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 .
Claims 1-8 are pending and examined on the merits.
Priority
Acknowledgement is made of this continuation application of Non-provisional Application No. 18/352,719, filed on 7/14/2023, which claims foreign priority under 35 U.S.C. 119(a)-(d) to Chinese Patent Application No. CN202210841210.8, filing date 7/18/2022. The certified copy has been filed in the instant application.
Drawings
The Drawings filed on 7/14/2023 are not accepted by the Examiner. The drawings are objected to under 37 CFR 1.83(a) because they fail to show the figure legends (not legible or visible) of figure 2, A-F as described in the specification. Any structural detail that is essential for a proper understanding of the disclosed invention should be shown in the drawing. MPEP § 608.02(d). Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Claim Objections
Claim 1 is objected to because of the following informalities in claim language:
Applicant’s recitation of S. Enteritidis in lines 1 and 4 should recite S. enteritidis with the first letter of the genus lower case.
Applicant’s recitation of ‘step 1: selection of strains selecting S. Enteritidis CVCC 1806, wherein…’ in line 3 should recite ‘step 1: selecting strain S. enteritidis CVCC 1806, wherein…’.
Applicant’s recitation of ‘step 2: preparing culture medium, cooling the medium to room temperature…’ in line 7 should recite ‘step 2: preparing a culture medium and cooling the medium to room temperature…’
Applicant’s recitation of ‘step 3: preparation of bacterial suspension washing bacterial cells…’ in lines 9-10 should recite ‘step 3: preparation of bacterial suspension by washing bacterial cells…’
Applicant’s recitation of ‘step 4: sodium hypochlorite stress treatment diluting NaClO stock solution…’ in lines 13-14 should recite ‘step 4: sodium hypochlorite stress treatment by diluting NaClO stock solution…’
Applicant’s recitation of ‘…sterile saline for 3 times…’ in line 10 should recite ‘… sterile saline 3 times…’
Applicant’s recitation of ‘step 5: counts of VBNC state cells counting culturable cells…’ in lines 19-20 should recite ‘step 5: counting culturable VBNC state cells…’ Appropriate correction is suggested.
Claim 1 is objected to because of the following informalities: the recitation of the abbreviation BHI. It is recommended that Applicant spell out the name associated with the abbreviation. Appropriate correction is suggested.
Claim 3 is objected to because of the following informalities in present and past-tense references within the claim language: Applicant’s recitation of ‘100 µL 1.5% Na2S2O3 is added to terminate the reaction’ in lines 4-5 should recite ‘…adding 100 µL of 1.5% Na2S2O3 to terminate the reaction.’ Applicant utilizes present tense language throughout the instant application claims. It is recommended that Applicant change the claim language of claim 3 to be the same as the instant application claims. Appropriate correction is suggested.
Claim 4 is objected to because of the following informalities: the term PCA is abbreviated. Appropriate correction is required.
Claim 5 is objected to because of the following informalities in claim language: Applicant’s recitation of ‘…wherein, in step 5…’ in line 2 should recite ‘… wherein, the counting of culturable cells in step 5 further comprises the following steps:…’ Furthermore, it is recommended that Applicant correct spacing issues between the terms ‘wherein’ and ‘in’ in line 2. In addition, it is recommended that Application delete the recitations ‘S5.2.1, S5.2.2, S5.2.3, S5.2.4 in lines 3-6 and replace it with a, b, c, d as it is unclear what Applicant is referencing with the recitations ‘S5.2.1, S5.2.2, S5.2.3, S5.2.4.’ Appropriate correction is suggested.
Claim 6 is objected to because of the following informalities in claim language: Applicant’s recitation of ‘… wherein the control reagent comprises negative control reagent and positive control reagent …’ in lines 2-3 should recite ‘… wherein the control reagent further comprises a negative control reagent and a positive control reagent …’ Appropriate correction is suggested.
Claim 6 is objected to because of the following informalities in claim language: Applicant’s recitation of ‘1ml of 1mL of bacterial liquid heated at 90℃ for 15min’ to ‘3 µ L of PI dye to 3 µ L mixed dye per 1mL of disinfectant treated bacterial solution.’ It is unclear if said claim limitation is referring to separate solutions or if the solutions are one in the same. In the interest of compact prosecution, Examiner will interpret said recitations to be ‘adding 3µl per 1ml of disinfectant treated bacterial solution of mixed dye, containing PI, per 1ml of disinfectant treated bacterial solution that is heated at 90C for 15min and mixing;’ Appropriate correction is suggested.
