LDETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 04/20/2026 has been entered.
Priority
Acknowledgement is made of Applicant’s claim to priority from US Applications 63/138,341; 63/138,323; 63/138,337; 63/138,321; 63/138,320; 63/138,318; 63/138,314; 63/138,312 and 63/138,310 filed 01/15/2021, from US Applications 63/138,316 filed 01/20/2021 and from US Application 63/148,527 filed 02/11/2021.
Application Status
Claims status: Claim 1 is currently amended. Claims 13 and 16 are cancelled. Claims 1-12, 14-15 and 17-22 are pending. Therefore, claims 1-12, 14-15 and 17-22 are under consideration in this Office Action.
Any objection or rejection not reiterated herein has been overcome by Applicant’s amendments.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 05/19/2026 was filed after the mailing date of the Office Action on 04/27/2026. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
The following rejections are new:
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
A. Determining the scope and contents of the prior art.
B. Ascertaining the differences between the prior art and the claims at issue.
C. Resolving the level of ordinary skill in the pertinent art.
D. Considering objective evidence present in the application indicating obviousness or non-obviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 3-4, 6, 9-11 and 17-22 are rejected under 35 U.S.C. §103 as being unpatentable over Lo (Lo, S.-J. et al. “Molecular-level dengue fever diagnostic devices made out of paper”. Lab On a Chip, Vol. 13 (2013), pp: 2686-2692), in view of Wang (Wang, L.-X. et al. “On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box”. Talanta, Vol. 208 (2020), p: 120407), Garcia-Bernalt Diego (Garcia- Bernalt Diego, J. et al. “Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test”. Scientific Reports, vol. 9 (2019), p: 14744), Wolf (Wolf, A. et al. WO 2019/073049 A1, published April 18, 2019; previously cited), Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf (both downloaded from Internet on 12/01/2024; previously cited), Miyamoto (Miyamoto, S. et al. US 2011/0117549 A1- published May 19, 2011), and Agarwal (Agarwal, C. et al. “Recent advances in paper-based analytical devices: a pivotal step forward in building Next-generation sensor technology” chapter; In: Inamuddin, T.S., Kumar Mishra, R., Asiri, A. (eds) “Sustainable Polymer Composites and Nanocomposites”. Springer Nature (2019), pp: 479-517- [Springer, Cham. https://doi.org/10.1007/978-3-030-05399-4_18]).
Regarding claim 1, Lo teaches a “Lab-on-Chip” method developed with probes for colorimetric and fluorescent assays on paper (see title and abstract; see “Introduction” section, page 2687, left column, lines 26-37).
Lo teaches a composition for RT-LAMP comprising dNTP mix, primers, betaine, Magnesium sulfate, Bst polymerase (New England Biolabs), AMV-reverse transcriptase, and ethidium bromide, DAPI and U-safe, i.e., fluorescent dyes (page 2687, left column, “Materials and methods” section).
Lo teaches that a colorimetric-based assay was previously set-up using biotin-streptavidin-HRP system, as a colorimetric indicator, on Whatman qualitative paper, however, the system was not found to be stable or precise for DNA quantitation (see page 2687, right column, “results and discussion” section).
Lo teaches the use of Whatman paper and a wax printer to manufacture paper devices (see page 2687, left column, “Materials and methods” section).
Lo teaches using ethidium bromide, however, the intensity profile is found to be better with U-safe, which alternative also removes the handling of a mutagen with high toxicity (see page 2690, left column, first paragraph).
Lo teaches the use of fluorescent dyes, however, endogenous proteins of samples might interfere with the fluorescent signal (false positives), therefore Lo uses U-safe as probe in RT-LAMP after the amplification product are purified and re-dissolved in ddH2O, i.e., a re-solubilization agent (see page 2690, left column, second paragraph and right column, first paragraph, lines 1-3).
Lo teaches that washing reagent can influence the signal intensity, i.e., PBS versus ddH2O, especially when using ethidium bromide (see page 2690, right column, second paragraph).
Lo also teaches that “Expanding upon these efforts could very well lead to the functional development of a paper-based diagnostic device for the diagnosis of dengue fever through a combination of three-dimensional microfluidic paper analytical devices (paper platform for the diagnosis of dengue fever) and small-scale heating devices (the performance of RT-LAMP), i.e., switching from building a practical procedure via using paper-based 96-well plates to making a portable, paper-based diagnostic device”.
Lo also teaches that a paper-based device via LAMP amplification has several advantages, the main two being: 1) the total diagnosis time was only 100 minutes, 60 minutes for RT-LAMP reaction, and 40 minutes for paper diagnosis; 2) the specimen amount needed for diagnosis was 2 µl for each zone, so the process is suitable for tiny amount point-of-case diagnoses. Lo concludes stating “we believe these promising results would provide impetus for the creation of specific disease diagnosing devices at the molecular level using only tiny sample amounts” (see page 2691, “Conclusions” section, left column).
