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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 Dec. 24, 2025 has been entered.
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on Dec. 24, 2025. Claims 1-2 and 5-19 are pending and currently examined.
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 of this title, 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 and 5-19 are rejected under 35 U.S.C. 103 as being unpatentable over Muhammed et al. (PLoS ONE, 2017, 12(3): e0174223), Ly-Chatain et al. (International Journal of Microbiology Volume 2011, Article ID 594369, 9 pages), Verreault et al. (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2011, p. 491–497, Vol. 77, No. 2), GenBank: KP793114.1 (Lactococcus phage 936 group phage Phi17, complete genome. Dated Mar. 2, 2015), and Thornton et al. (BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Vol. 39, No. 2, pp. 145–154, 2011).
Claims 1-2, 5-7 and 16-19 are directed to a quantitative polymerase chain reaction (qPCR) kit for detection and quantification of phage DNA from a lactic acid bacterium-infecting phage in a dairy sample, comprising a first primer pair having a robustness of a delta Cq lower than 1.0 cycle when tested in a temperature range of 55.0 - 70.0 degrees Celsius, directed to a lactococcal phage from the subgroup 936, c2 or P335 or a streptococcal phage from the subgroup pac. Claims 8-15 are directed to a method for detecting and quantifying phage DNA from lactic acid bacteria infecting phage in a dairy sample with a qPCR kit of claim 1.
Muhammed teaches that the authors optimized a high-throughput qPCR system that allows simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from ~1.1 x 105 to ~1.1 x 101 phage genomes per reaction, which corresponds to ~9 x 107 to ~9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother-bulk-cheese vat system. High levels of 936 and P335 phages were detected in the mother culture and the bulk starter, but also in the whey samples. Low levels of phages were detected in the cheese milk samples. See Abstract.
Muhammed teaches that primers were designed using CLC Genomics Workbench primer tool (Qiagen, Aarhus) and synthesized at Integrated DNA Technologies (IDT, Germany). Databases were screened for conserved genes in the genomes of the phages. Sequenced genomes, available in public databases and obtained from in-house sources, were used to perform multiple sequence alignment. At least 90% primer-target identity was used to qualify primers. The primers' specificity was tested in silico using primer-BLAST (NCBI, USA) with strict parameters (above four primer-unintended target mismatches within the last 10 bp at the 3' end, above six mismatches in total). The primers designed and optimized in this study are summarized in Table 1 (shown below). It teaches that the primers annealing temperature was tuned with gradient PCR (SureCycler 8800, Agilent Technologies, USA) using 2x PCR Master Mix (Thermo Fisher Scientific, USA). See page 3.
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Ly-Chatain teaches that, to prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups of Lactococcus bacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 102 PFU/mL. The curve slopes were −3.49, −3.69, and −3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient (R2) of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. See Abstract.
Ly-Chatain presents the primers in Table 2 (shown below) and teaches that, to determine the optimal concentration of primer in PCR, preliminary tests were performed by combining the primers in three final concentrations: 200, 400, and 800nM at different hybridization temperatures: 55, 58, and 62◦C. See page 2, right column, para 2.
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Ly-Chatain teaches that amplification and detection were performed with the Minicon system (Bio-Rad, France). The PCR reaction mixture contained 12.5 μL of SYBR Green PCR master mix (Quiagen, France), 9.5 μL of water, 1 μL of each primer (0.4μM as final concentration), and 1 μL of DNA in a 25μL final volume. The reaction was done under the following conditions: 98◦C for 30 sec and 40 cycles of 95◦C for 15 s, 62◦C for 30 sec. See page 2, right column, para 3.
Verreault teaches a study on detection of Lactococcal bacteriophages in cheese manufacturing plants. Verreault teaches that samples were then analyzed for the presence of two Lactococcus lactis phage groups (936 and c2), and quantification was done by quantitative PCR (qPCR). Both lactococcal phage groups were found on most swabbed surfaces, while airborne phages were detected at concentrations of at least 103 genomes/m3 of air. The NIOSH sampler had the highest rate of air samples with detectable levels of lactococcal phages. This study demonstrates that virulent phages can circulate through the air and that they are ubiquitous in cheese manufacturing facilities. See Abstract.
