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 .
Election/Restrictions
Applicant’s election without traverse of Group I, SEQ ID NO: 1, 2, 9, 12, 13, 20, 24, 25, 34, 39, 40, 49, 53, 54, and 63 in the reply filed on 10/15/25 is acknowledged.
Improper Markush Grouping
Claims 1-4, 7-12, 14-19, and 21 are rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush grouping of primers or probes identified by SEQ ID NO: alternatives (for example any one of SEQ ID NO: 1, 3, 5, and 7 or sequences with 1 or 2 mismatches)) is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: the oligonucleotides do not share a common structure.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
In the response filed on 3/18/2026 applicant pointed out that the forward and reverse primers are capable of generating amplicons, and so share a common use. The response did not address the second requirement that the oligonucleotide share a common structure. The rejection is maintained.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1, 4, 7, 8, 10, and 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Alam et al. (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2006, p. 2849–2855) in view of Mendes (Journal of Microbiological Methods 72 (2008) 191–196), Yu et al. (Biosensors andBioelectronics70(2015)282–288), GenBank KF551993 (Vibrio cholerae strain PTCC1611 cholera toxin A subunit (ctxA) gene; 15 Sept 2013; 2 pages), GenBank X59554 (V.cholerae, genes for rfbA-rfbT, ompX, orf1-3, 18 April 2005; 11 pages) , GenBank KY660230 (Mutant Vibrio cholerae O139 strain MO10 aspargine synthetase (vcm8); 9 May 2017; 2 pages), and GenBank KJ722608 (Vibrio cholerae O1 strain N16961 OmpW (ompW) gene, complete cds - Nucleotide – NCBI; 29 May 2014; 2 pages).
Alam et al. teaches a method in which Vibrio cholerae is detected by multiplex PCR employing primers directed to the V. cholerae species specific gene ompW, a gene specific to V. cholerae O1, a gene specific to V. cholerae O130, and the ctxA gene encoding subunit A of the cholera toxin. The method includes contacting said sample with a plurality of pairs of primers, generating amplicons if said sample comprises one or more of the targets, and determining the presence of the amplicons as an indication of the presence of one or more of V. cholerae, serogroup o1, serogroup 0139, and the gene encoding cholera toxin in said sample. See Abstract, p. 2850, 2nd column, and Table 1.
The reference does not teach the primers set forth and elected for the ompW and ctxA genes, nor does the reference teach amplifying with primers for rfbN and wbfR.
At the time the invention was made, Mendes taught amplification of rfbN gene for the detection of Vibrio cholerae O1 in a sample. Yu et al. taught amplification of wbfR to identify V. cholerae O139.
It would have been obvious to have modified the method taught by Alam et al. so as to have substituted primers for amplification of rfbN and wbfR as the substitution of one known method for another, that is to provide alternate methods for detecting the toxic O1 and O139 serotypes in samples. One would have been motivated to do so because these were known serotype specific amplification targets, as exemplified by Mendes and Yu.
Alam et al., Mendes et al., and Yu et al. do not teach primers having SEQ ID NO: 1, 2, 12, 13, 24, 25, 39, or 40, as elected.
The sequences of each of the relevant target genes were known, and the claimed primers had 100% identity to known sequences.
The sequence of the ompW gene was taught in KJ722608, and instant SEQ ID NO: 1 was identical to nucleotides 553-572, and instant SEQ ID NO: 2 was identical to nucleotides 624-642.
The sequence of the V. cholerae O1 strain rfbN gene had been taught in Genbank X59554, see nucleotides 12384-14861. Instant SEQ ID NO: 12 and 13 are fragments of this gene, see nucleotides 14385-14406 (SEQ ID NO: 12) and nucleotides 14462-14484 (complement of SEQ ID NO: 13).
The sequence of the O138 wbfR gene was known in KY660230, and instant SEQ ID NO: 24 is identical to nucleotides 1524-1547 of the known sequence, and instant SEQ ID NO: 25 is the complement of nucleoitdes 1663-1682.
The sequence of V. cholorae ctxA had been taught in GenBank KF551993. Instant SEQ ID NO: 39 is identical to nucleotides 146-166. Instant SEQ ID NO: 40 is the complement of nucleotides 211-229.
Further, Mendes taught designing primers using commercially available primer design software (section 2.2, Primers).