Lo is silent on ammonium sulfate, hygroscopic agent, and does not teach whether the colorimetric indicator does undergo a color change when the composition is applied and dried.
Lo switches to fluorescent dyes, and does not teach further colorimetric-based assays using systems different from the biotin-streptavidin-HRP system.
However, Miyamoto teaches different methods and device and kit for detecting a nucleic acid amplified by a nucleic acid amplification method (see title and abstract).
Miyamoto teaches that examples of dyes, i.e., fluorescent and colorimetric indicators, that can be used to detect nucleic acid amplification; examples of colorimetric indicators are Bromophenol blue, Phenol Red, Crystal Violet, etc.… (see [0053]-[0062]).
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date of the claim invention to have substituted a composition using the biotin-streptavidin-HRP system as taught by Lo, with a composition comprising another nucleic acid- specific dye/colorimetric indicator as taught by Miyamoto. One with ordinary skills in the art motivated in an affordable and portable system using a paper-based RT-LAMP, could have performed the substitution with a reasonable expectation of success and arrived at the claimed invention.
Indeed, Wang teaches a method that is an “on-chip RT-LAMP”, i.e. a LAMP analysis method on a solid phase medium (see title and abstract). Wang teaches a colorimetric detection of markers using on-chip RT-LAMP (see title and abstract).
Wang teaches a composition comprising a LAMP mix reaction buffer, Bst 2.0 polymerase, dNTPs, MgSO4, LAMP inner primers (FIP and BIP) and outer primers (F3 and B3), and LAMP loop primers (LF and LB), M-MLV reverse transcriptase, RT-reaction buffer and RNA (see page 3, sections 2.5-2.6). Wang teaches calcein as a colorimetric indicator, loaded directly onto the paper for detection (see page 3, left column, section 2.2, “Fabrication of the chip and integrated box” paragraph; and see Figure 2).
Garcia-Bernalt Diego teaches progresses in LAMP assay for detection of Schistosoma mansoni DNA and a ready-to-use test (see title).
Garcia-Bernalt Diego teaches a composition comprising 1M betaine, isothermal amplification buffer 1X, MgSO4, Bst polymerase 2.0 Warmstart® (New England Biolabs), dNTPs, FIP/BIP and F3/B3 primers, and SYBR Green as a detection dye (see pages 2-3, “Material and Methods” section, “Conventional SmMIT-LAMP” paragraph).
Garcia-Bernalt Diego teaches that using betaine delays the reaction by 10 minutes, and in their hand, betaine has no negative impact on the reaction of RT-LAMP. However, Garcia-Bernalt Diego also teaches that betaine may make a difference in preventing secondary structure formation in GC-rich region and pH dependent ion exchange (see Figure 1).
Therefore, Examiner interprets that betaine might be important for nucleic acid amplification in organisms in which GC-rich regions and secondary structures in nucleic acids are prevalent, and in paper-based reactions where pH needs to be controlled.
Garcia-Bernalt Diego teaches a stabilization procedure comprising drying by desiccation the composition, i.e., the LAMP reagents, using Trehalose without glycerol, i.e., less than 1.0 w% hygroscopic agent (see page 3, “Drying by desiccation” section.
Garcia-Bernalt Diego also teaches that using this process of drying-desiccating without centrifugation allows for optimum stability of the LAMP reagent. Garcia-Bernalt Diego teaches that the subsequent reconstitution of reagents worked well and amplification results were observed both by color change and in agarose gels electrophoresis (see page 5, first paragraph).
Garcia-Bernalt Diego also teaches a reconstitution agent, i.e., a re-solubilization agent, that is water, stating “for later real-time reactions, the pellets were reconstituted in different volumes depending on the reagents included in the pre-desiccation process: (i) 25 µL water when including all LAMP components (counting with S. mansoni DNA); (ii) 23 µL water and 2 µL DNA template of S. mansoni; and (iii) 22.6 µL water, 2 µL DNA template of S. mansoni and 0.40 µL EvaGreen”.
Wolf teaches solid-phase LAMP that can successfully and specifically amplify and detect small amounts of DNA (see “[S]hort conclusion” ¶, page 43 of 96, lines 17-19). Wolf teaches a Solid phase, see page 10, lines 18-35 and page 11, lines 1-9; page 23, lines 4-14; page 32, lines 1-8).
Wolf also teaches exact amounts of reagents in Tables 3 to 5 (pages 41 to 42), see below:
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In Table 3 as example, Wolf teaches: a couple of primers (primer E (F3) and primer F (B3) specific to target sequences of interest. (see page 9, line 8); a DNA polymerase, i.e. WarmStart® Bst 2.0® DNA polymerase from New England Biolabs (NEB); a solubilization agent, i.e. water; and Betaine.
