Patent Grant
8546127
The present invention relates to a device and method for collecting and lysing microbiological species to release nucleic acids such as RNA and DNA.
Detection and control of microorganisms are important in many fields including health care, environmental regulation, bio-warfare, pathogen identification, food and drug testing and in a variety of industrial systems. In industry, presence of undesirable microorganisms decreases the efficiency of operating equipment and ultimately increases the cost of associated goods or services. Furthermore, since microorganisms multiply rapidly, the presence of microbial activity also causes health risks to the public. There is an increasing concern with pathogenic organisms infecting water and process and creating increased human, animal and environmental health risk.
In cooling towers, for example, water borne pathogenic microorganisms, such as Legionella sp. may be present. If not properly treated with preferred biocides, aerosolized particles containing the microorganisms can create extreme health concerns from inhalation of the aerosolized microorganisms leading to disease such as Pontiac fever or the sometimes fatal Legionnaire's disease caused by Legionella pneumophila. Detection of this and other microorganisms is difficult, especially in the case of open recirculation water systems such as cooling towers because low concentration represent serious health risk and large water volumes must be concentrated into smaller sample volumes in order to perform the desired analytical test and obtain accurate and reproducible results.
In one aspect, the present invention provides an apparatus for collection of a target material such as a nucleic acid from a liquid sample comprising target species, such as cellular material or viruses, that contain the target material therein. A fluid flow conduit is provided and a filter medium is disposed in contact with the fluid flow conduit. The filter medium has a pore size adapted to retain the target species thereon and pass, as filtrate, lysate containing the target material. Initially, the passage of filtrate is stripped of the target species and would carry non-concentrated quantities of the target material. A lysing agent conduit communicates with the fluid flow conduit and is adapted to deliver lysing agent to the filter medium to lyse the target species thereby releasing the target material.
As used herein, the term “target species” can comprise microbiological organisms such as cellular material and viruses. The cellular material may, for example, be chosen from bacteria, algae, fungi, prokaryotes, etc. As is known, in the cellular organisms, the target material, nucleic acids, such as DNA or RNA, is located inside the cell. In a virus, the target material nucleic acid is located within a protein coat. “Lysing” is then a rupturing of the cell or protein coat to release the target nucleic acid.
Another exemplary embodiment pertains to a method for collecting a desired target material from a liquid sample comprising target species that contain the desired target material therein. The liquid sample is passed through a filter membrane having a pore size adapted to retain the target species thereon and to pass, as filtrate, lysate containing the target material. A lysing agent is brought into contact with the filter membrane and target cells retained thereon thereby rupturing the target cells or protein coat in the case of viral matter to release the target material, such as RNA or DNA. The desired target material passes as filtrate through the filter membrane and may then be collected for analysis.
The invention will be further described in conjunction with the appended drawing.
Turning to
The filter cartridge houses a filter medium 18 therein. As shown, the cartridge comprises a housing 20 preferably composed of a rigid plastic and having an inlet fitting 22 and an outlet fitting 24. The fittings 22, 24 are adapted for snug reception into the upstream fitting 8 and downstream fitting 10 of the main flow conduit. In some cases, it may be desirable to provide a co-or insert molded cartridge wherein the filter is co-or insert molded in the housing.
A recirculation conduit 30 is provided preferably of rubber or similar flexible material. Preferably, the conduit is penetratable by a hypodermic needle or the like. The recirculation conduit 30 sealingly communicates with the main flow conduit 4 at upstream conduit port 32 and downstream conduit port 34. A collection area or reservoir 36 may be provided in the recirculation conduit and is adapted for penetration by a hypodermic syringe or the like so that material may be extracted from the recirculation conduit thereat. Alternatively, a valve may be provided downstream of the filter to provide connection to an extraction tube. A pump 40 is provided in the recirculation conduit and is preferably of a bi-directional peristaltic pump type. Many varied types of pumps can be mentioned as exemplary. A non-direct contact pump is desirable as the recirculation loop may be disposed of after use.
