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 Status and Action Summary
This action is in response to the papers filed on November 26, 2025.
Claims 1-2 and 4-10 are under examination. Claim 3 was canceled by applicant. No other claims are currently pending.
Any objections and rejections not reiterated below are hereby withdrawn.
The objections to the specification have been withdrawn in view of the substitute specification filed November 26, 2025.
The 112(b) indefiniteness rejections of record have been withdrawn in view of the amendments to the claims.
Priority
The present application was filed on April 25, 2023 and claims foreign priority to CHINA 202211528367.1, filed on November 30, 2022.
Information Disclosure Statement
No Information disclosure statement has been filed in the present application as of the date of this office action.
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.
Claims 5-10 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
This is a new grounds of rejection necessitated by the amendments to the claims.
Claims 5-10 each recite the limitation "wherein the optimizing the LAMP reaction system comprises:". As amended, claim 1 (upon which claims 5-10 depend) does not recite a step of “optimizing the LAMP reaction system”. Therefore, there is insufficient antecedent basis for this limitation in the claim.
Applicant is reminded that no new matter may be added.
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.
Claims 1 and 5-10 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., AU 2021103313 A4, published August 5, 2021 in view of Biswas et al., “The Development of DNA Based Methods for the Reliable and Efficient Identification of Nicotiana tabacum in Tobacco and Its Derived Products” International Journal of Analytical Chemistry Volume 2016, Tanner et al., “Loop-Mediated Isothermal Amplification for Detection of Nucleic Acids” Current Protocols in Molecular Biology 15.14.1-15.14.14, January 2014,
This rejection has been updated as necessitated by the amendments to the claims.
Because of the indefiniteness issues discussed above for claims 5-10 regarding the requirement for a step of “optimizing the LAMP reaction system”, claim 1 has been interpreted herein as requiring this step in the interest of compact prosecution.
Regarding claim 1, Zhang et al. teach methods for a rapid test of tobacco targeting a tobacco-specific Ntsp151 genomic sequence comprising: extracting genomic DNA of the tobacco, establishing a PCR system using the primers Ntsp151F: 5’-ATTTGGCTTTGGCTATGGAA-3’ and Ntsp151R: 5’-CGGAGACAAGAGACCCAAGT-3’, and analyzing the result by electrophoresis (Zhang et al., paragraphs 0006-0016).
Zhang et al. do not explicitly teach detecting this tobacco-specific Ntsp151 genomic marker sequence using a LAMP reaction system, wherein the LAMP primers comprise the sequences of SEQ ID NO: 1-5, as recited by amended claim 1.
However, Biswas et al. teach a sensitive and specific visual LAMP method for rapid identification of tobacco comprising extracting genomic DNA from tobacco, designing LAMP primers and establishing a LAMP reaction (Biswas et al., abstract and page 2, column 2, paragraph 4-page 3, column 1, paragraph 3). Biswas et al. do not teach step(s) comprising optimizing the LAMP reaction system.
However, Tanner et al. teach LAMP is a widely cited isothermal technique for rapid point of use detection of specific nucleic acids (Tanner et al., abstract). Tanner et al. teach critical parameters and troubleshooting for optimization of LAMP reactions and that such optimization is desirable to improve amplification efficiency, reduce amplification of off-target products, and reduce the total time required to run the assay (Tanner et al., pages 12-14).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the teachings of Zhang et al. comprising detection of the tobacco-specific genetic marker Ntsp151 by PCR with the teachings of Biswas et al. comprising a sensitive and rapid LAMP assay for the presence of tobacco and the teachings of Tanner et al. that LAMP assays may be optimized by changing a number of parameters compared to the recommended starting conditions supplied by Biswas et al. and/or Tanner et al.