Claim 7 is objected to because of the following informalities in claim language: Applicant’s recitation of ‘…detecting bacterial sample comprises incubating mixture…’ in line 2 should recite ‘…detecting a bacterial sample comprises incubating a mixture...’ Furthermore, the recitation ‘…detecting the detect bacterial samples…’ in line 4 should recite ‘…detecting the bacterial samples…’ Appropriate correction is suggested.
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 1-8 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.
Regarding claim1 (claims 2-8 dependent thereof) , the phrase "step 1: selection of strains " in line 3, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. It is unclear if the claim encompasses just S. enteritidis or more than just S. enteritidis due to the recitation ‘strains.’ See MPEP § 2173.05(d). Appropriate correction is suggested.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. The omitted steps are: the heating of the culture medium. Applicant claims in step 2 of the instant application a step of cooling the medium to room temperature, however, Applicant has not provided any recitation upon which the medium is a higher temperature than room temperature to require a step of ‘cooling the medium.’ Appropriate correction is suggested.
Regarding claim 3, the phrase "systems were obtained for processing " in line 3, renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. It is unclear which ‘systems’ and ‘processing’ Applicant is claiming. See MPEP § 2173.05(d). Appropriate correction is suggested.
Regarding claim 3, the phrase "a uniformly mixed reagent" in line 2 renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. It is unclear what reagent Applicant is referencing with the recitation ‘a.’ See MPEP § 2173.05(d). Appropriate correction is suggested.
Claim 3 recites the limitation "a uniformly mixed reagent" in line 2. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of a uniformly mixed reagent. Appropriate correction is suggested.
Regarding claim 4, the phrase "proper volume " in line 4 renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. It is unclear what volume Applicant is referencing with the recitation ‘proper volume.’ See MPEP § 2173.05(d). Appropriate correction is suggested.
The term “continuously” in claim 4 line 3 is a relative term which renders the claim indefinite. The term “continuously” 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. It is unclear to limitation at which continuously is referencing. Appropriate correction is suggested.
Claim 4 recites the limitation " the PCA plate" in line 4. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of ‘PCA plate.’ Appropriate correction is suggested.
Regarding claim 5 (claims 6-8 dependent thereof), the phrase "control reagent " in line 4 renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. It is unclear what the ‘control reagent’ consists of. See MPEP § 2173.05(d). Appropriate correction is suggested.
Claim 6 recites the limitation "… wherein the control reagent comprises negative control reagent and positive control reagent …" in lines 2-3. There is insufficient antecedent basis for this limitation in the claim. There is no prior recitation of ‘negative control reagent’ and ‘positive control reagent’ It is suggested that Applicant amend the claim to recite ‘wherein the control reagent further comprises a negative control reagent and a positive control reagent…’ Appropriate correction is suggested.
Regarding claim 6, the phrase "an obtained mixture" in lines 5 and 7 renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. It is unclear what mixture Applicant is referencing with the recitation ‘an obtained mixture.’ See MPEP § 2173.05(d). Appropriate correction is suggested.
Claim 7 contains the trademark/trade name BD Biosciences, USA. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe FACS Verse flow cytometry and, accordingly, the identification/description is indefinite. Appropriate correction is suggested.
Claim 8 contains the trademark/trade name BD-AccuriC6. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe flow cytometry and, accordingly, the identification/description is indefinite. Appropriate correction is suggested.
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 1-4, are rejected under 35 U.S.C. 103 as being unpatentable over Highmore et al (2018, mbio, Examiner cited) {herein Highmore} in view of Arvaniti et al (Date of Publication: 15 December 2021, Examiner cited) {herein Arvaniti).
