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 .
Priority
The instant claims are entitled to an effective filing date of 02/28/2023.
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 43 is new. Claims 1-2, 5, 7, 10, 12, 14-15, 17-18, 24, 26-27, 31, 33-34, 39, 41 and 43 are pending and under consideration.
The rejection of claims 1-5, 7, 14-15, 17-18, 26-27 and 33-34 under 35 U.S.C. 102(a)(1) as being anticipated by Murphy with evidence from Culpin is withdrawn in light of the claim amendment. However, a new rejection under 102(a)(1) is applied below.
The rejection of claim 41 under 35 U.S.C. 102(a)(1) and 35 U.S.C. 102(a)(2) as being anticipated by Zegrati is withdrawn in light of the claim amendment.
The rejection of claim 10 under 35 U.S.C. §103 over Murphy in view of Zegrati is withdrawn in light of the amendment.
The rejection of claim 12 under 35 U.S.C. §103 over Murphy in view of Biswas is withdrawn in light of the amendment.
The rejection of claim 24 under 35 U.S.C. §103 over Murphy in view of Shi is withdrawn in light of the amendment.
The rejection of claim 31 under 35 U.S.C. §103 over Murphy in view of Seehusen is withdrawn in light of the amendment.
The rejection of claim 31 under 35 U.S.C. §103 over Murphy in view of Miyazaki is withdrawn in light of the amendment.
Claim Objections
Claims 1 and 43 are objected to because of the following informalities:
Claim 1 recites “comprising a target bacteria”, which should be replaced with “comprising target bacteria” because the article “a” is singular, but the term “bacteria” is plural.
Claim 43 recites “Listera” in line 5, which is a misspelling of “Listeria”.
Appropriate correction is required.
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 1-2, 5, 7, 10, 12, 14-15, 17-18, 24, 26-27, 31, 33, 34, 39, 41 and 43 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 1 recites “determining the presence or absence of the target bacterial species and/or the control bacterial strain in the one or more assay samples simultaneously” in lines 14-15, which is indefinite because it is unclear which occurrences are required to be simultaneous. Consequently, there are multiple reasonable interpretations. In the first interpretation, the claim requires the determination step to be simultaneous across the one or more assay samples. In the second interpretation, the claim requires simultaneously determining whether the target bacterial species and/or control bacterial strain are present or absent.
Claim 1 recites “the amplified test sample PCR product” in the last line. There is insufficient antecedent basis for this limitation in the claim. It is unclear which amplified test sample PCR product is being referenced because there is no earlier recitation for an amplified test sample PCR product but an amplified target PCR product. Therefore, one could reasonably interpret the amplified test sample PCR product as referring to the amplified target PCR product. Alternatively, the claim could refer to a separate test sample PCR product.
Claims 2, 5, 7, 10, 12, 14-15, 17-18, 24, 26-27, 31, 33, 34 and 39 depend from claim 1 and are rejected for the reason set forth above.
Claim 41 requires “one or more control bacterial strains of the same genus as the target bacteria detected by the one or more PCR primer sets, wherein the control bacterial strain is engineered to express a reporter molecule not naturally present in the bacteria”, which is indefinite for three reasons. First, it is unclear whether the one or more control bacterial strains are required to be of the Salmonella, Listeria or Escherichia genus, or whether the Escherichia genus is limited to the E. coli species. To clarify, the claim requires the one or more control bacterial strains to be of the same genus as “the target bacteria”, however there is no earlier recitation of target bacteria in claim 41, so it is unclear whether the target bacteria are Salmonella, Listeria or E. coli. Second, it is unclear whether “the control bacterial strain” in line 5 refers to one or all of the “one or more control bacterial strains” recited earlier in the claim. Third, claim 42 recites “the bacteria” in line 6, which is indefinite because it is unclear whether the recitation is in reference to the one or more control bacterial strains, or the target bacteria.
Claim 43 depends from claim 41 and is rejected for the reason set forth above.
Claim 43 recites “the one or more PCR primer sets”, which is indefinite because it is unclear which one or more PCR primer sets are being referenced and, consequently, there are multiple reasonable claim interpretations. In the first interpretation, claim 43 limits two components of the kit, i.e. the one or more PCR primer sets capable of detecting Salmonella, Listeria or E. coli, and the one or more PCR primer sets capable of detecting the one or more control bacterial strains. This first interpretation is reasonable because options (i), (ii) and (iii) include PCR primer sets capable of detecting Salmonella, Listeria, or E. coli, and a PCR primer set capable of detecting a GFP reporter molecule (SEQ ID NOs: 7-8). In the second interpretation, claim 43 limits one component of the kit, i.e. either the one or more PCR primer sets capable of detecting Salmonella, Listeria or E. coli, or the one or more PCR primer sets capable of detecting the one or more control bacterial strains. This second interpretation is reasonable because the claim recites the “one” or more PCR primer sets, which indicates that the claim may only intend to limit one PCR primer set. However, options (i)-(iii) all require more than one PCR primer set. As such, the metes and bounds of option (iv) are indefinite because it is unclear whether “any combination thereof” is limited to combinations of (i), (ii) and/or (iii), or whether “any combination thereof” encompasses any combination of one or more of the listed PCR primer sets.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 39 and 43 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 39 depends from claim 34, which requires one or more PCR primer sets capable of detecting E. coli. Claim 39 broadens the scope of the one or more PCR primer sets because claim 39 encompasses the following PCR primer sets that are not specific for E. coli: Shiga toxin 1 (SEQ ID NOs: 19-20), Shiga toxin 2 (SEQ ID NOs: 22-23) and intimin (SEQ ID NOs: 25-26).
