Improved diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis using polymerase chain reaction-enzyme-linked oligosorbent assay (PCR-ELOSA).
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 (OD(450)) 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 > or =0.375 was established using the mean OD(450)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.
Zhang
P
P
Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln 68583-0905, USA.
Gebhart
C J
CJ
Burden
D
D
Duhamel
G E
GE
D000818:Animals; D015139:Blotting, Southern; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004798:Enzymes / Q000737:chemistry; D004996:Ethidium; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D020639:Lawsonia Bacteria / Q000235:genetics / Q000302:isolation & purification*; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011504:Protein-Losing Enteropathies / Q000175:diagnosis / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology
D004798:Enzymes; D009841:Oligonucleotides; D004996:Ethidium