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 .
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on 8/23/2022, 5/04/2023, and 8/06/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDSs are being considered by the examiner.
Claim Status
Amended claims 1-27 were filed on 8/5/2021 and are under consideration.
Specification Objection
The use of the terms FAM and HEX found on at least Page 28, and VITEK and Qiagen, found on page 37, also CYTO and SYBR, are trade names or marks used in commerce and have been noted in this application. The terms, and any other trade names or marks should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 11, 17,19, 20-22, 26, and 27 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.
Claim 11 is indefinite in the intent of the meaning of set of broad-taxonomic range amplification primers is a set of “universal bacterial amplification primers”. Applicant could intend to mean that the broad-range primers have universal amplification ability for bacteria, or applicant could intend a literal reference to what is conventionally understood to be universal primers, such as M13 or universal primers for ITS, such as ITS1F, or applicant may intend something different entirely. Further clarification is required. Notably, claim 24 depends from claim 11, but is definite because claim 24 discloses two sequences that are not found to be publicly available in the art.
Claim 17 is indefinite in the use of the method according to claim 1, wherein set of broad-taxonomic range amplification primers is unclear regarding whether there is a lack of antecedent basis here and/or whether this makes reference to the exact same primers used in claim 1, or additional primers that have broad taxonomic range. Claim 26 and 27 depend directly or indirectly from claim 17 and are indefinite for the same reason.
Claim 19 is indefinite in the meaning of “essentially all strains or species” and essentially all strains ..from a microbial… Kingdom or Domain, since two ordinary artisans may come to different conclusions regarding what constitutes essentially all strains, and since it markedly unclear how one would know when they had met the limitation of essentially all strains, from for example a Kingdom or Domain when the vast majority of microbes remain uncharacterized, and even within the human body, novel taxa are still being identified (Kowarsky et al 2017 114:9623-9628). Claims 20, 21, 22 depend from and are indefinite in claim 19.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 1-27 are 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 Written Description rejection.
The MPEP § 2163 states that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Nature of the Invention:
The invention is directed to a method of microbial identification based upon DNA analysis, where Claim 1 recites “A method for identifying a microorganism at species or strain level in a biological sample by polymerase chain reaction (PCR), said method comprising the steps of: a) providing a biological sample suspected of comprising microbes, and optionally isolating nucleic acid sequences comprised in said biological sample; b) PCR amplifying at least one microbial rRNA internal transcribed spacer (ITS) region comprised in said, optionally isolated, nucleic acid sequences using a set of broad-taxonomic range amplification primers for amplifying said at least one microbial rRNA ITS, to thereby generate PCR amplicons; c) recording a high resolution melting curve for the PCR amplicons generated in step b), and recording the length of the PCR amplicons generated in step b) by capillary electrophoresis or sequencing; d) comparing the high resolution melting curve recorded in step c) with a database comprising high resolution melting curves of reference amplicons generated from reference microbial species or strains of known taxonomic identity using the same set of amplification primers, to thereby obtain a first taxonomic identity indicator of a microorganism present in said sample; e) comparing the length of each PCR amplicon having a distinct length recorded in step c) with a database comprising PCR amplicon lengths of reference amplicons generated from reference microbial species or strains of known taxonomic identity using the same set of amplification primers, to thereby obtain a second taxonomic identity indicator of a microorganism present in said sample; f) identifying a microorganism present in said sample to the species or strain level if the first and second taxonomic identity indicator match.”
Specifically relevant to this rejection, claim 1b addresses:
amplifying at least one microbial rRNA ITS region, using a set of broad-taxonomic range amplification primers for amplifying said at least one microbial rRNA ITS, to thereby generate PCR amplicons
This description encompasses a very large genus of primers, namely any primers that are capable of amplifying one or more microbial rRNA ITS regions, where the primers have a “broad-taxonomic range”, or wide range at a taxonomic level, which can be any taxonomic level.