Claim 43 depends from claim 41, which requires one or more PCR primer sets capable of detecting Salmonella, Listeria, or E. coli. Claim 43 broadens the scope of the one or more PCR primer sets required in claim 41 because claim 43 encompasses the following PCR primer sets that are not specific to Salmonella, Listeria, or E. coli: Shiga toxin 1 (SEQ ID NOs: 19-20), Shiga toxin 2 (SEQ ID NOs: 22-23) and intimin (SEQ ID NOs: 25-26).
Evidentiary reference Wang (Microorganisms, 2004, 12(4), 687) states that Shiga toxin can be grouped into two types, Stx1 and Stx2, among which a variety of variants and subtypes have been identified in various bacteria and host species. See the abstract. For example, Wang discloses that a strain of Aeromonas was shown to produce both Stx1 and Stx2. See section 3.5. Furthermore, evidentiary reference Yang (Scientific reports, 2020, 10(1), 3275) discloses that intimin is an importance virulence factor of other bacteria such as E. albertii and Citrobacter rodentium. See the first full sentence on page 2. The instant specification is silent as to whether (SEQ ID NOs: 19-20), (SEQ ID NOs: 22-23) and (SEQ ID NOs: 25-26) are specific to E. coli. Thus, claim 43 broadens the scope of the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Interpretation
Claim 1 requires 3 active method steps and one conditional step. First, lyse a test sample that potentially includes a target bacterium and a control sample that comprises a bacterial strain in the same genus as the target bacteria but engineered with a reporter molecule (e.g. green fluorescent protein (GFP)). Second, contact the test sample lysate and the control sample lysate with a mixture comprising one or more bacterial detection PCR primer set and a control detection PCR primer set (e.g. forward/reverse primers for a gfp gene) to generate one or more assay samples. Third, detect the presence or absence of a Cq value for an amplified target PCR product and/or an amplified control PCR product in each of the one or more assay samples simultaneously, such that the presence or absence of the target bacterial species and/or the control bacterial strain is simultaneously determined. Fourth, if target bacteria are present in the test sample, then determine if cross-contamination of the test sample occurred by detecting if the amplified control PCR product is present in the test sample when 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 test sample PCR product. This fourth step is interpreted as being conditional because the claim recites “if target bacteria are present in the test sample”. Furthermore, Ct and Cq are interpreted as being synonymous because, according to the instant specification, the number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle (Ct) or according to the MIQE guidelines quantification cycle (Cq). See [0087].
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-2, 5, 14-15, 17 and 33-34 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Murphy (hereafter Murphy2009 (PhD thesis, University of Greenwich, 2009).
Regarding claims 1, 17 and 33-34, Murphy2009 teaches developing E. coli green fluorescent protein (GFP) as a process control for the detection of pathogens including Escherichia coli O157:H7. See the first paragraph of 7.2.2. Murphy2009 teaches constructing a GFP-expressing strain of E. coli BLR by integrating the gfp gene. See section 4.2 on page 125. Murphy2009 teaches developing a Lenticule encased E. coli GFP strain for use as a control. See section 4.3.1 on page 130. Murphy2009 teaches preparing enrichment cultures by homogenizing food samples with a dilutant, such as modified Tryptone Soy broth (mTSB) for E. coli O157:H7. See 2.12.1 on page 82. Serial dilutions of the culture are prepared and one ml of each serial dilution is subjected to MagNa PureTM DNA extraction, and 5 µl of the resulting DNA is amplified. See section 2.13.2. For MagNa PureTM extraction, bacterial isolates are emulsified directly into lysis buffer DNA is extracted from lysed samples. See section 2.5.1 on page 49. For the duplex assay, a Lenticule disc (control) is added to the culture dilutions (test) prior to DNA extraction [which entails lysing] and the resulting DNA is subjected to the eaeγ:gfp PCR assay. Each dilution is amplified and a standard curve of mean Ct values (i.e. synonymous with Cq) against CFUml-1 is produced. See section 2.13.5. For the process control duplex PCR assay, Murphy2009 teaches combining qPCR mastermix, template DNA, and each forward and reverse oligonucleotide for the target pathogen with concentrations of forward and reverse gfp oligonucleotides. See section 2.9.9 on page 72. As shown in table 2.6 on page 43, the forward and reverse PCR primers include eaeγF and eaeγR2 [eaeγ is intimin gamma] for E. coli O157:H7 (bacterial detection PCR primer set), and gfpF and gfpR2 for the E. coli GFP (control detection PCR primer set). Murphy2009 teaches 5 mTSB enrichment cultures inoculated with goat cheese samples for the detection of E. coli O157:H7. None of the samples are found to be positive by the eaeγ gene PCR. See section 7.3.2 on page 213 and table 7.7. Where an E. coli GFP lenticule disc is included Ct values of 27-29 are detected, indicating no PCR inhibition. See the first passage on page 206.