As shown, a recirculation check valve 70 may be provided in the upstream portion of the recirculation conduit. A check valve 50 is also provided at inlet of the main flow conduit and another check valve 60 is provided at the outlet of the conduit 4 and is biased in the closed position via spring 62. The valve 50 may be provided with a spring bias to prevent backflow in the event the recirculation loop was to expand due to any pressure developed to overcome the bias on check valve 60. The position of the exit conduit 14 could be varied from that shown in the drawing to ensure a portion of filtrate, before lysing, is collected in the reservoir to provide a media to be pumped. This way a concentrated lysing agent could be added to the reservoir for injection into the system.
The filter medium 18 is provided with pore sizes chosen so that the desired target species containing the desired target material is captured as retentate thereon. However, after the target species is lysed or ruptured, the target material, such as RNA released from the cells or from a protein coating passes through the membrane as filtrate.
The filter medium 18 can be any of a variety filter membranes designed to retain the desired biological target species thereon. Non-limiting examples of the membrane material comprise nylon, stainless, cellulose ethers, and esters, PTFE, grass fiber, polypropylene, polyvinyl chloride, hydrophilic acrylic copolymer, polyether sulfone, and polycarbonate, etc. Pore sizes of these filters may be on the order of about 10 nm to about 5.0 um. One especially preferred membrane for use in collection of Legionella pneumophila cells is a glass fiber membrane available from Millipore having a pore size of about 2.7 um.
It should be mentioned that in another exemplary embodiment of the method, a prefiltering step may be provided wherein, prior to entry into the device 2, the fluid sample may be passed through one or more pre-filters to remove inorganic and large biological components while passing the target species as filtrate. These membranes may, for example, be composed of the same type of materials set forth above for use as the filter medium 18, but pore sizes should be larger, on the order of about 1 to 100 um, more preferably about 1 to 50 um. A tandem filter pair with an upstream filter pore size of about 20 um and a downstream filter pore size of about 11 um may be mentioned as exemplary.
In operation, approximately 500 ml of sample fluid containing target species that in turn, encapsulates the target material, enters through the inlet tube and is passed through the filter capsule. A vacuum or the like may be utilized to assist in drawing the fluid through the filter by applying the vacuum at the outlet port. In one exemplary embodiment, the filter medium is sized to retain Legionella bacteria cells yet allow passage of lysed RNA there through.
After the sample is passed through the device, a lysing agent is applied to the system and through the recirculation conduit 30 propelled by the driving action of pump 40. The lysing agent may be admitted through the conduit inlet 12 or it may simply be injected into the recirculation conduit 30 via a syringe or the like. The pump may run in either direction or the pump can be used in combination with an additional check valve to prevent the initial sample from bypassing the filtration device. After a period of time, the lysing buffer or lysing solution from the recirculation conduit ruptures the target species material captured on the filter medium and the lysed cell RNA may then be removed from the device either by using a syringe drawing the fluid directly from the recirculation conduit such as for example at the collection area 36, or, the fluid may be drawn out of the tube outlet. The RNA may then be analyzed by any of a variety of conventional assay techniques. The lysing agent may be applied in several ways. The lysing agent may be inserted through 12, injected into the reservoir 36, or inserted through an additional port added to the loop. If large volumes of lysing agent are applied, check valve 60 may open resulting in loss of target material unless the recirculation loop is evacuated first through a vacuum means as set forth above.
The check valve at the inlet prevents fluid from flowing out of the inlet during the recirculation cycling step and a spring biased check valve at the exit allows the outlet to seal unless slight pressure is applied to draw the fluid out. A spring relief on check valve 50 may also be desirable. Pump 40 is operated so that the pressure generated thereby is such that it does not overcome the outlet spring relief check valve.
As to the lysing agent that is employed, any one that has the effect of rupturing the cell membrane or proteinaceous coating of the virus may be used. Detergents are a non-limiting example of chemicals that are commonly used to disrupt a lipid double layer membrane to release cell contents and lyse membrane protein, and non-limiting examples of lysing chemicals are lithium dodecyl sulfate, CHAPS, Tween-20E, NP40, CTAB, PVPP, Triton X series detergents, sodium cholate, and sodium deoxycholate, guanidinium chloride, or caustic. Chaotropic agents like guanidiunium salts can also act as lysing agents in this system. The lysing efficiency of detergents and alternate lysing agents is dependent on the cell types and specific applications, but all tested materials show adequate lysing within the time requirements for this test method. Enzymes such as lysozymes, mutanolysin, Labiase, lysostaphin, lyticase, proteinase K, endolysin and achromopeptidases may be included as lysing reagents or additives to enhance lysing. Organic solvents, such as DMSO, DMF could be also included as lysing reagents or additives to enhance lysing. A variety of bioscience suppliers offer a wide collection of lysing buffers suitable for cell lysis application. Any physical methods such as shaking, heating cutting and homogenizing, etc., could be added in the process to enhance the lysing efficiency. At present a lysing solution comprising a dilute concentration of lithium lauryl sulfate, nonyl-phenol (40 moles ethoxylation) and DMSO may be mentioned as an exemplary lysing solution that is recirculated through the device.
