DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. This action is in response to the amendment filed on 05 April 2026. Applicant's arguments and amendments to the claims have been fully considered but do not place the application in condition for allowance. All rejections and objections not reiterated herein are hereby withdrawn.
3. Claims 1-6, 8-15, 17, 18 and 20 are pending.
Claims 1-3 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 4-6, 8-15, 17, 18 and 20 read on the elected invention and have been examined herein.
Claim Rejections - 35 USC § 103
4. 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.
Claim(s) 4-6, 8-15, 17, 18 and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable Singer et al (U.S. 20200331938; cited in the Office action of 12/05/2025) in view of Nelson et al (Cell Reports. Feb 2019. 26: 2227-2240, e1-e5, and Supplementary Information p. 1-20, 40 pages total; cited in the Office action of 12/05/2025) and further in view of Lee et al (bioRxiv. 2016. Available via URL: <biorxiv.org/content/10.1101/048603v1.full.pdf>, p. 1-20; cited in the Office action of 12/05/2025).
Singer et al teaches a method for enriching a target microbial / bacterial nucleic acid in a sample (e.g., para [0009] and [0011]), comprising: providing a sample comprising a target microorganism and a non-target cell, wherein the target microorganism and the non-target cell originate from different species (e.g., a sample comprising eukaryotic cells and microbial cells (para [0011]); selectively lysing the non-target eukaryotic cells using one of the eukaryotic cell lysis agents listed at para [0153] (which step is considered to be a step of lysing non-target cells by a cell lysis unit of a diagnostic system) to release non-target eukaryotic nucleic acids from the non-target eukaryotic cells (see also para [0014], [0021] and [0151-0152]; and depleting the non-target eukaryotic nucleic acid from the sample treated with the eukaryotic lysis agent by contacting the sample with an anionic-exchange resin / microparticle (which is considered to be a target nucleic acid enrichment unit of a diagnostic system; see, e.g., para [0021], [0057], [0142], [0295-0296]), thereby enriching the target nucleic acid of the target microorganism in the sample.
Note that neither the specification nor the claims define what constitutes a “unit of a diagnostic system” as it applies to a “cell lysis unit of a diagnostic system” or a “target nucleic acid enrichment unit of the diagnostic system.” Nor do the claims require any connection or interaction between the units. The claims as broadly recited encompass methods wherein cell lysis is performed using a cell lysis agent and separately a target enrichment step is performed using an anion-exchange microparticle or other resin. Further, Singer teaches that the methods can be performed using an automated integrated device (e.g., para [0166], [0433] and thereby teaches that the lysing step and enrichment step are part of an integrated detection device.
Regarding the limitation that the cell lysis unit comprises a non-ionic surfactant including saponin, polysorbate (TWEEN™) or octylphenol ethoxylateaponin (or TRITON™), Singer teaches lysis of the non-target eukaryotic cells using a solution that comprises, for instance, the non-ionic surfactants of saponin, TWEEN™ (i.e., polysorbate) or TRITON™ (i.e., octylphenol ethylate; see e.g., para [0153]).
Regarding the limitation that “the enriched nucleic acid has at least 2,000 nucleotides in length,” Singer does not specify the length of the enriched target microbial DNA. However, in the absence of evidence to the contrary, the resulting enriched microbial DNA is considered to be at least 2,000 nucleotides in length since Singer teaches that amplicons produced from this microbial DNA may be between about 400 to 4000 bp in length (para [0323]). That is, since the amplicons are up to 4000 bp in length, the enriched microbial DNA that is used to produce the amplicons must be at least 4000 bp in length (which meets the claim limitation of at least 2,000 nucleotides). Note also that Singer teaches that the selective lysis of eukaryotic cells/ leukocytes in a sample did not affect the integrity of bacterial DNA from Borrelia burgdorferi (para [0459]).
Singer teaches sequencing the enriched nucleic acid using a sequencing assay to identify microorganisms present in the sample (e.g., para [0329]).
Singer does not teach comparing sequencing data with a microbial genome database or resistance gene database and identifying a microorganism based on the result of the comparing.
