All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The present invention relates to methods and apparatus for nucleic acid amplification and detection. More particularly, the present invention relates to methods and apparatus for point-of-care nucleic acid amplification and detection.
Polymerase Chain Reaction (PCR) is considered the gold standard for nucleic acid amplification and detection because the specificity and sensitivity of PCR are considerably higher than that of analogous Enzyme-Linked Immuno-Sorbent Assay (“ELISA”) tests. However, PCR systems are costly and require very clean samples. Point-Of-Care (POC) PCR systems generally are not fully disposable, are not appropriate for unskilled use, require substantial power and/or contain complicated microfluidic processing and readout. Thus, PCR traditionally has been limited to high resource, centralized laboratory settings.
In view of the foregoing, it would be desirable to provide methods and apparatus for point-of-care nucleic acid amplification and detection that overcome the drawbacks of previously known methods and apparatus.
Several embodiments of the present invention will be apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout, and in which:
Although this disclosure is detailed and exact to enable those skilled in the art to practice the disclosed technologies, the physical embodiments herein disclosed merely exemplify the various aspects of the invention, which may be embodied in other specific structures. While the preferred embodiments are described, the details may be changed without departing from the invention, which is defined by the claims.
Preferably, sample preparation is fully or partially achieved using heat treatment and/or using a filter paper 20, such as a chemically treated filter paper, e.g., Flinders Technology Associates (“FTA”) cards available from Whatman (Kent, UK). The apparatus preferable utilizes an isothermal nucleic acid amplification technique, e.g., loop-mediated isothermal amplification (“LAMP)”, to reduce the instrumentation requirements associated with nucleic acid amplification. Detection of target amplification may be achieved, for example, via detection of a color shift and/or fluorescence in one or more dyes, such as hydroxynaphthol blue, picogreen, and/or SYBR green, added to the amplification reaction. Such colorimetric and/or fluorescent detection may be performed visually by an operator and/or may be achieved utilizing an imaging technique, such as spectrophotometric and/or fluorescence imaging, as described below.
As seen in
In order to collect sample S with sample collector 30, filter paper 20 may, for example, be dipped or placed into one or more sample matrices of interest. Additionally or alternatively, one or more drops of one or more sample matrices of interest may, for example, be placed or deposited onto filter paper 20. Additionally or alternatively, filter paper 20 may, for example, be swabbed or wiped across one or more sample matrices of interest.
After collection of sample S, all or a portion of sample collector 30 and filter paper 20 may be inserted through insertion slot 50 into nucleic acid amplification instrument 40, as seen in
During insertion of paper 20 and sample collector 30 into instrument 40 of apparatus 10, filter paper 20 optionally may pass, contact and/or otherwise interact with one or more rollers, brushes, dispensers, sprayers or other elements 60 that prepare the sample for nucleic acid amplification by a skilled or unskilled practitioner. As seen in
In addition or as an alternative to sample preparation element(s) 60, sample S may be prepared via heat treatment. For example, sample S may be heated to a temperature higher than required for isothermal amplification via LAMP (e.g., higher than about 60° C.-65° C.), thereby preparing the sample for LAMP via heat treatment. In some embodiments, sample S may comprise whole blood, which may, for example, be heat treated at about 99° C., e.g., for about 10 minutes, to achieve sample preparation. In some embodiments, sample S may not require preparation and/or placement of sample S on filter paper 20 may be sufficient to prepare the sample for nucleic acid amplification.
As seen in
Punch stage 80 is configured to translate relative to linear bearings 130 attached to bottom enclosure 110. As seen in
As seen in
Master mix 170 or enzyme 160 optionally also may comprise one or more dyes to facilitate detection of nucleic acid amplification. In some embodiments, master mix 170 may comprise a colorimetric dye, such as hydroxynaphthol (“HNB”) blue. Detection of target amplification may be achieved, for example, via detection of a color shift in the colorimetric dye in the presence of amplicon, e.g., due to a shift in free magnesium (Mg2+) concentration during LAMP amplification. Such colorimetric detection may be performed visually by an operator or may be achieved utilizing spectrophotometric imaging, as described below. In addition or as an alternative to colorimetric amplification detection with a colorimetric dye, a fluorescent dye, such as picogreen or SYBR green, may be utilized to detect amplification via fluorescence.
