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 .
DETAILED ACTION
Claims 3-4 6, 8-9, 11, 13, 16, 19-23, 25, 28-30, 32, 35-38, 40 and 42 are canceled. Claim 44 is new. Claims 1-2, 5, 7, 10, 12, 14-15, 17-18, 24, 26-27, 31, 33-34, 39, 41 and 43-44 are pending and under consideration.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 05/04/2026 has been entered.
Priority
The instant claims are entitled to an effective filing date of 02/28/2023.
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.
Claims 1-2, 5, 7, 10, 12, 14-15, 17, 18, 26-27, 33-34, 41 and 43 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 claims contain 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, at the time the application was filed, had possession of the claimed invention.
The claims are drawn to a genus of reporter molecules that can be expressed in a control bacterial strain of the same genus as the target bacteria, and are not naturally present in the target bacteria. Claims 1-2, 5, 7, 10, 12, 14-15, 17, 18, 26-27, 33-34, 41 and 43 do not substantially limit the structure of the molecule required to function as a reporter. The specification does not disclose a representative number of species of the claimed genus by reduction to practice, and does not provide adequate guidance with regard to the structural features of the reporter molecule required. Therefore, one of skill cannot immediately envision which reporter molecules will have the required functional characteristics, and one could not conclude that Applicant was in possession of the claimed genus at the time the filing.
For claims drawn to a genus, MPEP § 2163(3)(a)(ii) indicates 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. A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
The instant specification reduces to practice one example of a reporter molecule. In example 1, the specification teaches utilizing a Salmonella quality control strain engineered with GFP, and discloses that it can be used to determine if any potential positives are caused by cross-contamination. See [0101]. In example 2, the specification teaches utilizing a Listeria quality control strain tagged with GFP. See [0144]. In example 3, the specification teaches detecting E. coli in raw beef trim, raw ground beef, leafy greens, dried hemp flower and spunbonded polyolefin. See [0174]. The labeled strain, UVQC E. coli (O157:H7) containing a GFP marker, is used. The specification teaches that the GFP marker amplifies with the E. coli in the SIMUL-qPCR E. coli with GFP Assay, allowing easy, immediate differentiation upon detection of the target pathogen. See [0175]. In example 4, the specification teaches a Salmonella and STEC (shiga toxin producing E. coli) assay in which GFP serves as the reporter. See [0204]. In example 5, the specification teaches comparing the Cq values between E. coli 0157: H7 Target of Interest and the GFP Target. See [0247]. As such, examples 1-5 of the instant specification reduce to practice one GFP reporter molecule.
MPEP 2163(3)(a)(ii) states that “the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are "representative of the full variety or scope of the genus," or by the establishment of "a reasonable structure-function correlation. Such correlations may be established "by the inventor as described in the specification," or they may be "known in the art at the time of the filing date”. Considering the lack of guidance provided in the specification, one would appraise support from the state of the art to extrapolate the correlation between molecule structure and the claimed reporter function.
With respect to the state of the art on PCR reporter molecules, Boulin (The Online Review of C. elegans Biology, 2006) states that a crucial aspect of gene expression studies is choosing an adequate type of reporter for the purposes of the experiment. Each reporter differs in the amount of information it provides about the expression of a gene. The three general types of reporter gene constructs are: 1) transcriptional reporters, 2) translational reporters, and 3) "smg-1-based" transcriptional reporters. See Boulin p. 1 section 1.2 first paragraph. Boulin discloses that a large number of vector backbones and reporter gene derivatives are available. See p. 3 section 1.5.2 first paragraph. Thus, Boulin illustrates the breadth and unpredictability in the art of reporter molecules.
In view of the prior art, the instant disclosure does not satisfy the written description requirement because the species disclosed do not adequately represent the substantial variation within the claimed genus. The breadth of potential structures encompassed by the claims is substantial. As evidenced by Boulin, reporter molecules vary functionally in terms of the information it provides. The instant specification reduced to practice one example of reporter molecule capable of being expressed in a control bacterial strain of the same genus as a target bacterium. This represents a fraction of the possible number of species within the breadth of the claims. Accordingly, one of skill could not conclude that Applicant was in possession of the claimed genus at the time the application was filed.
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.
Claim 33-34, 39, 41, and 43-44 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.
Claims 33-34, 41, and 43 recite “Escherichia coli (E. coli), including E. coli that carry a Shiga toxin 1 gene, a Shiga toxin 2 gene, and/or an Intimin gene”, which renders the claims indefinite because it is unclear whether the limitations following the term “including” are part of the claimed invention or merely exemplary. Consequently, there are multiple reasonable claim interpretations. In one interpretation the claims exclude E. coli that do not carry Shiga toxin 1 gene, a Shiga toxin 2 gene, and/or an Intimin gene; and under an alternative interpretation the claims require any E. coli such as E. coli that carry a Shiga toxin 1 gene, a Shiga toxin 2 gene, and/or an Intimin gene.
Claims 39 and 44 depend from claim 34 and 41 respectively and are rejected for the reason set forth above.
