Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis.
We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 microg, thereby avoiding false-negative results.
Morata
P
P
Department of Biochemistry and Molecular Biology, Málaga University, Málaga, Spain. Morata@uma.es
Queipo-Ortuño
M I
MI
de Dios Colmenero
J
J
D002002:Brucella / Q000302:isolation & purification; D002003:Brucella abortus / Q000302:isolation & purification; D002006:Brucellosis / Q000097:blood / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000097:blood* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods; D006801:Humans; D007962:Leukocytes / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D013030:Spain
D004269:DNA, Bacterial