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 Claims
Applicant' s amendment filed 09/30/2025 is acknowledged. Claims 1, 7, 9, 11-14, 16, 18-19, 23-24, and 26-27 have been amended. Claims 40 and 41 have been added. Claims 6, 15, 17, 21-22, 25, 28, and 34 have been cancelled. Claims 1-5, 7-14, 16, 18-20, 23-24, 26-27, 29, 20-33, and 35-41 are pending in the instant application and claims 1-5, 7-14, 16, 18-20, 23-24, 26-27, 29, and 40-41 are the subject of this final office action.
All of the amendments and arguments have been reviewed and considered. Any rejections or objections not reiterated herein have been withdrawn in light of amendments to the claims or as discussed in this office action.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Previous Rejection
Status of Prior Rejections/Objections:
The claim objections to claims 1, 11-12, 24, and 27 are withdrawn in view of the claim amendments.
The previous 112(b) rejections directed to claim(s) 1-5, 7-20, 23-24, 26-27, and 29 is/are withdrawn in view of the amendments to the claims with the exception of that that now directed to claim 40 and further clarified.
The prior art rejection(s) are withdrawn in view of the amendments. See new rejections below.
New Ground(s) of Rejections
The new ground(s) of rejections were necessitated by applicant’s amendment of the claims.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Objections
Claim 12 is objected to because of the following informalities:
Claim 1 contains quotations around “the at least two promiscuous probes”. These are not needed and should be removed.
Claim 12: The claim recites “(vii) or any …”. The “or” should come before the Roman numeral.
Appropriate correction is required.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms
have been given their broadest reasonable interpretation (BRI) consistent with the
specification, as understood by one of ordinary skill in the art, as outlined in MPEP 2111.
Regarding claims 1, 13, and 19, “permissive” is defined as “a condition, such as a temperature, which allows hybridization of a probe to more than one polynucleotide sequence, but at different hybridization efficiency, thereby accommodating at least one mismatch” (para [0074]).
Regarding claim 19, “about 50:50” is defined as “within plus/minus 20%” (para [0027]). Therefore, “about 50:50” was interpreted to encompass ratios between 30:70 and 70:30.
Regarding claims 1 and 18, “amplitude” is defined as “magnitude or amplitude of fluorescence” (para [0068]). It is also interpreted to be synonymous with “intensity” (para [0059 and 0061]: “2D plot of fluorescent intensity (amplitude)”).
Claim Rejections - 35 USC § 112(b)
Claim 1-5, 7-14, 16, 18-20, 23-24, 26-27, 29, and 40-41 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 1-3, 9, and 20, the claims recite “the at least one nucleotide difference” (claims 1: “thereby detecting … the at least one nucleotide difference) or, for claim 20, “the at least one nucleotide sequence difference”.
There is insufficient antecedent basis for this limitation in the claims. Claim 1 recites “for each of the at least two different loci, measuring a fluorescence output generated by the promiscuous probe bound … to target polynucleotide sequences with and without the at least one nucleotide difference”. Thus, there are at least two different “at least one nucleotide difference” in the body of claim 1 since the steps are being performed for at least two different loci with distinct “at least one nucleotide difference”. Therefore, it is unclear which is being referred to in the subsequent references to “the at least one nucleotide difference”.
Further, in claim 3, the claim recites “contacting each of the positive reaction mixture constituents individually with … the promiscuous probe” and “validating the method by measuring a … output generated by the promiscuous probe”. It is unclear which promiscuous probe is being referred to.
Claims 2-5, 7-14, 16, 18-20, 23-24, 26-27, 29, and 40-41 are indefinite for depending from claim 1 or a dependent thereof and not rectifying the deficiency.
It is also noted that the preamble of claim 1 recites “detecting … an at least one nucleotide difference in a target polynucleotide sequence”. While it would be understood by the artisan that a polynucleotide sequence inherently contains multiple loci, clarity would be improved by introducing the loci in the preamble. For example, “…polynucleotide sequence for at least two different loci”.