Claims 1-4 are drawn to a method for inducing Salmonella enteritidis (S. Enteritidis) into viable but non-culture (VBNC) state by sodium hypochlorite, which comprises the following steps: step 1: selection of strains selecting S. Enteritidis CVCC 1806, wherein the S. Enteritidis CVCC 1806 is obtained from the China Microbiological Culture Collection Center, stored below - 80℃ and passed for 1-5 times; step 2: preparing culture medium, cooling the medium to room temperature, then carrying out an ultraviolet sterilization for 15min, where the medium is BHI medium ; step 3: preparation of bacterial suspension washing bacterial cells with 0.85% (w/v) sterile saline for 3 times, collecting the bacterial cells by centrifugation, and then resuspending the bacterial cells in 0.85% (w/v) sterile saline to obtain 10^9 CFU/mL bacterial suspension; step 4: sodium hypochlorite stress treatment diluting NaClO stock solution of NaClO disinfectant solution containing 56.8 mg/mL chlorine to 100 mg/L with sterilized sterile water for later use, taking 0.5 ml of the obtained bacterial suspension (10^9 CFU/mL) and the 100 mg/L NaClO solution respectively into a sterile EP tube, and vortex-mixing products in the sterile EP tube, and the stress concentration in the final system is 50 mg/L; step 5: counts of VBNC state cells counting culturable cells, determining number of the culturable bacteria in system after stress treatment with 50 mg/L NaClO for different times in step 4 by plate counting method; courting live cells, adjusting the concentration of bacterial suspension to 10^7-10^8 CFU/mL, and distinguishing live, dead and injured bacteria.
With respect to claim 1, Highmore teaches a method of inducing Salmonella enterica to enter a viable-but-nonculturable (VBNC) state by exposure to chlorine (abstract). Highmore further teaches that expositing pathogens to UV irradiation results in VBNC induction (page 7, para 3) and the utilization of UV as sanitizers (page 7, para 3). Examiner is interpreting ‘pathogens’ taught by Highmore to include Salmonella enterica as it is known by those of ordinary skill in the art that Salmonella enterica is a pathogen. Examiner is interpreting the sanitization via UV to be the same as sterilization via UV as both result in a reduced number of pathogens. Although the reference of Highmore does not explicitly teach the limitations of claim 1 (UV sterilization for 15min), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the amount of time cells are UV irradiated depending on the particular application. It would be routine for one to arrive at the time for UV irradiating time for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious. In addition, Highmore teaches a study conducted by the United Kingdom, spanning 17 years, determined that in food-borne outbreaks, Salmonella spp. were responsible for the highest number of disease cases (page 2, para 2). Evidentiary reference of Wang is cited to demonstrate that S. enteritidis strain CVCC 1806 is a serovar of Salmonella enterica and can survive extreme food processing environments (abstract). As such, it would be obvious to one of ordinary skill in the art to try using any serovar of S. enterica, including S. enteritidis strain CVCC 1806 as one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success. Furthermore, one of ordinary skill in the art would seek to find a mechanism to reduce the pathogenicity of S. enterica, especially strains associated with food-borne outbreaks. MPEP 2143.I.E states ‘The rationale to support a conclusion that the claim would have been obvious is that "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense. In that instance the fact that a combination was obvious to try might show that it was obvious under § 103."KSR, 550 U.S. at 421, 82 USPQ2d at 1397. Furthermore, Highmore teaches cells were cultured in brain heart infusion broth (BHIB) at 37C (page 10, para 1), of which is the same as the claimed BHI medium (instant application claim 1). Highmore further teaches leaf samples were inoculated with BHIB containing Salmonella enterica and incubated at 22C (page 10, para 2). Examiner is interpreting the BHIB medium as ‘cooling’ to room temperature (22C) from 37C. Cells were then washed with saline (page 10, para 3). Highmore further teaches samples of bacteria were concentrated by centrifugation (page 10, para 5). Samples were subsequently resuspended in saline (page 10, para 5). Examiner is interpreting the saline utilized by Highmore to be sterile as it is routine in the art to utilize sterile medium for microbial studies to reduce the probability of microbial contamination. Although the reference of Highmore does not explicitly teach the limitations of claim 1 (washing bacterial cells with 0.85% (w/v) sterile saline for 3 times; resuspending cells in 0.85% sterile saline), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the concentration of saline utilized to wash the cells, resuspend the cells in an appropriate concentration of saline and adjust for the appropriate number of times cells are washed depending on the particular application. As such, it would be routine for one to arrive at the concentration of saline utilized to wash the cells, resuspend the cells and the number of times to wash the cells for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious. Since the art teaches the structure of the bacterial cell in saline, it is the Examiners interpretation that the bacterial suspension in saline would necessarily have a 10^9 CFU/ml bacterial suspension. Furthermore, Highmore teaches a stock solution of free chlorine was produced (page 10, para 3). Bacterial suspension of 10^8 CFU/ml in saline was inoculated into 50ml ddH2) to which 50 ml chlorine was added as well (page 10, para 3). Bacterial cells were exposed to difference concentration of chlorine (figure 3). The sample was shaken vigorously (page 10, para 3). Examiner is interpreting the sample being shaken vigorously as being the same as the sample being vortexed as both process cause the sample to be agitated. The sample was then filtered via membrane filtration (page 10, para 3). The bacteria retained on the membrane were subjected to direct visual count (DVC) (page 10, para 3). The final concentration of bacterial cells was 10^6 CFU/ml (page 10, para 3). Examiner is interpreting the teaching of 10^6 CFU/ml to be indicative of the plate method as one would need to count the number of colonies on a plate to determine CFU. Since said cells grew on the plate, Examiner is interpreting them as being culturable cells. Furthermore, it is known by those of ordinary skill in the art that the plate method allows for one to distinguish between live and dead cells. As such, Examiner is interpreting the plating method taught by Highmore to distinguish between live and dead cells.
With respect to claim 2, Highmore teaches samples of bacteria were concentrated by centrifugation (page 10, para 5). Although the reference of Highmore does not explicitly teach the limitations of claim 2 (wherein in step 3, the centrifugation is conducted at ambient temperature of 4℃ with the centrifugation rate of 10000 rpm for 5 min), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the amount of time, rpm and temperature at which the cells are centrifuged depending on the particular application. It would be routine for one to arrive at the time, rpm and temperature for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious.
With respect to claim 3, Highmore teaches bacteria and chlorine were combined is shaken vigorously (page 10, para 3). Examiner is interpreting the action of ‘mixing vigorously’ the bacteria and chlorine taught by Highmore to be the same a uniformly mixed reagent. The samples were subsequently taken after 2min of shaking vigorously for culture and direct viable count (page 10, para 3). Examiner is interpreting the 2min of shaking vigorously, taught by Highmore, to be the same as stress treatment for up to 3hrs since the stress test of the instant application encompasses cells being exposed to stress inducing chemical reagent and the 3hrs taught by Highmore fit within the 2min recited in the instant application. Examiner is unclear what Applicant is referencing with the recitation of ‘systems were obtained for processing every 0.5h.’ As such, Examiner is interpreting said systems to be ‘samples were taken at 0.5h intervals.’ Furthermore, Examiner is interpreting the recitation ‘processing’ in claim 3 of the instant application to be removing a sample, after shaking vigorously for processing via direct viable count of bacteria as taught by Highmore (abstract). Highmore teaches a method wherein samples were taken at intervals of once a day and their percent survival was analyzed over the course of 13 days (fig. 6). Although the reference of Highmore does not explicitly teach the limitations of claim 3 (systems were obtained for processing every 0.5h), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the processing intervals depending on the particular application. It would be routine for one to arrive at the processing intervals for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious.
With respect to claim 4, Highmore teaches aliquots of treated samples were serially diluted in sterile solution and spread in duplicates on plates and incubated at 37°C (page 13, para 9). Examiner is interpreting the recited ‘proper volume’ to be an efficient volume of cells with sterile solution to spread evenly onto the plates taught by Highmore. Examiner is interpreting the recitation of serially diluted to be the same as continuously diluted as both result in the dilution of cells over time. Although the reference of Highmore does not explicitly teach the limitations of claim 4 (diluting 1 mL of sterile sample with 0.85% (w/v) sterile physiological saline, and inoculating the obtained diluent in proper volume on the PCA plate, and then culturing at 37℃ for 24 hours), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the dilution of the sterile samples, types of plates they are spread on and the duration of culturing depending on the particular application. It would be routine for one to arrive at the dilution of the sample, plate spreading and duration of culturing for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious.
However, Highmore does not teach the method of claim 1 of sodium hypochlorite; step 1: selection of strains selecting S. Enteritidis CVCC 1806, wherein the S. Enteritidis CVCC 1806 is obtained from the China Microbiological Culture Collection Center, stored below - 80℃ and passed for 1-5 times; step; step 4: sodium hypochlorite stress treatment diluting NaClO stock solution of NaClO disinfectant solution containing 56.8 mg/mL chlorine to 100 mg/L with sterilized sterile water for later use, taking 0.5 ml of the obtained bacterial suspension (10^9 CFU/mL) and the 100 mg/L NaClO solution respectively into a sterile EP tube, and vortex-mixing products in the sterile EP tube, and the stress concentration in the final system is 50 mg/L; determining number of the culturable bacteria in system after stress treatment with 50 mg/L NaClO; distinguishing injured bacteria cells (claim 1). The method of claim 3, wherein 100μL 1.5% Na2S2O3 is added to terminate the reaction (claim 3).
With respect to claim 1, Arvaniti teaches a method of inducing VBNC state in Listeria monocytogenes utilizing sodium hypochlorite (abstract) as a stress test. To prepare the sodium hypochlorite stress test, sodium hypochlorite was mixed with chlorine (page 13, para 6). Although the reference of Arvaniti does not explicitly teach the limitations of claim 1 (diluting NaClO stock solution of NaClO disinfectant solution containing 56.8 mg/mL chlorine to 100 mg/L with sterilized sterile water), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the concentration of chlorine depending on the particular application. It would be routine for one to arrive at the concentration of chlorine for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious. Arvaniti further teaches 10^9 CFU/ml of bacteria was centrifuged twice and resuspended in Ringer’s solution (page 13, para 7). Approximately 10^9 CFU/ml of bacteria were resuspended in solution containing the stress agent (page 13, para 7). Examiner is interpreting the stress agent to be sodium hypochlorite. Although the reference of Arvaniti does not explicitly teach the limitations of claim 1 (taking 0.5 ml of the obtained bacterial suspension and the 100 mg/L NaClO solution), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the amount of bacteria utilized in the stress test and the concentration of the NaClO depending on the particular application. It would be routine for one to arrive at the amount of bacteria to utilize for the stress test and concentration of NaClO for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious. It would be obvious to one of ordinary skill in the art to conduct said experimentation in sterile tubes to limit the possibility of contamination. As such, Examiner is interpreting the tubes being utilized by Arvaniti to be sterile. In addition, Arvaniti teaches at 40 ppm, after 3 h of exposure, the whole
population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state (abstract). Examiner is interpreting 40 ppm to be equivalent to 40mg/L. Since Arvaniti teaches induction of VBNC state at 40 mg/ml, it would be obvious to one of ordinary skill in the art that a stress concentration in a final system of recited 50 mg/ml of NaClO would result in a state of VBNC since 50 mg/ml is greater than 40 mg/ml. Arvaniti further teaches that the total culturable and non-culturable bacteria were determined (fig 2). Detectable injured cells were detectable after exposure to NaClO (page 6, para 3).
With respect to claim 3, Arvaniti teaches after the stress test, cells were resuspended in 100 µl of Ringer’s solution (page 14, para 2). Evidentiary reference of HIMEDIA is cited to demonstrate that Ringer’s solution contains sodium thiosulphate (page 1, para 1). As such, Examiner is interpreting the Ringer’s solution taught by Arvaniti to be the same as the sodium thiosulphate taught by Arvaniti as Arvaniti teaches Ringer’s solution stops the stress exposure, which is the same as recited ‘sodium thiosulphate’ is added to terminate the reaction.’ Although the reference of Arvaniti does not explicitly teach the limitation of claim 3 (1.5% Na2S2O3 (sodium thiosulfate)), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the concentration of sodium thiosulfate depending on the particular application. It would be routine for one to arrive at the concentration of sodium thiosulfate for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious.
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to apply the teachings of Highmore et al of a method of inducing Salmonella enterica to enter viable-but-nonculturable (VBNC) state by exposure to chlorine (abstract) or combine the teachings of Arvaniti because Arvaniti teaches a method of inducing VBNC state in Listeria monocytogenes utilizing sodium hypochlorite (abstract) as a stress test.
One of ordinary skill in the art would be motivated to either use the teachings of Highmore et al. by itself or combine the teachings of Arvaniti because Arvaniti provides the motivation for Highmore to use NaClO because Arvaniti teaches when L. monocytogenes is exposed to 5 ppm NaClO, at time 0, there is a highly significant decrease in total culturable population levels (page 6, para 3). One of ordinary skill in the art knowing the benefit to reducing the pathogenicity of food borne pathogens such as Salmonella enteridis based on the teachings of Highmore and Arvaniti would have a reasonable expectation of success to combine NaClO with S. enterridis as an effective means of induing said cells into viable but non-culture as Arvaniti teaches the induction of said state in L. monocytogenes. MPEP 2143.I.E states ‘The rationale to support a conclusion that the claim would have been obvious is that "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense. In that instance the fact that a combination was obvious to try might show that it was obvious under § 103."KSR, 550 U.S. at 421, 82 USPQ2d at 1397.’ As such, since it is known by those of ordinary skill in the art that both L. monocytogenes and S. enteridis are food borne pathogens, based on the teachings of Arvaniti, one of ordinary skill in the art would try reducing the pathogenicity of S. enteritis by exposing said cells to NaClO as said chemical was found to have profound effects on the culturability of L. monocytogenes.
One of skill in the art would have a reasonable expectation of success to make and use the claimed method for inducing Salmonella enteritidis (S. enteritidis) into viable but non-culture (VBNC) state by sodium hypochlorite because Highmore provides the basic method of inducing Salmonella enterica to enter viable-but-nonculturable (VBNC) state by exposure to chlorine (abstract), while Arvaniti provides the basic teaching of NaClO for inducing cells into a viable but non-culture (VBNC) state. Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 5-8 are rejected under 35 U.S.C. 103 as being unpatentable over Highmore et al (2018, mbio, Examiner cited) {herein Highmore} in view of Arvaniti et al (Date of Publication: 15 December 2021, Examiner cited) {herein Arvaniti and in further view of King et al (2000, Journal of Immunological Methods, Examiner cited) {herein King}.
Claim 5 is drawn to the method for inducing Salmonella enteritidis into VBNC state by sodium hypochlorite according to claim 1, wherein, in step 5 further comprises the following steps: S5.2.1 preparing dyes; S5.2.2 preparing control reagent; S5.2.3 detecting bacterial samples; S5.2.4 conducting a flow cytometry (FCM) test.
Claim 6 is drawn to the method for inducing Salmonella enteritidis into VBNC state by sodium hypochlorite according to claim 5, wherein the control reagent comprises negative control reagent and positive control reagent, the negative reagent is prepared by adding 3 µ L mixed dye per 1mL of disinfectant treated bacterial solution, adding 1mL of bacterial liquid heated at 90℃ for 15min to 3 µ L of PI dye, and mixing an obtained mixture; and the positive reagent is prepared by adding 1mL of bacterial liquid without disinfectant treatment to 3 µ L of SYTO9 dye and mixing an obtained mixture.
Claim 7 is drawn to the method for inducing Salmonella enteritidis into VBNC state by sodium hypochlorite according to claim 5, wherein a step of detecting bacterial sample comprises incubating mixture at room temperature and in dark for 15 min, filtering an incubated sample through a flow tube, and detecting the detect bacterial samples by using BD Biosciences FACS Verse flow cytometry (BD Biosciences, USA).
Claim 8 is drawn to the method for inducing Salmonella enteritidis into VBNC state by sodium hypochlorite according to claim 5, wherein the FCM test is performed by using BD-AccuriC6 flow cytometer equipped with 488nm and 640nm laser.
The teachings of Highmore, in view of Arvaniti as applied to claims 1-4 are set forth in the 103 rejection above.
With respect to claim 7, Highmore teaches cells were filtered through a membrane to achieve a final concentration of 10^6 CFU/ml (page 10, para 3).
However, Highmore, in view of Arvaniti do not teach the method of claim 5 of conducting a flow cytometry (FCM) test (claim 5). The method of claim 6, wherein the control reagent comprises negative control reagent and positive control reagent, the negative reagent is prepared by adding 3 µ L mixed dye per 1mL of disinfectant treated bacterial solution, adding 1mL of bacterial liquid heated at 90℃ for 15min to 3 µ L of PI dye, and mixing an obtained mixture; and the positive reagent is prepared by adding 1mL of bacterial liquid without disinfectant treatment to 3 µ L of SYTO9 dye and mixing an obtained mixture. The method of claim 7 of a flow tube, and detecting the detect bacterial samples by using BD Biosciences FACS Verse flow cytometry (BD Biosciences, USA) (claim 7). The method of claim 8, wherein the FCM test is performed by using BD-AccuriC6 flow cytometer equipped with 488nm and 640nm laser (claim 8).
With respect to claim 5, Arvaniti teaches a method wherein cells are stained with dye to visualize them by microscopy (page 12, para 2). Examiner is interpreting the staining of cells with dye to be an indication that dyes were prepared as recited in claim 5 of the instant application. Arvaniti further teaches untreated cells were suspended in sterile Ringer’s solution (page 14, para 2). Examiner is interpreting the recitation ‘control reagent’ as a reagent that is used at the same concentration throughout experimentation. Since Arvaniti teaches the utilization of the same concentration of Ringer’s reagent at 1.5%, Examiner is interpreting the concentration taught by Avaniti as being controlled. In addition, Arvaniti teaches bacterial samples are detected via florescent microscopy (page 14, para 3).
With respect to claim 6, Arvaniti teaches a method wherein Ringer’s reagent is added to both positive and negative controls (page 14, para 2). Examiner is interpreting the recitation ‘control reagent’ as a reagent that is used at the same concentration throughout experimentation. Since Arvaniti teaches the utilization of the same concentration of Ringer’s reagent at 1.5%, Examiner is interpreting the concentration as being held constant. As such, Examiner is interpreting the control reagent (Ringer’s reagent) as positive and negative control reagents (page 14, para 2). The negative reagent is prepared by adding 10 mM 5(6)-CFDA (Sigma) solution in dimethyl sulfoxide and in 29.92 mM propidium iodide (PI) dye to treated cells (page 14, para 2). Examiner is interpreting 10 mM 5(6)-CFDA (Sigma) solution in dimethyl sulfoxide and in 29.92 mM propidium iodide (PI) dye to be mixed dye, comprised of, PI and additional compounds. In the interest of practicing compact prosecution, Examiner is interpreting the recitation ‘the negative reagent is prepared by adding 3 µ L mixed dye per 1mL of disinfectant treated bacterial solution, adding 1mL of bacterial liquid heated at 90℃ for 15min to 3 µ L of PI dye’ in claim 6 to be the same as ‘adding 3µl per 1ml of disinfectant treated bacterial solution of mixed dye (PI dye) per 1ml of disinfectant treated bacterial solution and heated at 90C for 15min and mixing,’ of which Examiner is interpreting Avaniti to teach with the following teaching of ‘the negative reagent is prepared by adding 10 mM 5(6)-CFDA (Sigma) solution in dimethyl sulfoxide and in 29.92 mM propidium iodide (PI) dye to treated cells (page 14, para 2).’ Although the reference of Arvaniti does not explicitly teach the limitation of claim 6 (3 µ L mixed dye per 1mL of disinfectant treated bacterial solution, adding 1mL of bacterial liquid heated at 90℃ for 15min to 3 µ L of PI dye), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the negative reagent depending on the particular application. It would be routine for one to arrive at the amount of dye, bacterial liquid and conditions of the bacterial liquid for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious. Arvaniti further teaches positive controls consisted of 100 µl of untreated mid-stationary-growth-phase cells in sterile Ringer’s solution stained with CFDA (page 14, para 2). Evidentiary reference of biotium is cited to demonstrate that CFDA, taught by Arvaniti, is a dye used for florescent microscopy (page 1, para 1). As such, it would be obvious to one of ordinary skill in the art that the recited SYTO9 dye (claim 6) or CFDA, taught by Arvaniti could be utilized interchangeably to dye cells as both dyes are readily utilized in staining of cells. Although the reference of Arvaniti does not explicitly teach the limitation of claim 6 (the positive reagent is prepared by adding 1mL of bacterial liquid without disinfectant treatment to 3 µ L of SYTO9 dye), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the positive reagent depending on the particular application. It would be routine for one to arrive at the amount of dye and bacterial liquid for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious.