Regarding claim 2, Murphy2009 teaches culturing E. coli GFP on MacConkey Agar (i.e. enrichment media) for 24h as part of the Lenticule disc preparation. See the last passage on page 60. Furthermore, Murphy2009 teaches preparing enrichment cultures by homogenizing food samples with dilutant. For E. coli O157:H7, the dilutant is mTSB. The enrichment cultures are incubated. See section 2.13.1 on page 82. For the duplex assay, a Lenticule disc (control containing E. coli GFP) is added to the culture dilutions (test) prior to DNA extraction. See section 2.13.2 and 2.13.5. DNA is extracted from lysed samples. See section 2.5.1 on page 49.
Regarding claim 5, Murphy2009 teaches 5 mTSB cultures inoculated with goat cheese samples (food) for the detection of E. coli O157:H7. See section 7.3.2 on page 213 and table 7.7.
Regarding claim 14, Murphy2009 discloses that the CT value is defined as the PCR cycle number at which an increase in fluorescence is first detected for each amplification plot. See the last paragraph of page 2.9.5 and see figure 1.2 for the methods of fluorescence detection.
Regarding claim 15, for the process control duplex PCR assay, Murphy2009 teaches combining qPCR mastermix, template DNA, and each forward and reverse oligonucleotide for the target pathogen with concentrations of forward and reverse gfp oligonucleotides. See section 2.9.9 on page 72. As shown in table 2.6 on page 43, the forward and reverse PCR primers include eaeγF and eaeγR2 [eaeγ is intimin gamma] for E. coli O157:H7 (bacterial detection PCR primer set), and gfpF and gfpR2 for the E. coli GFP (control detection PCR primer set). As shown in table 7.7 the gfp gene is detected as an internal control.
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 1-5, 7, 14-15, 17-18, 26-27 and 33-34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119) in view of Pinheiro (US 2013/0065297) with evidence from Culpin (MagNA Pure LC DNA Isolation Kit 1 From Roche Applied Science Biocompare.com, 2009). Murphy and Culpin have previously been relied upon. Underlined text below is relevant to the amendment.
Regarding claim 1, Murphy teaches detecting Salmonella enterica or Listeria monocytogenes 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. Murphy teaches encapsulating the E. coli BLR-GFP in LENTICULE discs (control). See section 2.8. Murphy teaches adding a single LENTICULE disc containing 104 E. coli- GFP cells (control) to 1 ml of enrichment broth culture immediately prior to DNA extraction by MagNa Pure™ LC. The enrichment cultures include Salmonella spp. and Listeria spp. See section 2.9. Evidentiary reference Culpin teaches that MagNA Pure™ entails complete cell lysis in the third paragraph on page 2. Thus, Murphy teaches that an E. coli-GFP control sample is added to two different enrichment broth test samples: one with a Salmonella target, and the other with a Listeria target prior to cell lysis. Murphy teaches applying a PCR assay to DNA isolated from enrichment broths with lenticulated E. coli-GFP. The real-time PCR assays contain 12.5 µl qPCR mastermix, 5 µ of template DNA, 300 nM of each forward and reverse primer, and 100nM probe. See section 2.3. As shown in table 1, forward and reverse PCR primers include: hlyAF and hlyAR for the L. monocytogenes haemolysin, hlyA, gene (i.e. a bacterial detection PCR primer set); iroBF and iroBR for the S. enterica c-glycosyltransferase, iroB, gene (i.e. a bacterial detection PCR primer set); and gfpF and gfpR for the green fluorescent protein gene, gfp (i.e. a control PCR primer set). Murphy teaches detecting PCR products by monitoring the increase in fluorescence of the reporter dye at each PCR cycle. Murphy teaches that the software determines the threshold cycle (Ct) value [which is synonymous with instant Cq]. See section 2.3. Murphy teaches analyzing 393 enrichment broths inoculated with either a food sample or an environmental sample for the detection of Listeria or Salmonella using either the hlyA:gfp or the iroB:gfp PCR assay. The iroB gene fragment of S. enterica is detected with mean Ct values between 24 and 34. The hlyA gene fragment is detected with Ct values between 18 and 33. When E. coli-GFP (e.g. control) is added to enrichment broths prior to DNA extraction, the mean Ct values are between 27-29. See section 3.3 and table 4.
Murphy does not teach a control sample that comprises a control bacterial strain of the same genus as the target bacteria.