Regarding claim 26, the claim recites “the single well comprises two fluorescent color channels”. It is not clear what is meant by a “well” comprising fluorescent color channels. Is this intended to indicate that the well comprises “fluorescence” detectable in two color channels (i.e., the “optical output of [a] well” is detectable in at least two channels; see para [0059] and Fig. 13D)? Or is the well being used to indicate, in non-standard use of the term, a part of the microscope housing itself? If the latter, it is noted that there is no definition of “well” identified in the disclosure.
Regarding claim 40, the claim recites:
“a binding site to the at least one nucleotide difference that is positioned either: (i) in a middle region of the promiscuous probe length, wherein the middle region is defined in a central 50% portion of the probe length, or (ii) at an alternative location at least partially outside the middle region so long as promiscuous binding at a permissive temperature is maintained”.
It is not clear what is meant by “a binding site to the at least one nucleotide difference”. The “at least one nucleotide difference” may encompass multiple nucleotide differences, e.g., two or more SNPs in a general proximity to one another (e.g., as given as an example in para [0070]: “at multiple distinct positions along the polynucleotide sequence”). It is not clear if this limitation is intended to require that (a) any portion (e.g., a SNP of) of the at least one nucleotide difference to be within the central 50%, (b) at least the mid-point of the “at least one nucleotide difference”, (c) all the “at least one nucleotide difference” be within the middle region, or (d) something else.
Likewise, the claim recites “or (ii) in a region outside the middle region”. It is therefore not clear how at least one nucleotide differences that span over the 50% region are to be treated.
Further, it is not clear whether the central 50% portion is meant to be inclusive or exclusive when the probe length is odd.
Therefore the metes and bounds are not clear to one of ordinary skill in the art.
Claim Rejections - 35 USC § 112(a)
Claim 12 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection.
Regarding claim 12, claim 12 is amended to recite (i) SNPs, (ii) indels, (iii) the presence, absence, or abundance or viruses, (iv) the presence, absence, or abundance of … bacterial, fungi, or non-invasive species, (v) soil biome characterization, (vi) gut biome characterization, (vii) or “any combination of (i)-(vi)”.
The original claim and the specification (e.g., para [0020] [0093]) recite “SNPS … indels … presence, absence, and/or abundance or viruses … and/or gut biome” (emphasis added). Provisional applications 63160432 and 63172839, to which the instant application claim priority, recite the same in, respectively, para [0081] and para [0085].
No redefinition of the term “and/or” was identified to encompass “one or more of”.
For this reason, contrary to the amendment reciting “any combination of (i)-(vi)”, the disclosure only provides sufficient support for “all of (i)-(vi)” or “any one of (i)-(vi)”.
Thus, the claim amendment adds new matter by reciting limitations that were not disclosed in the specification as filed, and now change the scope of the instant disclosure as filed. Such limitations recited in the present claims, which did not appear in the instant specification nor in the provisional or PCT applications to which priority is claimed, introduce new concepts and do not comply with the written description requirements under 35 USC 112.
Claim Rejections - 35 USC § 103
Claim(s) 1-5, 7-14, 16, 18-19, 26-27, and 40-41 is/are rejected under 35 U.S.C. 103 as being unpatentable over Regan (WO 2014/152054; published 09/25/2014; as cited in the IDS dated 03/08/2023) in view of Rowlands (Rowlands V, et al. Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA. Sci Rep. 2019 Sep 2;9(1):12620) and Belousov (US 2009/0087922 A1; published 04/02/2009; as cited in the IDS dated 03/08/2023), as evidenced by Kent (Kent J. In-Silico PCR [Internet]. UCSC Genome Browser; [cited 2025 Mar 17]. Available from: https://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=2488317949_IJNUYdKiaZAenB83BYTPtAgKgl6I).
