Research Article: Development of a species-specific TaqMan-MGB real-time PCR assay to quantify Olsenella scatoligenes in pigs offered a chicory root-based diet

Date Published: June 16, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Xiaoqiong Li, Bent Borg Jensen, Ole Højberg, Samantha Joan Noel, Nuria Canibe.


Olsenella scatoligenes is the only skatole-producing bacterium isolated from the pig gut. Skatole, produced from microbial degradation of l-tryptophan, is the main contributor to boar taint, an off-odor and off-flavor taint, released upon heating meat from some entire male pigs. An appropriate method for quantifying O. scatoligenes would help investigating the relationship between O. scatoligenes abundance and skatole concentration
in the pig gut. Thus, the present study aimed at developing a TaqMan-MGB probe-based, species-specific qPCR assay for rapid quantification of O. scatoligenes. The use of a MGB probe allowed discriminating O. scatoligenes from other closely related species. Moreover, the assay allowed quantifying down to three target gene copies per PCR reaction using genomic DNA-constructed standards, or 1.5 × 103 cells/g digesta, using O. scatoligenes-spiked digesta samples as reference standards. The developed assay was applied to assess the impact of dietary chicory roots on O. scatoligenes in the hindgut of pigs. Olsenella scatoligenes made up < 0.01% of the microbial population in the pig hindgut. Interestingly, the highest number of O. scatoligenes was found in young entire male pigs fed high levels of chicory roots. This indicates that the known effect of chicory roots for reducing skatole production is not by inhibiting the growth of this skatole-producing bacterium in the pig hindgut. Accordingly, the abundance of O. scatoligenes in the hindgut does not seem to be an appropriate indicator of boar taint. The present study is the first to describe a TaqMan-MGB probe qPCR assay for detection and quantification of O. scatoligenes in pigs.

Partial Text

Skatole (3-methylindole) is the main compound responsible for boar taint, which is an off-odor and off-flavor meat trait released upon heating of meat from some entire male pigs (Wesoly and Weiler 2012). Boar tainted meat is disagreeable to most consumers and has therefore a negative economic impact for the meat industry (Jensen et al. 2014). Skatole is produced by anaerobic microbial degradation of l-tryptophan (TRP) via decarboxylation of indol-3-acetic acid (IAA) in the hindgut of pigs. It is produced in both sexes, but increased in some entire male pigs, probably due to a reduced skatole metabolism in the liver of these animals (Babol et al. 1999; Zamaratskaia et al. 2004). Dietary supplementation with easily fermentable carbohydrates with low ileal digestibility has been shown to reduce skatole production in the hindgut (Jensen et al. 1995; Knarreborg et al. 2002; Rideout et al. 2004; Lösel and Claus 2005; Hansen et al. 2006; Øverland et al. 2011; Vhile et al. 2012; Zammerini et al. 2012). The most effective carbohydrate seems to be purified inulin or inulin-rich feed components like chicory root or Jerusalem artichoke (Jensen et al. 2014).

The use of TaqMan-MGB qPCR enabled specific enumeration of the skatole-producing bacterium O. scatoligenes in pig hindgut. One of the challenges in the development of qPCR assays is designing primers that specifically target the species of interest in samples containing closely related bacteria. So far, only one 16s RNA gene-based nested PCR protocol has been established to detect Olsenella species, yet this method has failed to discriminate O. uli from O. profusa (Rôças and Siqueira 2005). Our results (Fig. 2a, b) showed that conventional PCR and SYBR Green qPCR alone was not sufficient to distinguish O. scatoligenes from O. profusa and O. uli, due to high sequence homology in the variable regions of the 16s rRNA gene among the Olsenella species. Due to significantly improved hybridization properties of MGB probes (Kutyavin 2000), we thus opted to employ a TaqMan-MGB probe. The MGB stabilizes A/T rich duplexes, allowing to use shorter probes with higher melting temperature compared to ordinary DNA probes, and the increased specificity of an MGB probe allows discrimination with a single-base mismatch (Van Hoeyveld et al. 2004; Mingxiao et al. 2013). Therefore, in order to increase the specificity of the qPCR reaction for targeting O. scatoligenes, we designed a MGB probe with 1–3 mismatches to the closest related species. The Ct difference between O. scatoligenes and the closest related species, O. profusa was equivalent to at least two log units of cells. Thus, signals of unspecific targets in gut samples were considered not to compromise the specific enumeration of O. scatoligenes. Our results supported that MGB probes can be more sequence specific than unmodified 16S DNA probes, especially for single-base mismatches.




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