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 .
Response to Amendment
The amendment filed 07/27/2026 is acknowledged. Regarding the Office action mailed 05/06/2026:
The rejections of claims 64-48 under 35 USC 112(b) and (d) are withdrawn in view of the amendments to these claims.
The rejections over the prior art of Fraley (Nucleic Acids Research 41(18):e175, 2013) in view of Kwang (US 2016/0310949) and Yang (US 2013/0217588), and further in view of Smith (US 2011/0207115), and further in view of Vernel-Pauillac (AAC 53(3):1264-7, 2009) are withdrawn in view of the amendment to claim 63, incorporating the limitations of claim 71, which the Examiner indicated as free of the prior art.
After further search and consideration, the Examiner discovered an oversight, in that another prior art reference, cited on an earlier IDS, precludes patentability at this time. The Examiner apologizes for this oversight. The new grounds of rejection are set forth below. This Office action is NON-FINAL.
Per MPEP 2153.01(a): “Specifically, Office personnel may not apply a disclosure as prior art under AIA 35 U.S.C. 102(a)(1) if the disclosure: (1) was made one year or less before the effective filing date of the claimed invention; (2) names the inventor or a joint inventor as an author or an inventor; and (3) does not name additional persons as authors on a printed publication or joint inventors on a patent.” In this instant situation, Fraley (2016) names additional authors not listed as inventors on the instant application.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claim(s) 63-67, 69-70, 72-75 and 77-78 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fraley (Nucleic Acids Research 41(18):e175, IDS reference) in view of Kwang (US 2016/0310949, IDS reference, hereinafter “Fraley 1”), Fraley (Scientific Reports 6:19218, 2016, IDS reference, hereinafter “Fraley 2”) and Wang (Annals of Clinical Microbiology and Antimicrobials 13:3, 2014).
Regarding claims 63 and 64, Fraley 1 taught:
A method of profiling multiple taxonomic units of organisms in a sample comprising nucleic acid, the method comprising:
Page 2, first paragraph (citations omitted): “We have developed a novel technique, universal digital high-resolution melt (U-dHRM)…This new technique unites and reinvents aspects of limiting dilution/digital PCR, broad-based/universal amplification, high-resolution melt (HRM) and microfluidic theory…To demonstrate the utility of U-dHRM, we consider two applications: polymicrobial sepsis diagnosis…”.
Page 2, third paragraph: “U-dHRM for pathogen detection is not hindered by contamination from environmental microbes, contamination within PCR reagents or multi-species polymicrobial infections…”.
Page 2, paragraph spanning columns: “Herein, as a proof of concept, we demonstrate application of the U-dHRM approach for broad-based detection in heterogeneous samples containing multiple pathogens and contaminants commonly involved in polymicrobial sepsis…”.
Page 7, left column, bottom paragraph: “Here, we demonstrate that U-dHRM with broad-based bacterial 16s rDNA primers targeting the hypervariable V6 region and DNA intercalating dye can resolve each bacteria within a mixture of pathogens involved in polymicrobial sepsis and common clinical contaminants (Table 2).”
partitioning the sample…;
Figure 1, panel (a): “U-dHRM uses a diluted sample input such that either zero or one copy of the target molecule is distributed per reaction well.”
Paragraph spanning pages 9-10: “In a real heterogeneous sample where unknown sequences are expected and starting concentrations of targets may be unknown, millions of broad-based UdHRM reactions will ensure enough dynamic range for successful single molecule detections (Table 3).”
performing an amplification reaction, wherein the amplification reaction comprises…amplification primers and one or more DNA intercalating dyes to produce one or more amplicons;
Page 7, left column, bottom paragraph: “Here, we demonstrate that U-dHRM with broad-based bacterial 16s rDNA primers targeting the hypervariable V6 region and DNA intercalating dye can resolve each bacteria within a mixture of pathogens involved in polymicrobial sepsis and common clinical contaminants (Table 2).”
performing melt analysis by simultaneously heating and imaging the partitions, whereby melt curve(s) for the one or more amplicons are produced for each partition;
Figure 1, panel (a): “Broad-based primers amplify all targets individually giving a single melt curve for each target/reaction and allowing quantification and identification of all species, including unknown or unexpected species, in the mixture.”