It would have been prima facie obvious for one of ordinary skill in the art at the time of invention to conduct the V. cholerae detection method of Alam, Mendes, and Yu using oligonucleotide primers selected from the known gene sequences, and comprising SEQ ID NO: 1, 2, 12, 13, 24, 25, 39, or 40. An ordinary artisan would have been motivated to do so with a reasonable expectation of success, since: (i) Alam, Mendes, and Yu expressly taught employing primers that were fragments of known sequences (ii) the complete sequences of each of the target genes was known in the art at the time of the invention, and (iii) Mendes disclosed a publicly available program that uses rules to design primers from any known target nucleic acid sequence.
The combined teachings of the cited references would have suggested a finite number of possible PCR primer pairs that could be designed from the known ompW, rfbN, wbfR, and ctxA sequences to the ordinary artisan, and, the ordinary artisan would have expected predictable results in obtaining and using these oligonucleotides V. cholerae detection method of Alam to produce an amplicons indicative of V. cholerae, toxin and toxic serotypes. Thus, absent any evidence of unexpected results with respect to particular primers the claimed method is prima facie obvious in view of the combined teachings of the cited references.
Furthermore, regarding claim 7, Yu teaches detecting V. cholerae in spiked stool samples. It would have been obvious to have modified the method taught by Alam to apply it to the detection of V. cholerae in stool samples to achieve the benefit of providing a way to diagnose patients who have “rice watery” stools (see section 3.3 and 3.7 of Yu).
Claim(s) 14, 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Alam et al. in view of Mendes, Yu et al., GenBank KF551993, GenBank, GenBank KY660230, and GenBank KJ722608 as applied to claims 1, 4, 7, 8, 10, and 12 above, and further in view of Hu et al. (Journal of Clinical Microbiology February 2012 Volume 50 Number 2 p. 288–293).
The teachings of Alam et al. in view of Mendes, Yu et al., GenBank KF551993, GenBank, GenBank KY660230, and GenBank KJ722608 are given previously in this Office action and are fully incorporated here.
These do not teach a method where the primers have exogenous sequences attached.
Hu teach a PCR assay where during the first few cycles of PCR, During the first few cycles of PCR, amplification is carried out by chimeric forward and reverse primers. In the later stages of PCR, amplification is predominantly carried out by universal forward and reverse primers.
This method is carried out using primers with a target specific portion and an exogenous, universal primer specific portion (p. 289, 1st column, figure 3). The universal primers are labeled, enabling a quantitative real time PCR assay.
It would have been prima facie obvious to have modified the method taught by Alam et al. in view of the additional references so as to have employed a quantitative real time multiplex assay such as the one taught by Hu et al. One would have been motivated to do so in order to detect many sequences at once in a highly sensitive manner, as demonstrated by Hu et al.
Claim(s) 16, 17, 18, 19, and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Alam et al. in view of Mendes, Yu et al., GenBank KF551993, GenBank, GenBank KY660230, and GenBank KJ722608 as applied to claim 1, 4, 7, 8, 10, and 12 above, and further in view of Ward et al. (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 2031–2042).
The teachings of Alam et al. in view of Mendes, Yu et al., GenBank KF551993, GenBank, GenBank KY660230, and GenBank KJ722608 are given previously in this Office action and are fully incorporated here.
These do not teach contacting the one or more amplicons with a plurality of oligonucleotide probes, wherein the oligonucleotide probes comprise SEQ ID NO: 9, 20, 34, or 49, consonant with the election, or a probe having only one or two mismatches relative to these.
Each of the claimed probe sequences were known in the sequences of the respective genes, as shown in the GenBank records cited in this rejection. Particularly, SEQ ID NO: 9 is nucleotide 578-603 of KJ722608, SEQ ID NO: 20 is the complement of nucleotides 14428-14459 of X58544, SEQ ID NO 34 is the complement of nucleotides 1630-2657 of KY66020, and SEQ ID NO: 49 is the complement of nucleotides 179-205 of KF551993.
Ward et al teaches multiplexed real-time PCR in which PCR and detection with a probes labeled with different fluorophores occurs in a single reaction. Each probe has a fluorescence emitter moiety and a fluorescence quencher moiety attached. The assay provides a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays. See abstract, Table 1, and throughout.