Regarding claims 1, 14, and 16, Regan teaches a method for detecting an allelic variant of target polynucleotide molecules using a single probe, wherein the method comprises:
(a) performing an amplification reaction on a sample comprising a plurality of target polynucleotide molecules, wherein each of a plurality of reaction volumes of the amplification reaction comprises:
(i) a forward primer that is complementary to a first sequence of the target polynucleotide molecules, wherein the first sequence is 5' of a target locus;
(ii) a reverse primer that is complementary to a second sequence of the target polynucleotide molecules, wherein the second sequence is 3' of the target locus, and on the opposite strand as the forward primer; and
(iii) a detection probe capable of hybridizing to a third sequence of the target polynucleotide molecules that is located between the first sequence and the second sequence, comprising a signal reporter and a quencher, and that hybridizes to the potentially variant target locus (para [00259]; also: Fig. 1 and para [00354-355])
wherein the detection probe may hybridize at different efficiencies depending on the presence of a variant at a permissive temperature (para [00354]: “The difference in probe binding to a PM [perfect match] target (MU) and MM [mismatch] target (WT) affects the fluorescence of the droplets…The PM probe has a higher Tm for a matched template, whereas this same probe will have a lower Tm to a mismatched template”; para [00264]: “at the annealing temperature…significantly less wild-type probe…gets cleaved during each cycle of PCR for droplets containing only mutant sequences which lowers the fluorescence of these droplets”; para [00258]; instant claim 14).
Regan teaches that the sample reaction mixture may further comprise PCR reagents and that the probe is fluorescently-labelled (para [00355]:” 5'-FAM-TTGGTCTAGCTACAGAGAAAT-MGB-3' (MU)… Bio-Rad® 2x ddPCR Supermix”; instant claim 16).
Regan teaches that the amplification is performed at a permissive temperature (Fig. 1; Fig. 2K) and that fluorescent output generated by the promiscuous probe bound to the sample amplicons is measured, such that the difference between hybridization efficiencies results in an amplitude difference between the promiscuous probe bound to sequence with and without the at least one nucleotide difference (para [00354]; para [00264]; Fig. 1; Fig. 2K).
Regan teaches that the number of detection probes may equal or exceed the number of optical channels, such that multiple loci can be genotyped simultaneously in the same reaction (para [00176]). Regan teaches the use of two optical channels with detection probes (instant claim 26; e.g., Fig. 3; Fig. 18).
Regan further optimizing the reaction efficiency for a particular target to result in a difference in signal level to allow different probes to be distinguished from one another, such that additional targets may be detected in the same multiplexed reaction (para [00258]). Regan teaches that optimizing sequence, length, and binding location to achieve appropriate annealing and melting properties, wherein making such design choices are well known in the art (para [00167]; see also para [00154-157]).
Regan teaches that a second pair of primers may be used in the amplification reaction (para [00119]) and may have identical melting temperature, wherein design and optimization options are known in the art (para [00135]).
Regan teaches two probes used on the same amplicon (Fig. 20), and on different amplicons (Fig. 15), wherein one is a non-promiscuous reference probe.
Regan teaches that the assay may comprise ddPCR (e.g., para [00287] and [00297]).
However, Regan fails to explicitly teach an embodiment with multiplexing using at least two promiscuous probes at one temperature.
Rowlands teaches a systematic approach to the development and optimization of multiplex ddPCR assays for the detection of point mutations so as to ensure extremely low false positives while retaining high sensitivity (entire document, e.g., Abstract; Fig. 3; Fig. 6). Rowlands teaches that multiplexing enables rapid and cost-effective monitoring for a limited number of mutations according to the Poisson distribution without the need for external reference standards or controls (pg. 1, para 2, spanning pg. 2).
