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Development of a multiplex qPCR for detection and quantitation of pathogenic intestinal spirochaetes in the faeces of pigs and chickens

journal contribution
posted on 2023-05-20, 08:20 authored by Yong SongYong Song, Hampson, DJ
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.

History

Publication title

Veterinary Microbiology

Volume

137

Issue

1-2

Pagination

129-136

ISSN

0378-1135

Department/School

Menzies Institute for Medical Research

Publisher

Elsevier Science Bv

Place of publication

Po Box 211, Amsterdam, Netherlands, 1000 Ae

Rights statement

Copyright 2008 Elsevier B.V. All rights reserved.

Repository Status

  • Restricted

Socio-economic Objectives

Other health not elsewhere classified

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