The ordinary artisan would have been motivated to modify the method taught by Zhang et al. with the teachings of Biswas et al. and Tanner et al. because of the express teachings of Biswas et al. that LAMP detection methods of tobacco-specific markers are particularly useful for on-site assays (Biswas et al., page 6, column 1, paragraph 1), and that the sensitivity of LAMP detection of tobacco-specific genomic markers is less sensitive to the degree of processing of particular tobacco products. Biswas et al. teach PCR methods for detection of tobacco-specific genomic markers, but not LAMP methods for detecting said markers, are sensitive to (i.e. negatively affected by) the presence of inhibitors and genomic DNA breakdown particular to processed tobacco (Biswas et al., page 4, column 2, paragraph 2- page 5, column 1, paragraph 1).
The ordinary artisan would have been motivated to modify the method taught by Zhang et al. and Biswas et al. with the teachings of Tanner et al. comprising optimizing LAMP reactions, because of the express teachings of Tanner et al. that optimization of several parameters in the LAMP reaction system are desirable improve amplification efficiency, reduce amplification of off-target products, and reduce the total time required to run the assay (Tanner et al., pages 12-14).
Regarding the requirement for the specific LAMP primers recited by claim 1, alignment of the claimed outer primers “F3” (SEQ ID NO: 1), “B3” (SEQ ID NO: 2), and the PCR primers Ntsp151F and Ntsp151R taught by Zhang et al. to a tobacco reference genome, it is apparent that the claimed LAMP primers fall within the tobacco-specific Ntsp151 amplicon utilized for specific PCR detection of tobacco taught by Zhang et al. (See below)
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Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the methods taught by Zhang et al. for PCR detection of the Ntsp151 marker by searching for suitable LAMP primers within the ~300 bp genomic interval identified by Zhang et al. as a sensitive and specific marker for tobacco.
The ordinary artisan would have been motivated to modify the teachings of Zhang et al. comprising detecting the tobacco-specific Ntsp151 marker by PCR with the teachings of Biswas et al. in view of Taylor et al. comprising detecting a different tobacco-specific marker by qPCR or LAMP using LAMP primers designed using an earlier version of the same publicly-available software used by the present inventors because of the predictable advantages of LAMP over PCR-based detection taught by Biswas et al. and Tanner et al. (LAMP is less sensitive to the extent of processing of a given tobacco sample, (Biswas et al., page 5-6 bridging paragraph), LAMP requires fewer consumable materials and no complex thermocycling, and can be deployed at the point of need (Tanner et al., page 11). The ordinary artisan would therefore have been motivated to detect the tobacco-specific marker taught by Zhang et al. using LAMP primers designed to detect the same ~300 bp genomic marker previously identified by Zhang et al. by publicly available software as taught by Biswas et al. and Tanner et al. to predictably obtain the advantages described above provided by LAMP over PCR-based detection of the sensitive and specific Ntsp151 marker identified by Zhang et al.
Regarding claim 5, Tanner et al. teaches optimizing reaction temperature for LAMP reactions comprises performing a temperature gradient between 60 and 65 °C (Tanner et al., page 12, column 1, paragraph 4.
Regarding claim 6, the selection of an optimum temperature (i.e. 63°C) within the gradient taught by Tanner et al. for the specific primers suggested by Zhang et al. in view of Biswas et al. and Tanner et al. would have been prima facie obvious to the ordinary artisan as part of routine optimization of conditions for a LAMP reaction as specifically taught by Tanner et al.
Regarding claim 7, Tanner et al. teaches optimizing reaction condition for LAMP reactions comprises performing a titration of Mg2+ from 2 to 10 mM, with “8 mM Mg2+ likely optimal for most reactions” (i.e. substantially overlapping with 0-12 mM recited by the claim) (Tanner et al., page 12, column 1, paragraph 2).
Regarding claim 8, the selection of an optimum Mg2+ concentration (i.e. 6 mmol/L; 6mM) within the titration taught by Tanner et al. for the specific primers suggested by Zhang et al. in view of Biswas et al. and Tanner et al. would have been prima facie obvious to the ordinary artisan as part of routine optimization of conditions for a LAMP reaction as specifically taught by Tanner et al.
