DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Objections
Claims 1, 8, 10, 16, and 18 are objected to because of the following informalities:
Claim 1, L4: At the first recitation of an abbreviation, it should be accompanied by its full name. This is the first recitation of “PMA” and should be accompanied by its full name “propidium monoazide”.
Claims 8 and 16: the comma after “human microbiome” should be removed.
Claim 10 and 18: “…bacteria from a surface…”
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 7, 14, and 15 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.
The term “standard” in claims 7 and 15 is a relative term which renders the claim indefinite. The term “standard” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Many DNA isolation kits are taught in the art; however, there are not agreed upon kits which would be suitable across all applications. Instead, kits are picked based upon the assay being performed and the specific goals of the artisan. Therefore, what would qualify as a “standard” DNA isolation kit is unclear. For the purposes of prior art, any kit or collection of reagents used to perform DNA isolation will be considered to read upon the instant claims.
Claim 14 recites a limitation regarding comparing values of quantified DNA. However, the parent claim (claim 11) does not comprise a quantification step(s). Therefore, there is insufficient antecedent basis for this limitation in the claim.
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 11-13 and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Ni [Ni J. et al. Microbial ecology. 2020 May;79(4):925-32] in view of Mhuireach [Mhuireach GÁ et al. Developments in the Built Environment. 2021 Jul 1;7:100055].
Ni describes a propidium monoazide (PMA)-PCR-based 16S rRNA gene sequencing analysis method which is capable of uncovering the viable microbiome present in complex environmental samples [Ni, abstract].
To perform this method, sludge samples from five sewage treatment plants were collected. Upon collection, 12 samples collected from anaerobic digesters (DS) were split into two subsamples: a first subsample, which was used for PMA treatment (DS_PMA), and a second subsample, which was washed with PBS alone (DS). PMA working solution was prepared by diluting 2.5uL of the stock solution (20 mM) to achieve a final PMA concentration of 0.1mM. This working solution was then mixed with the first subsamples and incubated for five minutes in the dark. Cross-linking of PMA with DNA was conducted by exposing the first subsamples to blue light for 15 minutes. The first subsamples were then washed of excess PMA. Finally, both subsamples were subjected to DNA extraction using the ISOIL for Beads Beating Kit, quantified, and sequenced [Ni, p926-927].
Sequencing was conducted using primers specific for the V3-V4 regions of the 16S rRNA gene during library preparation and analyzing the amplicons on the Illumina MiSeq sequencer. Among other analyses, relative abundance was calculated for each sample from the resultant data. By comparing the results of the two samples, Ni was able to evaluate the effects of PMA treatment on microbial community structure, depicted by Figure 2 (below) [Ni, p926-928].
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Ni does not teach that the subsamples are of equal volume.
Mhuireach teaches the ubiquity with which diverse bacteria, fungi and other microorganisms are found on and within building materials. While many are generally regarded as neutral in their effect on human health, a number of studies have suggested that exposure to diverse microbiota during childhood is critical for proper immune system development. Additionally, little is known regarding the influence of different building materials on bacterial community structure. To investigate this, multiple samples were collected from the surface of four different materials: gypsum, timber, earthen paster, and concrete. Each sample was homogenized and divided into two equal aliquots wherein one aliquot was treated with PMA and one was not. Samples were then extracted, quantitated via qPCR, and sequenced.
The skilled artisan would recognize that samples of unequal volume would skew the quantification results towards the sample that began with a greater initial concentration Using Mhuireach’s sampling scheme, the skilled artisan could avoid this bias by starting with as close to identical samples as possible. Therefore, one of ordinary skill in the art prior to the effective filing date of the claimed invention would have been motivated to incorporate the sampling scheme of Mhuireach into the viability assay of Ni in order to improve the accuracy of the assay and support conclusions drawn from comparisons between the untreated and treated subsamples. Applying a known technique to a known device (method or product) ready for improvement to yield predictable results is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, D.).
Claims 16-18 are rejected under 35 U.S.C. 103 as being unpatentable over Ni and Mhuireach as applied to claim 11 above, and further in view of Brooks [Brooks B. et al. Microbiome. 2018 Jun 20;6(1):112].
Ni is applied to the relevant teachings of claim 11 as discussed above and is incorporated herein by reference. Ni does not disclose that the sample is from a human microbiome or a built surface, or that the bacteria within that sample originates from human skin, saliva, the nasal cavity or the gut.
Brooks describes a method for evaluating the identity, persistence, and absolute abundance of neonatal intensive care unit (NICU) room-associated bacteria using droplet digital PCR (ddPCR) and 16S rRNA gene surveys [Brooks, abstract]. Samples were collected from a variety of surfaces including the floor, sink basin, and hand rail, as well as hand samples and feces samples from the infants [Brooks, Fig. 1, p2]. These samples were extracted, quantified with ddPCR, and sequenced. For both ddPCR and sequencing, the target was the 16s rRNA gene [Brooks, p3].
The results of this testing demonstrated that biomass density could vary across NICU surfaces by four to five orders of magnitude and that 5-10 OTUs account for most of the sequencing data, the majority of which are typically associated with the skin, nose, or feces. Additionally, a finding related to the change in biomass and microbial community structure of the sink basins over the course of a week was also highlighted in which discovered that the sink biomass was the highest and the community structure was relatively uneven on Mondays in comparison to the other days of the week, hypothesizing that this is likely due to less frequent cleanings on the weekends [Brooks, p9].
