and probes (Kong et al., 2002). We also designed one GBS-specific primer pair and two GBS-specific probes (Ke & Bergeron, 2001). Thus, in all, we designed one species-specific (cfb) primer pair, two species-specific (cfb) probes, nine serotype-specific primer pairs and 18 serotype-specific probes for this study (Table 3).

Primer design. The primers were designed to have similar physical characteristics in order to allow simultaneous amplification in a multiplex reaction; their lengths were between 18 and 27 bp, their melting temperatures were between 58.08 and 64.91 °C and they were designed so that amplicon sizes were in the range 241–366 bp (Table 3). To minimize cross-hybridization with other regions of the GBS genome or unrelated genes, FASTA searches were performed to compare primer sequences with GenBank. All multiplex PCR primers were 5' biotinylated to allow detection of hybridization with a streptavidin-peroxidase substrate.

Probe design. To allow optimal hybridization under the common conditions, probes were also designed to have similar physical characteristics, with lengths between 19 and 27 bp and melting temperatures between 58.00 and 62.10 °C (Table 3). FASTA searches were performed to compare their sequences with GenBank. All probes had a 5' amine group to facilitate covalent linkage to the nylon membrane and to allow membranes to be stripped and reused repeatedly.

Multiplex PCR reaction system. The 10-primer-pair multiplex PCR mixture was prepared as follows: 5 µl template DNA, 0.25 µl each of all forward (50 pmol µl^–1) and reverse (50 pmol µl^–1) primers, 1.25 µl dNTPs (2.5 mM of each dNTP), 2.5 µl 10x PCR buffer, 3 µl 25 mM MgCl₂ (4.5 mM final), 0.1 µl Qiagen hotstart Taq polymerase (5 units µl^–1) and water to 25 µl.

PCR programme. PCR was performed according to the Qiagen Hotstart Taq polymerase kit instructions: 95 °C for 15 min; 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min; 72 °C for 10 min; and 22 °C hold. A total of 8 µl of each PCR product was separated by electrophoresis on a 1.5 % agarose gel to confirm successful amplification (Kong et al., 2002). The remaining PCR products were used for RLB hybridization (5 µl of PCR product for each).

RLB hybridization. The RLB hybridization assay was based on a method described previously (van den Brule et al., 2002; Wang et al., 2004), except that the hybridization temperature was 60 °C, the conjugate used was streptavidin-peroxidase (Roche Diagnostics) diluted 1 : 4000 in 2x SSPE (SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA [pH 7.7]) with 0.5 % SDS, and the time of exposure to X-ray film (Hyperfilm; Amersham) was 7 min. RLB results were regarded as positive when both probes for a particular sequence gave positive results. To optimize hybridization conditions, the probes were tested at several twofold dilutions, starting at a concentration of 0.6 pM and with final labelling concentrations of between 0.3 and 0.2 pM. In our laboratory, membranes labelled with probes have been stripped and reused at least 20 times without any significant loss of signal (data not shown).

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Comparison of mPCR/RLB, MS and CS

CS has been most commonly used for GBS, but the proportion of non-typable isolates is significant and has increased over time (Borchardt et al., 2004), which could distort the recorded serotype distribution. MS identification is an attractive alternative (Kong et al., 2002; Borchardt et al., 2004). Our previous study showed 100 % typability for 206 isolates studied by partial sequencing of cpsE–cpsF–cpsG and six serotype-specific PCRs targeting cpsH and, in some cases, downstream genes (cpsI or cpsM) (Kong et al., 2002). Borchardt et al. (2004) used DNA dot blot hybridization, which allowed 99 % of 306 isolates to be typed, compared with 89 % by CS, and 95 % agreement between methods for isolates that were typable by both (Borchardt et al., 2004; Kong et al., 2002). Our newly developed mPCR/RLB showed 100 % typeability and 100 % agreement with CS and MS (Kong et al., 2002) (Tables 1 and 2, Fig. 1).

