Hi-fidelity bioaerosol condensation capture directly into genomic preservatives

Abstract

This invention relates generally to characterizing bioaerosols and, more particularly, to a system for recovering, quantifying, identifying, and assessing the metabolic activities of bioaerosols based on their major biopolymer profiles (lipids, carbohydrate and protein) and more specific their genetic materials (DNA/RNA), such as airborne viruses, bacteria, fungi and pollens.

Claims

1. A method of sampling, recovering and stabilizing bioaerosol materials comprising; a) providing; i) a condensation growth tube; and ii) an aerosol stream comprising water vapor and bioaerosol materials; b) directing said aerosol stream into said tube-so that said vapor condenses on said bioaerosol particles so as to form microdroplets; and c) collecting individual and/or a conglomerate of said microdroplets into at least one individual, sterile DNA-free container containing a genomic, transcriptomic or proteomic preservative, wherein said microdroplets are simultaneously exposed to said preservative.

2. The method of claim 1, wherein the method further comprises step d) recovery of bioaerosol particles.

3. The method of claim 2, wherein said bioaerosol particles comprise microbial nucleic acids.

4. The method of claim 1, wherein preservation of said bioaerosol particles is quantitative.

5. The method of claim 1, wherein said condensation growth tube is RNA-free and DNA-free prior to use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying figures, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The figures are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

(2) This invention is described in preferred embodiments in the following description with reference to the Figures, in which like numbers represent the same or similar elements.

(3) FIG. 1 shows one embodiment of the condensation capture process using a condensation growth tube capture (CGTC) apparatus (U.S. Pat. No. 6,712,881 [19], U.S. Pat. No. 7,736,421 [20], U.S. Pat. No. 8,801,838 [21], and U.S. Pat. No. 9,610,531 [22]).

(4) FIG. 2 shows the recovery of whole cell airborne bacteria from CGTC into microwells filled with preservative used for microbiological analysis of DNA by quantitative polymerase chain reaction (qPCR , pink), in parallel with direct microscopy (, blue), direct fluorescent particle counts (cytometry , red); and standard culturing (, green).

(5) FIG. 3 shows the recovery of whole cell airborne bacteria from CGTC into microwells filled with preservative used for microbiological analysis of DNA by quantitative polymerase chain reaction (qPCR ) as compared to concurrent sampling of the same bioaerosol with conventional filtration and the most common liquid impinger (SKC Biosampler), used for the bioaerosol capture.

DETAILED DESCRIPTION OF THE INVENTION

(6) The described features, structures, or characteristics of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are recited to provide a thorough understanding the preferred of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

(7) A known method of detecting and identifying bioaerosols is disclosed in U.S. Pat. No. 6,806,464 [23] (herein incorporated by reference). An aerosol time-of-flight mass spectrometer using fluorescence techniques is used to ionize selected bioaerosol particles. Laser radiation using a wavelength which is specific to substances affects fluorescence. A fluorescence detector is used to select the bioaerosol particles, and a second laser is used to emit light of a wavelength that effects the ionization of the bioaerosol particles selected by the fluorescence detector. Such a method of detecting and identifying a bioaerosol is rather complex, relying on relatively expensive and complex equipment. Furthermore, this is a destructive method which cannot provide information regarding microbial viability and has not been demonstrated to be able to accurately provide genetically-based taxonomic information regarding genera or species, in practical applications.

(8) Other methods for bioaerosol sampling rely on impaction or impingement. This is accomplished using inertial forces either by impaction on plates, such as that used in an Anderson Impactor (Copely Scientific), loaded with agar or by impingement into a liquid, such as that used in an All Glass Impinger (ACE Glass Incorporated) or a BioSampler (SKC Inc.) (U.S. Pat. No. 5,902,385 [24]) (herein incorporated by reference). Because inertia is a function of particle size, particle size plays a critical role in determining the ability to sample and quantify bioaerosols; in general, the larger the size, the higher the collection efficiency.

(9) An impactor is a device with nozzles that direct air flow carrying aerosol toward impaction plates or filters which serve as a collection media. The inertia of the aerosol particles drives its impaction, and therefore its collection efficiency decreases as particle size decreases. The collection efficiency can be increased by applying pressure or by applying a higher velocity. Filtration is a method of separating particles from the carrier gas by collecting the particles on filter media as the gas passes through open pores or structures of the filter material. Particles make contact with the filter media; and other particles previously deposited on the media, by impaction, interception or diffusion, with each removal mechanism being strongly dependent on particle size. While impaction and filtration can be highly efficient at collecting particles, these approaches stress airborne microbes through high velocity impact and desiccates cells as they are collected; the physiological effects of impact, shearing and desiccation associated with these types of aerosol recovery devices introduce tremendous artifacts regarding cellular damage and thus cannot be used for viability or quantitative genetic analyses with any reasonable degree of certainty. Therefore impaction and filtration cannot be used for observing viable bioaerosols or quantitation using genetic methods.

