Simultaneous detection of 33 Streptococcus suis serotypes using the luminex xTAG® assay™

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Highlights

  • A serotype-specific xTAG luminex assay for the 33 serotypes of Streptococcus suis simultaneous detection

  • Less than 40 min following amplification to assay a 96-well plate

  • The sensitivity was better than any previous methods.

  • It has potential to increase the multiplicity in a single reaction and meet the current and upcoming requirement of molecular laboratory for high-throughput serotyping.

Abstract

We report the development and evaluation of a serotype-specific xTAG luminex assay (SSA) that allows detection of the 33 serotypes of Streptococcus suis (S. suis). This assay is based on wzy gene targets directly involved in the cps biosynthesis and can be completed 40 min post-PCR amplification. The assay correctly and specifically identified the serotype of all 209 isolates tested, in comparison with two serotyping multiplex PCR methods previously developed. The sensitivity was higher than that of the previously described methods. The SSA system described here provides an easy-to-use, high-throughput system for rapid detection of S. suis serotypes.

Introduction

Streptococcus suis (S. suis) is one of the most important swine pathogens worldwide and has significant impacts on the swine industry. Furthermore, it is an emerging zoonotic agent: humans can be infected when in close contact with pigs or following consumption of raw or undercooked pork products (Gottschalk et al., 2007, Wertheim et al., 2009). S. suis serotyping is a valuable phenotypic subtyping tool that is required for a better understanding of the epidemiology of this important zoonotic pathogen. Thirty-five serotypes (type 1 through 34, and 1/2) of S. suis have been identified based on antigenic differences in their capsular polysaccharide (CP) (Gottschalk et al., 1991a, Gottschalk et al., 1991b, Gottschalk et al., 1989, Perch et al., 1983). The CP synthesis (cps) genes are clustered in a single locus on the S. suis chromosome. The cps clusters for the 35 reference serotypes have been published (Okura et al., 2013). S. suis serotypes are routinely identified using the agglutination or co-agglutination tests with serotype-specific antisera. However, these techniques are laborious, time-consuming, and relatively expensive. The problems associated with the production of the antisera and testing have led to the development of different molecular approaches, of which multiplex PCR (mPCR) with amplification of serotype-specific cps genes are an attractive alternative to the existing serological tests. In fact, three mPCR assays were developed to identify S. suis serotypes (Kerdsin et al., 2014, Liu et al., 2013, Okura et al., 2014). However, two or three sets of mPCR amplification are needed when using these assays. Furthermore, the interpretation of the data may be difficult due to the similar sizes of the PCR products for some serotypes.

Here, we developed a 32-plex S. suis serotyping assay (SSA) that simultaneously detects 33 serotypes using the luminex xTAG universal array technology. The Streptococcus orisratti strains originally classified as the reference strains for S. suis serotypes 32 (strain EA1172.91) and 34 (strain 92-2742) were not included in the present study (Hill et al., 2005). Although additional serotypes 20, 22 and 26 have recently been suggested to belong to a bacterial species different from S. suis (Nomoto et al., 2015, Tien le et al., 2013), these serotypes are still commonly isolated from diseased animals and considered as S. suis by diagnostic laboratories (Gottschalk et al., 2013). For that reason, they have been included in the development of the present test. The approach used in this study combines amplification of the target genes using mPCR with 32 pairs of primers that have a unique ‘TAG’ sequence incorporated upstream of the primer and a biotinylated downstream primer with a multiplexed bead-based suspension array detection system. The method and sero-specific validation of the SSA are herein presented.

Section snippets

Bacterial strains

Eighty-four serotypable S. suis strains, 19 other Streptococcus spp. strains and one Klebsiella pneumoniae strain from our previous study were used (Liu et al., 2013). One hundred-three additional S. suis field strains from clinically healthy pigs, 22 strains from diseased pigs, whose serotypes were determined by two previously developed mPCR (Liu et al., 2013, Okura et al., 2014), and 42 other non-S. suis strains were included in this study (Table 1). The strains were grown overnight on

Development, evaluation and applications of the SSA

We first performed simplex PCRs to determine the specificity of each primer pair using template DNA extracted from the 33 reference strains by sequencing each amplicons. Each pair of primers amplified the predicted PCR product specifically from the DNA samples. The SSA was then designed based on the simplex PCR assays above. Cross hybridization and non-specific hybridization between sequences were not observed.

Three independent experiments were performed to establish the sensitivity of the SSA

Discussion

S. suis surveillance depends on the accuracy of serotype identification. Conventional serotyping techniques are laborious, time-consuming and are relatively expensive. Additionally, expression of the CPS can be phenotypically lost such that these strains do not react with any of the antisera. Therefore, we developed a more practical and easier serotyping method. Determination of S. suis serotypes based on DNA markers provides an alternative to the traditional serotyping method. For analysis of

Conclusions

We developed a xTAG SSA that is a rapid, sensitive and specific molecular serotyping detection system that can be easily implemented in any laboratory equipped with the appropriate devices.

Acknowledgments

This work was supported by grants (2013ZX10004221, 2013ZX10004216-001-002) from the Ministry of Science and Technology, P. R. China.

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