Wolf is silent on ammonium sulfate. Examiner interprets that Wolf’ reagents do not include ammonium sulfate.
Regarding the hygroscopic agent, In the Specification, Applicant defines
“hygroscopic agents” as “include glycerol, ethanol, Methanol, calcium chloride, potassium chloride, calcium sulfate and combinations thereof” (see [0015]). And according to Applicant’s definition of “substantially free” mentioned above, it “refers to the complete or nearly complete extent of degree of an action, characteristic, property, state, structure, item, or result” (see instant Specification ¶ [0090]).
Regarding the amount of hygroscopic agents, Wolf add a hygroscopic agent,
i.e. glycerol, in the LAMP mix since the stock solution of the DNA polymerase contains glycerol. Wolf add the enzyme at a final concentration of 8 Units in 25 µl of reaction (see Table 3, page 41 of 96). This dilution uses the enzyme at a final effective concentration of 0.32 U/µl, since the total is 8 units of enzyme. The stock solution being at 120 U/µl, according to Bst 2.0® DNA polymerase-New England Biolabs pdf, only 0.067 µl was needed, therefore, 0.034 µl of glycerol were carried over, in a total volume of 25 µl. There is 0.14% of a hygroscopic agent in the composition taught by Wolf, which is less than what is required by claim 1.
The WarmStart® RTx Reverse Transcriptase-NEB pdf teaches a specific reverse transcriptase adapted for RT-LAMP reaction. Regarding the amount of hygroscopic agent, i.e. glycerol carried over when using this reagent, WarmStart® RTx Reverse Transcriptase-NEB pdf also teaches glycerol as a component of the storage buffer for the RTx enzyme (50% glycerol; see pdf). However, the enzyme is also offered in a glycerol-free format.
Lo, Wang, Garcia-Bernalt Diego and Wolf do not teach adding ammonium sulfate in their LAMP assay, and colorimetric indicator undergoing a color change when the composition is applied and dried.
However, Agarwal teaches recent advances in paper-based analytical devices (see title), therefore one of ordinary skills interested in developing paper-based RT-LAMP, would consider the teachings of Agarwal.
Agarwal teaches that a color change of a pH-sensitive dye like bromophenol blue coated on paper occurs in presence of ammonia, which acts as a Lewis base and induces the color change due to the release of hydrogen (see page 501, lines 2-4).
Agarwal teaches that ammonia is a volatile nitrogenous compound, that can be detected on paper-based Gas sensor method (see page 500, last sentence, page 501 first sentence).
Agarwal’s teachings also suggest that ammonium ions derivatives that can form ammonia in water should also be removed when developing a paper-based assay.
Therefore, Agarwal teaches that sensing methods for ammonia on paper substrate based on pH change exist; this teaching suggests that using ammonia derivatives might interfere with pH-based colorimetric detections. Thus, not using ammonia and derivatives in a LAMP reaction that is based on a colorimetric detection with pH change would prevent from having artefacts and background color.
Therefore, Agarwal teaches away from adding ammonia on a paper-based assay, since desiccating might also change the ammonia derivative’s structure and the release of Hydrogen.
In KSR Int 'l v. Teleflex, the Supreme Court, indicated that “The principles underlying [earlier] cases are instructive when the question is whether a patent claiming the combination of elements of prior art is obvious. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 likely bars its patentability”. KSR Int'l v. Teleflex lnc., 127 S. Ct. 1727, 1740 (2007).
Therefore, it would have been prima facie obvious to a person of ordinary skill in the art, before the effective filing date of the claimed invention, since Lo published their Lab on Chip and suggested improvement towards a portable paper-based assay in 2013, to have combined the teachings of Lo with the teachings of Wang, Garcia-Bernalt Diego, Wolf and Agarwal, and transform the composition for solid-phase RT-LAMP of Lo into a composition for a paper-based colorimetric RT-LAMP optimized to have less false positives and background staining. One with ordinary skills in the art motivated in performing a RT-LAMP reaction with less false-positives and artefactual staining on the paper device would have applied the modifications taught by Wang, Garcia-Bernalt Diego, Wolf and Agarwal, reducing the level of hygroscopic agents in drying-desiccating the reagents for depositing onto the paper device, removing ammonia and ammonium ions derivatives that can form ammonia in water, from reaction buffers avoiding false-positive and background stains. One with ordinary skills in the art could have performed these modifications with a reasonable expectation of success based on results obtained by Lo, Wang, Garcia-Bernalt Diego, Wolf and Agarwal, would have optimized the assay accordingly, and arrived at the claimed invention.
Regarding claim 3, Agarwal teaches that nitrogenous compounds such as ammonia are volatile compounds that can be used on paper devices for sensing/indicating meat spoilage in the Food Industry (see page 500, last sentence, and page 501, first sentence).