Claim 41 recites “wherein each control bacterial strain is configured to be present only in a control sample and not intentionally added to a test sample” in lines 7-9, which renders the claim indefinite because it is unclear how the limitation structurally limits the claimed kit product. Furthermore, claim 41 recites “wherein the one or more first PCR primer sets and the one or more second PCR primer sets are configured for multiplex real-time PCR such that amplified target PCR product and amplified control PCR product are detected in different fluorescence channels of a real-time PCR instrument, and wherein the one or more first primer sets and the one or more second primer sets are optimized such that cross-contamination of the test sample is capable of being determined when: (i) an amplified control PCR product is present in the test sample; and (ii) a Cq value of the amplified control PCR product occurs within 0 to 4 PCR cycles of, or prior to, a Cq value of the amplified PCR product of the test sample” in the last 10 lines. It is unclear how this recitation structurally limits the kit product claim. In one interpretation the claim requires the structure of a real-time PCR instrument, and under an alternative limitation the recitation is an intended use of the kit. Furthermore, the term “optimized” renders the claim indefinite because the term is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
Claim 43 depends from claim 41 and is rejected for the reasons set forth above.
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.
Claims 1-2, 5, 7, 10, 12, 14-15, 17 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023), in view of Noah (J Food Prot. 2005 Apr;68(4):680-6. doi: 10.4315/0362-028x-68.4.680. PMID: 15830656), with evidence from Microbiologics (Microbiologics SCA, 2024)
Regarding claim 1, Biswas teaches a method for determining the presence or absence of target bacteria in a sample comprising (i) providing a sample; (ii) contacting an aliquot of the sample with a lysis mixture under conditions to lyse at least a portion of cells, thereby generating a lysate; (iii) contacting an aliquot of the lysate with a detection mixture, thereby generating an assay mixture; (iv) amplifying target cDNA; (v) detecting presence or absence of the amplified target. See claim 74 of Biswas. Biswas teaches one or more positive control samples used alongside test samples. See [0060]. Biswas teaches lysing and amplifying samples by adding dilutions to cluster tubes (i.e. physically separate sample tubes) and adding lysis enzyme solution. Biswas teaches adding lysed samples (i.e. lysate) to PCR tube strips containing reaction mixtures. See [0225]. As shown in table 12, the reaction mixture includes a Salmonella forward primer, a Salmonella reverse primer (i.e. bacterial detection PCR primer set) and control RNA. See [00225] for table 12. Biswas teaches a detection mixture comprising a primer having specificity for a single-stranded nucleic acid region comprising a nucleic acid sequence of the target RNA (i.e. bacterial detection PCR primer set). See [0185]. The detection mixture components further comprise a control RNA (i.e. control primer set) and a control probe able to detect the control RNA. See [0186]. Biswas teaches quantifying the nucleic acid presence (i.e. detecting presence or absence) based on the signal cycle threshold (i.e. Cq) of the amplification reaction. See [0166].
Biswas does not teach a control sample comprising a control bacterial strain of the same genus as the target bacteria but engineered to express a reporter molecule not naturally present in the target bacteria, the control bacterial strain being present only in the control sample and not intentionally added to the test sample.
Noah teaches engineering enterohemorrhagic Escherichia coli O157:H7 and Salmonella Typhimurium to express the gene for a modified green fluorescent protein (GFP) (i.e. reporter molecule) and evaluating their potential use as positive controls in sample analyses. See the abstract. Noah teaches the following samples: three feta cheese, one cooked shrimp, one raw shrimp and one cooked crawfish. Each set of samples includes an uninoculated control (e.g. a test sample), one sample inoculated with strains E. coli O157:H7 ATCC 35150 and Salmonella Cerro ATCC 10723, and one inoculated with the combination of the E. coli O157:H7 ATCC 35150 or Salmonella Cerro ATCC 10723 strain and the GFP-positive strain (i.e. control sample). See p. 682 left column last passage. As shown in table 2, the uninoculated samples, i.e. “None”, showed no presence of E. coli O157:H7 (i.e. target bacteria). As shown in table 3, the uninoculated samples showed no presence of Salmonella serotype Cerro ATCC 10723 (i.e. target bacteria).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Noah’s uninoculated test sample for Biswas’s sample, and to further substitute Noah’s control sample containing the GFP-positive bacteria strain for Biswas’s positive control sample. One of ordinary skill in the art would be motivated to use Noah’s uninoculated test sample because Biswas suggests detecting Salmonella from environmental samples including for food safety testing and environmental monitoring. (see [0003]); and Noah teaches food samples without additional added bacteria. There would have been a reasonable expectation of success because Biswas demonstrates designing primers specific to Salmonella enterica (see [0220]), and Noah demonstrates detecting S. enterica (i.e. the Salmonella serotype Cerro ATCC 10723) on the uninoculated test samples (see table 3). One of ordinary skill in the art would have been further motivated to use Noah’s control sample because Noah suggests that the Salmonella GFP-positive strain should not occur naturally and can be easily differentiated as a control strain in the event of cross-contamination; thus, the Salmonella GFP-positive strain may offer an advantage as a replacement control strain for analyzing food samples (see p. 686 second to last paragraph). There would have been a reasonable expectation of success because Biswas teaches using a positive control alongside a test sample (see [0060]).
