The genus Yersinia belongs to the family Enterobacteriaceae and comprises 18 species (http://www.bacterio.net/xz/yersinia.html). Among these, some species like Yersinia pseudotuberculosis, and Yersinia enterocolitica are enteropathogens that cause foodborne illness. While most of the attention is focused in these species, several other less familiar Yersinia species (Yersinia aldovae, Yersinia bercovieri, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia mollaretii, Yersinia rohdei and Yersinia ruckeri), isolated from food and environment, are considered opportunistic pathogens [1].

Conventional methods for the identification of Yersinia spp., such as a biochemical profiling and serological tests, are time-consuming and limited by their poor ability to differentiate strains within some species and sometimes by the low reproducibility [2].

Recently, many genotypic methods have been developed for the identification of human pathogenic species like Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica [3].

Furthermore rapid, specific and multitarget methods focusing on less studied species are needed [4].

Minisequencinq is a novel technology for quickly detecting and identifying multiple Single Nucleotide Polymorphisms (SNPs). In particular, this technique is based on the simultaneous analysis of several SNPs by means of multiplex primer-extension reaction (PER) in conjunction with a genetic analyser [5].

The aim of the study was the development of a minisequencing test to differentiate several Yersinia species frequently isolated from food samples, including both human pathogens and opportunistic pathogens (Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. frederiksenii, Y. intermedia, Y. kristensenii, Y. mollaretii, Y. pseudotuberculosis, Y. rohdei and Y. ruckeri).

All reference strains used to develop the assay are listed in Table 1. Bacterial strains belonging to phylogenetically related genera were also tested to verify possible cross-reactions (Table 1).

Table 1 Bacterial strains used to develop the multiplex PER assay
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Moreover in order to evaluate the possible applicability of the proposed assay, 18 wild isolates (WI) obtained from meats of large game animal hunted in Italian Alps were used (six Y. rohdei, six Y. kristensenii, one Y. enterocolitica, four Y. intermedia, one Y. aldovae) (Table 1).

All the isolates were grown overnight at 31 °C in tryptone soya broth (TSB, Acumedia). DNA was extracted by boiling procedure and quantified using a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific).

The tufA (elongation factor Tu) sequences obtained from the GenBank database (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.nih.gov), corresponding to Y. aldovae (accession no. EF113987), Y. bercovieri (accession no. EF113993), Y. enterocolitica (accession no. EU566885), Y. frederiksenii (accession no. EF114009), Y. intermedia (accession no. EF114012), Y. kristensenii (accession no. EF114016), Y. mollaretii (accession no. EF114019), Y. pseudotuberculosis (accession no. EU566891), Y. rohdei (accession no. EF114027) and Y. ruckeri (accession no. EF114030) and other species belonging to genera correlated, were aligned with the ClustalV program [6] for the detection of SNPs to be used as diagnosis positions.

The tufA gene was selected because it has been proven to be valuable for accurate evaluation of genetic relationships among closely related microorganism such as the members of the family Enterobacteriaceae. In particular within the Yersinia genus, the tufA gene has a remarkable level of interspecies variability (16 %) [7].

Analysis of the alignment of the reference sequences showed that eight SNPs at positions 72, 93, 130, 171, 417, 474, 501, and 531 (numbering referred to GenBank accession no. EU566891), could differentiate the Yersinia species most commonly isolated from food and environment, providing specific minisequencing profiles (Table 2).

Table 2 Minisequencing profiles on the basis of the alignment of the reference sequences
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All the sequences available for each Yersinia species in GenBank were examined to confirm the absence of intraspecific variations of the diagnosis sites that have been selected.

Primers for the preliminary PCR have been designed upstream and downstream the diagnosis sites in well-conserved regions within the Yersinia genus (Table 3). Sequencing primers (SP) were designed immediately flanking the diagnostic sites and had varying lengths of poly(dT) non-homologous tails attached to the 5′ end (Table 3).

Table 3 Primers for preliminary PCR and PER
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Preliminary PCR reactions were performed in 50 μL volumes consisting of 20 mM Tris–HCl, 1 U of recombinant Taq DNA polymerase (Invitrogen), 0.2 mM each of dNTPs (Invitrogen), 2 mM MgCl2, 25 pmol of each primer and 50 ng of DNA. PCR conditions were 3 min at 94 °C followed by 35 cycles at 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min and a final extension at 72 °C for 5 min.

Preliminary PCR allowed the amplification of a 587 bp fragment in all reference Yersinia samples. No cross-reaction was detected when the primers were tested with other genera phylogenetically related to Yersinia genus.

The obtained PCR products acted as templates for the PER reaction after the enzymatic clean-up with Exo-Sap (USB Europe GmbH).

Tailed sequencing primers were tested individually to assess their performance and validate migration size before the eight primers were tested in multiplex reaction.

The multiplex PER was performed according to the SNaPshot multiplex kit protocol (Applied Biosystems, Foster City, CA) and using the following concentrations of sequencing primers: 0.12 μM of SP1, 0.18 μM of SP2, SP5 and SP6; 0.24 μM of SP8; 0.30 μM of SP3 and SP4; 0.48 μM of SP7.

PER products were cleaned using 1 unit of calf intestine alkaline phosphatase (CIAP) (Fermentas) at 37 °C for 1 h and at 75 °C for 15 min.

Finally, samples were prepared by adding 1 μL of the post-PER product to 24.6 μL of formamide (Applied Biosystems) and 0.4 μL of GeneScan 120 LIZ size standard (Applied Biosystems). Each sample was loaded on the ABI 310 Genetic Analyser (Applied Biosystems). Electropherograms were analyzed using the GeneScan 4.0 software (Applied Biosystems).

To confirm multiplex PER results, amplification products of tufA gene were sequenced using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, version 1.1 (Applied Biosystems).

All of the Yersinia reference strains and WI gave rise to the expected species-specific patterns thus confirming the specificity and applicability of the multiplex PER technique (Fig. 1).

Fig. 1
figure 1

Species specific patterns observed in reference strains. In the boxes are indicated the positions of diagnosis sites interrogated by each sequencing primers (as referred to GenBank accession no. EU566891)

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In conclusion, the multiplex PER assay is as precise and reliable as the sequencing test and it has the advantage of being quicker allowing an immediate interpretation of the results [8]. In fact, the discrimination of the 10 species is possible by means of a single read of the generated pattern.

Furthermore, since the minisequencing developed test is a multiplex format assay, it would increase the analytical productivity by reducing analysis costs and reagent consumption [9].

For all these reasons, the test proposed in this paper, applied for the specific detection of Yersinia species most frequently isolated from food, represents the ideal solution for routine analysis in laboratories equipped with genetic analyzer. Moreover the developed test can be an important tool for monitoring the ecology of Yersinia spp. and as consequence improving the food quality.