Page 7, right column: “Next, a spiked mixture of S. aureus, E. faecalis and P. acnes was prepared, digitized, amplified by V6 broad-based primers and assessed by U-dHRM. Figure 6 contrasts a bulk HRMA experiment (red melt curve) and the U-dHRM results (all other melt curves) of the polymicrobial mixture.” See also figure 6.
and generating a profile of the multiple taxonomic units of organisms in the sample based on a comparison of one or more of the shape, peak height, peak height threshold of the melt curve(s) to at least one reference melt curve.
Abstract: “Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves.”
Page 4, left column, last paragraph: “The resulting digital, temperature calibrated and stabilized melt curves are reliably sequence specific and accurately identifiable by matching to a previously generated database of temperature calibrated melt curves.”
Page 7, right column: “Four curves were positively identified by matching to the database curves (Figure 6D).” See also figure 6.
Regarding claims 69 and 70, Fraley 1 taught (page 2, second paragraph, citations omitted): “Subsequently, HRM is performed using DNA saturating dyes or multiple color probes to generate melt signatures that uniquely identify specific bacterial species.” In the case where an intercalating dye was used (as was the case in Fraley’s working examples), detection would necessarily be in a single fluorescent channel. However, in the case where multiple color probes were used, detection in multiple fluorescent channels would be used.
Regarding claim 74, Fraley 1 taught (page 10, right column): “Also, characterization of the contaminants in Taq and other reagents by U-dHRM database building will allow nonrelevant targets to be rigorously identified and excluded from profiling analysis.”
Regarding claim 75, Fraley 1 used genomic DNA (gDNA) in the working examples; e.g., page 7, right column: “Input concentrations were adjusted such that the same amount of the polymicrobial gDNA was added to the bulk well as was distributed across the digital wells.”
Regarding claims 77 and 78, Fraley 1 used a database for identifying the taxonomic units in the sample. Abstract: “Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves.” Therefore, it would be obvious to “identify” a database to use for this purpose when practicing Fraley 1’s method.
The differences between Fraley 1 and the claimed invention are:
While Fraley 1 indicated that “millions of broad-based UdHRM reactions will ensure enough dynamic range for successful single molecule detections”, Fraley 1 did not actually do this many reactions, and therefore did not partition the sample into “at least 10,000 partitions”.
Fraley 1 did not generate amplicons of at least 500 bp or 1000 bp.
Fraley 1 used a single primer pair (thus, a single “set” of primers) for amplifying the 16S rRNA gene; page 2, right column, “Polymicrobial primer design”. The claims require “multiple sets of amplification primers”.
Kwang taught (paragraph [0005], citations omitted): “Systems and methods are described for performing digital PCR using digital microfluidics configured for precise movement of picoliter to nanoliter sized partitions which can be used for partition generation, movement through a temperature gradient for PCR and nucleic acid melting, and signal detection, all within a single consumable device. Additionally, the user can perform quantitative multiplexing using high resolution melting/melting curves techniques.”
Kwang taught (paragraph [0069]): “The melting profile enables quantitative multiplexing as well as aid in categorizing partitions that may be difficult to discriminate as positive or negative. This consumable can be analyzed in real-time through optical imaging to track each partition as it flows through the consumable device and capture data such as signal intensity from PCR amplification and melting profile analysis.”
Kwang taught (paragraph [0048]): “For example, the device described herein is configured to analyze a plurality of samples partitioned into at least 1,000,000.”
Fraley 2 taught (paragraph spanning pages 1-2, citations omitted, emphasis provided): “Since nucleic acid melting interactions become more complex for longer stretches of sequence, we hypothesized that long amplicons could produce additional distinguishing melt features beyond Tm alone. In addition, many genotyping applications require sequence stretches of a thousand base pairs or more to identify key sequence variants or relevant combinations of variations. For example, bacterial identification applications require sequence information spanning thousands of base pairs and multiple hypervariable regions of the bacterial 16 S rRNA genetic loci (16 S).”
Fraley 2 further taught (page 2, 2nd full paragraph, citations omitted, emphasis provided): “We and others have previously designed HRM assays for 16 S-based identification using multiple separate amplification reactions and primer sets to interrogate short sequence stretches within a genetic loci. This was successful for identifying a pure sample, but fails in non-pure samples where multiple genotypes present a mixture of distinct sequences contributing to a single melt curve10. To overcome this challenge, we subsequently developed Universal digital High Resolution Melt (U-dHRM) by diluting the starting material such that each genotype is distributed into its own digital PCR reaction and generates a pure melt curve. However, this approach is still limited because the amplicon’s Tm is typically the only significant differentiating feature of a short amplicon melt curve. To increase the profiling breadth and sequence information generated from a single primer set and to test the ability of HRM to resolve variations in long amplicons, we here explore the use of a ~1000 bp amplicon covering six hypervariable regions in the 16 S genetic loci for HRM curve-based identification.”