Verreault teaches that all samples were kept on ice after on site sampling and then stored at 4°C until analyses, which were performed within 48 h. PTFE and PC filters were eluted by placing 5 ml of sterile water with 0.01% Tween 20 directly into the three-piece cassettes and by shaking the filters on a vortexer for 20 min. The volumes of liquid in the BioSamplers and the Coriolis samplers were measured, and the liquid was stored at 4°C. For the NIOSH samplers, 250 ul of sterile water with 0.01% Tween 20 was placed in each tube. The 1.5-ml tubes were pulse shaken on a vortexer until the pellet was no longer visible. The 15-ml tubes were processed in the same way; however, the tubes were also shaken upside down to clean the upper part of the tube. No DNA extraction was necessary for qPCR analysis. Every sample was analyzed by qPCR in triplicate at a minimum. See page 493, left column, para 4.
Verreault further teaches that Primers for quantitative PCRs (qPCRs) were designed using the Beacon Designer 4.0 program (PREMIER Biosoft International, Palo Alto, CA) for SYBR green protocols. The primer pair used for the detection of the lactococcal 936 phage group has already been published. The targeted region was within orf6 of phage P008, a gene coding for a highly conserved structural protein within the 936 group. The reverse and forward primers used were 5-CCAGCAGTAGGGCGAACAAAG-3 (positions 5187 to 5167) and (5-TGAGGGAGACGGAACAAACGG-3 (positions 5035 to 5055), respectively. For the c2 group, they targeted the gene coding for the highly conserved major capsid protein. The reverse and forward primers used were 5-GCATTAAAGCCAACTGATAGC-3 (positions
9642 to 9662) and 5-AGTAAGAGGGATAGCGAACC-3 (positions 9432 to 9451), respectively. See page 493, left column, para 4.
Verreault further teaches that the protocol used for qPCR was 94°C for 3 min (hot start), followed by 35 cycles of (i) 94°C for 20 s, (ii) 60°C or 53°C for 30 s for 936 and c2, respectively, (iii) plate reading, and (iv) 72°C for 25 s. A melting curve was calculated with readings every 0.2°C from 50°C to 95°C, holding the temperature for 1 s. The cycle threshold (CT) for all plates was set at a fluorescence intensity of 0.011. See page 493, left column, para 6.
Accordingly, Muhammed, Ly-Chatain and Verreault teach qPCR systems for detection and quantification of lactococcal bacteriophages (including phages c2, 936, and P335) and primers and agents used in the qPCR assays. The references teach optimization for the qPCR assay, including e.g., primer designs, reagents and cycling conditions. However, they are silent on if the qPCR system has a robustness of a delta Cq lower than 1.0 cycle when tested in a temperature range of 55.0 - 70.0 degrees Celsius, which is expected to be related directly to the qPCR conditions (including primer designs, nature of samples, reagents used, etc.).
GenBank: KP793114.1 discloses the complete genome sequence of Lactococcus phage 936 group phage Phi17, comprising sequence of the primers recited in claim 5. Teachings of GenBank: KP793114.1 indicate that the phage sequence used in designing PCR primers is known at the time of invention.
Thornton teaches that Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR1 Green dyes and TaqMan1, Molecular Beacon or Scorpion probes. SYBR1 Green is very simple to use and cost efficient. As SYBR1 Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR1 Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR1 Green-based primers with free, online software. See Abstract. Teachings of Thornton indicate that qPCR is conventional at the time of invention and various tools are available for experimental design and optimization.
It would have been prima facie obvious to one of ordinary skill in the art at the time of invention to combine the teachings of Muhammed, Ly-Chatain, Verreault, GenBank: KP793114.1 and Thornton to arrive at the invention as claimed through routine experimental optimization. There is a reasonable expectation of success to develop a qPCR for detection and quantification of phages 936, c2 or P335 that has a robustness of a delta Cq lower than 1.0 cycles, as claimed, through routine experimentation based on the combined teachings of the cited references.