Pinheiro teaches a modified bacterium comprising a mutated green fluorescent protein (GFP) gene inserted into the chromosome. The bacterium is Escherichia coli, Salmonella sp. or Listeria sp. See claims 25 and 30 of Pinheiro. In example 2, Pinheiro teaches integrating unrepressed GFP gene cassette into E. coli and Salmonella strains. A new oligonucleotide primer, gfpEnt, is synthesized for PCR amplification of the unrepressed gfp gene cassette and PCR amplification using gfpEnt and gfpR0 primers is performed. See [0102]-[0107]. In example 4, Pinheiro teaches following a similar approach to generate fluorescent Listeria monocytogenes. The gfp gene present is amplified using the PCR primers Gfpmutf1 and Gfpmutr1. See [0111] and [0113]. Furthermore, Pinheiro suggests that laboratories can inadvertently contaminate a real sample with a quality control (QC) strain, which can result in a false positive. See [0006]. Rare species such as Salmonella salford can be used as a QC strain for identifying cross contamination. See [0008]. However, rare species may have physiological properties that are different than commonly isolated organisms. For example, S. salford does not grow on media routinely used for isolating Salmonella from food, so, according to Pinheiro, it is not a suitable QC. See [0009]. Pinheiro suggests that cells visibly altered by expression of a modified GFP protein make them useful as QC strains. See [0129].
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 replace the E. coli BLR-GFP in the LENTICULE discs of Murphy with either the Salmonella GFP or Listeria GFP of Pinheiro, and to replace the gfpF/gfpR primers of Murphy with either the gfpEnt/gfpR0 primers or Gfpmutf1/Gfpmutr1 primers respectively. One would be motivated to use the Salmonella GFP or Listeria GFP of Pinheiro, because Pinheiro suggests using quality controls that have similar physiological properties compared to the organism being isolated [which in the case of Murphy includes Salmonella and Listeria]. There would be a reasonable expectation of success because Murphy demonstrates constructing a GFP-expressing strain of E. coli to be used as an internal control; and Pinheiro demonstrates constructing GFP-expressing Salmonella and Listeria strains to be used as quality controls. One would be further motivated to replace the gfpF/gfpR primers of Murphy with the primers of Pinheiro, because Pinheiro discloses that the gfpEnt/gfpR0 primers can be used for PCR amplification of the Salmonella GFP and that the Gfpmutf1/Gfpmutr1 primers can be used for the PCR amplification of the Listeria GFP. There would be a reasonable expectation of success because Pinheiro demonstrates using the primers for the PCR amplification of the gfp genes.
Regarding claim 2, for LENTICULE disc preparation, Murphy teaches culturing E. coli BLR-GFP on MacConkey agar (e.g. enrichment medium) for 24 hr. One colony is then emulsified in nutrient broth and used to inoculate a nutrient agar slope which is incubated for 24 hours. See section 2.8. Murphy teaches primary enrichment broths that are provided for PCR analysis following incubation and comprise BPW for Salmonella spp., and half-strength Fraser broth for Listeria spp. See section 2.9. Murphy teaches adding a single LENTICULE disc containing E. coli -GFP to 1ml of enrichment broth culture immediately prior to DNA extraction by MagNa Pure™ LC, which entails cell lysis as evidenced by Culpin. See section 2.9 of Murphy and page 2 of Culpin. Thus, Murphy teaches incubating Salmonella or Listeria test samples and the E. coli BLR-GFP control sample in enrichment broths separately prior to lysing.
Regarding claims 5 and 7, Murphy teaches inoculating enrichment broths with 353 food samples, 117 egg and 236 other foods; and 6 environmental samples, surface swabs and vacuum cleaner dust. See the first full paragraph on page 117. Thus, Murphy teaches food test samples (relevant to instant claim 5), and environmental test samples (relevant to instant claim 7).
Regarding claim 14, Murphy teaches monitoring the increase in fluorescence of the reporter dye at each PCR cycle. Software is used to determine the threshold cycle (Ct) value, i.e. the PCR cycle number at which fluorescence increases above a defined threshold. See the fourth paragraph of section 2.3. For PCR detection of the gfp gene, Murphy teaches using a blocked assay in which PCR amplification is performed and the 122 bp PCR product is visualized by UV transillumination. See section 2.7. Murphy discloses that bacterial colonies expressing GFP can be easily identified and differentiated when excited with UV light. See the last full paragraph of the left column on page 111.
Regarding claim 15, Murphy teaches using E. coli- GFP as an internal control in PCR assays. See section 2.9 and the abstract. Murphy teaches individual real-time PCRs that contain 300nM of each forward and reverse primer. See section 2.3. The forward and reverse primers listed in table 1 include gfpF and gfpR [sic. gfpR] for the green fluorescent protein gene (e.g. an internal amplification control primer set).
Regarding claim 17, Murphy teaches detecting Listeria monocytogenes and Salmonella enterica (e.g. target bacterial species) by PCR. See the abstract and section 2.9.
