Rapid high resolution melting assay to differentiate Streptococcus suis serotypes 2, 1/2, 1, and 14

Abstract This rapid high resolution melting (HRM) assay allows distinguishing between Streptococcus suis serotype pairs 2 and 1/2 as well as 1 and 14, respectively, based on a single‐nucleotide polymorphism within capsular polysaccharide synthesis gene cluster K. This assay is easy to implement and identifies potential zoonotic serotypes.


| INTRODUC TI ON
Streptococcus suis (S. suis) is an important pathogen of pigs and considered to be responsible for various diseases such as septicemia with sudden death, meningitis, endocarditits, and arthritis (Gottschalk, Segura, & Xu, 2007). Currently, there are at least 29 S. suis serotypes (Okura et al., 2016) described based on a serological reaction against the capsular polysaccharide (CPS), which has been described to be a major virulence factor with antiphagocytic properties (Baums & Valentin-Weigand, 2009). S. suis serotype 14 and in particular heterogenous serotype 2 are emerging zoonotic pathogens often associated with disease in both pigs and humans worldwide (Goyette-Desjardins, Auger, Xu, Segura, & Gottschalk, 2014). Formerly, serological typing was performed with different antisera of known type, based on three different techniques: Neufeld's capsular reaction, capillary precipitation, and coagglutination tests (Gottschalk, Higgins, Jacques, & Dubreuil, 1992). In recent times, more frequently multiplex PCR assays are used targeting genes based on CPS (Okura et al., 2014), which are identical for serotype pairs 2 and 1/2, and 1 and 14 except for a single base pair substitution in codon 161 of cpsK gene (Athey et al., 2016). A challenge for diagnostic laboratories is the fact that available PCR tests do not allow resolving these aforementioned serotype pairs 2 and 1/2, and 1 and 14.
Recently, an in silico pipeline using whole-genome sequencing (WGS) short-read data was developed, which is able to differentiate these serotypes (Athey et al., 2016). Nevertheless, for a rapid identification of potential zoonotic strains with serotypes 2 and 14, a reliable inexpensive and feasible high-throughput approach for routine diagnostic laboratories would be a valuable tool. The aim of this study was to evaluate the potential of cpsK as target for differentiating S. suis serotypes 2, 1/2, 1, and 14 from pure culture using a novel high resolution melting (HRM) assay.

| MATERIAL S AND ME THODS
For this purpose, four reference strains comprising serotypes 2, 1/2, 1, and 14, one human, and 12 porcine S. suis isolates of serotypes 1 or 14 and 2 or 1/2 were used to develop a novel HRM assay (Table 1). Moreover, 58 S. suis isolates of other serotypes, as well as four further Streptococcus spp. isolates were included in the study. All strains were grown on Columbia agar with sheep blood (Thermo Fisher Diagnostics AG) and incubated at 37°C for 48 hr under aerobic conditions. Strains were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker). Genomic DNA was extracted using a standard heat lysis protocol (Sambrook & Russel, 2006).

F I G U R E 1
Representation of sequence alignment of the cpsK amplicon generated by the HRM-PCR using primers cpsK_for and cpsK_rev (illustrated as pink arrows) of serotypes 2, 14, 1, and 1/2. Single-nucleotide polymorphism (SNP) region is highlighted in red, whereas conserved nucleotides are shown in blue. The SNP indicated gives rise to different melting behavior in the HRM assay. Accession numbers of GenBank of corresponding sequences are indicated amplicon of 117 bp (Figure 1). Normalized and difference plots were generated. To normalize the results, the premelt and postmelt signals of all samples were set to uniform relative values from 100% to 0%. In order to generate difference plots, normalized fluorescence data of sample curves were subtracted from the curve of reference strain serotype 1/2 to visually accentuate differences in a greater resolution.
The HRM assay was evaluated, and its specificity was determined. To examine the intra-and interassay variability of the melting temperatures (T m ), representing the repeatability of the developed HRM assay, all isolates were tested. The variability assays were performed in triplicates in three independent runs at three different days.

| RE SULTS
The HRM assay clearly divided the 17 S. suis strains into two clusters grouping serotype 2 with 14 and serotype 1/2 with 1 ( Figure 2). By combining serotype determination obtained by PCR (Kerdsin et al., 2014) into serotype pairs 2 and 1/2 or serotype pairs 1 and 14 with the obtained results of the HRM assay, it is possible to separate and F I G U R E 2 Representation of a high resolution melting (HRM) assay of 12 Swiss porcine Streptococcus suis strains, one human S. suis strain of serotype 14, and four reference strains with serotypes 14, 2, 1, and 1/2 obtained by triplicate-testing of each strain for intraassay variability determination. The two groups of melting curves obtained allow rapid identification of human pathogenic S. suis serotypes (serotypes 2 and 14). Serotype 1/2 (red), serotype 14 (blue), serotype 1 (green), and serotype 2 (pink) are illustrated in each plot. (a) qPCR amplification plot; (b) melting curves of the HRM step; (c) normalized plot; (d) difference plot in relation to reference strain serotype 1/2 correctly assign the corresponding serotype to each S. suis isolate.
Based on our results, the target was 100% specific for the four serotypes tested as none of the Streptococcus spp. or S. suis with different serotypes (Table 1) yielded a PCR positive result. The intra-assay coefficients of variability (CVs) of T m were between 0.008% and 0.03% and interassay CVs, respectively, between 0.04% and 0.07% illustrating a highly reproducible and stable assay. Serotypes 1 and 1/2 yielded a T m of 75.9 ± 0.1°C, whereas serotypes 2 and 14 yielded a T m of 76.2 ± 0.1°C, respectively (Figure 1).

| D ISCUSS I ON
In this report, a novel HRM assay based on SNP detection in PCR products is described. HRM analysis is a rapid and low cost genotyping method simply convertible in laboratories as a singleplex method with a low risk of contamination (Reed, Kent, & Wittwer, 2007

| CON CLUS ION
To conclude, we have developed a specific HRM assay distinguishing between serotype pairs 2 and 1/2 and 1 and 14, respectively, based on a SNP within cpsK. This assay allows a rapid separation of these S. suis serotypes in routine diagnostic laboratories using molecular based serotyping in order to assess a zoonotic risk of emerging strains.

ACK N OWLED G M ENTS
The authors wish to thank the laboratory staff of the Department of Veterinary Bacteriology, Vetsuisse Faculty, University of Zurich, for excellent technical assistance.

CO N FLI C T O F I NTE R E S T S
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw datasets from intra-and interassay variability runs are available upon request.