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Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.Human exposure to bioaerosols has been associated with a variety of diseases, and dissemination of microbial contaminants in indoor environments has been linked to the development of a cluster of symptoms that include eye and sinus irritation, sore throat, headache, fatigue, and dizziness (11). Contamination and subsequent dispersal of biocontaminants in the workplace and living quarters provide an environment that increases the possibility of occupant exposure and adverse health effects ranging from lost productivity to severe illness. Detection and measurement of biocontaminants in indoor environments are needed to assess contamination levels and to estimate the resulting exposure of occupants. However, monitoring is hampered by a lack of methods that provide precise, accurate, and representative exposure estimates for bioaerosols and microbe-contaminated surfaces (3). Conventional biocontaminant monitoring relies on collection of airborne and surface microorganisms and analysis of samples either by culturing on artificial growth media or by micro...

The PCR technique has potential for use in detection of low concentrations of airborne microorganisms. In this study, the sensitivity of PCR and its susceptibility to environmental interference were assessed with Escherichia coli DH1 as the target organism. Air samples, containing environmental bioaerosols, were collected with AGI-30 samplers and seeded with E. coli DH1 cells. Parallel studies were performed with cells seeded into the sampler prior to collection of air samples to determine the effects of environmental inhibition and sampling stress on the PCR assay. Baseline studies were also performed without environmental challenge or sampling stress to compare two protocols for cell lysis, solid phase and freeze-thaw. Amplification of a plasmid target sequence resulted in a detection limit of a single bacterial cell by the freeze-thaw and solid-phase methods within 5 and 9 h, respectively. With a genomic target, the sensitivity of the solid-phase method was 10-fold lower than that of freeze-thaw. Samples which contained 10 3 to 10 4 CFU of environmental organisms per m 3 inhibited amplification; however, a 1/10 dilution of these samples resulted in successful amplifications. No difference in sensitivity of the PCR assay was obtained as a result of sampling stress, although a 10-fold decrease in culturability was observed. A field validation of the protocol with genomic primers demonstrated the presence of airborne E. coli and/or Shigella spp. in outdoor samples. This study indicates that the PCR method for detection of airborne microorganisms is rapid and sensitive and can be used as an alternative method for air quality monitoring.

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m 2 ) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ؎ 5.8 CFU/m 2 for wet sampling and 100.5 ؎ 10.2 CFU/m 2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre-and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10 5 to 10 6 CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.