Rowlands teaches optimizing probes using factors including melting temperature using LNA bases, similar modifications, probe length, and cross-hybridization of sequences (pg. 4, ddPCR probe design, spanning pg. 5). Rowlands teaches optimization of annealing temperatures to arrive at a single temperature such that thresholds could be selected to separate positive and negative droplets in two channels, including for more than one mutation (pg. 5, Optimisation of the annealing temperature using a thermal gradient, spanning pg. 6). Rowlands further teaches that multiplexing of multi-site assays was achieved by modifying probe concentrations and the combination of FAM and HEX fluorescent labels (pg. 6, para 2; pg. 6, para 4; Fig. 7; Fig. 9). Rowlands teaches further optimizations to increase separation (pg. 10, para 4 through pg. 11, para 4).
Rowlands teaches that such multiplex ddPCR assays enable very rapid and cost-effective monitoring of mutations in plasma samples, and such assays can be very informative for serial analysis after target mutations are identified via sequence profiling of tumor tissue or baseline ctDNA sample (pg. 1, para 2, spanning pg. 2).
Belousov teaches further optimization methods for probes targeting polymorphisms that enable the artisan to either increase or narrow the melting temperature difference between target sequences with and without polymorphisms using nucleotide analogs (entire document, e.g., para [0123-133], [0050] and Example 7). Belousov teaches that melting temperature for the probes is a function of thermodynamic parameters of the duplex, concentration of the probe and target, nearest neighbors and probe length (para [0127]). Belousov teaches that conditions for hybridization are well-known in the art and can be varied within relatively wide limits, wherein hybridization stringency correlates with a lower tolerance for mismatched hybrids and is affected by temperature, pH, ionic strength, and concentration of organic solvents and are well-known in the art (para [0131] and [0125]).
Belousov teaches that if annealing temperatures (Tms) are known, a hybridization temperature (at a fixed ionic strength, pH, and probe/target concentration) can be chosen that is below the Tm of the desired duplex and above the Tm of an undesired duplex, wherein determination of the degree of hybridization is accomplished by simply testing for the presence of the hybridized probe (para [0133]).
Belousov teaches that the oligonucleotides uses may comprised “locked” sugars [i.e., LNAs] (para [0085]) or PNA and DNA/DNA chimeras to balance Tms and provide modified oligonucleotides having improved mismatch discrimination (para [0087]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have multiplexed the promiscuous probes of Regan in view of Rowlands and Belousov to at least two different loci optimized for a single permissive temperature use in the annealing step in view of Rowlands, motivated by the desire to improve the speed and cost of applying it to multiple mutation targets in clinical samples, as taught by Rowlands. Such additional targets represents a “duplication of parts”, wherein choosing a permissive temperature common to all loci would have represented routine optimization. See MPEP 2144.04(IV)(B) and 2144.05(II).
Regan, Rowlands, and Belousov each teach various methods for optimizing probes that contain nucleotide analogs to better discriminate targets from one another. There would have been a strong expectation of success as each is directed to the detection of mutations and wild type samples via amplification, wherein detection is performed using a fluorescent oligonucleotide probe that comprises nucleotide backbone and/or nucleotide analog substitutions to alter annealing temperatures.
Regarding claim 2, in the method of Regan in view of Rowlands and Belousov, Reagan teaches quantifying the concentration of the mutant and wildtype ratios [in the sample] (Fig. 4; para [00359]).
Regarding claim 3-5 and 19, in the method of Regan in view of Rowlands and Belousov, Rowlands teaches that every ddPCR run included negative template controls and positive template controls (pg. 3, para 3). Rowlands teaches that the annealing temperature gradient separates positive and negative droplets (i.e., is a mixture of the first and second polynucleotide) (Fig. 4; pg. 5, Optimisation of the annealing temperature using a thermal gradient, para 1).
It follows that if each reaction was run with said negative and positive template controls, the optimization reaction was also run with these controls [i.e., each of the positive control reaction mixtures constituents was contacted individually with the PCR primers and the promiscuous probe] (instant claim 3).
Rowlands teaches performing this for multiple probes (Fig. 4; pg. 5, Optimisation of the annealing temperature using a thermal gradient, para 1).