Therefore, by eliminating ammonium ions or ammonia from reagents in the RT-LAMP assay, one with ordinary skills in the art would have removed volatile compounds from the assay.
Lo, Wang, Garcia-Bernalt Diego, and Wolf do not use volatile agents such as ethanol to resuspend reagents.
Instant Disclosure’s definition of “substantially free” is described above , and it “refers to the complete or nearly complete extent of degree of an action, characteristic, property, state, structure, item, or result” (see instant Specification ¶ [0090]).
Therefore, the combination of references teaches a composition “substantially free of volatiles agents” since no volatile agent is used. The obviousness of the combination of references teachings the claimed invention’s elements are discussed above.
Regarding claims 4 and 6, in the Specification, Applicant defines “hygroscopic agents” as “include glycerol, ethanol, Methanol, calcium chloride, potassium chloride, calcium sulfate and combinations thereof” (see [0015]). The instant Specification’s definition of “substantially free of hygroscopic agents”. Instant Disclosure’s definition of “substantially free” is described above , and it “refers to the complete or nearly complete extent of degree of an action, characteristic, property, state, structure, item, or result” (see instant Specification ¶ [0090]).
The combination of references teaches the use of the WarmStart® Bst 2.0® DNA polymerase from New England Biolabs (NEB), and specifically at a final concentration of 8 Units in 25 µl of reaction for Wolf (see Table 3, page 41 of 96). This dilution uses the enzyme at a final effective concentration of 0.32 U/µl, since the total is 8 units of enzyme. The stock solution being at 120 U/µl, according to Bst 2.0® DNA polymerase-New England Biolabs pdf, only 0.067 µl was needed, therefore, 0.034 µl of glycerol were carried over, in a total volume of 25 µl. There is 0.14% of a hygroscopic agent in the composition taught by Wolf.
The WarmStart® RTx Reverse Transcriptase-NEB pdf also teaches glycerol as a component of the storage buffer for the RTx enzyme (50% glycerol; see pdf). However, the enzyme is offered in a glycerol-free format.
Wolf teaches a concentration of hygroscopic agents that is closer to zero and much less than 1%.
The combination of references do not teach ethanol, methanol, calcium chloride nor calcium sulfate in the RT-LAMP reaction.
WarmStart® Bst 2.0® DNA polymerase from New England Biolabs pdf teaches glycerol only in enzyme stock solution.
Therefore, Examiner interprets that the combination of references does not teach adding these reagents in the reactions, which satisfies the claim’s limitations.
Regarding claims 9-11, Lo teaches the Dengue virus, which is an RNA virus (see page 2688, left column, second paragraph, and figure 1).
Wolf teaches target sequences originating from a multitude of different
species, including, but not limited to, Salmonella spp, Campylobacter jejuni, verotoxin producing Escherichia coli O157:H7, Listeria monocytogenes, Shigella dysenteriae, Avian influenza Virus and NoroVirus be used in the detecting method (see page 15, lines 10-15). Wolf teaches more specifically influenza virus and norovirus as viral pathogens (see page 27 of 96, line 3; page 43 of 96, lines 21-32).
The obviousness of the combination of references over the claimed elements are discussed above.
Regarding claims 17, 21 and 22, the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal renders the composition of claim 1 obvious.
Lo, Wang and Wolf teaches RT-LAMP methods and compositions (see titles and abstracts). Garcia-Bernalt Diego teaches LAMP compositions and dry-desiccation of the compositions for storage and preservation (see abstract and page 3, “stabilization procedures of LAMP reagents for conventional and real-time tests” section).
Wolf also teaches that some polymerase enzymes has strand displacement activity at moderate temperatures, around 20-37⁰C, while others are active at elevated temperatures around 65⁰C (see page 24 of 96, lines 30-34).
Wolf teaches coupling the SP-primer (solid phase-primer) onto the solid support to enable amplification in the solid support ( see page 23 of 96, lines 25-27). Wolf further teaches that the SP-LAMP (solid phase-LAMP) method includes not only a sample and suitable primers for amplification thereof, but also LAMP amplification mixture that is mixed with the sample and the primer set . The amplification mixture comprises all the building blocks necessary for nucleotide amplification and suitable polymerase enzymes to facilitate the amplification (see page 24 of 96, lines 16-20).
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention, to have used the composition as taught by the combination of Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal, and designed and optimized a colorimetric, RT-LAMP method for the analysis of viral nucleic acid molecules on solid phase. One with ordinary skills in the art, motivated in designing a rapid, specific, portable and affordable RT-LAMP could have performed these modifications with a reasonable expectation of success and would arrived at the claimed invention.
Regarding claims 18 and 19, Lo teaches the specimen as serum ( see Figure 3).
