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. 2018 Jun 16;8(1):99.
doi: 10.1186/s13568-018-0627-y.

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

Xiaoqiong Li  1   2 , Bent Borg Jensen  1 , Ole Højberg  1 , Samantha Joan Noel  1 , Nuria Canibe  3
Affiliations

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

Xiaoqiong Li et al. AMB Express. .

Abstract

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.

Keywords: Chicory root; MGB probe; Olsenella scatoligenes; Skatole; TaqMan; qPCR.

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Figures

Fig. 1
Fig. 1
Sequence targeted by the O. scatoligenes-specific MGB probe and primers, taking into account analogous sequences in the closely related Olsenella species and Atopobium species. Mismatches are marked in red
Fig. 2
Fig. 2
Specificity of the primer pair alone and TaqMan primer–probe pair for Olsenella scatoligenes SK9K4T using genomic DNA targeting a specific region of the 16s rRNA gene sequence. a Conventional PCR with the primer pair alone. 1, O. scatoligenes; 2, O. uli; 3, O. profusa; 4–12, non-specific cultures; 13, distilled water. b SYBR Green real-time PCR with the primer pair alone. c TaqMan-MGB real-time PCR with the primer–probe pair
Fig. 3
Fig. 3
A representative amplification plot (a) and standard curve (b) of O. scatoligenes Taqman-MGB qPCR assay with tenfold dilutions of O. scatoligenes SK9K4T genomic DNA ranging from 3 × 106 to 3 × 100 copies per reaction by using ViiA™ 7 RUO software version 1.2.1
Fig. 4
Fig. 4
Amplification plots (a, c) and standard curves (b, d) of O. scatoligenes Taqman-MGB qPCR assay with tenfold dilutions of DNA from cecal (a, b) or colonic digesta (c, d) spiked with 1.5 × 1010 cells/g O. scatoligenes SK9K4T (ranging from 3 × 107 to 3 × 102 cells per reaction) by using ViiA™ 7 RUO software version 1.2.1
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