Development of a polymerase chain reaction assay for quantification of Lawsonia intracellularis.
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 x 10(2) L. intracellularis/ml. A concentration of between 2.55 x 10(4) and 2.55 x 10(3) 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.
Wattanaphansak
Suphot
S
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA.
Gebhart
Connie J
CJ
Anderson
Janet M
JM
Singer
Randall S
RS
D005243:Feces / Q000382:microbiology; D020639:Lawsonia Bacteria; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013696:Temperature