One or more of the reagents 150 optionally may be lyophilized, e.g., to facilitate long-term storage. Additionally or alternatively, one or more of the reagents may be temporarily sequestered from one or more of the other reagents prior to nucleic acid amplification via instrument 40. Such temporary reagent sequestration may facilitate long-term storage of the reagents and/or may forestall reagent mixing (and, thus, nucleic acid amplification) until desired, e.g., until the reagents have been exposed to sample S. For example, enzyme 160 may be sequestered from master mix 170, as shown in
In some embodiments, one or more of the reagents 150 (e.g., enzyme 160) may be temporarily sequestered within one or more temporary sequestration vessels 180 (see
As best seen in
Upon establishment of fluid tight seal, each reaction chamber 70 with sample-containing punch 140 and reagents 150 is configured to amplify a nucleic acid target sequence of interest, if contained in the sample S. Different chambers 70 may utilize different primers to facilitate amplification and detection of different target sequences of interest (i.e., to facilitate multiplexed nucleic acid amplification and detection). A fraction of the chambers 70 may serve as positive controls. Additionally or alternatively, a fraction of the chambers 70 may serve as negative controls. Negative and/or positive control chambers 70 optionally may be pre-sealed prior to sealing of the remaining chambers 70 with sample-containing punches 140. Negative control chambers 70 may not comprise punches 140 or may comprise punches 140 that contain no sample S. Positive control chambers 70 may contain one or more target nucleic acid sequences of interest that are expected to amplify during nucleic acid amplification (e.g., the positive control chambers may comprise punches 140 containing the one or more target nucleic acid sequences of interest).
In the manner described above, each (non-control) reaction chamber 70 may be loaded and sealed with (an optionally prepared) sample-containing filter paper punch 140 and reagents 150, thereby facilitating nucleic acid amplification, e.g., isothermal nucleic acid amplification. The loaded and sealed chambers 70 may be heated, e.g., isothermally heated, to amplify the one or more target nucleic acid sequences of interest. When conducting isothermal amplification via LAMP, the contents of chambers 70 may be heated in the range of about 60° C.-65° C. for about 15-70 minutes. Such heating may be achieved via a healing element utilizing any of variety of techniques, including (but not limited to) electrical, chemical and electrochemical techniques. For example, chambers 70 may be resistively heated via a hearing element, e.g., via a coating on an imaging sensor as described below.
As discussed previously, detection of target amplification optionally may be achieved via detection of a color shift (i.e. a wavelength shift) and/or fluorescence (i.e., an intensity shift) in one or more dyes in the presence of amplicon. Such colorimetric and/or fluorescence detection may be performed visually by an operator and/or may be achieved utilizing an imaging technique, such as spectrophotometric and/or fluorescence imaging. In the embodiment of
Sensor 200 optionally may comprise coating 210, such as Indium Tin Oxide (“ITO”) coating 210, which may be utilized to resistively heat the contents of each reaction chamber 70 to achieve target nucleic acid amplification. As discussed previously, when conducting isothermal amplification via LAMP, the contents of chambers 70 may be heated in the range of about 60° C.-65° C. for about 15-70 minutes. When one or more of the reagents 150 are sequestered in one or more temporary sequestration vessels 180 that comprise one or more thermal encasement material(s), the vessel(s) 180 may partially or completely melt, become more porous or otherwise release the sequestered reagents 150 (e.g., release sequestered enzyme 160) upon heating with ITO coating 210. After release of the sequestered reagent(s) 150 from the vessel(s) 180, all of the reagents 150 (including enzyme 160 and master mix 170), mix with each other in the presence of sample-containing filter paper punch 140, and isothermal amplification proceeds.