Regarding claim 18, Murphy teaches individual real-time PCRs that contain 300nM of each forward and reverse primer. See section 2.3. The forward and reverse primers listed in table 1 include iroBF and iroBR, for the C-glycosyltransferase, iroB, gene of Salmonella enterica (e.g. PCR primer set capable of detecting Salmonella); and gfpF and gpfR [sic. gfpR] for the green fluorescent protein gene (e.g. PCR primer set capable of detecting the gfp reporter molecule).
In example 2, Pinheiro teaches integrating unrepressed GFP gene cassette into E. coli and Salmonella strains. A new oligonucleotide primer, gfpEnt, is synthesized for PCR amplification of the unrepressed gfp gene cassette and PCR amplification using gfpEnt and gfpR0 primers is performed. See [0102]-[0107]. Thus, Pinheiro teaches primer sets capable of detecting a GFP reporter in a Salmonella control.
Regarding claim 26, Murphy teaches detecting Listeria monocytogenes. See the abstract and section 2.9.
Regarding claim 27, Murphy teaches individual real-time PCRs that contain 300nM of 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 (e.g. PCR primer set capable of detecting Listeria); and gfpF and gpfR [sic. gfpR] for the green fluorescent protein gene (e.g. PCR primer set capable of detecting the gfp reporter molecule).
In example 4, Pinheiro teaches following a similar approach to generate fluorescent Listeria monocytogenes. The gfp gene present is amplified using the PCR primers Gfpmutf1 and Gfpmutr1. See [0111] and [0113]. Thus, Pinheiro teaches primer sets capable of detecting a GFP reporter in a Listeria control.
Regarding claim 33, Murphy teaches establishing a model system for the extraction of DNA from a simple food matrix. E. coli-GFP is grown in LB and a 10-fold dilution series is prepared in maximum recovery dilutant (MRD) (e.g. control). One ml of each dilution is added to 9ml volumes of semi-skimmed milk (e.g. test sample comprising E. coli target) giving a final concentration range of 1 to 106 CFU/ml. DNA is extracted from 1ml of each dilution in both MRD and milk by MangNa Pure DNA extraction (e.g. entails lysing), and used as a target in the real-time gfp PCR. See section 2.5. Murphy discloses that there was no difference detected between the Ct values obtained using DNA extracted from MRD or milk. See the last paragraph of section 3.1. Thus, Murphy teaches real-time PCR assay that entails E. coli-GFP as a control in MRD and as a target in semi-skimmed milk.
Regarding claim 34, Murphy teaches applying the real-time PCR assay for the gfp gene to DNA extracted from decimal dilutions of E. coli BLR-GFP in both MRD (e.g. control) and milk (e.g. target). See the last paragraph of section 3.3. Murphy teaches individual real-time PCRs that contain 12.5 µl qPCR mastermix, 5 µ of template DNA, 300 nM of each forward and reverse primer, and 100nM probe (e.g. assay mixture). See section 2.3. As shown in table 1, the forward and reverse primers include gfpF and gfpR for the green fluorescent protein, gfp, gene of E. coli BLR-GFP (e.g. control), and gfpF and gfpRX for the gfp gene in E. coli BLR-GFP (e.g. target). Thus, Murphy teaches two different primer sets capable detecting E. coli BLR-GFP and a GFP reporter molecule. As such, Murphy teaches that (i) one primer set, e.g. the gfpF and gfpR, is capable of detecting E. coli and (ii) one primer set, e.g. gfpF and gfpRX, is capable of detecting a reporter molecule.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119) in view of Pinheiro (US 2013/0065297), as applied to claims 1-2, 5, 14-15, 17-18, 26-27, and 33-34 above, and further in view of Zegrati (US 2018/0258467).
The teachings of Murphy and Pinheiro with respect to instant claim 1 are discussed above.
Regarding claim 10, Murphy teaches inoculating enrichment broths with 353 food samples, 117 egg and 236 other foods; and 6 environmental samples, surface swabs and vacuum cleaner dust. See the first full paragraph on page 117. Murphy teaches analyzing 393 enrichment broths inoculated with either a food sample or an environmental sample for the detection of Listeria or Salmonella using either the hlyA:gfp or the iroB:gfp PCR assay. See section 3.3.
Murphy and Pinheiro do not teach a test sample that is a plant.
Zegrati teaches samples, such as food material, plant or animal material, vegetables, fruit, and salads. See [0133]. Zegrati teaches a method comprising (a) enriching a sample comprising one or more pathogens in a rich and nonselective media; wherein the rich and nonselective media comprises components to promote the growth of the one or more pathogens; (b) conducting a first sample lysis and a second sample lysis on the enriched sample or a portion thereof, wherein the second sample lysis is performed at a temperature higher than the temperature of the first sample lysis, thereby forming a lysed sample; (c) conducting an amplification with a set of amplification primers on the lysed sample, 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 pathogens, and wherein a second primer of the one or more primer pairs hybridizes to a sequence complimentary to the target nucleic acid; and (d) detecting the presence and/or absence of the one or more pathogens. See claim 4. The one or more pathogen comprises two or more of E. coli, Salmonella, or Listeria monocytogenes. See claim 49. Zegrati teaches one or more of Escherichia coli, Salmonella, or Listeria monocytogenes that are detected when one or more of Escherichia coli, Salmonella, or Listeria monocytogenes is inoculated in or on a food product, wherein the food product is lettuce. See claims 158 and 160.
