DETAILED 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 .
Status of the Application
The Amendment and Response filed July 14, 2025 is acknowledged.
Claims 1-3, 6-15 and 22-26 were pending. Claims 1-3, 6 and 8-13 are being examined on the merits. Claim 7 is canceled. Claims 14-15 and 22-26 remain withdrawn.
Response to Arguments
Applicant’s arguments filed July 14, 2025 have been fully considered.
The following objections and rejections are WITHDRAWN in view of Applicant’s arguments and amendments to the claims:
Objection to claim 8
Rejection of claim 7 under 35 USC § 112(d)
The following rejections are MODIFIED in view of Applicant’s amendments to the claims:
Rejection of claims under 35 USC § 101
Prior art rejections
Response to arguments regarding 35 USC § 101 rejections
Applicant argues that the 35 USC § 101 rejections should be withdrawn for several reasons. First, Applicant argues that the claim as a whole integrates the judicial exception (JE) into a practical application because the claims “provide an improved method for obtaining the concentration of a specific target RNA in a sample” and because various steps, e.g., the “amplification” and “capturing” steps, are “integral to the method as a whole” (Remarks, pp. 8-9).
The Examiner disagrees. First, it is not clear what technology or technical field Applicant asserts is being improved. Second, it is not clear what Applicant is referring to as the improvement in the technology or technical field. For example, if the technology or technical filed is intended to be molecular diagnostic testing, then using real-time LAMP techniques to test biological samples for the presence of some analyte is not an improvement in technology or a technical field, but rather is the current state of the art (e.g., see the teachings of Priye cited below in conjunction with the prior art testing, and Fig. 1(A)). The same analysis applies to the field of disease outbreak surveillance. The analysis as to patent eligibility, and in particular as to the consideration of an improvement in technology or technical field, does not collapse into whether there is some “benefit” in a generic sense to performing the steps of the instant method claims. Arguably, every method claim ever written in life sciences is directed to a method with some kind of benefit. Applicant does state that “the invention improves obtaining the concentration of the target RNA in a sample in terms of performance to cost values” (Remarks, p. 9), but does not provide any additional argument or evidence as to this assertion (e.g., the improvement is relative to what? fluorophore-based real-time LAMP? conventional PCR? As to the former, the specification merely suggests that colorimetric reagents may have the best performance to cost values (para. 5)). Thus, while Applicant has asserted that the claims are directed to an improvement in technology, they have not actually made an argument as to why this is the case or even identified the relevant technology. Further, as to whether the additional elements are “integral” to a particular machine or manufacture, the Examiner notes that the instant claims do not comprise a “particular machine or manufacture”. Also see MPEP 2106.05(b)(I), which states that “[i]t is important to note that a general purpose computer that applies a judicial exceptions, such as an abstract idea, by use of conventional computer functions does not qualify as a particular machine”.
Applicant additional argues that the step of capturing a photo of the well at regular intervals in a colorimetric assay “is integral to the invention” (Remarks, p. 9).
It is not clear what Applicant is referring to by “integral to the invention”. To the extent that Applicant is arguing that the “capturing” step integrates the JE into a practical application, the Examiner disagrees, because the “capturing” step is a data gathering step (see analysis below in conjunction with the 35 USC § 101 rejections). Such steps are identified in the MPEP 2106.05(g) as insignificant extra-solution activity.
Finally, Applicant argues that the claims do not recite a mental process because “transforming” step requires the use of a computer and “[t]he human eye cannot precisely quantify hue values in the way a scientific instrument can” (Remarks, p. 10).
As noted in the Non-Final Office Action mailed April 22, 2025, there is nothing in claim 1 that requires that the hue values be detectable or be detected at a higher resolution than is capable with the human eye. Thus, the “generating” step can practically be performed by the human mind. While claim 1 may also comprise embodiments where the “transforming” step is performed by a computer, that does not obviate the issue that claim 1 comprises embodiments that can practically be performed by the human mind.
Applicant argues that the steps of “capturing a photo of a well at regular intervals in a colorimetric assay, wherein the regular intervals are one photo every minute after the temperature of the well reaches the amplification temperature, and wherein decreasing hue values (h) as a function of time (t) are detected during amplification” is not routinely used for obtaining the concentration of a target RNA in a sample (Remarks, p. 10). Applicant also refers to Example 31 of the May 2016 Guidance on Subject Matter Eligibility (Remarks, p. 11).