Imaging sensor 200 may measure a baseline color of reagents 150 prior to isothermal heating and a final color of the reagents after isothermal heating (e.g., after isothermal heating via ITO coating 210). Since the reagents 150 within each reaction chamber 70 may, for example, include a colorimetric (or fluorescent) dye that shifts in color, e.g., from purple to blue, upon amplification of a target nucleic acid sequence, any such shift in color within the reaction chambers may be detected by the imaging sensor 200 as a differential between the baseline and final color, and this differential may be indicative of target amplification. As seen in FIGS. 1 and 2A-2B, digital readout or display 220 may output detection results (and/or instructions) to the user, removing any risk of ambiguity. While the embodiment of
As seen in
Instrument 40 may automatically initiate nucleic acid amplification and detection upon sealing of chambers 70 with punches 140. For example, translation of punch stage 80 and/or sealing of chambers 70 may complete a circuit that activates logic chip 230. Additionally or alternatively, the user or operator of instrument 40 may initiate nucleic acid amplification and detection, e.g., via an on/off switch, button, toggle, etc.
Punch elements 90 and punch stage 80 can be seen in
The methods and apparatus of
Although preferred illustrative embodiments of the present invention are described above, it will be apparent to those skilled in the art that various changes and modifications may be made thereto without departing from the invention. It is intended in the appended claims to cover all such changes and modifications that fall within the true spirit and scope of the invention.
The present application claims priority and the benefit of the filing date of U.S. provisional patent application Ser. No. 61/475,257, filed Apr. 14, 2011, which is incorporated herein by reference in its entirety.
Number | Name | Date | Kind |
---|---|---|---|
5411876 | Bloch et al. | May 1995 | A |
5498392 | Wilding et al. | Mar 1996 | A |
5753186 | Hanley et al. | May 1998 | A |
20030013109 | Ballinger et al. | Jan 2003 | A1 |
20050106713 | Phan et al. | May 2005 | A1 |
20070141605 | Vann et al. | Jun 2007 | A1 |
20070292858 | Chen et al. | Dec 2007 | A1 |
20080176755 | Amundson et al. | Jul 2008 | A1 |
20100015621 | Chang et al. | Jan 2010 | A1 |
20110294112 | Bearinger et al. | Dec 2011 | A1 |
20110294199 | Bearinger et al. | Dec 2011 | A1 |
20120202211 | Ochoa Corona | Aug 2012 | A1 |
Number | Date | Country |
---|---|---|
WO2009018473 | Feb 2009 | WO |
Entry |
---|
Jangam, S. R. et al., “Rapid, Point-of-Care Extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real time PCR.” Journal of Clinical Microbiology, Aug. 2009, vol. 47, No. 8, pp. 6363-2368. |
Menassa, N. et al., “Rapid detection of fungal keratitis with DNA-stabilizing FTA filter paper.” Investigative Ophthalmology and Visual Science, Apr. 2010, vol. 51, No. 4, pp. 1905-1910. |
Weigl, B. H. et al., “Non-instrumented Nucleic-Acid Amplification Assay.” Microfluidics, BioMEMS, and Medical Microsystems VI, Proc. of SPIE vol. 6886, 688604, 2008. |
LaBarre, P. et al., “Non-Instrumented Nucleic Acid Amplification (NINA): Instrument Free Molecular Malarai Diagnostics for Low-Resource Settings.” 32nd Annual International Conference of the IEEE EMBS, Buenos Aires, Argentina, Aug. 31-Sep. 4, 2010. pp. 1097-1099. |
Poon, L. L. M. et al., “Sensitive and Inexpensive Molecular Test for falciparum Malaria: Detecting Plasmodium falciparum DNA Directly for Heat-Treated Blood by Loop-Mediated Isothermal Amplification.” Clinical Chemistry 52, No. 2, 2006, pp. 303-306. |
Bearinger, J. P. et al., “Development and initial results of a low cost, disposable, point-of-care testing device for pathogen detection.” IEEE Transactions on Biomedical Engineering, Mar. 2011, vol. 58, No. 3. pp. 805-808. |
Number | Date | Country | |
---|---|---|---|
20120264116 A1 | Oct 2012 | US |
Number | Date | Country | |
---|---|---|---|
61475257 | Apr 2011 | US |