Wolf teaches that the term “sample” refers to any biological solution, entity or subject, including biological fluids (see page 9 of 96, lines 6-14). Wolf further teaches that the sample can be selected from blood, sputum (i.e., saliva), swabs and feces (see page 26 of 96, lines 15-20).
The obviousness of the combination of references over the claimed elements are discussed above.
Regarding claim 20, Lo teaches the Dengue virus, which is an RNA virus (see page 2688, left column, second paragraph, and figure 1).
Wolf teaches target sequences originating from a multitude of different
species, including, but not limited to, Salmonella spp, Campylobacter jejuni, verotoxin producing Escherichia coli O157:H7, Listeria monocytogenes, Shigella dysenteriae, Avian influenza Virus and NoroVirus be used in the detecting method (see page 15, lines 10-15). Wolf teaches more specifically influenza virus and norovirus as viral pathogens (see page 27 of 96, line 3; page 43 of 96, lines 21-32).
The obviousness of the combination of references over the claimed elements are discussed above.
Claims 2 and 12 are rejected under 35 U.S.C. §103 as being unpatentable over Lo (Lo, S.-J. et al. “Molecular-level dengue fever diagnostic devices made out of paper”. Lab On a Chip, Vol. 13 (2013), pp: 2686-2692), in view of Wang (Wang, L.-X. et al. “On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box”. Talanta, Vol. 208 (2020), p: 120407), Garcia-Bernalt Diego (Garcia- Bernalt Diego, J. et al. “Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test”. Scientific Reports, vol. 9 (2019), p: 14744), Wolf (Wolf, A. et al. WO 2019/073049 A1, published April 18, 2019), Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf (both downloaded from Internet on 12/01/2024; previously cited), Miyamoto (Miyamoto, S. et al. US 2011/0117549 A1- published May 19, 2011), and Agarwal (Agarwal, C. et al. “Recent advances in paper-based analytical devices: a pivotal step forward in building Next-generation sensor technology” chapter; In: Inamuddin, T.S., Kumar Mishra, R., Asiri, A. (eds) “Sustainable Polymer Composites and Nanocomposites”. Springer Nature (2019), pp: 479-517- [Springer, Cham. https://doi.org/10.1007/978-3-030-05399-4_18]), as applied to claim 1 above, and in further view of Ahn (Ahn, S.J. et al. “Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform”. BMC Infectious Diseases, Vol. 19 (2019), p: 676; previously cited), and Augustine (Augustine, R. et al. “Loop-mediated isothermal amplification (LAMP): a rapid, sensitive, specific, and cost-effective Point-of-Care test for Coronaviruses in the context of COVID-19 pandemic”. Biology, Vol. 9 (2020), p: 182).
The rejection of claim 1 is described above. The combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal renders elements of claim 1 obvious.
Regarding claim 2, reciting “the composition of claim 1, further comprising an antioxidant”, Miyamoto teaches that one of the dye to detect nucleic acid can be Phenol Red.
The combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal does not teach using Phenol Red specifically in a RT-LAMP.
However, Ahn teaches the use of Phenol Red as an indicator in RT-LAMP, which is both an antioxidant and an indicator in the RT-LAMP reaction (see page 3 of 12, right column, second paragraph).
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention to have modified the composition for RT-LAMP taught by the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal, and add the indicator and antioxidant Phenol Red to the composition as taught by Ahn. One motivated in a rapid colorimetric test using convenient reagents for a result directly observable by naked eyes could performed this modification. One with ordinary skills in the art motivated in using reagents already optimized and compatible with RT-LAMP, as taught by Ahn (see page 3 of 12, right column, second paragraph) could have performed this modification with a reasonable expectation of success, since Ahn also teaches a RT-LAMP using reagents such as WarmStart® reagents with success, and would arrived at the claimed invention.
Regarding claim 12, the combination of references does not teach the limitation “wherein the viral pathogen comprises H1N1, H2N2, H3N2, H1N1pdmO9, or SARS-CoV-2”.
However, Ahn teaches a colorimetric RT-LAMP diagnostic platform for the detection of multiple influenza viruses, including H1N1 and H3N2 (see title and abstract).
Augustine reviews knowledge specifically about LAMP for rapid, sensitive, specific and cost-effective point-of-care test for coronaviruses during COVID-19 pandemic (see title and abstract). Therefore, Augustine teaches about RT-LAMP, as applied to SARS-CoV-2 (see “Introduction” section, page 2, and figure 1).
Therefore, it would have been obvious to one of ordinary skills in the art, before the effective filing date of the claimed invention to have substituted the substrate to test, i.e., viral nucleic acid, as taught by Lo and Wolf, with a nucleic acid encoding for H1N1 viral proteins or for SARS-CoV-2 viral proteins as taught by Ahn or Augustine, within the method and platform for diagnostics as taught by the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal. One with ordinary skills in the art, motivated in analyzing a viral disease other than dengue fever or the Newcastle disease, could have performed this modification with a reasonable expectation of success and would arrived at the claimed invention.