To this end, Fraley 2 developed a new algorithm for analyzing long amplicon melt (LAM) curves, and disclosed (1st paragraph of Discussion, page 7): “Using the hypervariable bacterial 16 S gene as a model system, we found that long amplicons (~1000 bp) generate HRM curves of greater shape complexity, occasionally with multiple Tm peaks, than short amplicons (< 300 bp). For 37 clinically relevant bacteria, each with a unique 16 S gene sequence, we are able to automatically identify the bacteria by universally amplifying the gene, generating an HRM curve of the amplified sequence, and training our a nested OVO SVM algorithm to classify the melt curves.”
Fraley 2 concluded (page 8, 3rd paragraph, emphasis provided): “The results of this study suggest that the melting of long amplicons coupled with machine learning techniques can provide a rigorous, rapid, and high-throughput identification technique for long continuous DNA sequence stretches. In addition to the application for microbial identification demonstrated here, our technique can potentially impact many other areas…Because our technique is able to identify long sequence stretches, reassembly or combinations of multiple amplicons is avoided. This enables our LAM technique to scale to the digital/single cell format we previously developed, making it highly sensitive to low level targets and enabling it to be used for identification of heterogeneous genotypes (e.g. polymicrobial infection).”
It is noted that Fraley 1 also mentioned polymicrobial infections (page 11, left column, 1st paragraph, citations omitted, emphasis provided): “The ability of U-dHRM to quantify even low-level targets within hours may prove especially useful for diagnosing polymicrobial sepsis, which is associated with an even higher mortality rate than monomicrobial sepsis. The participation of other Gram-positive and Gram-negative bacteria, anaerobic bacteria, fungi or novel and emerging pathogens in infection can demand tailored treatments, making fast and accurate broad-based profiling to identify multiple microbes highly desirable.”
Wang disclosed a TaqMan probe-based real-time PCR for the simultaneous detection and discrimination of Gram positive (G+), Gram negative (G-) and Candida species which targets the bacterial 16S rRNA gene and the fungal 18S rRNA gene (Candida is a fungus); page 2, left column, 3rd paragraph. In Table 3, Wang compared the results of standard blood culture identification with the results of the multiplex TaqMan assay and correctly identified the infectious agent, including in cases of polymicrobial (mixed) infections. It is noted that in one case, the polymicrobial infection contained bacteria (Enterococcus) and fungi (Candida).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Fraley 1 by using the system taught by Kwang for the analysis, because Fraley 1 specifically suggested large numbers of partitions and Kwang’s system was capable of both generating such numbers of partitions as well as performing multiplex melting curve analysis on such partitions following PCR.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Fraley 1 by amplifying long amplicons of bacterial 16S of up to 1000 bp, as Fraley 2 taught this would leverage HRM curves of greater shape complexity for distinguishing between different species of bacteria.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method of Fraley 1 by using multiple primer sets for amplifying not only bacterial 16S, but also fungal 18S. One would have been motivated to do so because both Fraley 1 and Fraley 2 noted the importance of diagnosing polymicrobial infections, Wang demonstrated the occurrence of mixed infections of bacteria and fungi. Therefore, it would have been obvious to add primers to amplify the 18S rRNA of fungi, since the 16S rRNA primers for bacteria would not be effective to detect the presence of fungi. This would arrive at a method employing multiple sets of primers (one set for bacterial 16S rRNA and another set for fungal 18S rRNA).
Claim(s) 76 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fraley (Nucleic Acids Research 41(18):e175, IDS reference) in view of Kwang (US 2016/0310949, IDS reference, hereinafter “Fraley 1”), Fraley (Scientific Reports 6:19218, 2016, IDS reference, hereinafter “Fraley 2”) and Wang (Annals of Clinical Microbiology and Antimicrobials 13:3, 2014) as applied to claims 63-67, 69-70, 72-75 and 77-78 above, and further in view of Smith (US 2011/0207115, previously cited).
This disclosures of Fraley 1, Kwang, Fraley 2 and Wang have been discussed. Fraley did not disclose amplifying RNA; rather, Fraley 1 amplified DNA encoding ribosomal RNA.
