The use of a mimic to detect polymerase chain reaction-inhibitory factors in feces examined for the presence of Lawsonia intracellularis.
Lawsonia intracellularis is an intracellular organism that causes proliferative enteritis in pigs. This bacterium is difficult to culture, and antemortem demonstration of the microbe is therefore often performed on fecal samples by polymerase chain reaction (PCR). Polymerase chain reaction is sensitive and specific, but inhibitory factors in feces might cause false-negative results. This article describes the construction and use of an internal standard, a mimic. The mimic is amplified by the same primers as those used for L. intracellularis DNA and thus could indicate false-negative results in clinical samples. The amplicon was clearly visible when as few as 10 mimic molecules were added per amplification reaction and when no inhibitors werepresent. When fecal samples were spiked with the mimic, the detection limit was 10(2) molecules per PCR. Sixty clinical samples, 20 from wild boars, 20 from growing pigs with diarrhea, and 20 from pigs without diarrhea, were prepared by a boiling procedure and subjected to PCR together with 10(3) mimic molecules. Nine samples were positive, of which 7 originated from pigs with diarrhea and 2 from pigs without diarrhea. In 14 samples from wild boars, in 8 samples from pigs without diarrhea, and in 3 samples from pigs with diarrhea, neither the mimic nor the target DNA was visible. This indicated the presence of inhibitors in these samples. It is concluded that the mimic can be used as an internal control in the diagnosis of L. intracellularis to indicate inhibition of PCR.
Department of Large Animal Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, S75007 Uppsala, Sweden.
Magdalena
M
Jacobson
Stina
S
Englund
András
A
Ballagi-Pordány
D000818:Animals; D016477:Artifacts; D003001:Cloning, Molecular; D005243:Feces / Q000382:microbiology*; D020639:Lawsonia Bacteria / Q000235:genetics / Q000302:isolation & purification*; D018716:Molecular Mimicry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D013552:Swine / Q000382:microbiology; D013553:Swine Diseases / Q000175:diagnosis* / Q000382:microbiology