Rowlands also teaches defining a threshold (instant claim 4; Fig. 4a: pink line) and validating the multiplex specificity and sensitivity [i.e., the components of the method and definitions thresholds for the polynucleotides (instant claim 5; pg. 6, para 1).
Rowlands teaches that the tested temperatures span above and below the permissive temperature (Fig. 4).
Regan teaches that when the sample comprises a gene from a diploid source, a homozygous target polynucleotide with two alleles that have perfect match with the detection probe can generate a strong signal, a homozygous target polynucleotide with two alleles that have a mismatch with the detection probe can generate a weaker signal, and a heterozygous target polynucleotide can generate observable signals from two different populations of positive droplets (instant claim 3; para [00255]).
Regan also teaches that the methods are particularly suitable for threshold value amplification reactions, wherein the assay determines a threshold value such as for example the cycle number at which amplification of a particular target sequence above a threshold level is achieved (para [00249]; instant claims 4 and 5). Regan teaches that a calibration curve can be generated which can be used to determine presence or absence of a target molecule having the first allele and presence or absence of a target molecule having a second allele based on the signal of the first reporter emitted from the reaction volumes of the PCR (para [00255]). Regan further teaches partition positive for a particular target may produce a signal level or amplitude that is above a given threshold and/or within a given range (para [00256]; instant claims 4 and 5).
Regan teaches providing a sample that is homozygous mutant, homozygous wildtype, and a heterozygous [i.e., about 50:50] sample (Fig. 16).
Belousov teaches choosing a hybridization temperature below the Tm of a desire duplex and above the Tm of an undesired duplex, wherein determination of the degree of hybridization is accomplished by simply testing for the presence of the hybridized probe (para [0133]).
Thus, it follows that in the combined method, it would have been obvious, as part of the routinely optimization of the method, to applying the optimization scheme of Rowlands to the heterozygous sample of Regan (a substitution of a sample of known content, i.e., for the same purpose) and identify an annealing temperature suitable for the polynucleotide amplifications and low-off target optical detection, in view of Belousov and Rowlands (instant claim 19).
Regarding claim 7 and 11, in the method of Regan in view of Rowlands and Belousov, Regan teaches that the target polynucleotide sequence is from HT-29 cell lines (para [00355]) and that HT-29 is from a human [i.e., an animal] (para [00371]).
Regan further teaches the application of the methods provided herein to the diagnosis or prognosis of a condition, including cancer (para [00341]); to monitor or identify the origin of a disease (e.g., seasonal influenza, pandemic influenza); to monitor food safety (e.g., by analyzing an infectious agent, e.g., a bacteria, fungus, protist, or virus) (para [00352]); to monitor for a biosecurity threat, e.g., a bioterrorist attack using an agent such as influenza, anthrax, or smallpox (para [00351]). Regan teaches that samples may be taken from organisms including bacteria, archaea, or eucaryota or may be taken from an infectious agent (e.g., virus) (para [00216]). Regan teaches fetal or maternal genetic material and a biopsy (para [00207]).
Rowlands teaches the use of breast cancer patient samples (Fig. 8).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to apply the combine method to the additional targets in order to increase the applications and thus the market for the method. There would be a strong expectation for success as processing nucleic acids and designing primers/probes from different origins is well understood and would be within the skill set of a PHOSITA. Such also represents a simple substitution for the same purposes. See MPEP 2144.06(II).
Regarding claim 8, in the method of Regan in view of Rowlands and Belousov, Regan teaches extraction of DNA or RNA (pg. 41, F2; para [00226]), wherein the methods can be used in with reverse transcription-PCR (para [00230]). Regan teaches that the target polynucleotide can be cDNA generated from RNA through RT-PCR (para [00109]).
Belousov also teaches RNA targets (e.g., para [0087]).