The Examiner disagrees, and reiterates the comments from the Non-Final Office Action mailed April 22, 2025. Specifically, detecting hue values as a function of time during amplification forms the basis of real-time PCR. For example, in 2002, Mackay1 reviewed various detection chemistries, including fluorophores, that were commonly used in real-time PCR, and teaches detecting the fluorescent intensity (i.e., “hue value”) as a function of time during amplification (e.g., Fig. 1B; p. 1292-1298: amplicon detection section). In addition, a search of US Patent and Published Application databases for the term “real-time PCR” produces over 100,000 hits with a date prior to May 15, 2017, the effective filing date of the instant application. Thus, detecting hue values as a function of time during amplification has been performed routinely in the art for at least two decades. In addition, such real-time detection methods have been used in the context of LAMP, and/or with colorimetric detection reagents. For example, since at least 2016, New England Biolabs2 has produced a colorimetric LAMP master mix. The master mix protocol indicates that the LAMP reaction can be monitored with a color change from yellow to pink (to indicate a positive reaction), that reactions can be monitored at various time points during the reaction (i.e., in real time/as a function of time), and that the color change (or not) result can be photographed (p. 2, steps 7-8). Further, the fact that a molecular biology reagents company supplied a master mix for such a reaction in 2016 suggests that there was a corresponding demand for such a product in 2016. This, in turn, supports a finding that performing such a step was well-understood, routine and conventional at the time of filing.
Applicant argues that there are fundamental differences between a colorimetric assay and a fluorescence assay, in that colorimetric assays rely on measuring changes in light absorption at specific wavelengths, while fluorescence assays relay on measuring changes in light intensity (without changes in wavelengths) (Remarks, p. 12). Applicant further states that the instant colorimetric assay measures changes in “hue” (i.e., changes in color), while fluorescent assays measure changes in “fluorescence intensity” (perhaps, “brightness”), and that “hue” and “fluorescence intensity” are different and non-analogous variables (Remarks, pp. 11-12, 13-14).
The Examiner disagrees. Priye states that in fluorophores with emission wavelengths between 700 nm and 435 nm, the intensity and color data in the RGB color space is coupled (p. 7, para. 2). In Fig. 3(G), Priye also shows the results of a real-time LAMP using the RGB color space on a smartphone display, where there is a gradient from left to right of increasing fluorescent emission with time. As can be seen on the right side of the display, the higher concentration volume is both “more” red and brighter, while, on the left side of the display, the lower concentration volume is “less” red and darker. Thus, Priye teaches that in fluorescent assays, hue and intensity are not separable variables (in the RGB color space).
Applicant additionally argues that Mackay does not use colorimetric reagents and the NEB does not provide a real-time detection method (Remarks, p. 13).
The Examiner disagrees. Mackay was not cited for teaching the use of colorimetric reagents. As to NEB, as noted in the Non-Final Office Action mailed April 22, 2025 and above, the master mix protocol indicates that the LAMP reaction can be monitored at various time points during the reaction (i.e., in real time/as a function of time) (p. 2, steps 7-8). The ordinary artisan would understand that if an assay can be monitored as a function of time, then data generated from repeated monitoring steps over time can be used to construct a real-time amplification curve.
These arguments are not persuasive. The rejections are modified in view of the instant claim amendments.
Response to arguments regarding prior art rejections
Applicant argues that the prior art rejections should be withdrawn. Applicant discusses the prior art rejection of record, particularly as to the teachings of the Landers and Priye references as applied to instant claim 1, and then concludes “[t]hus, Priye relates to detecting fluorescence intensity and does not relate to detecting changes in hue at regular intervals” (Remarks, p. 16).
The Examiner disagrees. Landers was cited for teaching capturing a photo of the well to detect hue values, while Priye was cited for teaching that photos are taken at regular intervals. Thus, it is not relevant whether Priye teaches detecting fluorescence intensity or changes in hue values, as it was not cited for teaching that aspect of the detection scheme. Also, see the discussion above regarding hue and intensity coupling in fluorophore emissions.
Applicant additionally argues that Boggy, which is cited for teaching the equation in claim 1(d), does not teach generating an amplification curve which is a record of a decrease in average hue value as a function of time, because Boggy refers to detecting changes in fluorescence and “a change is fluorescence is a measurement of the light emitted by the fluorophore, whereas a colorimetric assay measures absorbance” (Remarks, p. 16).