State of Art: ITS primers have been widely used for many purposes, and notably, primers have been developed for multiple ITS regions in many taxa, though not so much for bacteria. Akhoundi et al (Molecular Aspects of Medicine, 57:1-29, 2017), addressed ITS use for leishmaniases studies, specifically considering species classification, and the sensitivity and specificity of markers to quantify variation from subgeneric levels (Abstract). Akhoundi pointed out that the most variable regions of rRNA genes, ideal for typing Leishmania species, are the ITS regions (Pg. 7 right col, 2nd full para, and final para). Cafarchia et al (Inf, Gen and Evol, 20:336-331, 2013) considered detection at the species level of pathogenic dermatophytic fungi of four Genus’ (Abstract). Here ITS use supported grouping by clinical traits, as well as species separation of geophilic species from other members of Arthrodermataceae (Pg 337 right col, final para; Table 2). Less common at the time of the filing was ITS use for bacterial species identification, though Andini (US20170231257 A1 published 11/9/2017) disclosed ITS primers for bacterial amplicon production [0006]. Thus, in sum, ITS primers are known in the literature, here used in line with the claims to identify species, but at this level, insufficient to be considered quite broad, particularly in line with the breadth of the claim. There is scant data regarding bacterial ITS amplification. Of significance as well, are the vast majority of microbes that we do not yet believe have been taxonomically identified. A fraction of this unknown volume is depicted in Kowarsky (PNAS 114: 9623-9628, 2017), who disclosed that even within human blood cell free DNA samples (inclusive of the microbes contained therein), many organisms (microbes and viruses) represent entirely novel taxa, just of the human microbiome alone (Abstract). Thus, we cannot necessarily even yet determine the entire Genus encompassed by all broad-taxonomic range amplification primers for rRNA ITS.
Guidance from Specification:
There is a Definitions section in the Specification (beginning on Pg 17), however “broad-taxonomic range” amplification primers are not defined.
Rather, the Specification disclosure on this topic is expansive:
Pg 4 addresses that ITS amplification is not species specific and Pg 5 states that universal DNA amplification for ITS can be at Genus, Family, Order, Phylum or Kingdom level.
On Pg 20, in an embodiment, these primers are for amplifying a microbial ITS region up to and including all strains or species from a Kingdom or Domain. However, “essentially all strains or species” preferably refers to more than 50%, up to 90+%, of species/strains in a genus, family, order, class, phylum, kingdom and/or domain. In another embodiment these primers are for at least one ITS region, amplifying 16S-23S rRNA ITS, then 23S-5S, and so on, with alternate regions.
Further “amplification primers” are defined broadly as “primers that hybridize to opposite strands of the target DNA sequence(s) and flank the region to be amplified”.
This description therefore is comprehensive, painted at high level of generality. The specification reveals SEQIDs for particular bacterial phyla (described below).
Specification Examples:
Example 1, prophetic, written in present/future tense at the start, addresses combining melting curves and 16S-23S IS fragment length data for species identification. The “high-level” writing lacking detail, indicates that DNA regions broadly conserved throughout taxonomical groups may be used when they have specific characteristics.
Example 1 continues with “strain selection and cultivation” which occurred for 12 bacterial species. The “DNA isolation” section disclosed exactly that; the “PCR” section disclosed phylum specific amplification of 16S-23S regions, with one SEQID for a forward primer targeting four Phyla: Firmicutes, Acintobacteria, Fusobacteria Verrucomicrobia, and another SEQID targeting one Phylum Bacteroidetes, plus three reverse primers providing universal coverage, SEQID 3-5, and additional primers with a subset of the aforementioned primers for the Proteobacteria phylum. The “third” PCR reaction mix was primers for a broad collection of bacteria, where the primer incorporated a suite of inosines to ensure broad reactivity.
Next, “species identification by melting curve analysis can be done…” was disclosed.
Next, “Fragment length analysis”; PCR, was performed with automated species calling by software linking peaks to IS-profile data for >500 microbial species.
Next,“Results” of melting curves and fragment analysis of eight species from three phyla (above), FIG3, where profiles are highly variable between species (Specification, Pg 40).
Example 2 addresses accurate identification for identical length fragments (e.g. similar melting curve or fragment length results for two species), Fig 4 depicts two different melting curves, or one comparison of two species, S pyogenes and C jeikeium.
Example 3 if the final example and addresses accurate identification when melting curves are the same, and reliance on 16S-23S fragment length profiles is discriminatory, and FIG5, depicts one comparison of two species, E. cloacae and K. pneumoniae.
