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Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays

Lane, H.S., Jones, J.B.ORCID: 0000-0002-0773-2007 and McDonald, W.L. (2017) Pooled sample testing for Bonamia ostreae: A tale of two SYBR Green real-time PCR assays. Journal of Veterinary Diagnostic Investigation, 29 (5). pp. 752-756.

Link to Published Version: https://doi.org/10.1177/1040638717717558
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Abstract

Pooled testing of samples is a common laboratory practice to increase efficiency and reduce expenses. We investigated the efficacy of 2 published SYBR Green real-time PCR assays when used to detect the haplosporidian parasite Bonamia ostreae in pooled samples of infected oyster tissue. Each PCR targets a different gene within the B. ostreae genome: the actin 1 gene or the 18S rRNA gene. Tissue homogenates (150 mg) of the New Zealand flat oyster Ostrea chilensis were spiked with ~1.5 × 103 purified B. ostreae cells to create experimental pools of 3, 5, and 10. Ten positive replicates of each pool size were assayed twice with each PCR and at 2 different amounts of DNA template. The PCR targeting the actin 1 gene was unable to reproducibly detect B. ostreae in any pool size. Conversely, the 18S rRNA gene PCR could reproducibly detect B. ostreae in pools of up to 5. Using a general linear model, there was a significant difference in the number of pools that correctly detected B. ostreae between each PCR (p < 0.01) and each pool size (p < 0.01). It is likely that the single copy actin 1 gene is more likely to be diluted and not detected by pooling than the multi-copy 18S rRNA gene. Our study highlights that validation data are necessary for pooled sample testing because detection efficacy may not be comparable to individual sample testing.

Item Type: Journal Article
Publisher: American Association of Veterinary Laboratory Diagnosticians
Copyright: © 2018 by SAGE Publications
URI: http://researchrepository.murdoch.edu.au/id/eprint/42718
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