However, Nelson teaches methods for identifying microorganisms present in samples obtained from human subjects (see abstract and p. 2228, col. 2). It is disclosed that following removal of human DNA, as well as extracellular DNA, from the sample obtained from a human subject, the enriched microbial DNA is analyzed by sequencing (see, e.g., abstract, p. 2228, col. 2 and p. e4-e5). The resulting sequencing data is then compared to sequencing information in databases, including an antibiotic resistance gene database and the SILVA database to identify particular microorganisms present in the sample (e.g., p. 2234, col. 1; p. e5 first full para; and p. 11 and 13-19 of Supplementary Information). Nelson teaches that this comparison of sequencing data aids in identifying the species of bacteria present in a sample (e.g., p. e5, first full para) and particularly aids in identifying antibiotic resistant microorganisms present in a sample (p. 2234, col. 2 to p. 2235, first para).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Singer so as to have compared sequencing data generated by sequencing the enriched microbial nucleic acids with sequencing data in microbial genome database or resistance gene database so as to have accomplished the objective set forth by Nelson of accurately identifying the species of microorganism(s) present in a sample from a subject and determining their resistance to antibiotics.
Secondly, regarding the amendment to claims 1 and 20 to recite “sequencing the enriched nucleic acid by a sequencing assay to generate sequencing data with at least 20 times the genome size of the target microorganism,” Singer does not specify the type of sequencing that is used to analyze the enriched microbial nucleic acids and does not teach this limitation.
However, Nelson teaches that the enriched microbial nucleic acids are sequenced by next generation sequencing to generate sequencing data for metagenomic analysis (p. e4). Nelson exemplifies sequencing methods in which “the mean coverage for detected resistance genes was 11.03, 17.83, 6.53, and 35.43 in samples 186, 205, 309, and 312, respectively” (p. 2234, col. 2). Nelson concludes that “(i)ncreased microbial sequencing coverage improves detection of important genes” (see “Highlights” on the coversheet).
Further, Lee teaches that the error rate inherent in sequencing methods can be reduced by sufficient coverage (p. 3, first para). Lee compared the read lengths and coverage required to sequence genomes using different sequencing methods with high levels of accuracy (e.g., p. 8-9). Lee (p. 11) concludes:
“while our analysis suggests that 20x coverage of a genome should be enough to well assemble a genome, we recommend researchers sample >75x when using the new long read sequencing technologies to make the error correction steps most effective and to ensure high coverage is available of the longest reads. Ideally, if the budget and sample materials allows, we recommend assembling 20x coverage of error corrected reads exclusively over 20kbp long, using haploid or inbred samples if possible.”
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further modified the method of Singer so as to have performed the sequencing assay at a high coverage, including at least 20x coverage of the genome, particularly when assembling a genome from the enriched microbial nucleic acids or performing long read sequencing assays in order to accomplish the objective set forth by Lee of ensuring the accuracy of the sequencing results.
Regarding claims 6 and 20, Singer teaches depleting the sample of non-target nucleic acids using an anionic-exchange microparticle or resin, which is considered to be a “solid phase adsorbent,” and removing the anionic exchange microparticle or resin to thereby enrich the target nucleic acid in the sample (e.g., para [0021], [0057], [0148] and [0235]).
Regarding claim 8, Singer exemplifies methods wherein “[t]he calculated removal rate was 99.95%±0.04% of the human DNA from 20 ml whole-blood” (para [0460]). Thereby, the method of Singer is one which results in at least a 10-fold enrichment of the target nucleic acid originally present within the sample. See also para [0463]
Regarding claims 9 and 10, Singer teaches that the target nucleic acid to be enriched is microbial nucleic acid and particularly is bacterial nucleic acid (e.g., para [0122]).
Regarding claims 11 and 12, as discussed above, Singer teaches that the non-target cell in the sample is a eukaryotic cell and particularly a mammalian cell (e.g., para [0010-0011], and [0013-0014]).
Regarding claims 13 and 14, Singer teaches that the sample is a biological sample from a subject suffering from an infection disease such as sepsis or pneumonia (para [0127]).
Regarding claim 15, Singer (para [0129]) states “the sample comprises a bodily fluid, bodily excretion, or bodily secretion, e.g., blood, urine, saliva, stool, or sputum. In some embodiments, samples are comprised of human blood.”