Biswas, Noah and Murphy do not teach when an amplified target PCR product is detected in the test sample, determining that cross-contamination of the test sample has occurred when: (a) an amplified control PCR product is present in the test sample; and (b) the Cq value of the amplified control PCR product occurs within 0 to 4 PCR cycles of, or prior to, the Cq value of the amplified target PCR product, if target bacteria are present in the test sample; and when cross-contamination of the test sample is determined to have occurred, designating any positive result for the target bacteria in that test sample as a false-positive due to cross-contamination based on the Cq values without additional culture-based or plate fluorescence confirmation.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Noah’s uninoculated test sample for Biswas’s sample, as discussed above, and in the process recognize that an amplified target PCR product necessarily would not be detected, because Noah teaches confirming biochemically and serologically there are no E. coli O157:H7 ATCC 35150 (see table 2 caption) and Salmonella Cerro ATCC 10723 (table 3 caption) present in the uninoculated test samples.
Regarding claim 2, Noah teaches adding inocula to food matrixes to enrichment broths during blending. See p. 682 left column second paragraph.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply Noah’s enrichment incubation step prior to Biswas’s lysis step. One of ordinary skill in the art would be motivated to do so because Noah suggests combining food matrix samples (i.e. uninoculated test sample and GFP-positive control samples) with enrichment media. There would have been a reasonable expectation of success because Biswas demonstrates culturing strains ([0224]) before lysing the sample (see [0225]).
Regarding claim 5, Noah teaches the following samples: three feta cheese, one cooked shrimp, one raw shrimp and one cooked crawfish. See p. 682 left column last passage.
Regarding claim 7, Biswas teaches a test environmental sample that is derived from food processing and/or beverage processing environmental sources. See [0077].
Regarding claim 10, Biswas teaches environmental sample collected from the environment, such as that of a manufacturing plant. See [0044]. Furthermore, Biswas teaches that foods that have the potential to be contaminated with Salmonella include sprouts and other vegetables, and fruits. See [0073].
Regarding claim 12, Biswas teaches a pre-treatment step comprising contacting an aliquot of a sample with a pre-treatment mixture under conditions to remove nucleic acids not associated with intact cells in the aliquot, and wherein the pre-treatment mixture comprises a nuclease that cleaves nucleic acids and the lysis mixture comprises a component that inactivates the nuclease. See claim 83 of Biswas.
Regarding claim 14, Biswas teaches probes that allow for visualization of the nucleic acid of interest. Biswas teaches fluorescent probes. See [0058]. Furthermore, Biswas teaches PCR and real-time quantitative PCR (i.e. fluorescent imaging). See, e.g., [0074].
Regarding claim 15, Biswas teaches control RNA (i.e. primer set) added to a detection mixture or an assay mixture for purposes of serving as a positive internal control for amplification. See [0041].
Regarding claim 17, Biswas teaches target bacteria of the genus Salmonella. See [0076].
Regarding claim 33, Noah teaches a E. coli O157:H7 ATCC 35150 (i.e. target bacteria). See table 2.
Evidentiary reference Microbiologics discloses that E. coli serotype O157:H7 is shiga toxin 1 and/or shiga toxin 2 positive. See p. 2 strain characteristics.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023) and Noah (J Food Prot. 2005 Apr;68(4):680-6. doi: 10.4315/0362-028x-68.4.680. PMID: 15830656), as applied to claims 1-2, 5, 7, 10, 12, 14-15, 17 and 33 above, and further in view of Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119, as previously relied upon)
Regarding claim 18, Biswas teaches a reaction mixture that includes a Salmonella forward primer, a Salmonella reverse primer (i.e. bacterial detection PCR primer set) and control RNA. See [00225] for table 12. Biswas teaches designing forward and reverse primers specific to Salmonella enterica. See [0220].
Noah teaches constructing GFP-expressing strain of Salmonella Typhimurium DT104 ATCC 700408. See p. 681 left column under the ‘materials and methods’ heading.
Biswas and Noah do not teach one or more PCR primer sets capable of detecting the reporter molecule.
Murphy teaches detecting Salmonella enterica in naturally contaminated food by real-time PCR using E. coli-GFP as an internal control. See section 2.9. Murphy teaches constructing a GFP-expressing strain of E. coli BLR by integrating the gfp gene. See section 2.6. For real-time PCR assays, Murphy teaches a qPCR mastermix (i.e. assay mixture) containing each forward and reverse primer. See section 2.3. As shown in table 1, forward and reverse PCR primers include: iroBF and iroBR for the S. enterica c-glycosyltransferase, iroB, gene; and gfpF and gfpR for the green fluorescent protein gene, gfp (i.e. PCR primer set capable of detecting a GFP reporter molecule).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Murphy’s assay mixture for Biswas’s reaction mixture. One of ordinary skill in the art would have been motivated to do so because Murphy suggests that the mixture can be used for the PCR detection of S. enterica and for detecting gfp reporter molecules. There would have been a reasonable expectation of success because Biswas teaches an assay mixture comprising forward and reverse primers for detecting S. enterica and control RNA; Noah teaches using GFP expressing strains as a control; and Murphy teaches an assay mixture comprising forward and reverse primers for detecting S. enterica and a control GFP reporter molecule.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023), Noah (J Food Prot. 2005 Apr;68(4):680-6. doi: 10.4315/0362-028x-68.4.680. PMID: 15830656), and Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119), as applied to claim 18 above, and further in view of Shi (CN101045943 as previously relied upon) and Miyazaki (WO2004005508 as previously relied upon).
