Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira species in swine

Abstract A novel TaqMan 5‐plex real‐time PCR using a combination of locked nucleic acid‐modified (LNA)‐ and minor groove binding (MGB)‐conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR‐based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross‐reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut‐off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5‐plex real‐time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single‐tube approach.

1998). B. innocens and B. murdochii, which can be encountered in pigs, chickens, and rats, have not been associated to any disease and are considered as harmless commensals (Stephens & Hampson, 2001). B. aalborgi is only found in humans and higher primates (Hovind-Hougen et al., 1982;Munshi et al., 2003). More recently, the emergence of two Brachyspira species has been described, which are capable of infecting birds and pigs, namely B. hampsonii (Chander et al., 2012) and B. suanatina (Rasback et al., 2007) both harboring strong hemolytic properties with clinical signs indistinguishable from swine dysentery.
Species identification is commonly performed by PCR assays, restriction fragment length polymorphism (RFLP) (Rohde & Habighorst-Blome, 2012), or by partial NADH oxidase gene (nox) sequencing (Atyeo et al., 1999). Most widely used targets for PCR assays detecting B. hyodysenteriae and/or B. pilosicoli include nox (Atyeo et al., 1999), 16S rDNA (La et al., 2003), 23S rDNA (Borgström et al., 2017;Leser et al., 1997), and tlyA (Fellström et al., 2001). It has been shown that some newly emerging B. hampsonii and B. suanatina strains may cross-react or stay undetected in some species-specific PCRs due to genetic similarities of target genes used for identification of involved strains thereby leading to a misidentification of B. hampsonii and B. suanatina (Burrough, 2017;Rohde et al., 2014). The strong hemolytic properties of these strains and the fact of causing a disease indistinguishable from swine dysentery drives the need of developing new routine diagnostic tests to rapidly uncover involved species.
Recently, it has been shown that sequencing of chaperonin cpn60 is superior to nox sequencing and revealed more reliable species identification for some isolates (Rohde et al., 2019).
Molecular chaperones are universally present in almost all eubacteria and archaea harboring phylogenetically more discriminative gene sequences for species identification than those of the traditionally used 16S rDNA target (Hill et al., 2004;Links et al., 2012). However, due to massive gene rearrangements within some Brachyspira species leading to a diversity of mosaic genomes (Hampson & Wang, 2018) or the presence of a great wealth of Brachyspira species (Johnson et al., 2018) it remains a challenge to assign the correct species for a certain minority of isolates independent from the chosen target gene.
To date, no qPCR assay distinguishing simultaneously between the main porcine pathogenic Brachyspira strains including B. hyodysenteriae, B. pilosicoli, B. hampsonii, and B. suanatina in one reaction mixture has been reported. The purpose of the present study was to develop a reliable and robust multiplex qPCR system that can be used to identify and differentiate all pathogenic Brachyspira species in swine. To evaluate the novel assay as a diagnostic tool, 503 samples were examined with the novel 5-plex qPCR and compared to different PCR-based assays targeting 23S rDNA, nox, and 16S rDNA. Given reliable monitoring of Brachyspira infections in pigs, it is of great advantage to have an efficient molecular tool for fast and accurate detection of all porcine pathogenic Brachyspira species in a one-tube approach.

| Culture and identification of clinical samples
Porcine rectal swabs were cultured on selective tryptose soy agar (TSA) and incubated at 42°C in an anaerobic environment (Trilab, Biomerieux, Marcy L'Etoile, France) for 4-6 days as described previously (Borgström et al., 2017;Dünser et al., 1997;Prohaska et al., 2014). Subcultures were performed if spirochetes were found by dark-field microscopy. The resulting colonies were washed off with 1 ml of ultrapure water and DNA was obtained through thermal lysis by boiling the bacterial cell suspension for 10 min at 99°C with a subsequent centrifugation step at 17,000 g for 3 min. 2 μl of the obtained supernatant containing DNA was used as a template in the PCR reaction. The concentration of the obtained DNA samples was in the range of 100-400 ng/μl. DNA samples were identified by multiplex qPCR targeting 23 s rDNA (Borgström et al., 2017). For further identification, DNA samples of a subset of epidemiologically non-linked clinical samples originating from different farms were chosen for a genus-specific PCR using primers targeting the Brachyspira nox gene (Rohde et al., 2002).