The Examiner disagrees. Boggy teaches that the model “describes the accumulation of amplicon DNA during PCR” and also teaches that variable in the equation that is associated with the fluorescence associated with dsDNA after “n” cycles can also be substituted with the amount of double-stranded DNA after “n” cycles (p. 2, para. 3). Thus, Boggy teaches that the model can be useful to apply to a variety of assays, and teaches that an amplification curve can be generated from data that reflects the accumulation of amplicon DNA. Since colorimetric reagents can also be used to reflect the accumulation of amplicon DNA, the ordinary artisan would understand that the Boggy equation would be useful for that purpose. Also, see the discussion above regarding hue and intensity coupling in fluorophore emissions.
Applicant additionally argues that “Subramian and Guescini are relied upon for teaching
that the amplification curves for real-time PCR and … LAMP are interchangeable” and later states “[t]his does not provide any information on how a colorimetric reagent changes color during the amplification” (Remarks, p. 17).
The Examiner disagrees. It is not clear what Applicant is arguing here as neither
Subramian nor Guescini are cited for teaching “how a colorimetric reagent changes color during the amplification”.
Applicant further argues that there is no reasonable expectation of success because the “ordinary artisan would not have been motivated to modify features of a fluorescent assay to a colorimetric assay with an expectation of success because they rely on different fundamental measurement principles” and also states …
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Remarks, p. 18.
The Examiner disagrees. The rejection does not substitute a fluorescent assay with a
colorimetric assay, as Applicant asserts. The primary reference, Landers, teaches an endpoint colorimetric assay. The teachings of Priye and Boggy are cited simply for teaching monitoring the amplification reaction over time (thus, converting it into a real-time assay) and applying those data into an algorithm to generate an amplification curve. Thus, the issue is not whether fluorescent and colorimetric assays “rely on different fundamental measurement principles”, but rather whether there would be a reasonable expectation of success in using colorimetric assays to generate data that reflects amplicon generation over time. Landers teaches that HNB generates a signal (color change) in a concentration-specific manner as amplicons accumulate (specifically, HNB changes color as free Mg2+ depletes in solution as amplification proceeds – p. 27, ll. 2-8). If HNB color change did not accurately reflect amplicon concentration in solution, then the Landers assay would not work. Thus, the ordinary artisan would understand that if the HNB hue in a reaction volume can be detected to accurately indicate concentration of amplicons in the reaction volume, then it would accurately indicate the concentration of amplicons regardless of when (at the endpoint or during) or how often (once or repeatedly) it is measured. Thus, the ordinary artisan would have a reasonable expectation of success of converting an endpoint colorimetric LAMP assay into a real-time colorimetric LAMP assay.
Finally, Applicant argues that the dependent claims are patentable for the same reasons
as instant claim 1 (Remarks, p. 19).
As noted, the Examiner disagrees with Applicant’s arguments regarding the patentability of claim 1.
These arguments are not persuasive. The rejections are modified in view of the instant claim amendments.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
For each rejection below, dependent claims are rejected similarly as not remedying the rejection, unless otherwise noted.
Claims 1-3, 6 and 8-13 are rejected under 35 U.S.C. 101 because the claimed inventions are directed to non-statutory subject matter. After consideration of relevant factors with respect to each claim as a whole, each claim is directed to one or more judicially-recognized exceptions to patentability (JEs), i.e. an abstract idea, a natural phenomenon, a law of nature and/or a product of nature, as identified below. Further, it is not clear that any element or combination of elements in addition to the JE(s), i.e. "additional elements," either integrate the identified JE(s) into a practical application and/or are non-conventional additional elements. Therefore, it is not clear that any claim is directed to significantly more than the identified JE(s).
MPEP 2106 organizes JE analysis into Steps 1, 2A (1st prong & 2nd prong) and 2B as analyzed below. MPEP 2106 and the following USPTO website provide further explanation and case law citations: www.uspto.gov/patent/laws-and-regulations/examination-policy/examination-guidance-and-training-materials.
Analysis of claims 1-3, 6 and 8-13
Step 1: Are the claims directed to a 101 process, machine, manufacture, or composition of matter (MPEP 2106.03)?
Independent claim 1 is directed to a 101 process, here a "method," with process steps such as "providing ...", “heating …”, “capturing …” and “obtaining …”.
