The test approved by the U.S. Food and Drug Administration for assessment of the barrier quality of medical exam gloves includes visual inspection and a water leak test. Neither method tests directly the ability of gloves to prevent penetration by microorganisms. Methods that use microorganisms (viruses and bacteria) to test gloves have been developed but require classical culturing of the organism to detect it. We have developed a PCR assay for bacteriophage X174 that allows the rapid detection of penetration of gloves by this virus. The method is suitable for use with both latex and synthetic gloves. The presence of glove powder on either latex or synthetic gloves had no effect on the ability of the PCR assay to detect bacteriophage DNA. The assay is rapid, sensitive, and inexpensive; requires only small sample volumes; and can be automated.In response to a growing need to protect health care workers and patients from the possibility of transmission of human immunodeficiency virus through contact with body fluids, the Occupational Safety and Health Administration issued standard precautions that state that gloves must be worn when "touching blood, body fluids, secretions, excretions, and contaminated items" (17). Consequently, the rate of use of exam gloves in clinical practice rose dramatically (7). To ensure consistent quality in the manufacture of gloves, the U.S. Food and Drug Administration (FDA) introduced a two-part testing protocol in which gloves are first visually inspected and then subjected to a water leak test (5). These tests, while convenient and inexpensive, suffer from a number of drawbacks. Both the visual test for defects and the water leak test, in which an inspector looks for leaks in a glove filled with a liter of water, are subject to operator error. It has also been observed that microorganisms can penetrate gloves that do not show visible water leaks (2, 9). Studies that use microorganisms to gauge the propensities of gloves to permit pathogen penetration examine the question more directly than water leak studies. However, the microbial assays described rely on classical microbiological culture techniques to detect microbial penetration and therefore are more labor-intensive, time-consuming, and expensive than the FDA-approved tests. This report describes an assay for the detection of glove penetration by a small virus, bacteriophage X174. The assay uses PCR to detect the bacteriophage. PCR offers the advantages of being highly sensitive and specific for the agent being detected. With the recent and rapid increase in the availability of laboratory robotics for molecular biology, this PCR assay also has the potential to be automated and therefore made useful in the glove manufacturing setting. Cultures of E. coli were maintained on solid NB medium, made as described above by the addition of 15 g of agar per liter and 1 ml of CaCl 2 and the omission of Tween 80. For plaque analysis of bacteriophage X174, the top agar composition was identical to that of the plate agar, except that the co...