The teachings of Biswas, Noah and Murphy are discussed above with respect to instant claim 18.
Regarding claim 24, Murphy teaches a qPCR mastermix (i.e. assay mixture) containing each forward and reverse primer. See section 2.3. As shown in table 1, forward and reverse PCR primers include: iroBF and iroBR for the S. enterica c-glycosyltransferase, iroB, gene; and gfpF and gfpR for the green fluorescent protein gene, gfp (i.e. PCR primer set capable of detecting a GFP reporter molecule).
Biswas, Noah, and Murphy do not teach one or more PCR primer sets capable of detecting Salmonella that are Salmonella 1 (SEQ ID NOs: 1-2) and/or Salmonella 2 (SEQ ID NOs 4-5) and the one or more PCR primer sets capable of detecting the reporter molecule is GFP (SEQ ID NOs: 7-8).
Shi teaches a PCR detection of Salmonella in the field of food safety. See [0002]. Shi teaches redesigning the detection primers based on the invA gene of Salmonella. See [0007]. Shi teaches a PCR detection method for Salmonella with an amplification internal standard, which includes: (1) using the specific gene sequence of Salmonella itself to construct an internal amplification standard and design specific primers, (3) performing PCR detection. See claim 1. SEQ ID NO: 1 of Shi is the invA gene and includes a subsequence that is 100% identical to instant SEQ ID NO: 1 and includes a subsequence that is the reverse compliment to instant SEQ ID NO: 2. See the office action appendix for the alignments.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Shi’s forward invA primer (i.e. instant SEQ ID NO: 1) and reverse invA primer (i.e. instant SEQ ID NO: 2) for Murphy’s forward and reverse iroB primers in the assay mixture. One of ordinary skill in the art would have been motivated to do so because Shi suggest using the invA gene for Salmonella detection in the field of food safety. There would have been a reasonable expectation of success because Biswas teaches detecting Salmonella from environmental samples including for food safety (see [0003]); and Shi teaches primers for the same purpose.
Biswas, Noah, Murphy and Shi do not teach one or more PCR primer sets capable of detecting the reporter molecule that is GFP (SEQ ID NOs: 7-8).
Miyazaki teaches cloning the GFP+ gene into a vector using two primers that amplify the GFP gene region by PCR. See [0035]. The GFP+ gene portion is shown in SEQ ID NO: 5. See [0036]. SEQ ID NO: 5 of Miyazaki includes a forward sequence that is 100% identical to instant SEQ ID NO: 7 and a reverse compliment sequence that is identical to instant SEQ ID NO: 8. Miyazaki teaches performing PCR using the full length of each complementary strand in a gene fragment as a primer. See [0005].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Miyazaki’s forward and reverse primers for the GFP+ gene (i.e. instant SEQ ID NOs: 7-8) for Murphy’s gfp forward and reverse primer set in the assay mixture. One of ordinary skill in the art would have been motivated to do so because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would have been a reasonable expectation of success because Noah demonstrates constructing control strains that express gfp, Murphy teaches an assay mixture comprising forward and reverse primers for gfp; and Miyazaki further teaches primers that serve the same purpose of detecting a GFP gene.
Claims 26-27 are rejected as being unpatentable over Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023) and Noah (J Food Prot. 2005 Apr;68(4):680-6. doi: 10.4315/0362-028x-68.4.680. PMID: 15830656), as applied to claims 1-2, 5, 7, 10, 12, 14-15, 17 and 33 above, and further in view of Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119, as previously relied upon) and Freitag (Infect Immun. 1999 Apr;67(4):1844-52).
Regarding claim 26, Biswas teaches methods used for lysing gram-positive bacteria, such as Listeria. See [0114]. Furthermore, Biswas teaches a method Biswas teaches a reaction mixture that includes a Salmonella forward primer, a Salmonella reverse primer (i.e. bacterial detection PCR primer set) and control RNA. See [00225] for table 12.
Noah suggests that GFP has been expressed in several bacterial pathogens including Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella. Noah references Freitag (reference 7) for teaching the GFP expression in L. monocytogenes. See p. 680 left column last passage. Noah teaches engineering E. coli O157:H& and Salmonella Typhimurium to express GFP for use as positive controls. See the abstract. Furthermore, Noah teaches the following samples: three feta cheese, one cooked shrimp, one raw shrimp and one cooked crawfish. Each set of samples includes an uninoculated control (e.g. a test sample), and one inoculated with the combination of the E. coli O157:H7 ATCC 35150 or Salmonella Cerro ATCC 10723 strain and the GFP-positive strain (i.e. control sample). See p. 682 left column last passage.
Biswas and Noah do not teach the target bacteria is Listeria; nor do Biswas and Noah teach a control bacterial strain of the same genus as the target bacteria but engineered to express a reporter molecule not naturally present in the target bacteria (as required in instant claim 1 from which claim 26 depends).