[Step 1: claim 1: YES]
Step 2A, 1st prong: Do the claims recite a judicially-recognized exception (JE), e.g. a law of nature, a natural phenomenon or product, or an abstract idea (MPEP 2106.04.II.A.1 & .04(a))?
MPEP 2106.I, 2nd para. identifies four court-recognized types of JEs: abstract ideas, laws of nature, natural phenomena and natural products.
MPEP § 2106.04(a)(2) further explains that abstract ideas may be grouped as:
• mathematical concepts (mathematical formulas or equations, mathematical relationships and mathematical calculations);
• certain methods of organizing human activity (fundamental economic practices or principles, managing personal behavior or relationships or interactions between people); and/or
• mental processes (procedures for observing, evaluating, analyzing/ judging and
organizing information).
The instant claims at Step 2A, 1st prong, at least preliminarily are interpreted as reciting JEs in the form of abstract ideas (claim 1), as follows.
At this step of the analysis, elements of independent claim 1 are interpreted as directed to the abstract idea of quantifying the concentration of a target nucleic acid, including the particularly recited JE steps/elements of "extracting …”, “transforming”, “generating..." and "obtaining ...," each of which, including all recitation within each listed element. The recited “capturing …” is interpreted as requiring physical measurement performed by a light source and a camera and therefore is not an abstract idea. The above-identified steps/elements are interpreted as directed to the abstract ideas identified below.
BRIs of the claims are analogous to the JE of an abstract idea in the form of a mathematical concept, including mathematical relationships and calculations. Instant examples of math concepts that cause the claim(s) to be directed to the above-identified JE(s) include the "generating an amplification curve [using] eq. (1)” as recited in claim 1. Case law, establishing the mathematical concept JE and to which the instant claims are analogized, is presented in MPEP 2106.04(a)(2).I, including examples of analogous mathematical concept JEs.
BRIs of the claims are analogous to the JE of an abstract idea in the form of a mental process, including obtaining and comparing intangible data. Instant examples of mental process that cause the claim(s) to be directed to the above-identified JE(s) include the “transforming” and “obtaining” as recited in claim 1. In a BRI, it is not clear that the claim embodiments are limited so as to require complexity precluding analogy to a mental process. Case law, establishing the mental process JE and to which the instant claims are analogized, is presented in MPEP 2106.04(a)(2).III, including examples of analogous mental process JEs.
[Step 2A, 1st prong: YES]
Step 2A, 2nd prong: Are the above-identified JEs integrated into a practical application (MPEP 2106.04.II.A.2 & .04(d))?
Generally regarding Step 2A, 2nd prong
MPEP 2106.04(d).I lists the following considerations for evaluating whether additional elements integrate a judicial exception into a practical application:
An improvement in the functioning of a computer, or an improvement to other technology or technical field, as discussed in MPEP §§ 2106.04(d)(1) and 2106.05(a);
Applying or using a judicial exception to affect a particular treatment or prophylaxis for a disease or medical condition, as discussed in MPEP § 2106.04(d)(2);
Implementing a judicial exception with, or using a judicial exception in conjunction with, a particular machine or manufacture that is integral to the claim, as discussed in MPEP § 2106.05(b);
Effecting a transformation or reduction of a particular article to a different state or thing, as discussed in MPEP § 2106.05(c); and
Applying or using the judicial exception in some other meaningful way beyond generally linking the use of the judicial exception to a particular technological environment, such that the claim as a whole is more than a drafting effort designed to monopolize the exception, as discussed in MPEP § 2106.05(e).
Additionally, the courts have also identified limitations that did not integrate a judicial exception into a practical application:
Merely reciting a phrase such as "apply it" (or an equivalent) along with the judicial exception, or merely including instructions to implement an abstract idea on a computer, or merely using a computer as a tool to perform an abstract idea, as discussed in MPEP 2106.05(f);
Adding insignificant extra-solution activity to the judicial exception, as discussed in MPEP 2106.05(g); and
Generally linking the use of a judicial exception to a particular technological environment or field of use, as discussed in MPEP 2106.05(h).
At this point in examination, it is not clear that the identified JEs are integrated into a practical application according to any of the "considerations" exemplified in MPEP 2106.04(d).
For example, relating to the first consideration at MPEP 2106.04(d)(1), it is not yet clear in the record that application of the above-identified JEs in all claimed embodiments must result in an improvement to the technology field in the form of, for example, improved detection and discrimination of nucleic acid targets (specification: [3, 6-7, 23-25]).