Regarding claim 18, Singer teaches extracting the microbial nucleic acids from microbial cells present in the sample depleted of eukaryotic cells prior to performing the sequencing step (e.g., para [0014], [0021] and [0329]). Nelson also teaches extracting the microbial nucleic acids from microbial cells present in the sample depleted of eukaryotic cells prior to performing the sequencing step (p. e4 “Phylogenetic composition from metagenomic shotgun sequencing”).Response to remarks regarding the prior rejections under 35 U.S.C. 102 and 103:
The response states that the limitation from claim 7 that the enriched nucleic acid has at least 2,000 nucleotides in length has been added to claims 1 and 20. It is argued that “Singer merely mentions the possible sizes of amplicons (i.e., amplified products) generated during subsequent amplification and detection steps. Such disclosure neither teaches nor suggests the molecular length, integrity, or continuity of the enriched nucleic acid molecules prior to amplification.”
This argument has been fully considered but is not persuasive. As noted by Applicant, Singer (para [0323]) states:
“In some embodiments, the amplicon is greater than about 400 bp. In some embodiments, the amplicon is between about 400 to 4000 bp, about 700 to 3700 bp, about 1000 to 3400 bp, about 1300 to 3100 bp, about 1600 to 2700 bp, about 1900 to 2400 bp, or about 2100 to 2200 bp. In some embodiments, use of amplicons of the lengths disclosed above are advantageous for downstream processing (e.g., detection and identification of microbial genetic materials) in the methods disclosed
herein.”
Since the amplified sequences are generated from the enriched nucleic acids, the enriched nucleic acids must be of at least the length of the amplified sequences - i.e., of lengths up to 4000 bp, including about “2100 to 2200 bp,” and thereby at least 2000 bp. Further, Singer does teach that the selective lysis of eukaryotic cells/ leukocytes in a sample did not affect the integrity of bacterial DNA from Borrelia burgdorferi (para [0459]: “Demonstrated herein in is that the selective lysis solution does not impact the integrity of Borrelia spirochetes”). Regarding the comment that Singer doesn’t teach the continuity of the enriched nucleic acids prior to amplification, this argument is not clear. To any extent that Applicant is arguing that Singer does not teach amplification of a full length sequence of at least 2000 bp, Singer teaches that it is the amplicons that are up to 4000 bp in length and thereby the amplicon’s produced in the method of Singer comprise a continuous sequence.
Applicant states: “Singer at paragraph [0153] lists numerous detergents or surfactants for eukaryotic cell lysis. Singer does not teach or suggest any non-ionic
surfactant as the eukaryotic cell lysis solution.” It is argued that Singer in Example 1 teaches using a Fos-Choline for lysis and that this is a zwitterionic detergent.
This argument has been fully considered but is not persuasive. As set forth in the rejection, Singer teaches “the eukaryotic cell lysis solution includes one or more detergents or surfactants. In some embodiments, the detergents or surfactants are non-ionic, anionic, cationic, zwitterionic, or non-detergent sulfobetaines. Detergents and surfactants, include, but are not limited to… saponin …. Triton X-100, Triton X-1 14, TWEEN 20, TWEEN 40, TWEEN 80…”. Thus, Singer clearly teaches lysis of the eukaryotic cells using non-target eukaryotic cells using saponin, polysorbate (TWEEN™) or octylphenol ethoxylate (TRITON™), which are non-ionic surfactants. The fact that Singer exemplifies or teaches that alternative agents can be used for performing lysis of eukaryotic cells does not negate the fact that Singer teaches that the eukaryotic cells are lysed with the non-ionic surfactants of saponin, polysorbate (TWEEN™) or octylphenol ethoxylate (TRITON™).
The response argues that “a person skilled in the art seeking to improve the metagenomic analysis described in Nelson would not be motivated to look back toward a PCR-specific sample preparation method as described in Singer, as it is neither designed for nor demonstrated to be compatible with the rigorous requirements of long-read sequencing and genome assembly.”
This argument is not persuasive because it is not directed to the rejection of record. The rejection is not based on a premise of modifying the method of Nelson by performing the lysing, enrichment, PCR and sequencing methods of Singer.
The response argues that “there is no reasonable expectation of success to combine Singer and Nelson in the alleged manner.”