Regarding claim 9, 12, and 27, in the method of Regan in view of Rowlands and Belousov, Regan teaches the at least one nucleotide difference is the B-raf V600E mutation (para [00355]). Regan teaches applying a probe that has a lower level of probe cleavage to mutations including a SNP (claims 9, 12, and 27; para [00354] and Fig. 20; see para [00112]).
Regarding claim 10, in the method of Regan in view of Rowlands and Belousov, Regan teaches that a target polynucleotide sequence that has a length of 136 bp, as evidenced by Kent. While Regan does not explicitly teach the length of the sequence amplified by the forward and reverse primers in para [00355], it is inherently taught because Regan teaches the sequences of the primers and that the template is human gDNA (para [00371]) and in the human genome, the pair of primers produces an amplicon with a length of 136 bp, as evidenced by Kent. While Regan does not explicitly teach that the pair of primers produce an amplicon of that length, it is implicitly taught because Kent teaches that those primer bind to the region chr7:140753289-140753424 of hg38, resulting in a 136 bp amplicon.
It is further noted that Regan teaches
Regarding claims 13 and 40-41, in the method of Regan in view of Rowlands and Belousov, Regan teaches a promiscuous probe with a length of 21 nt without a locked nucleotide, 38% GC content, the one nucleotide difference located at the 16th base [outside the central 50% portion if the 50% is interpreted to be exclusive; inside the central 50% if the 50% is interpreted to be inclusive] (instant claims 13 and 40; para [00355]). Said promiscuous probe therefore has a 95.2% binding region sequence complementary to a binding site of the target polynucleotide sequence for the wildtype template [i.e., <98%] and 100% for the mutant template [i.e.,>98%] (instant claim 41).
Rowlands teaches selecting an annealing temperature from within the range of 55-65 C (pg.5, Optimisation of the annealing temperature using a thermal gradient, para 1), wherein one such optimized annealing temperature for an assay was 59.2 C (instant claim 40; Fig. 4; Optimisation of the annealing temperature using a thermal gradient).
It follows, therefore, that the artisan in multiplexing the combined method would arrive at ranges for each probe that reflect those of the probes taught, as each of the claimed conditions is a matter of routine optimization, as discussed above.
Regarding claim 18, in the method of Regan in view of Rowlands and Belousov, Reagan teaches a median mutant amplitude of approximately 23000 units and a median wildtype amplitude of approximately 12500 units (Fig. 2K).
Rowlands teaches Ch1 amplitudes of ~9000, ~13000, ~18000, and ~27000 (Fig. 7a) and optimizing separation of droplets by amplitude (entire document, e.g., Fig. 6).
It follows, therefore, that the artisan in multiplexing the combined method would arrive at ranges for each probe as such represents a matter of routine optimization.
Regarding claim 26, in the method of Regan in view of Rowlands and Belousov, Regan teaches utilizing two fluorescent color channels (e.g., Fig. 16-17).
Rowlands teaches using two fluorescent color channels (e.g., Fig. 7)
Claim(s) 20 and 23-24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Regan (WO 2014/152054; published 09/25/2014; as cited in the IDS dated 03/08/2023) in view of Rowlands (Rowlands V, et al. Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA. Sci Rep. 2019 Sep 2;9(1):12620) and Belousov (US 2009/0087922 A1; published 04/02/2009; as cited in the IDS dated 03/08/2023), as applied to claim 1 above, and further in view of Guo (CN 110982945B; published 06/09/2020; as cited in the IDS dated 03/08/2023) .
Regarding claims 20 and 24, in the method of Regan in view of Rowlands and Belousov, Regan teaches the application of the methods provided to monitor or identify the origin of a disease (e.g., seasonal influenza, pandemic influenza) (para [00352]) and to monitor for a biosecurity threat, e.g., a bioterrorist attack using an agent such as influenza (para [00351]). Regan teaches that samples may be taken may be taken from an infectious agent (e.g., virus) (para [00216]).
Regan teaches PCR that incorporates reverse transcription, as cited above in claim 8.
