Specific detection of Stachybotrys chartarum in pure culture using quantitative polymerase chain reaction.

Research was conducted with laboratory cultures to establish a protocol for the rapid detection and quantitation of the toxigenic fungus Stachybotrys chartarum by means of polymerase chain reaction (PCR). Sequences for the 18 S rRNA gene of S. chartarum were obtained from GenBank and compared against all other available sequences on-line with the Basic Local Alignment Search Tool (BLAST). Two sets of TaqMan primers and one fluorescently labelled probe were designed and tested for selectivity, specificity and sensitivity of detection. A fluorogenic nuclease assay in conjunction with a sequence detector were used for the amplification and quantitation of S. chartarum. The primers designed amplified all S. chartarum isolates tested and did not amplify DNA extracted from other Stachybotrys species or 15 other fungal genera. The primer set selected had a sensitivity of <23 template copies. Many S. chartarum samples were initially negative after PCR amplification. Incorporation of an internal positive control in the PCR reaction demonstrated the presence of inhibitors in these samples. PCR inhibitors were removed by dilution or further purification of the DNA samples. The results of this research report on a quantitative PCR (QPCR) method for detection and quantitation of S. chartarum and demonstrate the presence of PCR inhibitors in some S. chartarum isolates.

Enhanced detection of surface-associated bacteria in indoor environments by quantitative PCR.

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.

Detection and quantitation of Aspergillus fumigatus in pure culture using polymerase chain reaction.

Research was conducted with laboratory cultures to establish a protocol for the rapid detection and quantitation of the thermophilic fungus, Aspergillus fumigatus, using genetic amplification. Oligonucleotide primers and a fluorescently labelled probe were designed for use with quantitative polymerase chain reaction (QPCR). Primers and probe were tested for selectivity, specificity and sensitivity of detection of the target organism using a fluorogenic nuclease assay and a sequence detector. The DNA extraction protocol consisted of enzymatic treatment and boiling of fungal spore suspensions followed by DNA concentration and purification. The primer set developed was specific for A. fumigatus and had a sensitivity of <20 template copies. These primers amplified all A. fumigatus isolates tested and did not amplify DNA extracted from other Aspergillus species or 15 other fungal genera. However, one A. fumigatus sample was initially negative after PCR amplification. Incorporation of an internal positive control in the PCR reaction demonstrated the presence of inhibitors in this and other samples. PCR inhibitors were removed by dilution or further purification of the DNA samples. This research resulted in a QPCR method for detection and quantitation of A. fumigatus and demonstrated the presence of PCR inhibitors in several A. fumigatus isolates.

Evaluation of portable air samplers for monitoring airborne culturable bacteria.

Airborne culturable bacteria were monitored at five locations (three in an office/laboratory building and two in a private residence) in a series of experiments designed to compare the efficiency of four air samplers: the Andersen two-stage, Burkard portable, RCS Plus, and SAS Super 90 samplers. A total of 280 samples was collected. The four samplers were operated simultaneously, each sampling 100 L of air with collection on trypticase soy agar. The data were corrected by applying positive hole conversion factors for the Burkard portable, Andersen two-stage, and SAS Super 90 air samplers, and were expressed as log10 values prior to statistical analysis by analysis of variance. The Burkard portable air sampler retrieved the highest number of airborne culturable bacteria at four of the five sampling sites, followed by the SAS Super 90 and the Andersen two-stage impactor. The number of bacteria retrieved by the RCS Plus was significantly less than those retrieved by the other samplers. Among the predominant bacterial genera retrieved by all samplers were Staphylococcus, Bacillus, Corynebacterium, Micrococcus, and Streptococcus.

PCR for bioaerosol monitoring: sensitivity and environmental interference.

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 m3 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.

Use of solid-phase PCR for enhanced detection of airborne microorganisms.

The solid-phase PCR (SP-PCR) was compared with a culture-based technique for the detection of aerosolized Escherichia coli DH1. Results with SP-PCR showed an increase in detection sensitivity over that of culture methods. Therefore, SP-PCR may be useful for the detection of airborne microorganisms which may be nonculturable because of aerosolization or sampling stress.

Monitoring airborne fungal spores in an experimental indoor environment to evaluate sampling methods and the effects of human activity on air sampling.

Aerobiological monitoring was conducted in an experimental room to aid in the development of standardized sampling protocols for airborne microorganisms in the indoor environment. The objectives of this research were to evaluate the relative efficiencies of selected sampling methods for the retrieval of airborne fungal spores and to determine the effect of human activity on air sampling. Dry aerosols containing known concentrations of Penicillium chrysogenum spores were generated, and air samples were taken by using Andersen six-stage, Surface Air System, Burkard, and depositional samplers. The Andersen and Burkard samplers retrieved the highest numbers of spores compared with the measurement standard, an aerodynamic particle sizer located inside the room. Data from paired samplers demonstrated that the Andersen sampler had the highest levels of sensitivity and repeatability. With a carpet as the source of P. chrysogenum spores, the effects of human activity (walking or vacuuming near the sampling site) on air sampling were also examined. Air samples were taken under undisturbed conditions and after human activity in the room. Human activity resulted in retrieval of significantly higher concentrations of airborne spores. Surface sampling of the carpet revealed moderate to heavy contamination despite relatively low airborne counts. Therefore, in certain situations, air sampling without concomitant surface sampling may not adequately reflect the level of microbial contamination in indoor environments.

Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae.

The Andersen six-stage impactor, the SAS (Surface Air System) impactor, the AGI-30 impinger, and gravity plates were evaluated for the retrieval of aerosol-released Pseudomonas syringae. The upper limits of the impactor samplers were exceeded at a spray concentration of 10 CFU/ml, indicating that these samplers are not appropriate for monitoring high airborne concentrations. Decreased cell concentrations were retrieved with increased sampling time for the Andersen and AGI samplers, indicating that a minimum sampling time is preferable for monitoring aerosolized vegetative cells.

Diagnosis of Corynebacterium pseudotuberculosis infections in sheep, using an enzyme-linked immunosorbent assay.

Lambs infected with Corynebacterium pseudotuberculosis (ovis) by injecting suspensions of live bacteria into the wool-free area of the axilla developed antibody against cell wall antigens and antitoxin, as measured by the enzyme-linked immunosorbent assay. The toxin was a better antigen for measuring infection than was the cell wall antigen. The enzyme-linked immunosorbent assay appeared to be as sensitive as the antihemolysin inhibition test for detecting antitoxin and was easier to perform.