Claim 5 is rejected under 35 U.S.C. §103 as being unpatentable over Lo (Lo, S.-J. et al. “Molecular-level dengue fever diagnostic devices made out of paper”. Lab On a Chip, Vol. 13 (2013), pp: 2686-2692), in view of Wang (Wang, L.-X. et al. “On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box”. Talanta, Vol. 208 (2020), p: 120407), Garcia-Bernalt Diego (Garcia- Bernalt Diego, J. et al. “Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test”. Scientific Reports, vol. 9 (2019), p: 14744), Wolf (Wolf, A. et al. WO 2019/073049 A1, published April 18, 2019), Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf (both downloaded from Internet on 12/01/2024; previously cited), Miyamoto (Miyamoto, S. et al. US 2011/0117549 A1- published May 19, 2011), and Agarwal (Agarwal, C. et al. “Recent advances in paper-based analytical devices: a pivotal step forward in building Next-generation sensor technology” chapter; In: Inamuddin, T.S., Kumar Mishra, R., Asiri, A. (eds) “Sustainable Polymer Composites and Nanocomposites”. Springer Nature (2019), pp: 479-517- [Springer, Cham. https://doi.org/10.1007/978-3-030-05399-4_18]), as applied to claim 1 above, and in further view of Lin (Lin, X. et al. “Influence of water evaporation/absorption on the stability of glycerol-water marbles”. RSC Advances, Vol. 9 (2019), p: 34465; previously cited).
The rejection of claim 1 is described above. The combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal renders elements of claim 1 obvious, however, it does not teach elements of claim 5, i.e. “the composition is substantially free of agents that absorb more than about 10 wt% when stored between about 40% and about 90% relative humidity at 25⁰ C”.
Regarding claim 5, the references combined, i.e. Wolf, Naik, modified by the NEB WarmStart® reagents pdfs, provide for a composition that is substantially free of hygroscopic agents (i.e. glycerol), in which the concentration of hygroscopic agent is less than 1 % of total volume, see above. However, the combination does not teach characteristics of glycerol, i.e. the only hygroscopic agent capable of absorbing water, in various conditions such as 40% to about 90% of relative humidity when stored at 25 ⁰C.
However, Lin teaches glycerol-water marbles and their stability in different environmental conditions, i.e. Relative Humidity (RH) from 26% to 82% (see Figure 3). In this figure 3, it is clear that the stability of the glycerol-water marbles depends on the original concentration/amount of glycerol in the marble. The pure water marble (0% of glycerol) decreased within 52 minutes at low RH indicating that almost no water was left in the marble, the water marble lost weight until complete collapse. However, higher RH, there was slower evaporation and better resistance to evaporation. When adding glycerol at 33.3%, there is weight loss in the marble, i.e. evaporation, with evaporation resistance at higher RH, but no mass gain. Li teaches mass gain for marbles containing 50 to 100% glycerol at 82% RH. There is mass gain when glycerol is at 71.4% or higher at 76% RH or higher. All measurements were performed in an air-conditioned laboratory at 297.2 +/- 1.0 K (which is approximately 24-25 ⁰C) (see page 2, left column, “Characterization” paragraph).
Therefore, since the amount of glycerol in the LAMP reaction is less than 0.15%, at RH from 40% to 82 %, the hygroscopic agent, glycerol, will not be able to absorb water and gain weight in an air-conditioned laboratory at 297.2 +/- 1.0 K (24-25 ⁰C).
It would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention to have combined the teachings of Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal, and measured the ability of the total hygroscopic agents’ amount in the mixture to absorb water, under the conditions taught by Lin, and tried and maintained the concentration of hygroscopic agents close to zero, as these are the appropriate conditions to ensure that there is no water absorption under storage conditions. Water absorption would modify the osmolarity of other dried components deposited onto the solid phase’s surface and modify their reactivity with samples. Garcia-Bernalt Diego teaches compositions for drying-desiccating comprising only trehalose and no glycerol (see page 3, “Stabilization procedures of LAMP reagents for conventional and real-time tests” section, “drying by desiccation” paragraph). One with ordinary skills in the art motivated in optimum storage conditions under 40% to 90% RH at 25 ⁰C, and maintaining the integrity and the reproducibility of the assays, would have tried and minimized the amount of hygroscopic agents capable of absorbing water as suggested by the teachings of Lin, with a reasonable expectation of success and arrived at the claimed invention.