[Step 2A, 2nd prong: claim 1: NO]
Step 2B: Do the claims recite a non-conventional arrangement of additional elements (i.e. elements in addition to any identified JE) (MPEP 2106.05)?
All elements of claim 1 are part of one or more identified JEs (as described above), except for elements identified here as conventional elements in addition to the above JEs:
The recited “providing …”, “heating …” and “capturing …” steps/elements (claim 1) are conventional elements of a laboratory and/or conventional data gathering/input elements, as exemplified by Landers (cited below in conjunction with the 35 USC § 103 rejections - p. 16, ll. 1-2, 11, 22-23, 28-30; p. 18, ll. 22-26; p. 17, ll. 1-3; p. 24, ll. 9-13) which describes a method of detecting a target nucleic acid in a sample by performing LAMP on a reaction mix in a well, comprising a sample and amplification reagents, and capturing a photo of the well to detect a signal. In addition, detecting a signal (here, capturing a photo) at regular intervals during the amplification reaction is also conventional data gathering, as taught by Priye (cited below in conjunction with the 35 USC § 103 rejections - p. 8, para. 8).
Finally, the limitation reciting “detecting hue values … as a function of time … during amplification” is also a conventional data gathering/input element, as exemplified by Mackay in 2002 in the context of real-time PCR3. Thus, generally, detecting hue values as a function of time during amplification has been performed routinely in the art for at least two decades. More specifically, such real-time detection methods are also conventionally used in the context of LAMP, and/or with colorimetric detection reagents4.
[Step 2B: claim 1: NO]
Summary and conclusion regarding claim 1
Summing up the above 101 JE analysis of claim 1, viewed as a whole and considering all elements individually and in combination, the claim does not recite limitations that transform it, finally interpreted as directed to the above-identified JE(s), into patent eligible subject matter.
Remaining claims 2-3, 6 and 8-13
Claims 6 and 8 add elements which also are part of the identified JEs for the same reasons described above and therefore do not provide the something significantly more necessary to satisfy 101.
Elements of the following claims are additional elements but nonetheless are conventional elements of a laboratory or computing environment, conventional data gathering elements or conventional post-processing elements:
claim 2: the recited “microwell” element, as evidenced by Landers (p. 16, l. 11; p. 18,
l. 22).
claim 3: the recited amplification “temperature” element, as evidence by Landers (p. 17, ll. 1-3; p. 26, ll. 1-7).
claims 9-12: the recited “ZIKV” target and “primers” directed to the “envelope protein coding region” steps/elements, as evidenced by Landers (p. 42, l. 32) and Priye (abstract; Table S1).
claim 13: the recited “HNB” “colorimetric reagent” element, as evidenced by Landers (p. 18, ll. 23-24).
Claim Rejections - 35 USC § 103
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.
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, 6, 8-9 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Landers5 (WO 2017/070571 A2) in view of Priye6 (A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses, Scientific Reports, 7: 44778, 1-11, March 20, 2017; and Supporting Information, pp. 1-7), Boggy7 (A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data, PLoS ONE, 5(8): e12355, 2010), GenBank Accession No. NC_012532.18 (Zika virus, complete genome, 2016), Subramian9 (An Empirical Approach for Quantifying Loop-Mediated Isothermal Amplification (LAMP) Using Escherichia coli as a Model System, PLoS ONE 9(6): e100596, 2014) and Guescini10 (A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition, BMC Bioinformatics, 9:326 – 1-12, 2008), and as evidenced by Abramoff11 (Image Processing with ImageJ, Biophotonics International, 11(7): 1-7, 2004).