View larger version (42K): [in this window] [in a new window]   Fig. 1. Results of multiplex PCR and reverse line blot (RLB) hybridization for 27 reference strains. Lanes 1 to 27 represent 27 reference strains in the following order: O90 (Ia), H36B (Ib), 18 RS 21 (II), M 781 (III), 3139 (IV), CJB 111 (V), SS 1214 (VI), 7271 (VII), JM9 130013 (VIII), A909 (Ia), BM110 (III), NZRM 908 (Ia), NZRM 909 (Ib), NZRM 910 (Ia), NZRM 911 (II), NZRM 912 (III), NZRM 2832 (IV), NZRM 2833 (V), NZRM 2834 (VI), NZRM 2217 (NT), O90 (Ia), A909 (Ia), 335 (Ia), 70339 (Ia), 65604 (3) (III), 15626/81 (IV), Pattison (NT).

`Hot start’ mPCR/RLB

Improvements in commercial PCR ‘hot start’ Taq polymerase technology (http://www1.qiagen.com/Products/Pcr/HotStarTaqSystem/HotStarTaq.aspx) have made it easier to obtain all expected amplification products from a large number of primers, which significantly promotes the use of multiplex PCR. In our assay, using the Qiagen Hotstart Taq polymerase kit, only minor modifications to a typical single PCR amplification protocol were required to obtain optimal results.

In order to maximize the speed and convenience and to increase the specificity and utility of the assay, hybridization targets were generated using ‘hot start’ multiplex PCR (Markoulatos et al., 2002). The short mPCR products (241–366 bp) span each of the oligonucleotide probes to which target sequences hybridize. Multiplex PCR requires (a) that all primer pairs amplify unique DNA sequences, individually and in combination, under identical reaction conditions and (b) that each amplification product can be individually identified among a mixture of products. In this study, we used RLB rather than gel electrophoresis (Kong et al., 2002) or real-time PCR (Wittwer et al., 2001) because it can simultaneously identify up to 43 targets. This means that the number of primer pairs in the mPCR could be further increased; we have successfully used 23 in another mPCR/RLB (unpublished data), and the use of up to 50 has been described (Goguet de la Salmoniere et al., 2004).

Advantages of mPCR/RLB over MS and CS

mPCR/RLB is easy to perform and does not require use of antisera; amplification products of 43 isolates can be tested simultaneously and the method would be suitable for high-throughput GBS epidemiological studies (Gold, 2003). Because PCR detects the presence of capsular genes, it can characterize isolates with low or absent capsule expression, which are non-serotypable by CS. The mPCR/RLB method also has cost and time advantages over our previous MS method (Kong et al., 2002) and the dot blot hybridization method of Borchardt et al. (2004), because it requires only a single PCR, the membrane can be reused more than 20 times and the assay can be completed within one working day. It also has the potential to be used directly on clinical specimens (Gold, 2003).

Because this method uses only 20 of 43 lanes in the MiniBlotter, additional targets can be added to both mPCR and RLB, such as selected protein antigen genes or mobile genetic elements, which are included in our three component genotyping system (Kong et al., 2003), and antibiotic-resistance genes.

Sensitivity and specificity

Some probes reacted (usually very weakly) with amplicons from more than one serotype. For example, amplicons from most serotype Ia strains hybridized weakly to VcpsHSp and those from most serotype V strains with probe IacpsHSp. However, the combined results of VcpsHAp/VcpsHSp and IacpsHAp/IacpsHSp clearly distinguished between serotypes Ia and V (Fig. 1). Otherwise, the sensitivity and specificity of the RLB method for confirmed GBS isolates are high, compared with CS and individual serotype-specific PCR. The only discrepancies between methods were for isolates non-typable by CS. All 25 CS non-typable isolates were assigned a capsular type by mPCR/RLB and the results were confirmed by serotype-specific PCR and sequencing. However, because this method identifies the serotype indirectly, isolates that have defective cps operons and thus are acapsular or produce an aberrant capsule could be misclassified.

In conclusion, we have developed a multiplex PCR and RLB assay as an alternative to CS that is sensitive and specific, and reduces misclassification of non-serotypable isolates. Application of mPCR/RLB in future epidemiological studies and for surveillance will facilitate the appropriate formulation of candidate GBS vaccines.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We thank our colleagues for providing GBS isolates other than those isolated in our laboratory. Twenty-seven GBS reference strains were kindly provided by the Drs Lawrence Paoletti and Catherine Lachenauer, Dr Diana Martin and Professor Johan Maelandas as indicated in Table 1. Dr Nicola Jones, Nuffield Department of Clinical Laboratory Sciences, Institute for Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK, kindly provided 83 GBS strains used in their previous MLST study.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES