INHIBITION OF PCR BY AQUEOUS AND VITREOUS FLUIDS

The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology, W hile much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicon carryover , relatively little attention has been given to the causes of false-ne gative PCRs, This report describes a PCR inhibitor in normal aqueous a nd vitreous fluids that can produce false-negative PCR results, As lit tle as 0.5 mu l of vitreous fluid and 20 mu l of aqueous fluid can com pletely inhibit DNA amplification in a 100-mu l PCR mixture. This inhi bition was not primer specific, nor was it due to chelation of Mg2+ io ns or DNase activity in the ocular fluid, The inhibitor was completely resistant to boiling for 15 min, However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extr action, The extent of PCR inhibition depended upon the type of thermos table DNA polymerase used in the reaction, Tag DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from T hermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completel y resistant, Thus, the inhibitory effects of intraocular fluids on PCR s can be removed by diluting the specimen, by chloroform extraction, o r by using Tth or Tfl DNA polymerases.