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Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens
Citation
Song, Y and Hampson, DJ, Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens, Veterinary Microbiology, 137, (1-2) pp. 129-136. ISSN 0378-1135 (2009) [Refereed Article]
Copyright Statement
Copyright 2008 Elsevier B.V. All rights reserved.
DOI: doi:10.1016/j.vetmic.2008.12.020
Abstract
Anaerobic intestinal spirochaetes of the genus Brachyspira include several important pathogenic species, particularly those infecting pigs and chickens. In this study a multiplex-quantitative polymerase chain reaction (M-qPCR) assay was developed based on amplification of a 198 base pair portion of the NADH oxidase gene, using TaqMan probes for detecting and quantifying the three main pathogenic species, B. hyodysenteriae, B. pilosicoli and B. intermedia. The specificity of the assay was validated using 130 spirochaete strains belonging to members of the seven officially named and two provisionally named Brachyspira species. The detection limit for all three targeted species was 1-10 viable cells and 10 fg DNA per reaction. Further detection limit testing was conducted on porcine and chicken faecal specimens that were spiked with spirochaete cells before DNA extraction. The assay could detect 102 to 103 cells per 0.2g of sample, giving an improved detection threshold compared to standard PCRs. The M-qPCR was further developed by incorporating a novel internal control (IC) that employed host cells as template DNA. This adaptation allowed monitoring of the quality of the extracted DNA and ensured that there was no inhibition of the PCR reaction. Use of the IC further improved the detection limits of the assay and increased confidence in being able to detect low numbers of pathogens in faecal samples. Taken together, the results indicate that the new M-qPCR assay is a valuable tool for detecting and quantifying low numbers of pathogenic intestinal spirochaetes in the faeces of pigs and chickens, and potentially other species.
Item Details
| Item Type: | Refereed Article |
|---|---|
| Keywords: | Brachyspira, spirochaete, qPCR, pigs, chickens |
| Research Division: | Medical and Health Sciences |
| Research Group: | Medical Microbiology |
| Research Field: | Medical Bacteriology |
| Objective Division: | Health |
| Objective Group: | Other Health |
| Objective Field: | Health not elsewhere classified |
| UTAS Author: | Song, Y (Dr Yong Song) |
| ID Code: | 135804 |
| Year Published: | 2009 |
| Web of Science® Times Cited: | 36 |
| Deposited By: | Menzies Institute for Medical Research |
| Deposited On: | 2019-11-14 |
| Last Modified: | 2019-12-12 |
| Downloads: | 0 |
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