Claims 7 and 8 are rejected under 35 U.S.C. §103 as being unpatentable over Lo (Lo, S.-J. et al. “Molecular-level dengue fever diagnostic devices made out of paper”. Lab On a Chip, Vol. 13 (2013), pp: 2686-2692), in view of Wang (Wang, L.-X. et al. “On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box”. Talanta, Vol. 208 (2020), p: 120407), Garcia-Bernalt Diego (Garcia- Bernalt Diego, J. et al. “Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test”. Scientific Reports, vol. 9 (2019), p: 14744), Wolf (Wolf, A. et al. WO 2019/073049 A1, published April 18, 2019), Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf (both downloaded from Internet on 12/01/2024; previously cited), Miyamoto (Miyamoto, S. et al. US 2011/0117549 A1- published May 19, 2011), and Agarwal (Agarwal, C. et al. “Recent advances in paper-based analytical devices: a pivotal step forward in building Next-generation sensor technology” chapter; In: Inamuddin, T.S., Kumar Mishra, R., Asiri, A. (eds) “Sustainable Polymer Composites and Nanocomposites”. Springer Nature (2019), pp: 479-517- [Springer, Cham. https://doi.org/10.1007/978-3-030-05399-4_18]), as applied to claim 1 above, and in further view of Battrell (Battrell, C.F. et al. US Patent 8,921,085 B2, published December 30, 2014).
The rejection of claim 1 is described above. The combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal renders elements of claim 1 obvious.
Regarding claims 7 and 8, Triton™ X-100 is taught by Wolf and is considered a non-ionic surfactant. Wolf teaches that the primers are mixed in a solution comprising this surfactant (See page 39 of 96, line 31).
Wolf also teaches using Bovine Serum Albumin (BSA) to cover all spots and block the surface of the slide (see page 39, lines 4-6).
However, Wolf and the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal, does not render obvious using a surfactant as a re-solubilization agent.
Battrell teaches compositions and methods for dehydrated storage of on-board reagents in microfluidic devices (see title and abstract).
Battrell teaches that surfactants such as Tween®-20, Triton® X-100, Nonidet® P40, PEG-8000 and BSA are used to reduce adsorption of biologicals to the plastic surfaces for passivity, to reduce aggregation of polymerase and often to increase activity of the polymerase (see column 10, lines 27-37).
Battrell teaches that to reconstitute, a volume of 15 µL containing target DNA and primers was used (see column 20, line 42-43). Battrell teaches that the dried reagents are stored in a dry form without lyophilization or freezing and reconstituted at the point of use with either a biological sample or a sample eluate at the point of use (see abstract).
Battrell teaches that other excipients such as surfactants may be needed to stabilize the highly folded structure of polymerase during dry storage, because the reagent material is subjected to interfacial adsorption and denaturation during drying and rehydration, i.e., re-solubilization. Passivation methods were also adopted (see column 6, lines 8-15).
Battrell teaches a variable excipient and a rehydration solution comprising DNA/primers in the composition for RT-PCR (see column 21, Table III).
Therefore, for lateral flow mobility and surface passivity, the teachings suggest that reconstitution/rehydration can be performed using a volume of liquid comprising a surfactant such as Tween®-20, Triton® X-100, Nonidet® P40, PEG-8000 and BSA.
It would have been obvious to one of ordinary skills in the art, before the effective filing date of the claimed invention to have modified the re-solubilization solution, i.e., water, as taught by the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal, and added an excipient/surfactant such as Tween®-20, Triton® X-100, Nonidet® P40, PEG-8000 or BSA to the rehydration/re-solubilization solution. One with ordinary skills in the art, motivated in preserving the polymerase activity while enhancing surface passivity and flow in the device, could have performed this modification with a reasonable expectation of success and would arrived at the claimed invention.
Claims 14 and 15 are rejected under 35 U.S.C. §103 as being unpatentable over Lo (Lo, S.-J. et al. “Molecular-level dengue fever diagnostic devices made out of paper”. Lab On a Chip, Vol. 13 (2013), pp: 2686-2692), in view of Wang (Wang, L.-X. et al. “On-chip RT-LAMP and colorimetric detection of the prostate cancer 3 biomarker with an integrated thermal and imaging box”. Talanta, Vol. 208 (2020), p: 120407), Garcia-Bernalt Diego (Garcia- Bernalt Diego, J. et al. “Progress in loop-mediated isothermal amplification assay for detection of Schistosoma mansoni DNA: towards a ready-to-use test”. Scientific Reports, vol. 9 (2019), p: 14744), Wolf (Wolf, A. et al. WO 2019/073049 A1, published April 18, 2019), Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf (both downloaded from Internet on 12/01/2024; previously cited), Miyamoto (Miyamoto, S. et al. US 2011/0117549 A1- published May 19, 2011), and Agarwal (Agarwal, C. et al. “Recent advances in paper-based analytical devices: a pivotal step forward in building Next-generation sensor technology” chapter; In: Inamuddin, T.S., Kumar Mishra, R., Asiri, A. (eds) “Sustainable Polymer Composites and Nanocomposites”. Springer Nature (2019), pp: 479-517- [Springer, Cham. https://doi.org/10.1007/978-3-030-05399-4_18]), as applied to claim 1 above, and in further view of Cordone (Cordone, L. et al. “Proteins in saccharides matrices and the trehalose peculiarity: Biochemical and Biophysical properties”. Current Organic Chemistry, Vol. 19 (2015), pp:1-23).