| Development of the 5-plex qPCR
Primers and probes were designed using CLC Main Workbench software 7.5.1 from alignments of available cpn60 sequences from the NCBI databank (Rohde et al., 2019). Additionally, cpn60 of  Table 2 with the respective labeled reporter dye. Probes for B. pilosicoli and B. hyodysenteriae (Thermo Fisher Scientific, Renfrewshire, UK) comprise minor groove binding (MGB) molecules at the 3′-end enabling relatively short probe sequences to be species-specific, thus increasing the specificity of the probes (Kutyavin et al., 2000). Targets  The specificity of both primer and probe sequences were confirmed by BLAST searches. Oligonucleotide primers were synthesized by Microsynth (Balgach, Switzerland).
For monitoring the potentially inhibitory behavior of each PCR reaction, an internal amplification control (IAC) was added to the master mix. Therefore, five femtogram (fg) of a plasmid containing enhanced green fluorescent protein (eGFP) gene was used to generate a 177 bp long amplicon with eGFP-specific primers eGFP_ forward (5′-GACCACTACCAGCAGAACAC-3′) and eGFP_reverse (5′-GAACTCCAGCAGGACCATG-3′) and detected by the eGFPprobe (5′-ATTO 647 N-AGCACCCAGTCCGCCCTGAGCA-BHQ3-3′) (Hoffmann et al., 2006). 18 reference strains (Table 1) were used to develop the 5-plex qPCR assay. The multiplex format was optimized regarding probe and primer concentrations by evaluating different concentration gradients. Data analysis was performed using Rotor-Gene Q Software 2.3.1 (Qiagen). Samples with a threshold cycle (C t ) value of ≤37 were considered positive. DNA samples with no detected fluorescent signal for IAC were repeated as 1:5 or 1:10 dilution to minimize potentially inhibitory features.

| Specificity
To determine the specificity of the 5-plex qPCR, an exclusivity panel consisting of 25 pathogenic bacteria was tested (Table A1).

| Analytical sensitivity
To determine the analytical sensitivities of the multiplex qPCR, four  To examine the intra-and inter-assay variability of the novel qPCR assay representing its repeatability, the above mentioned four reference strains were tested using tenfold dilution series in the linear range between 10 7 and 100 GE. The variability assays were performed in triplicates in three experiments.

| Efficiency
To calculate efficiencies of the multiplex qPCR for each target probe,

| Specificity
The tested exclusivity panel of 25 pathogenic bacteria resulted in negative results for all strains (Table A1). All reference strains including pathogenic and non-pathogenic Brachyspira spp. examined by qPCR correlated with the expected results (Table 1). Hence, the novel 5-plex qPCR assay had a specificity of 100%.

| Analytical sensitivity
The dynamic range of the standard curve was between 10 7 and 100 GE for all four tested Brachyspira reference strains. The concen-  (Figure 3), corresponding to a cut-off Ct value of 37. The results of the variability assays revealed a variation of CV% of <3% for the inter-assay variability and <4% for the intra-assay variability demonstrating the multiplex qPCR to be a highly reproducible and robust assay (Table A2).

| Efficiency
In the linear range of the tested dilution series between 10 7 and 100  Figure 4).  obtained DNA samples was necessary since PCR performance was surveilled by a simultaneously added exogenous internal control (eGFP). In rare cases of qPCR inhibition or unusual high background of amplification curves, the DNA samples were diluted 1:5.

| Comparison of the new 5-plex qPCR with other PCR assays
In the present study, only one B. suanatina strain from a

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

E TH I C S S TATEM ENT
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DATA AVA I L A B I L I T Y S TAT E M E N T
All data relevant to the study are included in the article and the ap-