Regarding independent claim 1, Landers teaches …
A computer-implemented method for real-time quantification of the concentration of a
target RNA in a sample using loop-mediated isothermal amplification (LAMP), the method comprising: providing a well comprising a sample in a reaction mix, (p. 16, ll. 1-2, 11, 28-30; p. 18, l. 22; p. 24, ll. 9-13; p. 29, ll. 26-30; p. 31, ll. 21-26);
the reaction mix comprising: three pairs of primers, wherein the three pairs of primers recognize six distinct regions in the target DNA (p. 28, ll. 15-16; p. 24, ll. 11-12: six primers – LF, LB, F3, B3, FIP, BIP);
reverse transcriptase; DNA polymerase; dNTP (p. 24, ll. 5-12: Bst polymerase is a DNA polymerase; p. 27, ll. 5-7);
and a colorimetric reagent that decreases in hue during amplification of target RNA (p. 24, ll. 22-23: HNB decreases in hue from purple to sky blue with successful amplification);
detecting a decrease in hue values (h) as a function of time (t) during amplification comprising the steps of: heating the well to an amplification temperature, wherein the amplification temperature is between 60°C to 68°C (p. 17, ll. 1-3: 65°C);
capturing a photo of the well after the temperature of the well reaches the amplification temperature (p. 16, ll. 22-23; p. 18, ll. 22-26);
extracting red, green and blue (RGB) values with a computer from at least one pixel in the photo of the well; transforming the RGB values with a computer from the at least one pixel in the photo of the well into hue, saturation, and value (HSV) values (p. 19, ll. 1-8 – in addition, Landers cites the extracting and transforming conversions as being performed with “ImageJ” imaging processing software; Abramoff teaches that ImageJ software runs on a computer: e.g., p. 2, middle col., para. 2).
Landers does not teach capturing a photo of the well at regular intervals, wherein the regular intervals are one photo every minute after the temperature of the well reaches the amplification temperature. However, Priye teaches this limitation (p. 8, para. 8).
Boggy teaches …
Generating an amplification curve, wherein the amplification curve is a record of a decrease in an average hue value (h) as a function of time (t) (p. 2, para. 2; equation 14: reflects changes in fluorescence (i.e., hue value) as a function of cycle (which increases over time));
and obtaining the concentration of the target RNA in the sample based on Tt on the amplification curve (p. 1, para. 2).
Boggy, as evidenced by Abramoff, teaches that the amplification curve is generated with a computer. That is, Boggy teaches that the amplification curve is generated by the MAK2 model, which is “mechanistic software analysis” (p. 4, para. 6). While Boggy does not recite that software is run on a computer, that is generally understood to be the case in the art, and, as noted above, Abramoff explicitly teaches that software is run on a computer (e.g., p. 2, middle col., para. 2).
Further, Boggy equation 14 teaches or suggests instant equation (1), as follows:
Regarding Hmin and Hmax, the Boggy curve starts at a background fluorescence level Fb and then adds positive values (Fmax-Fb) as cycles occur. As in the instant Fig. 3A-C, the instant equation 1 starts at positive Hmax and then subtracts negative values (Hmin-Hmax) over time according to a sigmoid relationship. The equations differ only because equivalent data has been plotted differently. So it would have been obvious to rearrange the equation in this way to reflect the orientation of the plots of instant Fig. 3.
S is a slope factor (Boggy’s “q” teaches the instant “S”)
Tt is the time in which h approaches the inflection point (Boggy’s (log(n)-log(r)) teaches the instant (Tt-t), and this also is discussed immediately following equation 14. Boggy’s “n” for cycle number and “r” teach the instant Tt and t for time).
The use of base 10 (here) instead of base “e” (in Boggy) is mathematically prima facie obvious as a mathematical equivalency.
Boggy’s “S” may be 1 as taught by Boggy in their discussion of equation 14 and so Boggy teaches instant equation 1, i.e., the lack of an exponent or the absent exponent effectively being “1” for the sum.
Finally, regarding the differences between the Boggy real-time PCR and the instant LAMP, Subramian and Guescini teach that the amplification curves for these types of amplification are interchangeable. Specifically, Guescini teaches that real-time PCR generates a sigmoidal amplification curve that can be accurately modeled with the Richards equation (p. 5, right col., para. 3; Fig. 1). Further, Subramian teaches that LAMP amplification also generates sigmoidal amplification curves that can likewise be accurately modeled with the same equation, and further teaches that time to positive in LAMP is analogous to the threshold cycle in real-time PCR (abstract; Figs. 3, 5; p. 9, left col., para. 2). Thus, the ordinary artisan would understand that the Boggy equation can be used to generate an amplification curve for both qPCR and LAMP.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to modify the Landers method with the Priye step of capturing photos at regular signals. Landers teaches the need for a portable nucleic acid detection system with improved throughput. The Priye step of capturing photos at regular intervals allows for real-time monitoring of the amplification reaction. The ordinary artisan would have been motivated to incorporate the real-time monitoring of Priye into the Landers method with the expectation that doing so would result in the advantage of earlier detection of positive results. The ordinary artisan would have had an expectation of success as real-time monitoring of amplification reactions is well-known in the art.
