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

• Published in: Molecular and cellular probes Vol. 14; no. 2; pp. 101 - 108
• Main Authors: Zhang, P, Gebhart, CJ, Burden, D, Duhamel, GE
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.04.2000
• Subjects: Animals; Blotting, Southern; Electrophoresis, Agar Gel - methods; enzyme-linked oligosorbent assay, Lawsonia intracellularis, PCR, proliferative enteropathy, swine; Enzymes - chemistry; Ethidium; Gram-Negative Bacterial Infections - diagnosis; Gram-Negative Bacterial Infections - microbiology; Gram-Negative Bacterial Infections - veterinary; Lawsonia Bacteria - genetics; Lawsonia Bacteria - isolation & purification; Oligonucleotides - chemistry; Polymerase Chain Reaction - methods; Protein-Losing Enteropathies - diagnosis; Protein-Losing Enteropathies - veterinary; Reproducibility of Results; Sensitivity and Specificity; Swine; Swine Diseases - diagnosis; Swine Diseases - microbiology; enzyme-linked oligosorbent assay, Lawsonia intracellularis, PCR, proliferative enteropathy, swine
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1006/mcpr.2000.0296

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 OD450of 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 Íinterpretation of ethidium bromide-stained PCR products after agarose gel electrophoresis.