Table1 lists a single forward primer for each Firmicutes and Bacterioidetes phyla, the 3 reverse primers (SEQID 1-5); a single forward primer for Proteabacteria Phylum and several reverse primers (SEQID 6-13). Next are one pair of primers described as universal bacterial ITS primers, with proline runs (SEQ ID 14-15).
What the disclosure does not teach:
There is not evidence of possession of the broad genus of primers that is any and all broad-taxonomic range amplification primers for amplifying said at least one microbial rRNA ITS, and which is also used also to meet additional relevant claim(s) limitations. This claim phraseology is much broader than the working examples depicting possession of one universal bacterial ITS primer set, three phyla-level bacterial primers that amplify <15 bacterial species, plus one comparison of two bacterial species distinguished by amplicon melting and one comparison of two bacterial species distinguished by fragment length.
Thus, a small subset of amplified bacterial species, of phyla, and of the range of 16S-23S for ITS, present evidence of possession of a fraction of this large, claimed genus of primers, but lacking is the breadth and variety that would encompass any and all broad-taxonomic range primers.
Conclusion: Taking into consideration the factors outlined above, including the nature of the invention, the state of the art, the limited guidance provided by the applicant and the specific example, it is concluded that Applicant does not indicate possession of the broad Genus of primers of the invention, as described.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, the claims will be given their broadest reasonable interpretation, in view of the specification, and as set forth at MPEP§ 2111.
For clarity of the record, interpretation, in view of the specification (Pg 14) initially in Claim1b, of “broad-taxonomic range amplification primers” for amplifying at least one ITS region, will be considered to mean primers that amplify sequences, the primers which may be operative at any taxonomic level, including for example Phylum or Family, or Genus.
In Claim 5, the use of multiple strains will be interpreted to mean more than strain, per Merriam-Webster.com definition of consisting of more than one.
Claim 17 will be interpreted to mean the same or additional broad taxonomic range primers as in claim 1.
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.
Claims 1, 2, 3, 5, 7, 17, 18, 26, 27 are rejected under 35 U.S.C. 103 as being unpatentable over Andini (Andini et al, US10,570,464B2, filed 5/8/17) in view of Budding (Budding et al., FASEB 24: 4556-4564 published 2010).
The instant claim 1 is directed to: A method for identifying a microorganism in a biological sample by PCR
b) PCR amplifying microbial rRNA ITS region using a set of broad-taxonomic range primers for microbial rRNA ITS, to generate PCR amplicons;
c) recording a high resolution melting curve for the PCR amplicons,
and recording the length of the PCR amplicons by electrophoresis or sequencing;
d) comparing the melting curve recorded with a database comprising high resolution melting (HRM) curves of reference amplicons from reference microbial species or strains of known taxonomic identity using the same primers, to obtain a taxonomic identity of a microorganism present in said sample;
e) comparing the length of each PCR amplicon having a distinct length recorded in step c) with a database comprising PCR amplicon lengths of reference amplicons generated from reference microbial species or strains of known taxonomic identity using the same set of amplification primers, to thereby obtain a second taxonomic identity indicator of a microorganism present in said sample;
f) identifying a microorganism present in said sample to the species or strain level if the first and second taxonomic identity indicator match.
Re: claim 1, 2, 3, 5, 7, 18, Andini disclosed a method of identifying bacteria in a biosample by isolating the bacterial DNA from the sample, amplifying a portion or more of ITS (Col 2, ln -9; claim 1, claim 4), using ITS primers with broad taxonomic range, amplifying bacterial ITS (Col 2, ln 25-45), generating ITS amplicons (by PCR, claim 7). These primer sets came from a group with high (.95) identity to particular sequences (Col 2, 25-35) required to hybridize to and amplifying bacteria (Co 2, ln 40-45).
