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Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis

The obligate intracellular bacterium Lawsonia intracellularis causes enteritis and poor growth in weaned pigs. Cultivation is difficult and diagnosis ante mortem is mainly based on techniques such as polymerase chain reaction. However, false negative results caused by the presence of PCR-inhibitory...

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Autores principales: Jacobson, Magdalena, Norling, Börje, Gunnarson, Anders, Aspan, Anna
Formato: Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777015/
https://www.ncbi.nlm.nih.gov/pubmed/19936108
http://dx.doi.org/10.1155/2009/410945
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author Jacobson, Magdalena
Norling, Börje
Gunnarson, Anders
Aspan, Anna
author_facet Jacobson, Magdalena
Norling, Börje
Gunnarson, Anders
Aspan, Anna
author_sort Jacobson, Magdalena
collection PubMed
description The obligate intracellular bacterium Lawsonia intracellularis causes enteritis and poor growth in weaned pigs. Cultivation is difficult and diagnosis ante mortem is mainly based on techniques such as polymerase chain reaction. However, false negative results caused by the presence of PCR-inhibitory factors constitute a problem. This study aimed to develop and evaluate a new technique, flotation, to separate L. intracellularis from inhibitors in faeces prior to PCR. The technique was evaluated by comparison to two previously evaluated and commonly used methods, preparation by boiling lysate combined with nested PCR and preparation by a commercial kit combined with conventional PCR. Continuous density centrifugation of faecal samples containing L. intracellularis suggested the buoyant density of the microbe to be between 1.064 and 1.077 g/mL. Several flotation setups were tested to achieve optimal separation of the microbe from inhibitors and faecal particles. The finally selected setup floated whole L. intracellularis from the application site at the bottom to the upper part of the gradient while inhibitory components mainly remained in the bottom. PCR was performed directly on material recovered from the upper interphase. The method was evaluated on 116 clinical samples. As compared to sample preparation by boiling combined with nested PCR, fewer samples were inhibited but also fewer positives were identified. In comparison to preparation by a commercial kit combined with conventional PCR, presently used for routine diagnosis, similar results were obtained. However, the new method was comparably faster to perform. The new method, based on flotation of Lawsonia intracellularis combined with conventional PCR, was well suited for routine diagnosis.
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spelling pubmed-27770152009-11-23 Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis Jacobson, Magdalena Norling, Börje Gunnarson, Anders Aspan, Anna Int J Microbiol Research Article The obligate intracellular bacterium Lawsonia intracellularis causes enteritis and poor growth in weaned pigs. Cultivation is difficult and diagnosis ante mortem is mainly based on techniques such as polymerase chain reaction. However, false negative results caused by the presence of PCR-inhibitory factors constitute a problem. This study aimed to develop and evaluate a new technique, flotation, to separate L. intracellularis from inhibitors in faeces prior to PCR. The technique was evaluated by comparison to two previously evaluated and commonly used methods, preparation by boiling lysate combined with nested PCR and preparation by a commercial kit combined with conventional PCR. Continuous density centrifugation of faecal samples containing L. intracellularis suggested the buoyant density of the microbe to be between 1.064 and 1.077 g/mL. Several flotation setups were tested to achieve optimal separation of the microbe from inhibitors and faecal particles. The finally selected setup floated whole L. intracellularis from the application site at the bottom to the upper part of the gradient while inhibitory components mainly remained in the bottom. PCR was performed directly on material recovered from the upper interphase. The method was evaluated on 116 clinical samples. As compared to sample preparation by boiling combined with nested PCR, fewer samples were inhibited but also fewer positives were identified. In comparison to preparation by a commercial kit combined with conventional PCR, presently used for routine diagnosis, similar results were obtained. However, the new method was comparably faster to perform. The new method, based on flotation of Lawsonia intracellularis combined with conventional PCR, was well suited for routine diagnosis. Hindawi Publishing Corporation 2009 2009-06-17 /pmc/articles/PMC2777015/ /pubmed/19936108 http://dx.doi.org/10.1155/2009/410945 Text en Copyright © 2009 Magdalena Jacobson et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Jacobson, Magdalena
Norling, Börje
Gunnarson, Anders
Aspan, Anna
Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis
title Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis
title_full Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis
title_fullStr Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis
title_full_unstemmed Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis
title_short Flotation—A New Method to Circumvent PCR Inhibitors in the Diagnosis of Lawsonia intracellularis
title_sort flotation—a new method to circumvent pcr inhibitors in the diagnosis of lawsonia intracellularis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777015/
https://www.ncbi.nlm.nih.gov/pubmed/19936108
http://dx.doi.org/10.1155/2009/410945
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