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. 2020 Nov 11;7(4):175.
doi: 10.3390/vetsci7040175.

Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types

Rejoice Nyarku  1 , Ayesha Hassim  1 , Annelize Jonker  1 , Melvyn Quan  1
Affiliations

Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types

Rejoice Nyarku et al. Vet Sci. .

Abstract

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.

Keywords: abomasal fluid; blood; brucellosis; diagnosis; milk; qPCR.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Multiple sequence alignment of the 16S-23S rDNA internal transcribed spacer (ITS) region of Brucella spp. and closely related Ochrobactrum spp. available on GenBank®. Primers and probe have been indicated by arrows and a rectangle respectively. X95889 was used for the nucleotide position numbering. Identical sequences have been grouped and labelled with a randomly selected sequence in the group. The number of identical sequences in a group is indicated in brackets after the name of the group.
Figure 2
Figure 2
Standard curves of a genus-specific 16S-23S rDNA internal transcribed spacer Brucella spp. PCR assay using different matrices [i.e., blood (red circles), milk (green triangles), and tissue (blue diamonds)]. Each dilution was run in triplicate. The equation of the regression line (red line for blood, green dash for milk and blue dash for tissue) is indicated, as well as the coefficient of determination (R2). One bacterium contains three copies of the 16S-23S rDNA internal transcribed spacer gene.
Figure 3
Figure 3
The 95% limit of detection (dotted lines) of a genus-specific 16S-23S rDNA internal transcribed spacer Brucella spp. PCR for tissue (diamond), blood (circle), and milk (triangle). One bacterium contains three copies of the 16S-23S rDNA internal transcribed spacer gene.
See this image and copyright information in PMC

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