As above noted, in other varying embodiments, the exit conduit can be positioned to allow air to escape from the reservoir and retain a portion of the fluid filtrate prior to lysing agent application. For example, the exit conduit could be positioned downstream from the filter along the right hand side of the apparatus as shown in the drawing, communicating with conduit 4 and extending angularly upwardly therefrom. Additionally, a separate valve means could be provided in communication with the fluid flow conduit, lysing agent conduit or reservoir to provide for extraction of the desired target material.
While I have shown and described herein certain embodiments of the invention, it is intended that there be covered as well any change or modification therein which may be made without departing from the spirit and scope of the invention as defined in the appended claims.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4080294 | Edwards et al. | Mar 1978 | A |
| 4137169 | El-Hindi | Jan 1979 | A |
| 4358535 | Falkow et al. | Nov 1982 | A |
| 4478714 | Blake et al. | Oct 1984 | A |
| 4518402 | Dargel | May 1985 | A |
| 4976876 | Diman et al. | Dec 1990 | A |
| 5017292 | DiLeo et al. | May 1991 | A |
| 5096474 | Miller, Jr. et al. | Mar 1992 | A |
| 5155018 | Gillespie et al. | Oct 1992 | A |
| 5376527 | Robson et al. | Dec 1994 | A |
| 5423989 | Allen et al. | Jun 1995 | A |
| 5456835 | Castino et al. | Oct 1995 | A |
| 5458782 | Hou et al. | Oct 1995 | A |
| 5620790 | Holzki et al. | Apr 1997 | A |
| 5658714 | Westfall et al. | Aug 1997 | A |
| 5669946 | Blair, Jr. | Sep 1997 | A |
| 5783686 | Gonzalez | Jul 1998 | A |
| 5808041 | Padhye et al. | Sep 1998 | A |
| 5972613 | Somack et al. | Oct 1999 | A |
| 6011148 | Bussey et al. | Jan 2000 | A |
| 6015493 | Smith et al. | Jan 2000 | A |
| 6312588 | Conrad et al. | Nov 2001 | B1 |
| 6383818 | Arai et al. | May 2002 | B1 |
| 6405875 | Cutler | Jun 2002 | B1 |
| 6479273 | Bogedain et al. | Nov 2002 | B1 |
| 6508936 | Hassan | Jan 2003 | B1 |
| 6544424 | Shevitz | Apr 2003 | B1 |
| 6544751 | Brandwein et al. | Apr 2003 | B1 |
| 6551642 | Trout | Apr 2003 | B2 |
| 6821757 | Sauer et al. | Nov 2004 | B2 |
| 6838272 | Bogedain et al. | Jan 2005 | B2 |
| 6939697 | Champluvier et al. | Sep 2005 | B2 |
| 6958392 | Fomovskaia et al. | Oct 2005 | B2 |
| 7220549 | Buzby | May 2007 | B2 |
| 7252801 | Coffey | Aug 2007 | B2 |
| 7378238 | Hilbrig et al. | May 2008 | B2 |
| 7385040 | Johansson et al. | Jun 2008 | B2 |
| 20020164410 | Ogden et al. | Nov 2002 | A1 |
| 20030064951 | Bussey et al. | Apr 2003 | A1 |
| 20030201229 | Siwak et al. | Oct 2003 | A1 |
| 20040076980 | Charlton et al. | Apr 2004 | A1 |
| 20040245163 | Lim et al. | Dec 2004 | A1 |
| 20050191619 | Davis et al. | Sep 2005 | A1 |
| 20050244299 | Dasgupta et al. | Nov 2005 | A1 |
| 20070003997 | Kemmochi et al. | Jan 2007 | A1 |
| Number | Date | Country |
|---|---|---|
| 1606621 | Apr 2005 | CN |
| 1886498 | Dec 2006 | CN |
| 101113437 | Jan 2008 | CN |
| 00845520 | Jul 1983 | EP |
| 0 958 037 | Aug 2003 | EP |
