PCR for bioaerosol monitoring: sensitivity and environmental interference

Abstract

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.