Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis

Suphot Wattanaphansak, Connie J. Gebhart, Janet M. Anderson, Randall S. Singer

Research output: Contribution to journalArticle

18 Scopus citations

Abstract

The objective of the present study was to develop a quantitative polymerase chain reaction (qPCR) assay using SYBR Green for quantification of Lawsonia intracellularis in cell culture and pig fecal samples. Specific primers were designed and tested using the aspartate ammonia-lyase (aspA) gene as a target. Serial 10-fold dilutions of cell culture samples and several sets of spiked feces were used for qPCR optimization. The lower limit of the linear range of the assay in cell culture was 5.1×102 L. intracellularis/ml. A concentration of between 2.55×104 and 2.55×103 L. intracellularis/g was the lower limit of the linear range when testing community DNA from spiked fecal samples. From both cell culture and fecal samples, L. intracellularis could be detected but not accurately quantified at levels approximately 1 log below the linear range. No cross-reactivity of qPCR was found when the assay was tested using the DNA extracted from 16 species of enteric bacteria commonly found in pig feces or closely related to L. intracellularis. The new qPCR assay might prove to be a sensitive, specific, precise, and accurate method for the detection and quantification of L. intracellularis in field samples.

Original languageEnglish (US)
Pages (from-to)598-602
Number of pages5
JournalJournal of Veterinary Diagnostic Investigation
Volume22
Issue number4
DOIs
StatePublished - Jan 1 2010

Keywords

  • Lawsonia intracellularis
  • Polymerase chain reaction
  • Proliferative enteropathy
  • Quantitative polymerase chain reaction

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