Murphy teaches detecting Salmonella enterica and Listeria monocytogenes (i.e. target bacteria). See the abstract and section 2.9. Murphy teaches a qPCR mastermix with each forward and reverse primer. See p. 112 right column lines 1-2. The forward and reverse primers include: iroBF and iroBR for the S. enterica c-glycosyltransferase, iroB, gene; hlyAF and hlyAR, for the haemolysine, hlyA, gene of Listeria monocytogenes; and gfpF and gpfR [sic. gfpR] for the green fluorescent protein gene. See table 1.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Murphy’s assay mixture for Biswas’s reaction mixture; and in the process target Listeria and Salmonella. One of ordinary skill in the art would have been motivated to do so because Noah teaches food samples that are not inoculated; and Murphy suggests that Listeria monocytogenes is food pathogen (see the abstract of Murphy). There would have been a reasonable expectation of success because Murphy demonstrates detecting Listeria and Salmonella.
Biswas, Noah and Murphy do not teach a control bacterial strain of the same genus as the target bacteria but engineered to express a reporter molecule not naturally present in the target bacteria (as required in instant claim 1 from which claim 26 depends).
Freitag teaches L. monocytogenes mutants containing gfp. See p. 1846 left column last passage. Freitag teaches the use of the green fluorescent protein (GFP) reporter gene system to monitor L. monocytogenes intracellular gene expression. See p 1844 right column last paragraph.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to further substitute L. monocytogenes mutant containing gfp for Noah’s GFP-positive control bacteria. One of ordinary skill in the art would have been motivated to do so because Freitag suggests that the L. monocytogenes gfp fusion mutants show no discernible bacterial growth effects or viability effects; thus, the patterns of gene expression are “probably more reflective of the natural environment than are expression patterns obtained by using mutants with decreased viability” (see p. 1851 second to last paragraph). There would have been a reasonable expectation of success because Noah demonstrates using GFP expressing strains as controls, and further references Freitag’s GFP expressing Listeria (see p. 680 left column last passage and reference 7 of Noah).
Regarding claim 27, Murphy teaches a qPCRs mastermix containing each forward and reverse primer. See section 2.3. The forward and reverse primers listed in table 1 include hlyAF and hlyAR, for the haemolysine, hlyA, gene of Listeria monocytogenes; and gfpF and gpfR [sic. gfpR] for the green fluorescent protein gene.
Claim 31 is rejected as being unpatentable over Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023), Noah (J Food Prot. 2005 Apr;68(4):680-6. doi: 10.4315/0362-028x-68.4.680. PMID: 15830656), Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119, as previously relied upon) and Freitag (Infect Immun. 1999 Apr;67(4):1844-52), as applied to claims 26-27 above, and further in view of Seehusen (NCBI, accession number EU139438 submitted 09/05/2007; previously cited as NIH) and Miyazaki (WO2004005508)
Regarding claim 31, Murphy teaches a qPCRs mastermix containing each forward and reverse primer. See section 2.3. The forward and reverse primers listed in table 1 include hlyAF and hlyAR, for the haemolysine, hlyA, gene of Listeria monocytogenes; and gfpF and gpfR [sic. gfpR] for the green fluorescent protein gene.
Biswas, Noah, Murphy, and Freitag do not teach one or more PCR primer sets capable of detecting Listeria spp. (SEQ ID NO: 10-11), and/or Listeria monocytogenes (SEQ ID NOs: 13-14), and one or more PCR primer sets capable of detecting the reporter molecule is GFP (SEQ ID NOs: 7-8).
Seehusen teaches a Listeria monocytogenes listeriolysin o (hlyA) gene which includes a subsequence that is 100% identical to instant SEQ ID NO: 13, and a subsequence that is 100% identical to the reverse compliment of instant SEQ ID NO: 14. See the office action appendix mailed 07/22/2025 for the alignments.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Seehusen’s hlyA forward and reverse primers for Murphy’s hlyAF and hlyAR. A person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would have been a reasonable expectation of success because the Seehusen’s hlyA sequences reasonably serve the same function as Murphy’s hlyAF and hlyAR.
Biswas, Noah, Murphy, Freitag and Seehusen do not teach one or more PCR primer sets capable of detecting the reporter molecule is GFP (SEQ ID NOs: 7-8).
Miyazaki teaches cloning the GFP+ gene into a vector using two primers that amplify the GFP gene region by PCR. See [0035]. The GFP+ gene portion is shown in SEQ ID NO: 5. See [0036]. SEQ ID NO: 5 of Miyazaki includes a forward sequence that is 100% identical to instant SEQ ID NO: 7 and a reverse compliment sequence that is identical to instant SEQ ID NO: 8. Miyazaki teaches performing PCR using the full length of each complementary strand in a gene fragment as a primer. See [0005].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Miyazaki’s forward and reverse primers for the GFP+ gene (i.e. instant SEQ ID NOs: 7-8) for Murphy’s gfp forward and reverse primer set in the assay mixture. One of ordinary skill in the art would have been motivated to do so because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would have been a reasonable expectation of success because Noah demonstrates constructing control strains that express gfp, Murphy teaches an assay mixture comprising forward and reverse primers for gfp; and Miyazaki further teaches primers that serve the same purpose of detecting a GFP gene.
