De la Viuda M, Fille M, Ruiz J, Aslanzadeh J
Department of Laboratory Medicine, University of Connecticut School of Medicine, Farmington 06030, USA.
J Clin Microbiol. 1996 Dec;34(12):3115-9. doi: 10.1128/jcm.34.12.3115-3119.1996.
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.
异补骨脂素（IP - 10）对扩增子的光化学灭活作用被认为是预防PCR产物污染的一种可能方法。为评估该技术，在每毫升含有0、25、50和100微克IP - 10的条件下，对伯氏疏螺旋体ospA基因的扩增子（10¹¹至10³）进行系列稀释扩增45个循环。PCR产物经紫外线照射15分钟以激活IP - 10并使扩增子灭菌。取1微升各灭菌样品再扩增45个循环。然后将PCR产物在琼脂糖凝胶中分离，转移至尼龙膜上，并用碱性磷酸酶偶联的化学发光探针进行杂交检测。尽管浓度为50和100微克/毫升的IP - 10能有效灭活高达10¹¹个扩增子，但该化合物对PCR有抑制作用。浓度为25微克/毫升的IP - 10对PCR有轻微抑制作用，且不能完全灭活所有扩增子。因此，在后续实验中用AmpliWax替代矿物油，并按上述方法对10⁹至10³个扩增子进行PCR扩增。扩增后，将PCR管冷却使AmpliWax凝固，并接种不同浓度的IP - 10。采用该技术，每毫升200微克的IP - 10能有效灭活多达10⁹个靶扩增子产生的PCR产物。同样，在康涅狄格州南部一个该病原体高度流行地区的62只蜱中检测伯氏疏螺旋体时，评估了PCR前后添加IP - 10（50微克/毫升）的效果。在每毫升含有50微克IP - 10的条件下进行PCR，凝胶电泳后在62只蜱中有17只（27%）检测到伯氏疏螺旋体特异性DNA，PCR产物经Southern印迹杂交后在62只蜱中有34只（55%）检测到。相比之下，PCR后添加IP - 10，凝胶电泳后在62只蜱中有31只（50%）检测到疏螺旋体特异性DNA，Southern印迹杂交后在62只蜱中有46只（64%）检测到。我们得出结论，用AmpliWax替代矿物油有助于消除IP - 10和其他灭菌剂对PCR产物扩增后灭菌的抑制作用。