Regarding claim 9, Tanner et al. teaches “a typical LAMP reaction requires 10 to 90 minutes, depending on the speed of amplification, target copy number, and level of non-template amplification” (i.e. optimizing comprises carrying out a LAMP reaction for 10 to 90 minutes) (Tanner et al., page 13, column 2, paragraph 2).
Regarding claim 10, the selection of an optimum reaction duration (i.e. 60 minutes) within the optimizable range taught by Tanner et al. for the specific primers suggested by Zhang et al. in view of Biswas et al. and Tanner et al. would have been prima facie obvious to the ordinary artisan as part of routine optimization of conditions for a LAMP reaction as specifically taught by Tanner et al.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al. in view of Biswas et al. and Tanner et al. as applied to claims 1 and 5-10 above, and further in view of HwangBo et al., “Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100” Plant Biotechnol Rep (2010) 4:49-52, published November 28, 2009 and Reyes-Escogido et al., “Purification of bacterial genomic DNA in less than 20 min using chelex-100 microwave: examples from strains of lactic acid bacteria isolated from soil samples” Antoni eval Leeuwenhoek (2010) 98:465-474, published June 14, 2010.
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 1, as described above, Zhang et al. in view of Biswas et al. and Tanner et al. teach LAMP methods for rapid detection of tobacco comprising extracting genomic DNA, designing LAMP primers within the ~300 bp amplicon in Ntsp151 identified by Zhang et al., establishing a reaction system, and optimizing the LAMP reaction system.
Regarding claim 2, Biswas et al. does not teach extracting genomic DNA comprises using the recited Chelex-100 (i.e. a chelating ion-exchange resin having iminodiacetate functional groups) method. Rather, Biswas et al. extract genomic DNA from tobacco using a commercial silica-based spin column kit (DNeasy Plant Mini kit) (Biswas et al., page 2 – page 3 bridging paragraph).
However, HwangBo et al. teach methods for rapid extraction of genomic DNA from a variety of plants including tobacco (HwangBo et al., page 50, column 1, paragraph 2) comprising adding 10-15 mg plant tissue to an Eppendorf tube (i.e. a microcentrifuge tube), grinding for 1 minute in a volume of 5% Chelex 100 (i.e. a chelating ion-exchange resin having iminodiacetate functional groups) solution, vortexing (i.e. shaking and suspending) for 10 seconds, incubating in boiling water for 5 minutes, vortexing for 10 seconds, and centrifuging at 13,000 rpm for 1 minute, after which the supernatant is taken for testing (HwangBo et al., page 50, column 1, paragraph 3). HwangBo et al. teach that treatment steps in complicated conventional methods such as CTAB with proteinase K and RNase A is not necessary for detection of DNA by PCR (HwangBo et al., page 49, column 1, and Figure 2h). Reyes-Escogido et al. teach a similar method comprising heating a suspension of bacterial cells in Chelex 100 supplemented with proteinase K and RNase A, demonstrating that while inclusion of RNase A and proteinase K may not be strictly necessary for extraction of genomic DNA from difficult to handle samples (i.e. plants, soil bacteria), inclusion of these steps is not detrimental to DNA purification using Chelex 100 resin (Reyes-Escogido et al., page 467, column 2). Both HwangBo et al. (HwangBo et al., figure 2 and page 52, column 1) and Reyes-Escogido et al. (Reyes-Escogido et al., page 466) teach DNA extraction using Chelex 100 resin is a rapid alternative to older methods comprising CTAB-phenol-chloroform-isoamyl alcohol extraction of nucleic acids from plants and bacteria that has several advantages including faster purification (<15 minutes compared to hours), elimination of organic solvent contamination and waste, better purity of DNA (Reyes-Escogido et al., page 469 and HwangBo et al., page 52, column 1, paragraph 2).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have modified the method comprising DNA purification for a LAMP assay for detection of tobacco by a commercially available “DNA Plant Mini Kit” taught by Biswas et al. in view of Tanner et al. to substitute the commercial silica-based column kit (used by Biswas et al.) for the method comprising Chelex-100 resin taught by Escogido et al. and HwangBo et al. The ordinary artisan would have been motivated to substitute these alternative methods of DNA purification from tobacco samples with a reasonable expectation of success because they are both known, equivalent alternatives in the art to the laborious and less safe CTAB and organic solvent extraction protocols that require significantly more hands-on time and expensive ventilation equipment to prevent exposure of the process user to hazardous phenol and chloroform fumes.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al., in view of Biswas et al. and Tanner et al., as applied to claims 1 and 5-10 above, and further in view of New England Biolabs “Typical LAMP Protocol (M0538)” and “Bst 2.0 WarmStart® DNA Polymerase (M0538) Certificate of Analysis” (April 26, 2016).