Claims 34 and 39 are rejected as being unpatentable over Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023) and Noah (J Food Prot. 2005 Apr;68(4):680-6. doi: 10.4315/0362-028x-68.4.680. PMID: 15830656), as applied to claims 1-2, 5, 7, 10, 12, 14-15, 17 and 33 above, and further in view of Yu (CN 103436619, and translation) and Miyazaki (WO2004005508), with evidence from Microbiologics (Microbiologics SCA, 2024)
Regarding claim 34, Biswas teaches a detection mixture (i.e. assay mixture) comprising a primer having specificity for a single-stranded nucleic acid region comprising a nucleic acid sequence of the target RNA (i.e. bacterial detection PCR primer set). See [0185]. The detection mixture components further comprise a control RNA (i.e. control primer set) and a control probe able to detect the control RNA. See [0186].
Noah teaches a E. coli O157:H7 ATCC 35150 (i.e. target bacteria). See table 2. Furthermore, Noah teaches a E. coli O157:H7 GFP-positive strain that is stable and robust and could be beneficial for use as an easily distinguishable positive control strain. See p. 686 left column last 4 lines.
Evidentiary reference Microbiologics discloses that E. coli serotype O157:H7 is shiga toxin 1 and/or shiga toxin 2 positive. See p. 2 strain characteristics.
Biswas and Noah do not teach the assay mixture comprising (i) one or more PCR sets capable of detecting E. coli including E. coli that carry a Shiga toxin 1 gene, a Shiga toxin 2 gene and/or an Intimin gene and (ii) one or more PCR primer sets capable of detecting the reporter molecule.
Yu discloses that foodborne illnesses caused by E. coli O157:H7 continue to occur. See [0006]. Yu teaches designing primers for intimin (encoded by eaeA) and PCR amplification. See claim 1.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Yu’s eaeA primers for the bacterial detection PCR primer set in the assay mixture of Biswas. One of ordinary skill in the art would have been motivated to do so because Yu suggests that E. coli O157:H7 causes foodborne illnesses. There would have been a reasonable expectation of success because Noah demonstrates testing the detecting E. coli serotype O157:H7 in food samples (see Noah table 2); and Yu suggests that the eaeA primers can be used for detecting E. coli including E. coli O157:H7.
Biswas, Noah and Yu do not teach (ii) one or more PCR primer sets capable of detecting the reporter molecule.
Miyazaki teaches cloning the GFP+ gene into a vector using two primers that amplify the GFP gene region by PCR. See [0035].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Miyazaki’s forward and reverse primers for the GFP+ gene for Biswas’s control RNA in the assay mixture. One of ordinary skill in the art would have been motivated to do so because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would have been a reasonable expectation of success because Noah demonstrates constructing control strains that express gfp, and Miyazaki further teaches primers that serve the same purpose of detecting a GFP gene.
Regarding claim 39, Yu teaches a nucleotide sequence 917 bp in length that includes a forward sequence ( 598-616bp) that is identical to the instant SEQ ID NO: 25 and a sequence to which the instant SEQ ID NO:26 is a reverse complement (702-679bp). See paragraph [0122] for the sequence of Yu and see the office action appendix for the alignment.
Miyazaki teaches SEQ ID NO: 5. See [0036]. SEQ ID NO: 5 of Miyazaki includes a forward sequence that is 100% identical to instant SEQ ID NO: 7 and a reverse compliment sequence that is identical to instant SEQ ID NO: 8.
Claim 41 is rejected under 35 U.S.C. 103 as being unpatentable over Zegrati (US 2018/0258467) in view of Pinheiro (US 2013/0065297).
Regarding claim 41, Zegrati teaches a kit for simultaneous[ly] detecting the presence and/or absence of one or more pathogen comprising: a set of amplification primers, wherein the amplification primers comprise one or more primer pairs, wherein a first primer of the one or more primer pairs hybridizes to a target nucleic acid sequence of the one or more pathogen, and wherein a second primer of the one or more primer pairs hybridizes to a sequence complimentary to the target nucleic acid (i.e. one or more first PCR primer sets). See claim 62. Zegrati teaches primers for determining a sequence of Shiga Toxin-Producing E. coli gene intimin (eaeA) by PCR. See claim 114. Zegrati teaches primers for determining a sequence of Shiga Toxin-Producing E. coli gene Shiga toxin 1 (stx1). See claim 116. Furthermore, Zegrati teaches primers for determining a sequence of an internal control by PCR. See claim 179. Zegrati teaches a kit comprising a positive PCR control. See claim 88. Thus, Zegrati teaches a kit comprising primer pairs for detecting eaeA and stx1 of an E. coli target, and Zegrati teaches a primer pair for detecting an internal control. Zegrati teaches multiplex PCR. See [0254].
Zegrati does not teach (b) one or more control bacterial strains, each control bacterial strain of the same genus as a corresponding one of the target bacteria detected by the one or more first PCR primer sets, wherein each control bacterial strain is engineered to express a reporter molecule not naturally present in its corresponding target bacteria, and wherein each control bacterial strain is configured to be present only in a control sample and not intentionally added to a test sample; and (c) one or more second PCR primer sets capable of detecting the one or more control bacterial strains. However, Zegrati does teach primers capable of detecting a control.