It would have been additionally obvious to further incorporate the Boggy amplification curve into the method of Landers to quantify the concentration of the target DNA, as Boggy teaches that the amplification curve is suitable for such a purpose. See MPEP 2144.07. Finally, it would have been obvious to rearrange the Boggy variables to arrive at the instant equation as the ordinary artisan would have understood that the Boggy equation and the instant equation are equivalents, and because choosing among equivalent data analysis methods is an arbitrary design choice. The ordinary artisan would have had an expectation of success as generating amplification curves is well-known in the art of nucleic acid amplification, and because Landers does not limit the types of data analysis that may be performed. Further, Subramian and Guescini teach that qPCR and LAMP amplification curves can be generated using the same equation.
Regarding dependent claims 2-3 and 13, Landers teaches a microwell (p. 16, l. 11; p. 18,
l. 22), as recited in claim 2, and that the amplification temperature is 65°C (p. 17, ll. 1-3), as recited in claim 3. Finally, regarding claim 13, Landers teaches the HNB colorimetric reagent (p. 18, ll. 23-24).
Regarding dependent claim 6, Priye teaches identifying a ROI from which to measure the average h (Supporting Information, Fig. S4). Priye does not teach that the ROI comprises all pixels with a saturation value of at least 0.1. However, Priye does provide various teachings and suggestions as to how to select appropriate ROIs, and how to account for background levels of signal (e.g., Figs. 3-4; p. 7, para. 2; p. 9, para. 3; and, Supporting Information: Figs. S3-S5).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to optimize the ROI identification and data analysis steps in the modified Landers method, discussed above, in order to quantify the concentration of the target DNA. The ordinary artisan would have been motivated to optimize the various data analysis steps through routine experimentation based on the teachings of Priye. The ordinary artisan would have had an expectation of success as image analysis is well-known in the art of nucleic acid amplification, and because Landers does not limit the types of data analysis that may be performed.
Regarding dependent claim 8, Boggy teaches or suggests that the step of obtaining the concentration comprises substituting the threshold cycle (Ct) value in qPCR with T (Boggy’s (log(n)-log(r)) teaches the instant (Tt-t). Boggy’s “n” for cycle number and “r” teach the instant Tt and t for time).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to further incorporate the Boggy data analysis steps into the modified Landers method, discussed above, to quantify the concentration of the target RNA, as Boggy teaches that amplification curve is suitable for such a purpose (see MPEP 2144.07). In addition, the ordinary artisan would have understood that the Boggy equation and the instant equation are equivalents, and choosing among equivalent data analysis methods is an arbitrary design choice. The ordinary artisan would have had an expectation of success as generating amplification curves is well-known in the art of nucleic acid amplification, and because Landers does not limit the types of data analysis that may be performed. Further, Subramian and Guescini teach that qPCR and LAMP amplification curves can be generated using the same equation.
Regarding dependent claim 9, Landers teaches that the target nucleic acid is a virus (p. 42, l. 32), but does not teach ZIKV specifically. However, Priye teaches that the target nucleic acid is ZIKV (e.g., abstract), and specifically, the ZIKV envelope protein coding region (Table S1: Env-984 primers which correspond approximately to nucleotides 984 through 1236 of GenBank Accession No. NC_012532.1; further, the entire envelope coding region spans nucleotides 977 to 2476).
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to further incorporate the Priye ZIKV envelope coding region into the modified Landers method, discussed above. Landers teaches that the method is useful for detecting viral nucleic acid sequences. It would have been obvious to the ordinary artisan to try the Priye target in the modified Landers method, as Landers teaches that the method is useful to detect such targets. The ordinary artisan would have had an expectation of success, as detecting ZIKV nucleic acid with various amplification methods is well-known in the art.
Claims 10-12 are rejected under 35 U.S.C. 103 as being unpatentable over Landers (WO 2017/070571 A2) in view of Priye (A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses, Scientific Reports, 7: 44778, 1-11, March 20, 2017; and Supporting Information, pp. 1-7), Boggy (A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data, PLoS ONE, 5(8): e12355, 2010), GenBank Accession No. NC_012532.1 (Zika virus, complete genome, 2016), Subramian (An Empirical Approach for Quantifying Loop-Mediated Isothermal Amplification (LAMP) Using Escherichia coli as a Model System, PLoS ONE, 9(6): e100596, 2014) and Guescini (A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition, BMC Bioinformatics, 9:326 – 1-12, 2008), and as evidenced by Abramoff (Image Processing with ImageJ, Biophotonics International, 11(7): 1-7, 2004), as applied to claims 1 and 9 above, and further in view of Eiken Chemical12 (A Guide to LAMP primer design, PrimerExplorer V4, 2005).