Smith taught (paragraph [0116]): “In some cases, to determine whether a sample contains live or dead microbial contamination, the probe nucleic acid can be designed to detect microbial ribosomal RNA (e.g., microbial 16S rRNA) as the target nucleic acid. The amounts of ribosomal RNA can be very high in growing microbial cells and can be at least an order of magnitude higher than those of genomic microbial DNA. In addition, the presence of microbial rRNA can indicate the presence of live microorganisms since RNA typically degrades quickly (e.g., within minutes of cell death).”
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method suggested by the combined disclosures of Fraley 1, Kwang, Fraley 2 and Wang by amplifying the RNA of 16S rRNA (and 18S rRNA in the case of fungi), rather than the DNA encoding it, as this would have offered the advantage of increased sensitivity in the case of actively growing bacteria, since Smith taught that the amount of RNA could be at least an order of magnitude higher than those of genomic DNA, and in addition offered the benefit of detecting live (versus dead) bacteria.
Claim(s) 68 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fraley (Nucleic Acids Research 41(18):e175, IDS reference) in view of Kwang (US 2016/0310949, IDS reference, hereinafter “Fraley 1”), Fraley (Scientific Reports 6:19218, 2016, IDS reference, hereinafter “Fraley 2”) and Wang (Annals of Clinical Microbiology and Antimicrobials 13:3, 2014) as applied to claims 63-67, 69-70, 72-75 and 77-78 above, and further in view of Vernel-Pauillac (Antimicrobial Agents and Chemotherapy 53(3):1264-7 (2009), previously cited).
The disclosures of Fraley 1, Kwang, Fraley 2 and Wang have been discussed. Fraley 1 taught distinguishing between different species using HRM analysis of amplicons of the 16S rRNA gene, but did not teach distinguishing between variants within a species.
Vernel-Pauillac disclosed use of HRM analysis to discriminate between variants within a species, specifically distinguishing between antimicrobial resistant and susceptible strains of Neisseria based on sequence variation in the gyrA and parC genes (see entire article).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the application to modify the method suggested by the combined teachings of Fraley 1, Kwang, Fraley 2 and Wang by incorporating additional primers to additional targets implicated in antimicrobial resistance, so that HRM analysis could be used to identify the presence of resistant pathogens as disclosed by Vernel-Pauillac, to provide the benefit of detecting such pathogens in a sample, allowing for the appropriate choice of antimicrobial agents for treating infections.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 63-65, 67, 73, 74 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 6 of U.S. Patent No. 11,915,795 in view of Wang (Annals of Clinical Microbiology and Antimicrobials 13:3, 2014).
‘795 claim 6 discloses:
partitioning the sample into at least 10,000 partitions;
“partitioning the reaction mixture into 10,000 or greater partitions”; because the sample was in the reaction mixture, this would also partition the sample
performing an amplification reaction,
“amplifying the nucleic acid in each partition to produce an amplification product”
and one or more DNA intercalating dyes to produce one or more amplicons,
“one or more DNA intercalating dyes…amplifying the nucleic acid in each partition to produce an amplification product”
wherein the one or more amplicons are at least 500 base pairs;
“one or more amplicons have a size greater than 500 base pairs”
performing melt analysis by simultaneously heating and imaging the partitions, whereby melt curve(s) for the one or more amplicons are produced for each partition;
“performing melt analysis of the amplification product in each partition, whereby a melt curve for the amplification product in each partition is produced”
and generating a profile of the [nucleic acid sequence] in the sample based on a comparison of one or more of the shape, peak height, peak height threshold of the melt curve(s) to at least one reference melt curve
“profiling the sequence of the nucleic acid…a comparison of a shape of one or more target amplification product melt curves to a plurality of melt curves from different nucleic acid sequences, and a comparison of a peak height of the one or more target amplification product melt curves to a peak height threshold”; the “melt curves from different nucleic acid sequences” constitute “reference” melt curves
‘795 claim 6 did not disclose that the sample contained multiple taxonomic units, profiling these multiple taxonomic units, or the use of “multiple primer sets”.
Wang disclosed multiplex PCR to simultaneously detect bacterial and fungal pathogens in a sample (page 2, left column, 3rd paragraph). Because the assay targeted the 16S bacterial rRNA and the 18S fungal rRNA, different sets of primers were needed for each.
It would have been obvious to modify the method of ‘795 claim 6 by using multiple sets of primers to detect both bacterial and fungal pathogens in a sample as disclosed by Wang (and, thereby, test for multiple taxonomic units in a sample).
Conclusion
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/SAMUEL C WOOLWINE/ Primary Examiner, Art Unit 1681
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