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 take a sample of the lettuce food product of Zegrati as a food sample in the method of Murphy modified by Pinheiro discussed above. Doing so is merely substituting known equivalents. One would be motivated to do so because Zegrati suggests that Salmonella and Listeria monocytogenes can contaminate lettuce. There would be a reasonable expectation of success because Murphy demonstrates analyzing food samples via PCR assays to detect L. monocytogenes and S. enterica, and Zegrati teaches conducting an amplification with a set of amplification primers on lysed samples and detecting one or more pathogens, such as Listeria and/or Salmonella.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119) in view of Pinheiro (US 2013/0065297), as applied to claims 1-2, 5, 14-15, 17-18, 26-27, and 33-34 above, and further in view of Biswas (US 2022/0315991; as cited in the IDS filed 06/27/2023).
The teachings of Murphy and Pinheiro with respect to instant claim 1 are discussed above.
Regarding claim 12, Murphy teaching inoculating enrichment broths with environmental samples and analyzing the enrichment broths for the detection of Listeria and Salmonella using either hlyA:gfp or iroB:gfp PCR assays. See section 3.3. Murphy teaches adding LENTICULE discs containing E. coli-GFP cells (e.g. control) to an enrichment broth culture prior to DNA extraction by MagNa Pure™ LC. See section 2.9.
Murphy and Pinheiro do not teach pre-treating the test sample and/or the control sample to remove nucleic acids not associated with intact cells with a pre-treatment mixture comprising a nuclease, thereby generating a pre-treated test sample and/or pre-treated control sample before said lysing, wherein said lysin is carried out with a lysis mixture comprising an agent that inactivates the nuclease.
Biswas teaches a method for determining the presence or absence of a target bacteria in a sample, wherein the sample is an environmental sample. See claims 74 and 77. 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.
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 the pre-treatment technique of Biswas to the environmental samples of Murphy, prior to the DNA extraction step of Murphy in the method of Murphy and Pinheiro discussed above. One would be motivated to do so because Murphy teaches environmental samples, and Biswas suggests pre-treating environmental samples. There would be a reasonable expectation of success because Biswas teaches that the technique can be used for Salmonella detection (e.g. see [0194]).
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119) in view of Pinheiro (US 2013/0065297), as applied to claims 1-2, 5, 14-15, 17-18, 26-27, and 33-34 above, and further in view of Shi (CN101045943) and Miyazaki (WO2004005508).
The teachings of Murphy and Pinheiro with respect to instant claims 1 and 18 are discussed above.
Regarding claim 24, Murphy teaches performing real-time PCR assays. The individual real time PCRs contain 12.5 µl qPCR mastermix, 5 µ of template DNA, 300 nM of each forward and reverse primer, and 100nM probe (e.g. assay mixture). See section 2.3. As shown in table 1, the forward and reverse primers include iroBF and iroBR, for the c-glycosyltransferase iroB gene of Salmonella enterica. Furthermore, Murphy teaches an internal control for the detection of S. enterica by PCR. See, for example, the abstract.
Pinheiro teaches producing cells that express modified GFP protein for use as quality controls. See [0129]. In example 2, Pinheiro teaches generating an unrepressed gfp gene cassette, and the chromosomal integration of unrepressed gfp gene cassette into E. coli and Salmonella strains. See [0101]-[0107]. Pinheiro suggests that the technique is applicable to other bacterial species and other promoter systems and marker genes can be used in a similar fashion. See [0130].
Murphy and Pinheiro do not teach one or more PCR primer sets comprising: Salmonella 1 (SEQ ID NOs: 1-2) and/or Salmonella 2 (SEQ ID NOs:4-5), nor do Murphy and Pinheiro teach one or more PCR primer sets capable of detecting the reporter molecule that is GFP (SEQ ID NOs: 7-8).
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.