| 0 979 237 | Nov 2004 | EP |
| 1 710 011 | Oct 2006 | EP |
| 0389 063 | Oct 2006 | EP |
| 1 624 950 | Jul 2007 | EP |
| 1873243 | Jan 2008 | EP |
| WO 0027510 | May 2000 | WO |
| WO 0236248 | May 2002 | WO |
| WO 03035234 | May 2003 | WO |
| 2005052137 | Jun 2005 | WO |
| WO 2005115595 | Aug 2005 | WO |
| WO 2007004263 | Jan 2007 | WO |
| WO 2007065229 | Jun 2007 | WO |
| Entry |
|---|
| Wikipedia Pinch Valve Brochure, printed Jun. 6, 2010, pp. 1-3. |
| International Search Report issued in connection with corresponding PCT Application No. PCT/US2009/047448 on Oct. 5, 2009. |
| MSDS No. 301041.233—APTIMA Unisex Swab Specimen Collection Kit, Manufacturer: Gen-Probe Incorporated, San Diego, CA. |
| MSDS No. 74385—Nonident P40 Substitute, Fluka, Manufacturer: Sigma-Aldrich, Shanghai, China. |
| Lambert, K.J., “Pretreatment of Cells of ‘Klebsiella pneumoniae’ with 50% (v/v) Dimethylsulfoxide Yields Purified Deoxyribonucleic Acid of Low Polysaccharide Content”, Agric. Biol. Chem., vol. 46, #12, 1982, pp. 3079-3080. |
| Lytle, C.D., et al., “Virus Passage through Track-Etch Membranes Modified by Salinity and a Nonionic Surfactant”, Applied & Environ. Microbiology, vol. 65, #6, Jun. 1999, pp. 2773-2775. |
| Taylor, M.T., et al., “Lysing Bacterial Spores by Sonication Through a Flexible Interface in a Microfluidic System”, Analytical Chemistry, ACS Publications, vol. 73, #3, Feb. 1, 2001, pp. 492-496. |
| Nishiguchi, M.K., et al., “DNA Isolation Procedures”, Methods and Tools in Bioscience and Medicine, 2002, pp. 249-287. |
| Law, K.A. et al., “Initial investigations into the ultrasonic lysis of microbial cells for the release of adenosine triphosphate”, Analytical Biochemistry, vol. 317, 2003, pp. 266-267. |
| Amari, M. et al., “A Comparison of the Effect of PTFE and UPE Membrane Filters on the Quality of Photoresist Developers”, Mykrolis, www.mykrolis.com, 2004, 9 pages. |
| VanBavel, E., “Effects of Shear Stress on Endothelial Cells: Possible Relevance for Ultrasound Applications”, Progress in Biophysics and Molecular Biology, vol. 93, 2007, pp. 374-383. |
| Morris, D., et al., “Lysate Clearance for Prokaryotic DNA Isolation Using the AcroPrep 96 Filter Plate” Pall Corporation, www.pall.com, 6 pages. |
| “Assay”, Wikipedia, http://en.www.wikipedia.org/wiki/Assay, Apr. 23, 2008, 3 pages. |
| “Legionella”, Wikipedia, http://en.www.wikipedia.org/wiki/Legionella, May 30, 2008, 4 pages. |
| “RNA”, Wikipedia, http://en.www.wikipedia.org/wiki/RNA, Jun. 6, 2008, 6 pages. |
| U.S. Appl. No. 60/011,148, filed Jan. 4, 2000, Bussey et al. |
| Search Report issued by the State Intellectual Property Office of the People's Republic of China sent Dec. 27, 2012 for Chinese Patent Application No. CN 200980125281.0 filed Jun. 16, 2009. |
| First Office Action issued by the State Intellectual Property Office of the People's Republic of China issued Jan. 11, 2013 for Chinese Patent Application No. CN 200980125281.0 filed Jun. 16, 2009. |
| Number | Date | Country | |
|---|---|---|---|
| 20090325269 A1 | Dec 2009 | US |