Regarding claims 3 and 4, since complete genome sequences of the target phages are known at the time of invention for design of primers, one of skill in the art would have found it obvious to select any region for PCR amplification based on experimental needs, including the region as claimed. E.g., a conserved sequence region would likely be selected for detections of multiple phages by one primer pair while a type-specific region would be selected for phage-specific detections.
Regarding claim 5, since complete genome sequences of the target phages are known at the time of invention for design of primers, one of skill in the art would have found it obvious to arrive at the primers as claimed through routine experimental optimization, unless there is evidence that the claimed primers produce unexpected results.
Regarding claims 16-19, the claims do not specify any primer sequences. Therefore, the claimed delta Cq values as well as the temperature ranges are considered as intended goals of a study that can be arrived via experimental optimization.
Response to Applicant’s Arguments
Applicant’s arguments filed on Dec. 24, 2025 have been fully considered and addressed as follows.
Applicant argues that the present invention is directed to a qPCR kit including primers with low delta-Cq over a temperature range, combined with a new and conserved phage amplicon [936] to enable quantitative detection directly in a dairy sample, i.e., without the extraction and purification of the DNA out of the dairy sample (see, e.g., claim 7). Applicant argues that this is achieved with just a sample dilution, allowing for a rapid detection method, and that given that the matrix can be inhibitive to the PCR reaction, there is a need for a robust qPCR assay with robust primers.
Applicant argues that Muhammed and Ly-Chatain use standard cycling conditions, and that they teach away from performing qPCR without the extraction and purification of the DNA out of the dairy sample, as they both employ DNA extraction to isolate DNA from the inhibitory matrix. Applicant argues that Muhammed and Ly-Chastain focus primarily on qPCR basics, aiming for a correlation between PFU phage titers and the Cq value of DNA detected in the extracted DNA sample, and that these extraction protocols are time-consuming, require more chemicals, and are quite intensive in terms of molecular biology, necessitating trained personnel.
Applicant argues that the Office, by noting that the genome sequence of Lactococcus phage 936 group phage Phi17 was known in the art via GenBank Accession No. KP793114.1, and making the phage sequence used in designing PCR primers known to a person of ordinary skill in the art, assumes that all genes are equivalent. Applicant argues that claim 1 recites SEQ ID NO:16 (structural protein 1), while Muhammed targets different regions like the terS gene. Applicant argues that the Office has not shown that targeting terS or other genes would inherently yield the claimed robustness.
Applicant argues that the Office argues that because Thornton teaches the existence of primer design software, any functional property achieved by primers that could be designed is "obvious as an intended result." Applicant argues that Thornton only provides general rules for primer design and does not teach a method for achieving the specific functional property of temperature-insensitive amplification, as claimed. Applicant argues that standard primer design, as taught by Thornton, focuses on optimizing for a single specific annealing temperature (Ta) to maximize efficiency and specificity. Applicant argues that Thornton never discusses how to set up a robust qPCR assay to directly detect DNA in a dairy matrix, and provides no motivation for a PHOSITA to do so.
Applicant argues that claimed kit and method produce unexpected results by allowing for direct qPCR testing on dairy samples after only a dilution step, resulting in rapid turnaround (≤ 60 minutes) compared to hours for extraction-based methods, reducing cost and labor, making the kit and method suitable for on-site or in-process monitoring, while preserving sample integrity and avoiding biases introduced by extraction efficiency, achieving robust amplification (DСq ≤ 0.5 across 58-68°C) and a detection limit comparable or superior to extraction-based methods. Applicant argues that there is no motivation shown by the prior art to design primers that maintain a nearly identical Cq across a wide 15-degree temperature gradient, as claimed. Applicant argues that the Office fails to explain why a PHOSITA would be motivated to seek temperature independence at all. The prior art phages taught by Muhammed and Ly-Chastain were detected in laboratory settings using precise thermal cyclers.
Applicant’s arguments above are not persuasive.
