The Polymerase Chain Reaction Applying dUTP-UDG Protocol for Detection of Mycobacterium tuberculosis / 대한임상병리학회지

ABSTRACT

BACKGROUND: The polymerase chain reaction(PCR) assay is rapid, sensitive analytical technique but has problem of high false-positive rate. We applied dUTP-UDG PCR (dU-PCR) method to prevent carryover contamination major source of high false positive in PCR assays, for detection of Mycobacterium tuberculosis. METHODS: The PCRs for detection of M. tuberculosis were performed with P1 and P2 primers based on IS6110 repeated sequence. FTC-2000 was used for capillary PCR and Uno-Thermoblock was used for heating block PCR. In order to evaluate the effect of dU-PCR controlling carryover contamination, PCRs were performed in the presence of UDG and the absence of UDG. To compare the sensitivity of usual dT-PCR with dU-PCR, chromosomal DNA of M. tuberculosis ranging 500pg to 0.5fg were amplified by dT-PCR and dU-PCR method using two different thermocycler, capillary and heating block type, respectively. RESULT: The dU-PCR using UDG prevented carryover contamination by amplicon DNA up to 500pg. By capillay PCR method, the lower limits of detectability of dT-PCR and dU-PCR were 0.5fg and 500fg, respectively, which indicates the sensitivity of dU-PCR was lower than dT-PCR. But by heating block method, the lower limits of detectability of both method of dU and dT-PCR were 0.5fg. So the sensitivity of dU-PCR was same as dT-PCR. CONCLUSION: The dU-PCR by heating-block method was sensitive test for detection of M. tuberculosis that effectively prevent carryover contamination by amplicon.