Re: claim 5 Andini disclosed broad range primers for rRNA ITS amplification (e.g. 30, 5-10) and results are found in the blast below of the ITS forward primer used by Andini retrieves blast hits from more than one Genus:
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Amplicon generation can be performed along with 16srRNA and 23S rRNA phylogenetic analysis (Col2, line 10). This biosample with eubacterial pathogenic microbes (Col 1, line 47) also meets instant claim 2, and the sample may be saliva or a lavage sample (Col 2, 54-60) meeting instant claim 7. The high-resolution melting (HRM) analysis was performed on the sample amplicons, where notably length impacted amplicon Tm (13, 50-52). Sample amplicons were aligned with reference library high resolution melting (HRM) curves for ITS from bacterial species, where high diversity in species curve shapes were found within a given Genus (e.g. Bacillus) (Col 26, 6-12, 19) (instant claim 5, 18). Bacterial species were identified based upon comparing data of HRM curves for ITS amplicons (curve shape and peak positions, amplicon Tm) to the ITS reference curves for the known bacterial species comprising the reference library database of species reference curves (Col 1 end to Col 2, Col 2, 1-7, claim 3, Figs 4-92, claim 22; Col 26, lines 5-25; claims 1,2,21-22). The bacteria may be sequenced (Para 3, 4-6).
At least a portion of ITS was amplified and sequenced for microbial identification, it would have been obvious to include this sequence in the relation database generated, with melting curve data and species identification, as further information such as type of infection, category of pathogen (e.g. gram negative) and more, could be included as additional fields, including flat files, which could be sequence (Col 20, 64 to Col 21, 22, instant claim 18), as Andini disclosed that amplicon length and base composition impact Tm (Col 13, 46-55). ITS region length heterogeneity as a function of species (Col 15, lines 1-5) and the importance of sequence differences (Col 15, 43-50) were indicated as the rationale for differences in species HRM curves (Col 15, lines 5-15; with Tm shape and temperature (Col 15, 40-60), length and sequence are the important factors driving melt curve shapes, Col 28, 45-50). The significance of length was clearly recognized so conceptually this point is not novel. Also addressed was the relevance of length in making ITS their actively targeted region of value (Col26, 1-10).
Andini disclosed that HRM alone was not as information rick as including sequencing (Col 24, 36-37). Andini also e.g., performed 16S sequencing on blood culture samples (27, 10-16) and depicted heteroduplex analysis of E coli ITS but was not preforming capillary electrophoresis or sequencing all of the ITS amplicons.
Andini did not disclose use of fragment length in the same manner as the instant claims, nor the recording amplicon length by electrophoresis or sequencing.
Re: claims 1, 3, 4, 6, 8, 13, 17, 26, 27, addressing instant claim 1, Budding disclosed PCR-based profiling, combining bacterial species’ differentiation by ITS length (16S-23S rDNA interspace region) with phylum-specific primer labelling (Abstract). Labelling of IS fragments enabled fragment sorting into clinical groups, (Pg. 4563, right col, para 2) with some species level identification (Pg 4562, right col, final para). Fragment analysis was conducted on PCR products (and analyzed with software, Pg 4558 right col para 1), where length of IS fragments was used (Pg 4558 right col, para 3). Fragment size was determined on gels with a ladder (ruler), and bands were extracted, reamplified, cloned, and IS fragments were also sequenced (Pg 4558, right col, para 4-5).
Re: claim 3, Budding disclosed reference amplicons, in vivo and in silico, from PCR ITS-based amplification of microbial species using the same ITS primers (Pg 4559-4560, right col final two para to left col para 1, left col final para; Pg 4561 right col para to on; Pg 4563).
Re: claim 4, Andini and Budding both considered pathogenic bacteria, and had length-melting curve data and Andini recognized the relevance of the this within taxa highly variable length and sequence data in ITS. Further, Budding obtained sequence data and used it with melting curve data, identifying species specific length polymorphism data combined with Phylum specific sequence polymorphisms of 16S and obtaining peak profiles for species within Phyla and species level length profiles (Pg 4557 left col para 3; (Pg 4557 right col, para 1, that was used with melting curve data). Database construction ensued, the technique for high-throughput profiling with capillary electrophoresis (Pg. 4557, left col, para 3).
Re: claim 6, Andini considered that ITS between 16S-23S rRNA use as a good single species level locus (Col 28, 27-30).
Budding explicitly used 16S-23S IS (Pg 4557, left col 2nd para). Budding articulated the extreme size variability and sequence making this more suitable for analysis of complex communities (Ppg 4557 left col, 2nd para).
Re: claim 8, Budding explicitly disclosed Bacteroidetes and Firmicutes since they comprise most of the intestinal microbiota that Budding was investigating.
