Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA)

P. Zhang, C. J. Gebhart, D. Burden, G. E. Duhamel

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Proliferative enteropathy (PE) caused by Lawsonia intracellularis is a major diarrheal disease affecting swine worldwide. Routine laboratory diagnosis of PE is done by amplification of L. intracellularis-specific DNA sequences by PCR followed by agarose gel electrophoresis and staining of PCR products with ethidium bromide. We report the development of an enzyme-linked oligosorbent assay (ELOSA) for specific identification of chromosomal L. intracellularis 328-bp PCR amplified products. The ELOSA involved determination of optical density value at 450 nm (OD450) after hybridization of biotin-labelled PCR products with an amine-modified internal oligonucleotide capture probe immobilized in microwell plates, and avidin-biotin-peroxidase complex. A positive ELOSA cut-off value of ≥ 0.375 was established using the mean OD450 of negative control specimens plus three times the standard deviation. Using this value, the detection limit of PCR amplified L. intracellularis-specific products by ethidium bromide-stained agarose gel electrophoresis, Southern blot, and ELOSA were estimated to be 6.1 ng, between 0.8 and 3.0 ng, and 0.8 ng of DNA, respectively. Comparison of ethidium bromide-stained agarose gel analysis with ELOSA for detection of L. intracellularis-specific PCR products from 315 clinical specimens revealed 78% sensitivity, 100% specificity and 94% accuracy. The ELOSA produced a spectrophotometric signal that confirmed the authenticity of PCR products without subjective interpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)101-108
Number of pages8
JournalMolecular and Cellular Probes
Volume14
Issue number2
DOIs
StatePublished - Apr 2000

Keywords

  • Enzyme-linked oligosorbent assay
  • Lawsonia intracellularis
  • PCR
  • Proliferative enteropathy
  • Swine

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