Regan teaches that the length of the forward primer and the reverse primer can depend on the sequence of the target polynucleotide and the target locus and the length and/or Tm of the forward primer and reverse primer can be optimized (para [00120]).
Regan fails to teach SARS-CoV-2 as the target polynucleotide sequence, SARS-Cov-2 variants as the at least one nucleotide sequence difference, and probes/primers targeting SARS-CoV-2.
Guo rectifies this by teaching a nucleic acid composition, kit and method
for detecting 2019 new coronavirus [SARS-CoV-2] (pg. 3, Summary of Invention, para. 1). Guo teaches that timely and accurate detection of the new coronavirus is very important as some patients die in complication with other underlying diseases (pg. 3, Background technique, para 1). Guo teaches SEQ ID NO 4, which comprises 100% sequence identity to instant SEQ ID NO 5. Guo teaches that the nucleic acid composition including SEQ ID NO 4 has high sensitivity and specificity (Abstract).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modified the combined method to detect the novel coronavirus SARS-CoV-2 in view of Guo, motivated by the desire to identify and monitor alleles/variants in the virus in order to aid in tracking the origin of cases of the novel, globally significant disease, as taught by Guo. It further would have been obvious to start with one or more of the sequences of Guo in order given the high sensitivity and the specificity of the assay to avoid some routine testing while designing primers. In doing so, it further would have been obvious to modify the sequences of Guo as desired by a practitioner (e.g., by shortening them) in order to adjust Tm to the practitioner’s desired conditions. As such, the exact length/positioning of sequences is held to be a matter of routine optimization to achieve an optimal or desired Tm under MPEP 2144.05(II); this further is supported by SEQ ID NO 2 and 9 that differ only by one base at the 3’. There would have been a strong expectation of success in adapting the method of Regan for SARS-CoV-2 as a PHOSITA would be able design primers and probes to a target, so long as a sequence is known, particularly if one or more primers or probes is available as a starting point.
Regarding claim 23, in the method of Regan in view of Rowlands and Belousov, and further in view of Guo, it follows that, as the combined method detects at least one nucleotide difference for at least two different loci on the target nucleotide sequence each with corresponding a promiscuous probe, wherein Regan teaches applying to mutations, and it is obvious to apply the method to a target sequence from SARS-COV-2 comprising mutations, such that they are detected.
Regan teaches applying two PM detection probes two the two channels (i.e., probes with distinct fluorescence emission maxima) (Fig. 16; para [00373-374]). Thus, it would follow that in routinely optimizing in view of the combined method, the artisan would design the method such that they have distinct “fluorescence emission maximum”, interpreted here to be the same or the substantially similar fluorescent dye utilize for detection of the probe.
Additionally and/or alternatively, it would have been obvious to try to utilize another fluorescent channel (such that the utilized probes had different emission maxima by choosing a different dye) rather than utilizing the first if the routine optimization was not successful (e.g., unable to eliminate “rain” as described in Rowlands). There are a finite number of solutions (same or different) and Guo teaches the need of accurate and timely detection of SARS-Cov-2, wherein it would further be understood that identification of variants would enhance the accuracy of detection. Given the optimizations discussed, the artisan could have pursued such with a reasonable expectation of success as such represents the choice of a known dyes and further optimization, known techniques in the field.
Claim(s) 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Regan (WO 2014/152054; published 09/25/2014; as cited in the IDS dated 03/08/2023) in view of Rowlands (Rowlands V, et al. Optimisation of robust singleplex and multiplex droplet digital PCR assays for high confidence mutation detection in circulating tumour DNA. Sci Rep. 2019 Sep 2;9(1):12620) and Belousov (US 2009/0087922 A1; published 04/02/2009; as cited in the IDS dated 03/08/2023), as applied to claim 1 above, and further in view of Wu (Wu F, et al. SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases. mSystems. 2020 Jul 21;5(4):e00614-20).