The rejection of claim 1 is described above. The combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal renders elements of claim 1 obvious.
Regarding claims 14 and 15, Lo teaches ddH2O as main solute, and PBS buffer as a washing agent (See page 2687, right column, first paragraph). Lo does not teach any specific additive, but betaine (see “materials and methods” section, page 2687).
Wang teaches only adding MgSO4 to the LAMP mix buffer. Wang does not teach extra additives.
Wolf teaches “water” as main solute, which is a non-discoloration agent (see Table 3).
Garcia-Bernalt Diego teaches trehalose, i.e. a sugar known for its stabilizing capacity. Garcia-Bernalt Diego teaches that the physical properties of trehalose allow direct interaction with compound to protect it during the drying process and limit mobility of the compound via increasing its state of hydration (see page 2, lines 33-37).
The combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal does not teach whether Trehalose is a non-discoloration agent.
However, Cordone teaches the peculiarity of trehalose (see title and abstract).
Cordone teaches that trehalose is often used as an additive, especially in the food industry because of its stability and preserving ability. Trehalose is particularly effective in preventing discoloration and preserving the aroma in many preserved products, also preserving them from temperature fluctuations (see page 5, left column, lines 13-18). Cordone teaches that trehalose in low water content situation, suppresses the Maillard reaction between carbohydrates and proteins, which is known to cause enzyme inactivation (see page 14, right column, lines 23-26).
Therefore, it would have been obvious to one of ordinary skills in the art, before the effective filing date of the claimed invention to have modified the composition as taught by the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal, and added Trehalose, as taught by Garcia-Bernalt Diego, for its properties highlighted by Cordone. One with ordinary skills in the art, motivated in preserving the activities of enzymes needed for RT-LAMP in the composition during drying-desiccation process, could have performed this modification with a reasonable expectation of success and would arrived at the claimed invention.
Response to Arguments
Applicant's arguments filed 04/20/2026 with respect to claims 1-12, 14-15, and 17-22 have been considered but are moot because the new ground of rejection does not rely on the same combination of references applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Regarding Applicant’s arguments against Wolf on page 7 of 11 of Remarks, in
response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In this case, Lo teaches a colorimetric indicator in the Biotin- streptavidin HRP system; Wang teaches calcein as a colorimetric indicator on the solid phase; Miyamoto teaches alternatives for colorimetric indicators.
Wolf does not teach affirmatively the ammonium-sulfate-free, betaine-containing formulation recited, however, Garcia-Bernalt Diego gives rationale for using Betaine, which Wolf teaches positively, in a LAMP composition; and Agarwal teaches away from ammonia and its volatile compounds
Wolf may not teaches all other components claimed, however Wolf teaches precise amounts of components that render the evaluation of hygroscopic agents amounts possible.
Wolf may not teach all components in a RT-LAMP, but the combination of references Lo, Wang, Garcia-Bernalt Diego, Wolf, Bst DNA polymerase-NEB pdf and WarmStart® RTx Reverse Transcriptase-NEB pdf, Miyamoto and Agarwal renders elements of claim 1 obvious.
Wolf and the NEB references may not teach a colorimetric indicator in a solid phase RT-LAMP composition that remains color-stable when the composition is applied and dried, however, Garcia-Bernalt Diego teaches a method for drying-desiccating that protects components of a LAMP reaction, with good results after reconstitution. Agarwal teaches that sensing methods for ammonia on paper substrate based on pH change exist, suggesting that using ammonia derivatives might interfere with pH-based colorimetric detections. Therefore, not using ammonia and derivatives in a LAMP reaction that is based on a colorimetric detection with pH change would prevent from having artefacts and background color.
Ahn may not teach a solid-phase composition, but Anh teaches about using phenol red in a RT-LAMP reaction.
Lin does not teach a solid-phase RT-LAMP, but Lin teaches about glycerol-water mixes and humidity.
Solid-phase RT-LAMP is known and described by Lo. Lo suggests further improvements that have been made by Wang, Garcia-Bernalt Diego, and Wolf. Using paper as a solid phase in sensor biosystems is taught and reviewed by Agarwal and Miyamoto teaches alternatives for colorimetric indicators that can be used for nucleic acid detection. Therefore, the combination of references renders all the elements of the claims obvious.
Conclusion
No claim is allowed
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/A.D./Examiner, Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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