This rejection has been updated as necessitated by the amendments to the claims.
Regarding claim 1, as described above, Biswas et al. in view of Tanner et al. teach visual LAMP methods for sensitive, rapid, and specific identification of tobacco comprising extracting genomic DNA from tobacco, designing LAMP primers within the ~300 bp amplicon in Ntsp151 identified by Zhang et al., establishing a LAMP reaction (Biswas et al., abstract and page 2, column 2, paragraph 4-page 3, column 1, paragraph 3), and optimizing the LAMP reaction system (Tanner et al., pages 12-14).
Regarding claim 4, Zhang et al. in view of Biswas et al. and Tanner et al. teach visual LAMP methods were accomplished using SYBR Green I dye (i.e. a DNA-intercalating fluorescent dye) or 2% agarose gel electrophoresis (Biswas et al., page 3, column 1, paragraph 3). Tanner et al. teach performing standard LAMP reactions (before the previously described routine optimization steps suggested by Tanner et al.) comprise: 1x commercial Isothermal Amplification Buffer (NEB) (20 mM tris, pH 8.8, 50 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Tween 20, and 1.4 mM dNTPs supplemented with an additional 6 mM MgSO4) (Tanner et al., page 10-11) Tanner et al. further teach suggested 1x concentrations of each LAMP primer type: 1.6 µM FIP, 1.6 µM BIP, 0.2 µM B3, 0.2 µM F3, and 0.4 µM Loop B for use with 8 U Bst 2.0 DNA Polymerase (i.e. a thermostable strand-displacing DNA polymerase) and positive control template DNA (Tanner et al., page 3-4).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have followed the guidance provided by Tanner et al. to have optimized the LAMP reaction conditions for the tobacco specific marker taught by Zhang et al. in view of Biswas et al. and Tanner et al. and arrive at the presently claimed LAMP reaction system concentrations. The ordinary artisan would have been motivated to have optimized the known parameters by the routine methods taught by Tanner et al. for any LAMP assay to have attained the predictable benefits of reduced off-target amplification, reduced amplification time, and improved limit of detection (Tanner et al., page 12).
Response to arguments
The response argues that the requirement for the specific LAMP primers of SEQ ID NO: 1-5 are not disclosed in the prior art and argues that these primers would not have been obvious to one having ordinary skill in the art because of the teachings of Tanner that “LAMP is prone to non-template amplification due to the amount and number of primers, so care should be taken to avoid primer dimers or other effects”.
This argument has been thoroughly reviewed and is not persuasive for the reasons which follow.
It is acknowledged that LAMP primers for Ntsp151 having the specific sequences SEQ ID NO: 1-5 are not anticipated by the cited prior art.
However, as is discussed in the 103 rejection of record and made explicit in the updated 103 rejection of claim 1 over Zhang et al. in view of Biswas et al. and Tanner et al. (see above), the LAMP primers of SEQ ID NOs: 1-5 required by the claim fall within the known ~300 bp sequence amplified in the PCR assay taught by Zhang et al. In fact, one of the claimed outer primers substantially overlaps with one of the two primers for specific amplification of Ntsp151 taught by Zhang et al. (see alignment below, reproduced from 103 rejection for convenience).