Pinheiro teaches a modified bacterium comprising a mutated green fluorescent protein (GFP) gene inserted into the chromosome. See claim 25 of Pinheiro. The bacterium is Escherichia coli, Salmonella sp. or Listeria sp. See claim 30 of Pinheiro. Pinheiro suggests that cells visibly altered by expression of a modified GFP protein make them useful as quality control (QC) strains. See [0129]. In example 2, Pinheiro teaches integrating unrepressed GFP gene cassette into E. coli and Pinheiro teaches gfpEnt and gfpR0 primers. See [0102]-[0107].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to add the E. coli GFP bacterium of Pinheiro to the kit of Zegrati and to further substitute the Pinheiro’s gfpEnt and gfpR0 control primers for Zegrati’s control primers. One of ordinary skill in the art would have been motivated to include the E. coli GFP of Pinheiro in the kit of Zegrati, because Pinheiro suggests that the bacterium is useful as a quality control strain. There would be a reasonable expectation of success because Zegrati teaches a kit comprising a PCR control, and Pinheiro teaches a bacterium that can be used as a control. One of ordinary skill in the art would have been further motivated to use Pinheiro’s control primers because Pinheiro suggests that the primers are capable of detecting the unrepressed gfp gene in the E. coli transformant. There would have been a reasonable expectation of success because Zegrati teaches a kit comprising primers for detecting an internal control, and Pinheiro teaches primers for detecting the gfp in an E. coli bacterium that can be used as a control.
Zegrati and Pinheiro do not teach each control bacterial strain configured to be present only in a control sample and not intentionally added to a test sample; and one or more first PCR primer sets and the one or more second PCR primer sets are configured for multiplex real-time PCR such that amplified target PCR product and amplified control PCR product are detected in different fluorescence channels of a real-time PCR instrument, and wherein the one or more first primer sets and the one or more second primer sets are optimized such that cross-contamination of the test sample is capable of being determined when:(i) an amplified control PCR product is present in the test sample; and(ii) a Cq value of the amplified control PCR product occurs within 0 to 4 PCR cycles of, or prior to, a Cq value of the amplified PCR product of the test sample.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention that Pinheiro’s control bacterial strain can be configured to be present in only a control sample; and that Zegrati’s and Pinheiro’s PCR primer sets can necessarily be configured for multiplex real-time PCR. MPEP 2112.01(II) states that "products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990).
Claim 43 is rejected under 35 U.S.C. 103 as being unpatentable over Zegrati (US 2018/0258467) and Pinheiro (US 2013/0065297), as applied to claim 41 above and further in view of Yu (CN 103436619, and translation) and Brousseau (US 2004/0219530).
Regarding claim 43, Zegrati teaches a kit comprising a set of amplification primers. See claim 62. Zegrati teaches primers for eaeA and stx1. See claims 114 and 116.
Zegrati and Pinheiro do not teach a kit comprising one or more first PCR primer sets are selected from the group consisting of: (i) Salmonella 1 (SEQ ID NOs: 1-2) and Salmonella 2 (SEQ ID NOs:4-5); (ii) Listeria spp. (SEQ ID NOs:10-11) and L. monocytogenes (SEQ ID NOs:13-14); (iii) two or more of E. coli O157:H7 (SEQ ID NOs:16-17), Shiga toxin 1 (SEQ ID NOs:19-20), Shiga toxin 2 (SEQ ID NOs:22-23), Intimin (SEQ ID NOs:25-26), E. coli 0103 (SEQ ID NOs:28-29), E. coli 026 (SEQ ID NOs:31-32), E. coli 0145 (SEQ ID NOs:34-35), E. coli 0111 (SEQ ID NOs:37-38), E. coli 045 (SEQ ID NOs:40-41), E. coli 0121 (SEQ ID NOs:43-44), when the target bacteria is Escherichia coli including E. coli that carry a Shiga toxin 1 gene, a Shiga toxin 2 gene and/or an Intimin gene; and (iv) any combination of (i), (ii) and/or (iii).
Yu teaches a method for identifying atypical Escherichia coli in calf diarrhea, the method includes designing a diagnostic primer for intimin (encoded by eaeA) and PCR amplification. See claim 1. Yu teaches a nucleotide sequence that includes a forward sequence that is identical to instant SEQ ID NO: 25 and a sequence to which instant SEQ ID NO:26 is a reverse complement. See paragraph [0122] for the sequence of Yu and see the office action appendix for the alignment.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Yu’s eaeA primer (i.e. instant SEQ ID NOs: 25-26) for Zegrati’s eaeA primers. One of ordinary skill in the art would have been motivated to do so because Yu suggests that the eaeA gene is associated with the pathogenic bacterium E. coli O157:H7. There would have been a reasonable expectation of success because the eaeA primers of Yu reasonably serve the same function as the eaeA primers of Zegrati.
Zegrati, Pinheiro and Yu do not teach (iii) two or more of E. coli O157:H7 (SEQ ID NOs:16-17), Shiga toxin 1 (SEQ ID NOs:19-20), Shiga toxin 2 (SEQ ID NOs:22-23), Intimin (SEQ ID NOs:25-26), E. coli 0103 (SEQ ID NOs:28-29), E. coli 026 (SEQ ID NOs:31-32), E. coli 0145 (SEQ ID NOs:34-35), E. coli 0111 (SEQ ID NOs:37-38), E. coli 045 (SEQ ID NOs:40-41), E. coli 0121 (SEQ ID NOs:43-44), when the target bacteria is Escherichia coli including E. coli that carry a Shiga toxin 1 gene, a Shiga toxin 2 gene and/or an Intimin gene.