Claim 13, PCR amplicon length recorded by capillary electrophoresis or sequencing was disclosed by Budding (Pg 4558 left col last para to right col first para and third para; Pg 4558 right col last two para to Pg 4559, left col).
Re: claim 17, 26 and 27, Budding disclosed a set of broad primers for amplification of the 16S-23S interspace region (Pg 4557, left col, para 2), and selected phylum specific fluorescently labelled forward primers for Firmicutes and Actinobacteria and used unlabelled versions of these for amplification of the fragments that were used for sequencing (Pg 4558, left col, para 3-4).
Prior to the effective filing date of the application, it would have been prima facie obvious to one of ordinary skill in the art to have incorporated the methods of Budding into the work of Andini since this would have benefitted Andini’s pathogen detection ability and thus motivated Andini. Andini was interested in eubacteria in general and pathogen detection so that subjects could be diagnosed and treated for disease, and had Andini incorporated the Phyla and species Budding detected, including Firmicutes and Acintobacteria this would have expanded the library of Andini, and improved detection ability, and thus motivated Andini. Andini could have incorporated the ITS regions, primers and library data of Budding, but most clearly would have benefitted from the additional data obtained by incorporating Budding’s method of capillary electrophoresis, and amplicon sequencing which would have improved ability to identify/ confirm pathogen identity, which Andini had noted themselves (discussed above) was valuable, so motivation would have been high. Budding had disclosed that sample collection to data analysis was easy to implement and the method’s accessibility and high-throughput potential enabled the technique to be a clinical tool in analysis of large data sets (Pg 4563 final para), benefitting Andini, given their ultimate goal was improving human (or other subject) health through identification of disease-causing pathogens and reduction of suffering.
Claims 9 is rejected under 35 U.S.C. 103 as being unpatentable over Andini et al. (US10,570,464B2, filed 5/8/17) in view of Budding (FASEB 24: 4556-4564 published 2010) further in view of Hendrick (US2010/0129816 A1; published May 27, 2010).
The contributions of Andini and Budding have been discussed.
Re: Claim 9, amplification primers comprise SEQIDNO 2-5, Hendrick disclosed a method for analyzing microorganisms from different taxonomic groups (Abstract), having recognized disclosure of novel phylotypes found in human intestinal microbial flora, including particularly Firmicutes, Bacteriodetes, also Proteobacteria, Actinobacteria, Fusobacteria and Verrucomicrobia ([0003]-[0004]).
Human microflora health and disease was of interest to Hendrick ([0004]), in addition to pathogenic microbes in general ([0007]-[0008]), as was finding analysis methods not so challenging as extant ones [0006]. Bacterial identification methods at the time included amplifying 23SrDNA with degenerate primers, or 23S/5S rDNA with one primer for a genus and a second primer for a species ([0011]-[0012]).
Hendrick disclosed using first primers for a first conserved (homologous) location of a first taxonomic group (e.g. phylum, class), second primers for a second conserved location for a second group, third primers for a third conserved location but in the first taxonomic group, and fourth primers to a final conserved location specific for the second taxonomic group ([0017-0021][0037]). Amplification produces fragments with size differences, and different nucleotide sequences. The fragment size differences are detected, and assignable to distinct phyla, when the aforementioned primers are used, where combining primers increases the percentage of microorganisms identified in a taxonomic group ([0023]-[0026], [0051], [0081]-[0082]).
SEQID 1-5 are valuable, vetted primers demonstrated to be specific to particular taxonomic groups, and used for analysis of species in particular phyla ([0129][0132]). SEQID 1 would be for Firmicuta and Acintobacteria [0137]. Instant SEQID2 is Hendrick SEQID2 (BacISf) forward primer (Pg 8 right col top line); instant SEQID3-SEQID5 are the reverse primers, where Hendrick SEQID3 (DUISr1), SEQID4 (DUISr2) and SEQID5 (Pg8 right col [0141]) are the same. SEQID2 is specific for phylum Bacteriodetes [0137].
Prior to the effective filing date of the invention, it would have been prima facia obvious to one of ordinary skill in the art, to have employed the primers of Hendrick in the method of Andini as modified by Budding, to expand the identifiable phyla allowing for further use of the amplicon lengths for species identification. The motivation to combine Hendrik to the method of Andini as modified by Budding, would have been come from an ability to identify any additional taxa possible with these extant primers.
