Use of AmpliWax to optimize amplicon sterilization by isopsoralen.
The photochemical inactivation of amplicons by isopsoralen (IP-10) has been suggested as a possible means to prevent PCR carryover contamination. To evaluate the technique, serial dilutions of amplicons (10(11) to 10(3)) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 micrograms of IP-10 per ml for 45 cycles. The PCR products were exposed to UV light for 15 min to activate IP-10 and sterilize the amplicons. One microliter of each sterilized sample was reamplified for an additional 45 cycles. The PCR products were then resolved in an agarose gel, blotted onto a nylon membrane, and probed with an alkaline phosphatase-conjugated chemiluminescent probe. Although IP-10 at concentrations of 50 and 100 micrograms/ml effectively sterilized up to 10(11) amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 micrograms/ml had slight inhibitory effect on PCR and did not completely sterilized all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 10(9) to 10(3) amplicons as described above. Following the amplification, the PCR tubes were cooled to solidify the AmpliWax and inoculated with various concentrations of IP-10. With this technique, PCR products produced from as many as 10(9) target amplicons were effectively sterilized with 200 micrograms of IP-10 per ml. Similarly, the addition of IP-10 (50 micrograms/ml) before and after PCR was evaluated for the detection of B. burgdorferi in 62 ticks from a region of Southern Connecticut where the organism is highly endemic. PCR performed in the presence of 50 micrograms of IP-10 per ml detected B. burgdorferi-specific DNA in 17 of 62 ticks (27%) following gel electrophoresis and in 34 of 62 ticks (55%) following Southern blot hybridization of the PCR products. In contrast, post-PCR addition of IP-10 detected borrelia-specific DNA in 31 of 62 ticks (50%) following gel electrophoresis and in 46 of 62 ticks (64%) following Southern blot hybridization. We conclude that the replacement of mineral oil with AmpliWax can be useful in eliminating the inhibitory effects of IP-10 and other sterilizing agents for post-PCR sterilization of amplicons.
De la Viuda
M
M
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
Fille
M
M
Ruiz
J
J
Aslanzadeh
J
J
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001428:Bacterial Vaccines; D015748:Borrelia burgdorferi Group / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004340:Drug Contamination / Q000517:prevention & control; D011564:Furocoumarins; D005798:Genes, Bacterial; D018884:Ixodes / Q000382:microbiology; D008074:Lipoproteins; D010777:Photochemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D013242:Sterilization / Q000379:methods*; D014466:Ultraviolet Rays; D014885:Waxes
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D011564:Furocoumarins; D008074:Lipoproteins; C072045:OspA protein; D014885:Waxes; C011659:angelicin