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Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer
Dissertation (MSc)--University of Pretoria, 2019.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2020
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| _version_ | 1867613577519562753 |
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| access_status_str | Open Access |
| author2 | Quan, Melvyn |
| author_browse | Quan, Melvyn |
| author_facet | Quan, Melvyn |
| collection | Thesis |
| dc_rights_str_mv | © 2020 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc)--University of Pretoria, 2019. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/77428 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:38:21.763Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/77428 Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer Quan, Melvyn u17309337@tuks.co.za Hassim, Ayesha Nyarku, Rejoice E. UCTD diagnosis qPCR brucellosis blood, milk abomasal fluid Veterinary science theses SDG-01 Veterinary science theses SDG-03 SDG-01: No poverty SDG-03: Good health and well-being Dissertation (MSc)--University of Pretoria, 2019. Brucellosis is an economically important bacterial disease of both animals and humans. In sub-Saharan Africa, the diagnosis of the disease remains a challenge. Brucellosis is underreported in South Africa, due to inconsistency in reports of bacteriological and serological tests, which lack adequate sensitivity and specificity in the diagnosis of the disease. They also are ineffective in confirming brucellosis during early stages of the disease. The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid (rDNA) internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for early diagnosis of brucellosis and as a rapid screening tool. To achieve this, blood, milk and tissue samples were spiked with B. abortus biovar (bv.) 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The efficiency was 105% in tissue, 99% in blood, and 93% in milk. The 95% limit of detection (LOD) of the ITS qPCR assay was highest in tissue, followed by blood, then milk; thus (1.45, 13.30 and 45.54 bacterial genome copies/PCR reaction). Furthermore, the diagnostic performance of the assay was compared to the Brucella cell surface protein real time polymerase chain reaction (BCSP31 qPCR) assay. 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 were 87% and 95% respectively, compared to the 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The ITS qPCR gave earlier CT’s with a difference in CT (ΔCT) between ITS and BCSP31 ranging between 7.1 and 3.24. The assay was efficient, sensitive and specific. It detected as little as 1.45 bacterial genome copies/PCR reaction in tissue, making this assay a valuable tool in early detection of the presence of the Brucella pathogen. It is sensitive and specific in the diagnosis of brucellosis. Veterinary Tropical Diseases MSc Unrestricted 2020-12-21T09:53:43Z 2020-12-21T09:53:43Z 2020/04/22 2020 Dissertation Nyarku, RE 2020, Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer, MSc Dissertation, University of Pretoria, Pretoria, viewed yymmdd <http://hdl.handle.net/2263/77428> A2020 http://hdl.handle.net/2263/77428 en © 2020 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD diagnosis qPCR brucellosis blood, milk abomasal fluid Veterinary science theses SDG-01 Veterinary science theses SDG-03 SDG-01: No poverty SDG-03: Good health and well-being Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer |
| title | Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer |
| title_full | Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer |
| title_fullStr | Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer |
| title_full_unstemmed | Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer |
| title_short | Development and analytical validation of a genus-specific Brucella real-time PCR assay targeting the 16S-23S rDNA internal transcribed spacer |
| title_sort | development and analytical validation of a genus specific brucella real time pcr assay targeting the 16s 23s rdna internal transcribed spacer |
| topic | UCTD diagnosis qPCR brucellosis blood, milk abomasal fluid Veterinary science theses SDG-01 Veterinary science theses SDG-03 SDG-01: No poverty SDG-03: Good health and well-being |
| url | http://hdl.handle.net/2263/77428 |