One of ordinary skill in the art prior to the effective filing date of the claimed invention would have been motivated to employ the method of Ni to analyze the human microbiome and build surface samples described by Brooks because both use similar 16S rRNA sequencing workflows for characterizing microbial communities. Given the similarities between the methods, one would have reasonably expected Ni’s method to be suitable for Brook’s sample types resulting in more accurate characterization of the viable microbial community present in the NICU rooms due to the use of PMA. Applying a known technique to a known device (method or product) ready for improvement to yield predictable results is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, D.).
Claims 1, 2, and 4-10 are rejected under 35 U.S.C. 103 as being unpatentable over Ni in view of Brooks and Mhuireach.
Ni describes a propidium monoazide (PMA)-PCR-based 16S rRNA gene sequencing analysis method which is capable of uncovering the viable microbiome present in complex environmental samples [Ni, abstract].
To perform this method, sludge samples from five sewage treatment plants were collected. Upon collection, 12 samples collected from anaerobic digesters (DS) were split into two subsamples: a first subsample, which was used for PMA treatment (DS_PMA), and a second subsample, which was washed with PBS alone (DS). PMA working solution was prepared by diluting 2.5uL of the stock solution (20 mM) to achieve a final PMA concentration of 0.1mM. This working solution was then mixed with the first subsamples and incubated for five minutes in the dark. Cross-linking of PMA with DNA was conducted by exposing the first subsamples to blue light for 15 minutes. The first subsamples were then washed of excess PMA. Finally, both subsamples were subjected to DNA extraction using the ISOIL for Beads Beating Kit, quantified, and sequenced. Quantification was performed by quantitative PCR (qPCR) on a LightCycler 2.0 instrument using primers specific for the V3-V4 regions of the 16S rRNA gene. The quantification results of the subsamples were recorded and compared as shown in Table 1. Ni states that quantification of gene copy number allows for quantification of viable cells (below) [Ni, p926-927, Table S1].
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Ni does not teach that quantification is conducted via droplet digital PCR (ddPCR) nor that the subsamples are of equal volume.
Brooks describes a method for evaluating the identity, persistence, and absolute abundance of neonatal intensive care unit (NICU) room-associated bacteria using droplet digital PCR (ddPCR) and 16S rRNA gene surveys [Brooks, abstract]. These samples were extracted, quantified with ddPCR, and sequenced. For both ddPCR and sequencing, the target was the 16s rRNA gene [Brooks, p3]. The results of this testing demonstrated that biomass density could vary across NICU surfaces by four to five orders of magnitude.
Mhuireach teaches the ubiquity with which diverse bacteria, fungi and other microorganisms are found on and within building materials. While many are generally regarded as neutral in their effect on human health, a number of studies have suggested that exposure to diverse microbiota during childhood is critical for proper immune system development. Additionally, little is known regarding the influence of different building materials on bacterial community structure. To investigate this, multiple samples were collected from the surface of four different materials: gypsum, timber, earthen paster, and concrete. Each sample was homogenized and divided into two equal aliquots wherein one aliquot was treated with PMA and one was not. Samples were then extracted, quantitated via qPCR, and sequenced. The skilled artisan would recognize that this sampling scheme would result in as close to identical subsamples as possible and would thus allow for a more accurate determination of total DNA (untreated subsample) versus viable bacteria (treated subsample).
Therefore, one of ordinary skill in the art prior to the effective filing date of the claimed invention would have been motivated to modify the quantification step of Ni to be performed by ddPCR on samples collected by the sampling scheme of Mhuireach in order to provide precise and absolute quantification of the viable microbes within each sample. Ni’s method was developed in an attempt to provide a more accurate picture of the viable microorganisms in sewage samples and improving the method by analyzing equal subsamples via ddPCR represents an obvious next step towards this goal. Applying a known technique to a known device (method or product) ready for improvement to yield predictable results is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, D.).
Regarding claim 6, Ni compared the DNA concentration of the untreated subsamples to that of the treated subsamples and calculated a decreasing rate. It would have been obvious to one of ordinary skill in the art to express the comparison taught by Ni as a ratio of the treated sample to that of the treated sample, rather than a percent decrease, on order to directly express the relative amount of DNA remaining following treatment.
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Regarding claims 8-10, Brooks collected samples from of surfaces including the floor, sink basin, and hand rail, as well as hand samples and feces samples from the infants, and found that the majority of the NICU microbiome is typically associated with the skin, nose, or feces. [Brooks, Fig. 1, p2, p9].
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Ni, Brooks, and Mhuireach as applied to claim 1 above, and further in view of Soumet [Soumet C et al. Letters in applied microbiology. 1999 Feb 1;28(2):113-7].
Ni and Brooks are applied to the relevant teachings of claim 1 as discussed above and are incorporated herein by reference. Neither teach using primers specific to a flagellar gene in Salmonella typhimurium.
Salmonella is the most important pathogen reported from food-borne illness in France and leads to hospitalizations and death each year. To prevent this and ensure safe products for consumers, Salmonella control is necessary at all the key steps of food production. This requires rapid and reliable methods for Salmonella detection. Soumet developed a multiplex PCR system for the detection of all Salmonella serotypes and the specific detection of Salmonella Typhimurium and Enteritidis. For Salmonella Typhimurium, primers specific for the flicC gene (a flagellar) were used [Soumet, p113-114].
One of ordinary skill in the art prior to the effective filing date of the claimed invention, looking to adapt the methodology of Ni and Brooks to address the Salmonella control concerns described by Soumet, would have been motivated to adopt Soumet’s primers as they were already designed, tested, and shown to work for the same intended purpose of detecting a microbe via PCR.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Kara N Kovach whose telephone number is (571)272-8134. The examiner can normally be reached Monday - Friday, 9am - 3pm.
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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