Murphy, Pinheiro 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 apply the PCR technique of Murphy as modified by Pinheiro (discussed above) to the invA (SEQ ID NO: 1) of Shi; and to further replace the unrepressed gfp gene in the Salmonella GFP of Pinheiro and gfpEnt/gfpR0 primer thereof with the GFP+ gene (SEQ ID NO: 5) and primers of Miyazaki. One would be motivated to apply the method to the invA of Shi because Shi suggests that the invA gene is associated with food borne Salmonella pathogens (e.g. see [0004]). There would be a reasonable expectation of success because Murphy demonstrates performing real-time PCRs for the detection of Salmonella in food samples in the presence of an internal control; and Shi suggests using the invA in a PCR detection method with an amplification internal standard. One would be further motivated to use the GFP+ gene and primers of Miyazaki because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would be a reasonable expectation of success because Murphy, Pinheiro and Miyazaki demonstrate integrating GFP genes into vectors for expression and using the appropriate primers thereof for PCR amplification.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Murphy (International journal of food microbiology, 2007, 120(1-2), 110-119) in view of Pinheiro (US 2013/0065297), as applied to claims 1-2, 5, 14-15, 17-18, 26-27, and 33-34 above, and further in view of Seehusen (J Comp Pathol. 2008 Aug-Oct;139(2-3):126-9) and Miyazaki (WO2004005508) with evidence from “NIH” (Listeria monocytogenes listeriolysin O (hlyA) gene, partial cds – Nucleotide – NCBI (2025); hereafter referred to as NIH).
The teachings of Murphy and Pinheiro with respect to instant claims 1 and 27 are discussed above.
Regarding claim 31, Murphy suggests that the application of PCR-based technologies in clinical laboratories has vastly improved the detection of a wide range of pathogenic microorganisms. However, PCR assays have had a more limited application for testing food. See the introduction section on page 110. Murphy teaches detecting Salmonella enterica or Listeria monocytogenes in naturally contaminated food by real-time PCR using E. coli-GFP as an internal control. See section 2.9. Murphy teaches individual real-time PCRs that contain 12.5 µl qPCR mastermix, 5 µ of template DNA, 300 nM of each forward and reverse primer, and 100nM probe (e.g. assay mixture). 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 (e.g. PCR primer set capable of detecting Listeria).
Pinheiro teaches producing cells that express modified GFP protein for use as quality controls. See [0129]. In example 4, Pinheiro teaches generating fluorescent Listeria monocytogens by replacing the gfp gene mut1 in an E. coli/Listeria shuttle vector with triple mutant gfp gene. The gfp gene is amplified by the Gfpmutf1 and Gfpmutr1 primers. See [0111]. Pinheiro suggests that the technique is applicable to other bacterial species and other promoter systems and marker genes can be used in a similar fashion. See [0130].
Murphy and Pinheiro do not teach one or more PCR primer sets comprising: Listeria (SEQ ID Nos: 10-11) and/or Listeria monocytogenes (SEQ ID Nos: 13-14), nor do Murphy and Pinheiro teach one or more PCR primer sets capable of detecting the reporter molecule that is GFP (SEQ ID Nos: 7-8).
Seehusen teaches detecting L monocytogenes antigen in areas of necrosis in the ileum and liver [of a male alpaca], and diagnosing septicaemic listeriosis. Seehusen teaches preforming a PCR for amplifying specific Listeria spp. and L. monocytogenes specific gene fragments, for confirmatory purposes. See the right column of page 127. Seehusen suggests that this method has been previously used to verify the results of microbiological or histological investigations or as a rapid screening tool in epidemiological surveys or in food product monitoring. As evidenced by NIH, Seehusen teaches a Listeria monocytogenes listeriolysin o (hlyA) gene which includes a subsequence that is 100% identical to instant SEQ ID NO: 7, and a subsequence that is 100% identical to the reverse compliment of instant SEQ ID NO: 8. See the office action appendix for the alignments.
Murphy, Pinheiro and Seehusen 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 replace the hlyA primers of Murphy with primers for the hlyA gene of Seehusen, and to further replace the triple mutant gfp gene and Gfpmutf1/Gfpmutr1 primers of Pinheiro with the GFP+ and primers of Miyazaki. One would be motivated to use the hlyA of Seehusen because Seehusen suggests that specific L. monocytogenes gene fragments can be used to confirm a septicaemic listeriosis diagnosis. In other words, one would be motivated to detect an hlyA gene known to be associated with septicaemic listeriosis. Considering the structural and functional similarities between all hlyA genes, one would reasonably expect the hlyA gene of Seehusen to serve the same function as the hlyA gene of Murphy. One would be further motivated to use the GFP+ gene and primers of Miyazaki because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would be a reasonable expectation of success because Murphy, Pinheiro and Miyazaki demonstrate integrating GFP genes into vectors for expression and using the appropriate primers thereof for PCR amplification.
Claim 39 is rejected under 35 U.S.C. 103 as being unpatentable over Murphy (hereafter Murphy2009) (PhD thesis, University of Greenwich, 2009) in view of Yu (CN 103436619, and translation) and Miyazaki (WO2004005508).
The teachings of Murphy2009 with respect to instant claims 1, 33 and 34 are discussed above.