This argument is not persuasive because Singer teaches sequencing the enriched, amplified microbial nucleic acids obtained from samples in which eukaryotic cells were lysed and Nelson was cited for its teachings of sequencing microbial nucleic acids by performing next generation sequencing methods. Thus, the references are analogous prior art. Further, Nelson was cited (in part) for teaching methods for identifying microorganisms present in samples obtained from human subjects wherein the methods comprise removing human DNA, enriching microbial DNA, and then sequencing the enriched microbial nucleic acids. Thus, again, Singer and Nelson are analogous art. Nelson was further teaches comparing the resulting sequencing data to sequencing information in databases, including an antibiotic resistance gene database and the SILVA database to identify particular microorganisms present in the sample. Nelson teaches that this comparison of sequencing data aids in identifying the species of bacteria present in a sample (e.g., p. e5, first full para) and particularly aids in identifying antibiotic resistant microorganisms present in a sample (p. 2234, col. 2 to p. 2235, first para). Applicant’s response does not explain why one would not have had a reasonable expectation of modifying the method of Singer so as to have compared sequencing data generated by sequencing the enriched microbial nucleic acids with sequencing data in microbial genome database or resistance gene database. Nelson provides the guidance and motivation to have made this modification of the method of Singer.
The response argues “neither Singer nor Nelson teaches or suggests that replacing the endonuclease digestion of Nelson with the isolation process of Singer would result in any improved enrichment efficiency and the ability to generate long sequence reads for rapid genome assembly, not to mention that Singer features in utilizing the DIANAs-based PCR for detection of a specific pathogen.”
This argument is not persuasive because it is not directed to the rejection of record. The rejection does not indicate that the method of Nelson in which non-target nucleic acids are digested with a nuclease should be modified to use the enrichment method of Singer. The rejection is over the primary reference of Singer in view of Nelson and Lee.
The response argues:
“the claimed method renders unexpected results over the endonuclease digestion-based method disclosed in Nelson. Even assuming arguendo that Singer could be combined with Nelson, there is no basis to reasonably arrive at the claimed method that exhibits superior effectiveness in identifying bacterial species and resistance genes.”
This argument has also been fully considered but is not persuasive. Again, the rejection is not based on modifying the ;method of Nelson so as to enrich the nucleic acids using the method of Singer, as Applicant is arguing. Applicant has created their own rejection and then argued that rejection. Such arguments are not persuasive. Further, Applicant is relying on the teachings of Example 2 to show improved results over the method of Nelson. Applicant has not shown any unexpected, improved results over the method of Singer. The response cites Example 2 of the specification. This example compares methods in which “a human blood sample containing K. pneumoniae or S. aureus was pretreated with the immobilized adsorption of human nucleic acids or the commercially available kits (i.e., MolYsis Basic 5 Kit, NEBNext Microbiome DNA Enrichment Kit, and QIAamp BiOstic Bacteremia DNA Kit), and then subjected to qPCR, Nanopore sequencing, and identification of the bacterial species and resistance genes based on the sequencing data generated from the Nanopore sequencing.” The primary reference of Singer teaches the same method asserted to provide improved results for removing the eukaryotic (e.g., human) nucleic acids from the lysed sample. - i.e., a solid phase support comprising an anion exchange resin (e.g., see Singer at para [0021] “separating free eukaryotic DNA from the sample by contacting the sample with anionic-exchange microparticles”; and para [0295-0296] of Singer: “the sample with the lysed eukaryotic cells is passed through or contacted with the anion exchange resin… In some embodiments, the anion exchange resin is conjugated to a support substrate. Examples of a support substrate include, but are not limited to, a particle, a bead, a surface, or a sphere. In some embodiments, the support substrate is magnetic, e.g., a magnetic particle or bead”).
Also, Applicant’s arguments regarding the results obtained in Example 2 are not commensurate in scope with the claims since claim 1 generally recites “depleting the non-target nucleic acid by a target nucleic acid enrichment unit of the diagnostic system.” Thereby, claim 1 encompasses methods in which the lysed human / eukaryotic nucleic acids are removed by any means, including nuclease digestion. Further, Example 2 of the specification refers to “immobilized adsorption” of the nucleic acids but does not teach that any solid phase adsorbent, including each of the solid phase adsorbents listed in claim 20 (e.g., any column extraction membrane) provide an unexpected improvement over the alternative methods for removing the released, non-target (eukaryotic) nucleic acids.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682
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