As indicated in the previous Office action, the specified robustness is considered as intended results of the claimed invention. Even though they may be obtained by the primers and other conditions used in experiments shown in Examples 2 and 3 (those primers and conditions are not specified in the claims), there is no evidence that the specified robustness is unique to the structural protein 1 coding region in general. On the other hand, since the complete sequence of phage subgroup 936 is known, one of skill in the art would have found it obvious to design and test PCR primers annealing to any phage genome sequence to look for effective PCR designs through routine experimental optimization, unless there is evidence that the claimed region, i.e., the structural protein 1 coding region, is critical.
Instead of arguing against the above standings and providing factual evidence that the claimed target region (note: the claims are generic in the primer sequences except for the target (annealed) region which is the structural protein 1 encoding region) is unique comparing to other regions on the phage genome, Applicant focuses arguments on that the cited prior art references do not teach (or teach away from) doing PCR without the nucleic acid extraction process. First, claims 1-2, 5-7, and 15-19 are directed to a kit for detection and quantification of phage DNA comprising a primer pair. A nucleic acid extraction process is not a limitation for the kit as claimed. Secondly, the method claims a PCR method “comprising” “(iii) testing the, optionally diluted, sample with an qPCR kit according to claim 1”. By reciting “comprising”, the claims do not exclude a nucleic acid extraction/isolation process. Thirdly, Verreault explicitly teaches that samples from dairy production processes can be assayed for phage existence without nucleic acid extraction (see discussion in the art rejection above). Additionally, based on the teachings of Verreault, a skilled artisan would have found it obvious to omit the nucleic acid extraction step in order to simplify the overall phage detection process. Omission of an element and its function is obvious if the function of the element is not desired. See MPEP 2144.04 II.A.
As to Applicant’s argument about teaching away, MPEP section 2145 X.D relates to assertions that the art teaches away from the claimed invention. Such teachings are not considered to be a teaching away merely by indicating that something is in some manner inferior to another. In essence, a teaching away must criticize, discredit, or otherwise discourage the solution. Here, no prior art references effectively criticize, discredit, or otherwise discourage not extracting nucleic acid from a dairy sample before PCR.
As to Applicant’s argument that the Office has not shown that targeting terS or other genes by Muhammed (or other cited references) would inherently yield the claimed robustness, it is noted that Applicant has not provide evidence showing that the invention as claimed yield a robustness any better than qPCRs targeting other genome regions, such as those disclosed in Muhammed, Ly-Chatain and Verreault. The claims specify neither the primer sequences nor qPCR conditions used in obtaining the claimed “robustness”.
As to Applicant’s argument that Thornton does not discuss how to set up a robust qPCR assay to directly detect DNA in a dairy matrix, and provides no motivation for a PHOSITA to do so, teachings of Thornton suggest that designing of PCR primers is a routine in the art. One of skill in the art would have found it obvious design PCR primers based on the known phage sequence disclosed in GenBank Accession No. KP793114.1.
As to Applicant's arguments about unexpected results, as an initial matter, "the burden of showing unexpected results rests on he who asserts them. Thus it is not enough to show that results are obtained which differ from those obtained in the prior art: that difference must be shown to be an unexpected difference." In re Klosak, 455 F.2d 1077, 1080 (CCPA 1972) (citation omitted). Moreover, "[i]t is well settled that unexpected results must be established by factual evidence. Mere argument or conclusory statements in the specification does not suffice." In re De Blauwe, 736 F.2d 699, 705 (Fed. Cir. 1984) (citation omitted). Applicant's attention is directed to MPEP 716.02(b)-(e) for how unexpected results can be established. E.g., to evaluate if the claimed invention produces unexpected results, one must consider if the results produced by the claimed invention are commensurate in scope with the claims and how the results compare with the closest prior art. See MPEP Section 716.02(d) and (e). Here, the results presented by Applicant are not commensurate in scope with the claims and there is no comparison between the results with the closest prior art.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NIANXIANG (NICK) ZOU whose telephone number is (571)272-2850. The examiner can normally be reached on Monday - Friday, 8:30 am - 5:00 pm, EST. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, MICHAEL ALLEN, on (571) 270-3497, can be reached. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300. Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/NIANXIANG ZOU/Primary Examiner, Art Unit 1671