PCR-based method for detecting viral penetration of medical exam gloves

Abstract

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 phiX174 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.

FIG. 1.

Limit of detection of purified bacteriophage φX174 DNA by PCR. Bacteriophage φX174 DNA was purified from infected E. coli cells with a QIAprep spin column and diluted in sterile water. One microliter of each dilution was subjected to PCR amplification. The results shown are representative of those for two independent DNA preparations.

FIG. 2.

Detection of bacteriophage φX174 DNA in a host cell lysate. A stock suspension of bacteriophage was prepared by growth in liquid culture with E. coli C, which was then serially diluted. One-microliter aliquots of each dilution were subjected to PCR amplification and to determination of phage titers by plate counting. The number of PFU in each PCR mixture is indicated above each lane. The results shown are representative of those from eight experiments.

FIG. 3.

PCR amplification of bacteriophage DNA. Water from inside and outside of punctured gloves was sampled and serially diluted 10-fold. The titers in each of the dilutions were then determined by plate counting, and the dilutions were used as templates in the PCRs. The numbers above each lane indicate the numbers of PFU per microliter in each dilution; the left-most lane contains undiluted sample. One microliter of each dilution was used as the template for PCR. Data are representative of those for six to eight gloves for each type of glove material. Complete data for the experiment are summarized in Table 1. As a percentage of the total number of phage inside each glove, more bacteriophage were found to have leaked from punctured synthetic gloves than from latex gloves. PVC, polyvinyl chloride.