(10) An impinger is a container with nozzles and an aqueous collection medium. Air flow exiting the inlet nozzle(s) form bubbles in the liquid. Aerosol particles in the bubbles can leave the bubbles due to its inertia, and therefore the collection efficiency decreases as its particle size decreases. Available impingers such as All Glass impingers have less than 70% efficiency for particles less than 0.5 μm. The BioSampler, which is an improved version using swirling jets, still has only 80% efficiency for 0.3 μm. As described, either a viable impactor or an impinger has low efficiency for bioaerosols below 0.3 μm. According to Hogan et al. (“Sampling Methodologies and Dosage Assessment Techniques for Submicrometer and Ultrafine Virus Aerosol Particles”, Applied Microbiology, 99, p. 1422-1434, 2005 [25]), the efficiency of BioSamplers and All Glass Impingers for collecting MS2 bacteriophage is less than 10%. Further, liquid impingers have variable recovery efficiency where hydrophobic airborne microbes are concerned, including for example fungal spores, the bacteria belonging to the family of Actinomycetes, notably including Mycobacteria species. While bioaerosols impinged in liquid experience less impact stress than their counterparts collected in impactors, these devices also impart significant physiological stress. This stress is realized by bioaerosols approaching sonic speeds and large pressure drops through the collection nozzles, and once in the impinger reservoir, stress is realized by impinger reflux, rapid evaporation and cold temperature (<10 C), all of which introduce uncertainties in subsequent genetic and biochemical analysis of the impinger contents.

(11) Thus, there is a need to overcome these and other problems of the prior art and to provide a bioaerosol recovery system that has high capture efficiency, minimizes physiological stress and recovers airborne microbes directly into preservative(s), or onto surfaces saturated with sorbed to, or otherwise associating with preservatives, including but not limited to membranes and filters, that maintain biopolymers with hi-fidelity. Air filters, impactors, and liquid impingers are among the most common alternatives for sampling airborne microbes (bioaerosols). While these low-tech collection methods are cheap, easy and popular, they are fraught with problems for modern aerobiology analysis. Filters impart intense mechanical and desiccation stresses on airborne microbes upon collection. Further, they must elute and dilute samples for further processing that drastically affects sensitivity (PCR and or sequencing); they require tedious, time-intensive, multi-step manual processing; have low extraction efficiencies; and are prone to contamination. Because of low biomass yields, filter-based collection makes it impossible to recover time-resolved samples during periods that are relevant to observing microbial activity in-situ.

(12) One embodiment of the current invention device condenses humidity in a device that concentrates ambient bioaerosols directly into thin films and liquids that preserves genetic materials on contact. Although it is not necessary to understand the mechanism of an invention, it is believed that this condensation process stabilizes bioaerosol genetic materials as they are collected from air, in a small-volume convenient for subsequent DNA/RNA amplification and/or sequencing and (bio)chemical analyses.

(13) One embodiment of the current invention is shown in a schematic describing condensation growth tube capture (CGTC) apparatus in FIG. 1. Although it is not necessary to understand the mechanism of an invention, it is believed that this sampling method moderates temperature under laminar-flow, in a wet-walled tube to create a region of supersaturation in the aerosol stream. The supersaturated water vapor absorbs, adsorbs and/or otherwise condenses on bioaerosol particles enlarging them to form microdroplets, that can be analogous to a “fog”. The microdroplets may remain independent or agglomerate; but regardless, each droplet is then directly collected by gentle, low-velocity impingement into wells, microwells or any analytical tube or containment with a genomic (DNA and RNA), transcriptomic (RNA of any kind), protein and/r lipid preservative. Although it is not necessary to understand the mechanism of an invention, it is believed that this sampler efficiently collects >95% airborne microbes in the size range of viruses, bacteria and fungal spores and avoids the stress and analytical recovery problems introduced by filters and impactors.

(14) Genetic material can be aseptically recovered from CGCT wells in liquid preservatives used to prepare samples for DNA and RNA quantitation and sequencing on popular high throughput platforms. This CGCT device may be portable, and has use in the laboratory and in the field. In controlled bioaerosol chamber studies, total gene copy numbers as determined by qPCR with universal bacterial (16s rDNA) and have been quantitatively compared to direct microscopic counts with reproducible quantitative agreement.

(15) FIG. 2 shows the recovery of whole cell airborne bacteria from CGTC into microwells filled with liquid preservative used for microbiological analysis of DNA by quantitative polymerase chain reaction (qPCR ), in parallel with direct microscopy (), direct fluorescent particle counts (cytometry ); and standard culturing ().

(16) FIG. 3 compares the recovery of conventional filters, liquid impingers and CGTC collecting directly in liquid genomic preservative. In this comparison, the filter, liquid impinger and CGTC concurrently collected bioaerosol from the same calibrated source. As judged by quantitative PCR using 16s rDNA as the target amplicon, CGTC into liquid preservative has at least an order of magnitude better recovery performance when compared, side-by-side, with conventional filtration and liquid impingement.

(17) Since quantitative PCR is successful with between 100 and 2000 airborne cells captured in 10 minutes), the relative abundance of different DNA sequences using CTGC in this preservative capture scenario, can thus likely be reduced into microbial community structures from environmental samples using accepted statistical bioinformatics approaches developed for this purpose. Although it is not necessary to understand the mechanism of an invention, it is believed that being able to characterize airborne microbes and assess their activity, as they exist in-situ may lead to a high fidelity preservation of bioaerosol transcriptomes using CTGC in this scenario. It is further believed that the physical and temporal collection of artifacts that bias aerosol DNA sequencing, and previously prohibited or otherwise impacted RNA recovery, specifically mRNA recovery, are mitigated by condensation collection in this CGTC platform where genomic and/or transcripomic preservatives are used for terminal particle capture. Although it is not necessary to understand the mechanism of an invention, it is believed that in this configuration, CGTC facilitates non-damaging genome and/or transcriptome recovery from bioaerosols in a way that was not previously possible.

(18) Thus, specific compositions and methods of hi-fidelity bioaerosol condensation capture directly into genomic preservatives have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

(19) Although the invention has been described with reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all applications, patents, and publications cited above, and of the corresponding application are hereby incorporated by reference.

REFERENCES

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