Brousseau teaches a virulence gene selected from a group that includes stx1. See claim 11. Brousseau teaches a probe that comprises a nucleic acid sequence selected from a group that includes SEQ ID NO: 91. See claim 12 and table 1. Brousseau teaches DNA probes that successfully distinguish stx1 from stx2. See example 4 paragraph [0085]. SEQ ID NO: 91 of Brousseau includes a subsequence that is identical to instant SEQ ID NO: 19 and a reverse complement to instant SEQ ID NO: 20. See the office action appendix for the alignment.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Brousseau’s stx1 primers for Zegrati’s stx1 primers. One of ordinary skill in the art would have been motivated to do so because Brousseau suggests that it is capable of distinguishing stx1 from stx2. There would be a reasonable expectation of success because the stx1 primers of Brousseau reasonably serve the same function as the stx1 primers of Zegrati in the kit.
Claim 44 is rejected under 35 U.S.C. 103 as being unpatentable over Zegrati (US 2018/0258467) in view of Pinheiro (US 2013/0065297), as applied to claim 41 above, and further in view of Miyazaki (WO2004005508).
Regarding claim 44, Pinheiro teaches gfpEnt and gfpR0 primers. See [0102]-[0107].
Zegrati and Pinheiro do not one or more PCR primer sets comprising GFP (SEQ ID NOs: 7-8)
Miyazaki teaches cloning the GFP+ gene into a vector using two primers that amplify the GFP gene region by PCR. See [0035]. Miyazaki teaches SEQ ID NO: 5. See [0036]. SEQ ID NO: 5 of Miyazaki includes a forward sequence that is 100% identical to instant SEQ ID NO: 7 and a reverse compliment sequence that is identical to instant SEQ ID NO: 8.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Miyazaki’s GFP+ forward and reverse primers for Pinheiro’s gfpEnt and gfpR0 primers in Zegrati’s kit. One of ordinary skill in the art would have been motivated to do so because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would have been a reasonable expectation of success because Zegrati teaches a kit comprising a positive PCR control (see claim 88 of Zegrati), Pinheiro teaches a control bacterial strain containing GFP, and Miyazaki teaches primers for GFP detection.
Response to Arguments
Applicant's arguments filed 05/04/2026 have been fully considered to the extent that they apply to the new grounds for rejection, but they are not persuasive.
Applicant argues that Pinheiro does not expressly describe using GFP-labeled Salmonella and Listeria strains as a reporter target in a multiplex real-time diagnostic PCR assay, and it does not disclose any decision rule in which assay validity is determined based on the relative Cq values of a target and a control. See the remarks p. 13 first full paragraph.
This argument is unpersuasive, because Pinheiro is relied upon for teaching the one or more control bacterial strain (claim 41 (b)) and the one or more second PCR primer sets (claim 41(c)). Claim 41 is a product claim, not a method claim. Therefore, the claim 41 and dependent claims do not require an active step of multiplex real-time diagnostic PCR assay. Furthermore, Applicant has not set forth evidence indicating that Pinheiro’s primers would be incapable of being used in a multiplex real-time diagnostic PCR assay step.
Applicant argues that Zegrati does not disclose any control bacterial strain at all, much less a control strain of the same genus as the target bacteria. Zegrati’s internal control is a nucleic acid construct used to monitor amplification performance, not a bacterial control strain engineered with a reporter molecule. Pinheiro simply teaches GFP-labeled Salmonella and Listeria strains used as plate-based quality control organisms that are identified by fluorescence on agar; it does not teach packaging these control bacterial strains in a diagnostic PCR kit with reporter-specific primer sets. Neither Zegrati or Pinheiro teach or suggest a kit where each control bacterial strain is configured to be present only in a control sample and not intentionally added to the test sample, and where cross-contamination of a test sample is capable of being determined when an amplified control PCR product is present in the test sample and a Cq of the amplified control PCR product occurs within 0 to 4 PCR cycles of, or prior to, a Cq value of the amplified PCR product of the test sample. See p. 17 first paragraph
This argument is not persuasive because Zegrati is not relied upon for teaching the one or more control bacterial strain claim element (instant claim 41). Pinheiro is relied upon for teaching cells visibly altered by expression of a modified GFP protein make them useful as quality control (QC) strains ( [0129]). Applicant argues that Pinheiro simply teaches GFP-labeled Salmonella and Listeria strains used as plate-based quality control. However, Pinheiro is not limited Salmonella and Listeria, because Pinheiro discloses that the GFP gene can be inserted into bacterium is Escherichia coli, Salmonella sp. or Listeria sp. See claim 30 of Pinheiro. Applicant argues that Zegrati and Pinheiro do not teach a control bacterial strain configured to be present only in a control sample. This argument is not persuasive because Applicant has not pointed to a claimed structural element that distinguishes the claimed one or more control bacterial strains, from that of Pinheiro.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657
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