It would have been obvious to have modified the method taught by Alam and the additional references so as to have selected probes for detection of amplicons from the known sequences for the detection of amplicons. One would have been motivated to do so in order to take advantages of the real-time PCR method as taught by Ward et al.
Claim(s) 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Alam et al. in view of Mendes, Yu et al., GenBank KF551993, GenBank, GenBank KY660230, and GenBank KJ722608 as applied to claim 1, 4, 7, 8, 10, and 12 above, and further in view of Gubala et al. (2006. Appl Environ Microbiol 72: 6424-6428).
The teachings of Alam et al. in view of Mendes, Yu et al., GenBank KF551993, GenBank, GenBank KY660230, and GenBank KJ722608 are given previously in this Office action and are fully incorporated here.
These do not teach contacting the one or more amplicons with a plurality of oligonucleotide probes, wherein the oligonucleotide probes comprise SEQ ID NO: 9, 20, 34, or 49, consonant with the election, or a probe having only one or two mismatches relative to these, wherein each probe is flanked by complementary sequences at the 5’ end and the 3’ end wherein each complementary sequence is complementary to the other.
Each of the claimed probe sequences were known in the sequences of the respective genes, as shown in the GenBank records cited in this rejection. Particularly, SEQ ID NO: 9 is nucleotide 578-603 of KJ722608, SEQ ID NO: 20 is the complement of nucleotides 14428-14459 of X58544, SEQ ID NO 34 is the complement of nucleotides 1630-2657 of KY66020, and SEQ ID NO: 49 is the complement of nucleotides 179-205 of KF551993.
Gubala et al teaches multiplexed real-time PCR in which PCR and detection with a probes labeled with different fluorophores occurs in a single reaction. The probes are molecular beacons which have target sequences flanked by complementary sequences at the 5’ end and the 3’ end wherein each complementary sequence is complementary to the other. See Table 2. Each probe has a fluorescence emitter moiety and a fluorescence quencher moiety attached. The reference teaches that the assay using molecular beacons for the simultaneous detection of four target genes, resulted the specificity and sensitivity that surpassed those of the published PCR assays for the detection of V. cholerae. See p. 2467.
It would have been obvious to have modified the method taught by Alam and the additional references so as to have selected probes for detection of amplicons from the known sequences for the detection of amplicons, and to have used a molecular beacon design for multiplex detection. One would have been motivated to do so in order to take advantages of the real-time multiplex molecular beacon PCR method as taught by Gubala et al.
Response to Remarks
Any rejection not reiterated or addressed in this Office action was overcome by the amendment filed 3/18/2026.
Applicant traverses the rejection for obviousness. Applicant argues that there is no suggestion for combining the cited references for the detection of all of the presently cited target genes, and even if there were, there was no reasonable expectation of success.
The rejection clearly sets forth the rational for detecting rfbN and wbfR, namely because thse were taught to be specific for V. cholerae O1 and V. cholerae O139, and therefore could be motivated to use these targets for detecting these pathogens based on the teachings of Mendes and Yu. (Office action p. 5).
As to the argument for lack of expectation of success, since the methods of Mendes and Yu use different detection schemes than Alam. However, this is not persuasive because it does not provide any evidence that targeting these known pathogen specific genes by M-PCR as taught by Alam would not be expected to function. Multiplex PCR was widely practiced before the effective filing date, and as taught by Mendes at the time of the invention commercially available primer design software was in use by the skilled artisan. An absolute expectation of success is not required for a prima facie case of obviousness.
No unexpected results are shown for the claimed primer and probe sets, which are not limited to primers and probes consisting of the recited SEQ ID NO, as they can be longer due to the comprising language AND the claims allow for mismatches in each sequence. Applicant cites to the multiplex PCR method tested in specification ¶145, but this method is not commensurate in scope with any of the claims, nor does it show any comparison against the closest prior art of Alam which shows detection of all of the same types of Vibrio. Furthermore, most of the claimed methods are inclusive of methods for serial amplification, and do not require multiplex amplification. As noted in the rejection, commercial tools for developing primer sets were known before the effective filing date. There is no evidence that the selection and use of the instant primers was unexpected.
The prior art rejection over claims 2, 3, 9, and 11 is WITHDRAWN because the prior art does not teach or suggest amplifying the YaiO gene along with the other targets. The basis for the claims is to the parent application 11/5/2020.
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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Juliet Switzer
Primary Examiner
Art Unit 1682
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
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