Regarding dependent claims 10-12, this particular location and these recited primers are suggested by Priye, GenBank Accession No. NC_012532.1 and Eichen Chemical. Specifically, Priye teaches detecting the ZIKV envelope protein coding region (abstract; Table S1), which corresponds to nucleotides 977 to 2476 of the ZIKV genome (GenBank Accession No. NC_012532.1). In addition, SEQ ID NO: 1 corresponds to nucleotides 1350-1373 and 1301-1320 of GenBank Accession No. NC_012532.1, while SEQ ID NO: 2 corresponds to 1396-1417 and 1458-1477, SEQ ID NO: 3 corresponds to 1321 to 1340, SEQ ID NO: 4 corresponds to 1421 and 1441, SEQ ID NO: 5 corresponds to 1279 to 1297 and SEQ ID NO: 6 corresponds to 1479 to 1497. All of these regions are part of the ZIKV envelope protein coding region, according to GenBank Accession No. NC_012532.1. In addition, Eichen Chemical teaches how to design LAMP primers based on nucleotide sequences.
Prior to the effective filing date of the instant invention, it would have been prima facie obvious to use the instant primers to detect the recited ZIKV genomic location using the modified Landers method, discussed above. As noted, the modified Landers method teaches detecting the ZIKV nucleic acid corresponding to the envelope protein coding region using LAMP. The ordinary artisan would have been motivated to design LAMP primers and a corresponding LAMP assay to detect Zika virus, and would have customized the particular target location and the particular primers used through routine optimization. The ordinary artisan would have had the expectation that doing so would result in a ZIKV detection method that could be used in a point-of-care setting, and would have had an expectation of success as LAMP primer design is well-known in the art, as demonstrated by Eichen Chemical, and because Priye provides general principles for primer design (Supporting Information, p. 6).
Conclusion
Claims 1-3, 6 and 8-13 are being examined and are rejected. No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/CAROLYN L GREENE/Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681
1 Mackay, Real-time PCR in virology, Nucleic Acids Research, 30(6): 1292-1305, 2002 (cited in the PTO-892 Notice of References Cited mailed April 22, 2025).
2 New England Biolabs, WarmStart Colorimetric LAMP 2X Master Mix Typical LAMP Protocol (M1800), 2016 (cited in the PTO-892 Notice of References Cited mailed April 22, 2025).
3 In 2002, Mackay (Real-time PCR in virology, Nucleic Acids Research, 30(6): 1292-1305, 2002) reviewed various detection chemistries, including fluorophores, that were commonly used in real-time PCR, and teaches detecting the fluorescent intensity (i.e., “hue value”) as a function of time during amplification (e.g., Fig. 1B; p. 1292-1298: amplicon detection section).
4 Since at least 2016, New England Biolabs (WarmStart Colorimetric LAMP 2X Master Mix Typical LAMP Protocol (M1800), 2016) has produced a colorimetric LAMP master mix. The master mix protocol indicates that the LAMP reaction can be monitored with a color change from yellow to pink (to indicate a positive reaction), that reactions can be monitored at various time points during the reaction (i.e., in real time/as a function of time), and that the color change (or not) result can be photographed (p. 2, steps 7-8). Further, the fact that a molecular biology reagents company supplied a master mix for such a reaction in 2016 suggests that there was a corresponding demand for such a product in 2016. This, in turn, supports a finding that performing such a step was well-understood, routine and conventional at the time of filing.
5 Landers was cited in the Information Disclosure Statement submitted April 13, 2020.
6 Priye was cited in the PTO-892 Notice of References Cited mailed February 1, 2024.
7 Boggy was cited in the PTO-892 Notice of References Cited mailed February 1, 2024.
8 GenBank Accession No. NC_012532.1 was cited in the PTO-892 Notice of References Cited mailed September 19, 2023.
9 Subramian was cited in the PTO-892 Notice of References Cited mailed April 22, 2025.
10 Guescini was cited in the PTO-892 Notice of References Cited mailed April 22, 2025.
11 Abramoff was cited in the PTO-892 Notice of References cited mailed August 26, 2024.
12 Eiken Chemical was cited in the PTO-892 Notice of References Cited mailed September 19, 2023.