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As discussed in the 103 rejection above, Biswas et al. and Tanner et al. teach principles and tools for facile selection of candidate LAMP primers given a particular known target sequence and optimization of LAMP reactions for particular targets in particular samples.
Absent unexpected results particular to the claimed specific combination of LAMP primers, it is the position of the examiner that the guidance provided by Biswas et al. and Tanner et al. would have rendered obvious the selection, validation, and optimization of any of the small number of possible LAMP primer sets entirely contained within the very small (~300 bp) genomic interval established by Zhang et al.
The response further asserts that there is not a clear rationale to support the cited combination of references. The response argues that the ordinary artisan would not have been motivated to select the specific combination of primers recited by amended claim 1 because of the teachings of Tanner et al. that LAMP primer design is complicated by LAMP sensitivity to primer interactions.
This argument has been thoroughly reviewed and is not persuasive.
As discussed above and summarized here, the claimed LAMP primers are entirely contained within the 300 bp PCR amplicon taught by Zhang et al. as a sensitive and specific marker for tobacco detection by PCR. Biswas et al. specifically teaches detection of a different tobacco-specific marker by PCR and LAMP, and teaches that detection of a tobacco-specific marker by LAMP has several known and predictable advantages including requirement for fewer consumables, decreased sensitivity to inhibitors and DNA degradation particular to detecting DNA markers in processed tobacco samples, and the ability to deploy LAMP tests at the point of need (i.e. in supply chains or in import controls), and Tanner et al. teach particular known principles for optimizing LAMP reactions in general that would have led one of skill in the art to have selected and validated candidate LAMP primers within the known ~300 bp tobacco-specific genomic marker taught by Zhang et al. by said known principles and optimization procedures .
Therefore, it is the position of the examiner that the advantages (i.e. motivation and expected advantages) taught by Biswas et al. for detection of a tobacco-specific genomic marker by LAMP relative to detection of the same marker by PCR are particularly relevant to the claimed invention, wherein LAMP primers were selected within the known ~300 bp tobacco-specific genomic marker detected by PCR in the methods taught by Zhang et al.
The response further asserts that the inventor identified a problem others did not, namely, “prior to applicant’s invention there was no LAMP assay targeting the Ntsp151 locus… because the art either used LAMP for a different tobacco gene or used Ntsp151 only in PCR format.”
This argument has been thoroughly reviewed and is not persuasive. It is acknowledged that the cited prior art did not detect the Ntsp151 marker taught by Zhang et al. by LAMP, but rather by PCR. However, as discussed above, Biswas et al. provides motivation for detecting a different tobacco-specific genomic marker by LAMP rather than PCR, in particular, the known advantage that LAMP is less sensitive to inhibitors and DNA degradation particular to samples of processed tobacco products. The “problem” identified in the response that a LAMP-based method had not yet been developed for the Ntsp151 marker taught by Zhang et al. does not constitute a problem others had not identified. For example, Biswas et al. teach that LAMP detection of tobacco markers is desirable relative to PCR assays for point of need applications and is advantageously less sensitive to the presence of inhibitors and DNA degradation known as particular confounders in detection of DNA markers in processed tobacco products. The ordinary artisan would have expected these advantages to extend to detection of any particular tobacco-specific DNA marker because of the teachings of Tanner et al. that “LAMP has been performed on a wide variety of sample types and has been demonstrated to have a higher tolerance to typical… inhibitors than PCR” (Tanner et al., page 13, column 1, paragraph 3) and has been widely applied to diagnostic detection of hundreds of pathogens in clinical, plant, food, and animal samples since its publication in 2000. (Tanner et al., page 1, paragraph 2).
Finally, the response asserts that “dependent claims 2 and 4-10 are not obvious based on [the cited prior art] because limitations recited in claim 1 are not taught in the prior art references. This argument has been thoroughly reviewed and is not persuasive as is detailed in the 103 rejections updated as necessitated by the claim amendments and in the response to other arguments (see above).
Conclusion
No claim is allowed.
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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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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