Use of AmpliWax to optimize amplicon sterilization by isopsoralen

Abstract

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 (1011 to 103) from the Borrelia burgdorferi OSP A gene were amplified in the presence of 0, 25, 50, and 100 μg 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 μg/ml effectively sterilized up to 1011 amplicons, the compound was inhibitory to PCR. IP-10 at a concentration of 25 μg/ml had slight inhibitory effect on PCR and did not completely sterilize all of the amplicons. Therefore, in subsequent experiments AmpliWax was substituted for mineral oil, and PCR was performed on 109 to 103 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 109 target amplicons were effectively sterilized with 200 μg of IP-10 per ml. Similarly, the addition of IP-10 (50 μg/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 μg 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.