[Use of the in vitro enzymatic amplification method for the detection of Mycobacterium paratuberculosis in feces].
A polymerase chain reaction was developed, using as target sequence an insertion element of 1,451 base pairs (IS 900), specific for Mycobacterium paratuberculosis (15-20 copies per genome). The test was performed in three stages: (1) extraction of bacterial deoxyribonucleic acid (DNA), from faeces stored at +4 degrees C, -20 degrees C, in 70% ethanol or in a buffer solution; (2) amplification of the target DNA by means of thermostable DNA polymerase; (3) detection of the amplified DNA by electrophoresis, confirmed by dot blot assay after hybridisation with an internal labelled oligonucleotide of digoxigenin. Reproducible results were obtained with DNA extracted from faeces stored at -20 degrees C or in 70% ethanol. The sensitivity and specificity of the method used, particularly double amplification and hybridisation, are discussed by comparing the results obtained by bacterial culture from faeces.
Guillou
J P
JP
Centre national d'études vétérinaires et alimentaires, Laboratoire central de recherches vétérinaires, Maisons-Alfort, France.
Hénault
S
S
Ostyn
A
A
Thorel
M F
MF
D000818:Animals; D015139:Blotting, Southern; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D005243:Feces / Q000382:microbiology*; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial