Evaluation of a rapid method of extracting DNA from stool samples for use in hybridization assays.
The ability of the Extractor system (Molecular Biosystems, Inc., San Diego, Calif.) to isolate nucleic acid (NA) from stool samples for use in hybridization assays was investigated. Crude NA was recovered from 45 of 50 stool samples by using this system. The amount of NA recovered varied considerably depending on the microbial flora present in the sample (mean +/- standard deviation, 50.2 +/- 46.7 micrograms; range, 2 to 228 micrograms) but did not correlate with the consistency of the sample. Samples containing primarily gram-positive organisms or yeast cells gave lower yields of NA (less than 10 micrograms) than those containing gram-negative bacilli. The five samples which did not yield NA were sterile when cultured aerobically on blood agar plates. Samples of the 45 stools yielding NA were inoculated into broth and grown overnight, and a 10-microliters sample of broth was spotted onto nitrocellulose filters. The NA samples recovered from the Extractor column were applied to nylon membranes by using the Centri-dot system. The NA on the broth blots and the NA on the Centri-dot filters were hybridized with a 310-base-pair probe specific for the 2"-O-aminoglycoside adenylyltransferase [ANT(2")] resistance gene. The Extractor-Centri-dot system demonstrated 61.9% sensitivity and 95.8% specificity in detecting the ANT(2") gene in stool samples containing colonies demonstrating the ANT(2") phenotype. The positive and negative predictive values of the NA blot were 92.8 and 74.2%, respectively.
Coll
P
P
Hospital de la Santa Creu i Sant Pau, Universitat Autonoma de Barcelona, Spain.
Phillips
K
K
Tenover
F C
FC
D015342:DNA Probes; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D005243:Feces / Q000032:analysis / Q000382:microbiology*; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009693:Nucleic Acid Hybridization
D015342:DNA Probes; D004269:DNA, Bacterial