With respect to claim 7, Arvaniti teaches a method wherein stained cells were incubated at room temperature in the dark for 20 min (page 14, para 2). Although the reference of Arvaniti does not explicitly teach the limitation of claim 7 (in dark for 15 min), MPEP 2144.05 states"[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (MPEP 2144.05 IIA)." One of ordinary skill would desire to optimize the amount of time for incubating the cells depending on the particular application. It would be routine for one to arrive at the amount of time for incubation for the application they intend on using the cells. Therefore, the above invention would have been prima facie obvious.
However, Arvaniti does not teaches the method of claim 5 of conducting a flow cytometry (FCM) test (claim 5). The method of claim 7 of a flow tube, and detecting the detect bacterial samples by using BD Biosciences FACS Verse flow cytometry (BD Biosciences, USA) (claim 7). The method of claim 8, wherein the FCM test is performed by using BD-AccuriC6 flow cytometer equipped with 488nm and 640nm laser (claim 8).
With respect to claims 5, 7-8, King teaches a method wherein Flow cytometry is utilized to measure cell death and cell killing (abstract). Samples were transferred to tubes and analyzed by flow cytometry (page 8, column 1, para 1) utilizing a Coulter ELITE at 488nm (fig 1). Examiner is interpreting the ‘tube’ taught by King to be a flow tube as it is utilized in flow cytometry to measure the number of dead vs alive cells. It would be obvious to one of ordinary skill in that art that the recited ‘BD Biosciences FACS Verse flow cytometry (BD Biosciences, USA)’ (claim 7), ‘BD-AccuriC6’ (claim 8) or King taught Coulter ELITE could be utilized as instruments for the measurement of flow cytometry as each of the recited tools are readily utilized in the art to do so. Examiner is interpreting the recitation ‘equipped with 488nm and 640nm laser’ to be a conditional limitation of the claim. Examiner is interpreting that since King teaches Coulter ELITE, using 488nm, then it would necessarily be ‘equipped with’ a 640nm laser as Coulter ELITE has a wide range of lasers utilized for flow cytometry.
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to apply the teachings of Highmore et al of a method of inducing Salmonella enterica to enter viable-but-nonculturable (VBNC) state by exposure to chlorine (abstract) or combine the teachings of Arvaniti and King because King teaches a method wherein Flow cytometry is utilized to measure cell death and cell killing (abstract). Whereas, Arvaniti teaches a method of inducing VBNC state in Listeria monocytogenes utilizing sodium hypochlorite (abstract) as a stress test.
One of ordinary skill in the art would be motivated to either use the teachings of Highmore et al. by itself or combine the teachings of Arvaniti and King because King provides the motivation for Highmore to use flow cytometry to evaluate the effectiveness of inducing cells to enter a viable-but-nonculturable (VBNC) and teaches flow cytometry provides a powerful and versatile approach to the measurement of cell death and cell killing (abstract). One of ordinary skill in the art knowing the benefit of being able to accurately determine if cells are in a viable-but-nonculturable (VBNC) state based on the teachings of Highmore, Arvaniti and King would have a reasonable expectation of success to use flow cytometry as a measure of said state as King teaches flow cytometry can quickly perform numerous quantitative, sensitive measurements on each individual cell within a large, heterogeneous population (abstract). As such, utilizing flow cytometry as a tool for measuring the status of cells in a viable-but-nonculturable (VBNC) state would save the time and resources one would devote to having to repeat experimentations due to inaccurate measurements.
One of skill in the art would have a reasonable expectation of success to make and use the claimed method for inducing Salmonella enteritidis (S. enteritidis) into viable but non-culture (VBNC) state by sodium hypochlorite because Highmore provides the basic method of inducing Salmonella enterica to enter viable-but-nonculturable (VBNC) state by exposure to chlorine (abstract). The reference of Arvaniti provides the basic teaching of NaClO for inducing cells into a viable but non-culture (VBNC) state. Whereas, King provides the teaching of utilizing flow cytometry to measure cells in the viable-but-nonculturable (VBNC) state (abstract). Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Conclusion
Claims 1-8 are pending and examined on the merits.
Claims 1-8 are rejected
No claims are in condition for allowance.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656