Magnetic Bead Capture Eliminates PCR Inhibitors in Samples Collected from the Airborne Environment, Permitting Detection of Pneumocystis carinii DNA

Abstract

PCR detection methods are useful in studies of organisms not amenable to culture. Inhibitors in environmental samples can interfere with such assays. We describe a magnetic bead DNA capture protocol that removes inhibitors from outdoor air samples, maintaining the sensitivity of a 16S Pneumocystis carinii mitochondrial rRNA gene-based PCR.Pneumocystis carinii pneumonia (PCP) remains the most common opportunistic infection among individuals diagnosed with AIDS in the United States (5). However, basic knowledge of the ecology and epidemiology of the causative agent, P. carinii, is still lacking. The once widely accepted theory of PCP reactivation in severely immunosuppressed persons is currently being questioned in favor of the view that most episodes of PCP result from a de novo acquisition of the organism through inhalation of contaminated air (8, 11-13, 16, 18, 19, 21-24).There is anecdotal evidence supporting the hypothesis that patients with fulminant PCP are the source of infectious P. carinii, shedding large numbers of organisms into their immediate environment as is the case with tuberculosis (2,3,7,9,10,17). Alternatively, some studies suggest the existence of environmental reservoirs (20, 25), a transmission model which more closely resembles that of aspergillosis. Determining the source(s) of infectious P. carinii organisms is key to understanding when and how potential exposure control measures could be instituted.PCR-based detection methods used in conjunction with environmental sampling are key tools in understanding the ecology and epidemiology of pathogens, such as P. carinii, that are difficult or impossible to culture. However, PCR results may be compromised by the presence of inhibitors in environmental samples. Such inhibitors have been reported in air, soil, and water samples (1,4,15). These include humic acid, clays, various organics, and large amounts of nontarget DNA in the collected sample, although additional types of inhibitors are likely to exist (4). Here we describe a methodology that allows for successful amplification of a target P. carinii DNA sequence spiked onto on a membrane filter containing inhibitory materials collected from a suburban outdoor environment.Air samples were collected from an urban indoor environment and from a suburban outdoor environment during the autumn in the Southeastern United States. Air samples were collected over a 24-h period at approximately 0.4 liters per min using polyvinylidene difluoride filters (pore size, 0.45 m) for use with PCR analysis and at approximately 0.25 liters per min using polycarbonate filters (pore size, 0.45 m) for use with microscopic analysis. Indoor air sampling for viable fungi was accomplished using an Andersen impactor containing malt extract agar plates which were incubated at 25°C for approximately 1 week.Fungal concentrations (in CFU per cubic meter) in the indoor environment were performed to assess the potential for microbial contamination. The rationale for this was that a high indoor fungal concentration would be ind...

Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

“…Studies applying filtration for bioaerosol collection have used sampling times ranging from a few minutes to several hours (27,54), or even as long as 24 h (55,56). It has been shown that prolonged sampling periods by impaction-based samplers increase the risk of microorganism viability loss (16,57,58).…”

Use of an Andersen Bioaerosol Sampler to Simultaneously Provide Culturable Particle and Culturable Organism Size Distributions

“…Additionally, for pathogenic agents with low infectious dose units (e.g., Neisseria meningitidis and Mycobacterium tuberculosis with infectious doses of <10 organisms) it is important to know the number of CO especially for some pathogens, e.g., V. cholerae, where the major contributors to infection are the culturable cells as opposed to active but not culturable organisms, ABNC (Nelson et al 2008). An instrumental technique, quantitative polymerase chain reaction detection (PCR), has the potential to account for the ABNC as well as species that are more difficult to culture, so quantitative PCR is considered to be more comprehensive than culture analysis (Maher et al 2001). …”

Inhibition of quantitative PCR analysis of fungal conidia associated with indoor air particulate matter

“…Wilson (1997) provides an extensive list of sample substrates and associated inhibitors. Trace amounts of humic substances, heavy metals, constituents of bacterial cells, proteolytic enzymes, and high concentrations of non-target DNA have all been associated with the inhibition of the PCR in environmental matrices such as air, soil, water, sewage, and sediment (Alvarez et al, 1994(Alvarez et al, , 1995Keswani, Kashon, & Chen, 2005;Maher, Dillon, Vermund, & Unnasch, 2001;Roe et al, 2001;Wilson, 1997).…”