Regarding claim 29, in the method of Regan in view of Rowlands and Belousov, Regan teaches that the sample may be virus and that the methods are applicable to identifying infectious agent origins, including pandemic viruses, and the nucleic acid may be RNA, as cited above. Regan teaches quantifying the concentration of the mutant and wildtype ratios, as cited above.
Regan does not teach wastewater as a sample or the steps of filtering wastewater and concentrating virus.
Wu rectifies this by teaching a method of wastewater surveillance to measure the presence of emerging infectious diseases like the novel coronavirus SARS-CoV-2 (Abstract), wherein a viral enrichment protocol was applied to quantify viral titer in sewage using RT-qPCR, (pg. 2, Results, para 2) wherein the RT-qPCR is performed with probes (Table 1). Wu teaches that raw sewage samples were first filtered and precipitated with PEG-NaCl for viral enrichment, followed by RNA extraction, reverse transcription, and real-time PCR, wherein the protocol does not rely on expensive chemicals or materials and thus can be widely employed for viral detection in wastewater samples and further avoids competitive usage of commercial viral extraction kits utilized in clinical testing (pg. 4, Discussion, para 1, spanning pg. 5). Wu teaches that this data source can improve the precision of epidemiological modelling to understand the penetrance of SARS-CoV-2 in specific vulnerable communities and may be useful in modeling pandemic and future outbreaks (Abstract). Wu teaches identifying a mismatch in one sample using Sanger sequencing (Fig. 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed method to combine the combined method of monitoring variants, including in viral samples and/or RNA samples, with the method of preparing wastewater for analysis of RNA viruses Wu in order to monitor variants of SARS-CoV-2 in a relatively easily collectable sample that can be targeted to monitor vulnerable communities. Applying the steps of Wu would have been obvious in order to avoid use of commercial kits, which may be in short supply during pandemic outbreaks, as taught by Wu. There would have been a strong expectation for success as the methods are directed to forms of quantitative PCR utilizing probes and it would have been predictable to substitute a sample from Wu into the combined method and to design probe(s) in view of Wu for variants of interest to SARS-CoV-2 in the method of Regan.
Response to Arguments
Applicant' s arguments, see pg. 12-13, filed 09/30/2025, with respect to the rejection(s) of claim(s) 1-2, 7, 10, 13, 14, and 16-18 under 35 USC 102 have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of Rowlands and Belousov.
However, the Applicant's arguments filed 09/30/2025 regarding the 103 rejections in view of Alland have been fully considered but they are not persuasive. While other art has been applied, it is noted for the sake of compact prosecution that the claims require that the “at least one PCR reaction” be at the “single permissive temperature common to all loci” and that “each of the at least two promiscuous probes is promiscuous at the single permissive temperature”.
Alland teaches annealing and extension at 67 C (pg. 25 line 19) and at least one promiscuous probe that is promiscuous (i.e., differential binding efficiencies at the temperature) at 67 C (e.g., Fig. 2A-C). Further, Regan teaches that such differential binding efficiencies lead to fluorescence amplitude difference when that is measured.
All other arguments depend on those already discussed.
For these reasons, the arguments are not persuasive.
Conclusion
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Ahn – 2016 (Ahn JJ, et al. Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes. Mitochondrial DNA A DNA Mapp Seq Anal. 2016 Jul;27(4):2641-4.) teaches the use of two LNA designed to be 1-4 bp mismatches to discriminate between targets at two sites (Abstract; pg. 2644, Finding country-of-origin specific polymorphism; pg. 2644, LNA probe). Ahn – 2016 teaches annealing/extension at 60 C and that the FAM probe is promiscuous at this temperature (Fig. 2; pg. 2643, Asymmetric PCR). It is noted that the melting temperature curve of the HEX probe ends at 60 C for Rus and Nz1 [i.e., within what would expected to be an optimizable range].
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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/EMMA R HOPPE/Examiner, Art Unit 1683
/NANCY J LEITH/Primary Examiner, Art Unit 1636