Claims 10 is rejected under 35 U.S.C. 103 as being unpatentable over Andini (Andini et al, US10,570,464B2, filed 5/8/17) in view of Budding (Budding et al., FASEB 24: 4556-4564 published 2010), further in view of Budding-‘979 (Budding and Savelkoul, WO2015170979A1, published 12 Nov 2015). The contributions of Andini and Budding have been discussed.
Re: Claim 10, SEQID 6 and 7-13, Budding-‘979 disclosed instant SEQID 6 and 7-13, where instant SEQID6 is a 100% match to Proteobacteria 16S-23S rRNA23, a specific forward primer ProtISf (SEQ 6, claim 4), instant SEQID 7, a 100% match to Proteobacteria 16S-23S rRNA23, a specific reverse primer, DPISr1 (SEQ 7, claim 4), SEQ 8, a match to Proteobacteria 16S-23S rRNA specific reverse PCR primer DPISr2, SEQ 8, claim 4; SEQ 9: a match to Proteobacteria 16S-23S rRNA specific reverse PCR primer DPISr3, SEQ 9, claim 4; SEQ10: Proteobacteria 16S-23S rRNA specific reverse PCR primer DPISr4, SEQ 10, claim 4; SEQ 11, Proteobacteria 16S-23S rRNA specific reverse PCR primer DPISr5, SEQ 11, claim 4, Seq 12: Proteobacteria 16S-23S rRNA specific reverse PCR primer DPISr6, SEQ 12, claim 4, SEQ 13: Budding used this as a primer for ITS (claim 4 seq id 13) for Proteobacteria (e.g. GI disease and more) 16S-23S rRNA reverse PCR primers DPISr7 (SEQID13).
Prior to the effective filing date, it would have been prima facie obvious to one of ordinary skill in the art to use the primers of Budding-‘979 in the method of Andini as modified by Budding to amplify Proteobacteria with extant valuable, known primers. Andini was interested in detection of bacterial pathogens, thus would have been motivated to use these known, effective primers for Proteobacteria pathogen identification in their work, which could include Acinetobacter (baumannii) an opportunistic pathogen.
Claims 12, 14, 15, 25 are rejected under 35 U.S.C. 103 as being unpatentable over US10,570,464B2 (Andini et al, filed 5/8/17) in view of Gransee (Proc of SPIE Vol: 9487 published: 2015). The teachings of Andini with respect to the prior claims has been discussed.
Re: Claim 12, amplifying by qPCR, claim 14, recording melt curves on a miniaturized device, and claim 15, database of melting curves and PCR amplicon lengths generated from reference microbial species further comprises reference amplicons from human sequence as controls for aspecific amplicon generation,
Andini indicated that amplification by PCR included a wide-array of PCR types (Col 2, 15-20) and that their work could be a simple add-on to most qPCR with HRM capabilities as beneficial for early testing to aid in diagnosis of illness (Col 29, ln 54-60).
Gransee disclosed fast amplification qPCR integrated with melting curve analysis, performed using a lab-on-a-chip system (Fig 4 for B. subtilis)(meeting instant claim 25). The result is high quality qPCR amplification plots with sensitive melt curve analysis and internal controls to validate performance (Abstract). Gransee disclosed a control in the PCR amplification could be the detection of human DNA sequence in parallel to amplifying the targeted pathogen sequence (Pg 5, para 1, last few sentences), which aligned with human sample material with pathogens collected by Andini. The intended use of the Gransee invention is point of care medical diagnosis (and others) in alignment with the uses of Andini (Pg 1, para 1; Pg 6 penult para).
Prior to the effective filing date of the instant application it would have been prima facie obvious to use the sensitive and rapid method of Gransee, improving speed/sensitivity, in the method of Andini so that optimal results could be obtained as fast as possible, on-site for medical diagnoses where needed, potentially allowing for more rapid medical treatment and intervention that could decrease suffering due to bacterial infection and perhaps save lives.
Conclusion
All claims are rejected. Claim 16, 23 and 24 are free of the art.
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/LISA HORTH/ Examiner, Art Unit 1681
/GARY BENZION/ Supervisory Patent Examiner, Art Unit 1681