Regarding claim 39, Murphy2009 teaches the application of molecular techniques for the rapid and sensitive detection of gastrointestinal pathogens directly in food. See the title. Murphy2009 teaches developing E. coli green fluorescent protein (GFP) as a process control for the detection of pathogens including Escherichia coli O157:H7. See the first paragraph of 7.2.2. Murphy2009 teaches developing a Lenticule encased E. coli GFP strain for use as a control. See section 4.3.1 on page 130. Murphy2009 teaches a duplex assay, in which a Lenticule disc (control) is added to the culture dilutions (test) prior to DNA extraction [which entails lysing] and the resulting DNA is subjected to the eaeγ:gfp PCR assay. Each dilution is amplified and a standard curve of mean Ct values (i.e. synonymous with Cq) against CFUml-1 is produced. See section 2.13.2 and 2.13.5. As shown in table 2.6 on page 43, the forward and reverse PCR primers include eaeγF and eaeγR2 [eaeγ is intimin gamma] for E. coli O157:H7 (bacterial detection PCR primer set), and gfpF and gfpR2 for the E. coli GFP (control detection PCR primer set).
Murphy2009 does not teach one or more PCR primer sets that comprise one or more of E. coli 0157:H7 (SEQ ID NOs:16- 17), Shiga toxin gene(stx) 1 (SEQ ID NOs:19-20), Shiga toxin gene(stx) 2 (SEQ ID NOs:22- 23), Intimin gene (eae) (SEQ ID NOs: 25-26), E. coli 0103 (SEQ ID NOs:28-29) and E. coli 026 (SEQ ID NOs:31-32), E. coli 0145 (SEQ ID NOs:34-35), E. coli 0111 (SEQ ID NOs:37- 158971211v238), E. coli 045 (SEQ ID NOs:40-41), E. coli 0121 (SEQ ID NOs:43-44), nor does Murphy2009 teach one or more PCR primer sets capable of detecting the reporter molecule that is GFP (SEQ ID NOs:7-8).
Yu discloses that foodborne illnesses caused by E. coli O157:H7 continue to occur. E. coli can reside in the intestines of livestock resulting in livestock loss. See [0006]. 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 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.
Murphy2009 and Yu 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]. Miyazaki teaches transforming E. coli host cells with the recombinant plasmid. See [0025].
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 replace the eaeγ gene and primers thereof of Murphy2009 with eaeA gene and primers thereof of Yu, and to further replace the gfp and primers of Murphy2009 with the GFP+ and primers of Miyazaki. One would be motivated to use eaeA gene and primers Yu because Murphy2009 focuses on gastrointestinal pathogens, and Yu suggests that the eaeA is present in the intestines. There would be a reasonable expectation of success because Murphy2009 and Yu teach using the intimin gene and primers thereof for the identification of E. coli O157:H7. One would be further motivated to use the GFP+ transformant and primers of Miyazaki because Miyazaki suggests that the GFP+ gene encodes an active fluorescent GFP upon induction. There would be a reasonable expectation of success because both Murphy2009 and Miyazaki teach transforming the gfp gene into E. Coli hosts.
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. 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 does not teach one or more control bacterial strains of the same genus as the target bacteria detected by the one or more PCR primer sets, wherein the control bacterial strain is engineered to express a reporter molecule not naturally present in the bacteria, and one or more 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. The bacterium is Escherichia coli, Salmonella sp. or Listeria sp. See claims 25 and 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 replace the internal control primers of Zegrati in the kit with the gfpEnt and gfpR0 primers of Pinheiro. One would be 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, and Zegrati suggests that without appropriate controls, it may not be possible to determine whether an absence of the contaminating pathogen being detected is a result of the failure of the assay, or as a result of the absence of any contaminating pathogens in the sample [0241]. There would be a reasonable expectation of success because Pinheiro teaches kit comprising a positive PCR control, and Pinheiro teaches a bacterium that can be used as a control. One would be further motivated to replace the internal control primers in the kit of Zegrati with the primers of Pinheiro because Pinheiro suggests that the primers are capable of detecting the unrepressed gfp gene in the E. coli transformant. There would be 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.
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).
The teachings of Zegrati and Pinheiro regarding claim 41 are discussed above.
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 PCR primer sets are selected from the group consisting of: (i) Salmonella 1 (SEQ ID NOs: 1-2), Salmonella 2 (SEQ ID NOs:4-5), and GFP (SEQ ID NOs:7-8); (ii) Listeria spp. (SEQ ID NOs:10-11), L. monocytogenes (SEQ ID NOs:13-14), and GFP (SEQ ID NOs:7-8);(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), and GFP (SEQ ID NOs:7-8); and (iv) any combination thereof.
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.
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), and GFP (SEQ ID NOs:7-8).
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 replace the eaeA and stx1 primers of Zegrati with eaeA primers from the sequence taught by Yu and stx1 primers from the sequence of Brousseau. Doing so is merely substituting known equivalents. One would be motivated to use eaeA primers taught by Yu because Yu suggests that the eaeA gene is associated with the pathogenic bacterium E. coli O157:H7. There would be a reasonable expectation of success because the eaeA primers of Yu reasonably serve the same function as the eaeA primers of Zegrati in the kit. One would be further motivated to use the stx1 of Brousseau 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.
Response to Arguments
Applicant's arguments filed 11/21/2025 have been fully considered but they do not apply to the new grounds of rejection set forth above.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657