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 1-7 are pending and will be examined.
Note regarding specification and Application Papers
The names of the inventors do not include complete names, only initials for first names for each inventor. Clarification of complete inventorship information is requested.
Claim Objections
Claim 2 is objected to because of the following informalities: step 1 uses the term “denaturating”. The term should either read denaturing or recite a step that achieves denaturation. Appropriate correction is required.
Drawings - objection
The drawings filed on March 9, 2023 are acceptable subject to correction of the informalities indicated below. In order to avoid abandonment of this application, correction is required in reply to the Office action. The correction will not be held in abeyance.
Figure 1A, step 104, states “APREPARING” which appears to include a typographical error. Correction is requested.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-7 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Plichart et al. (Parasites & Vectors, 2013, 6:10, p 1-5).
With regard to claim 1, Plichart teaches a method of identifying a plurality of microorganisms from a sample using Nucleic Acid amplification Technology (NAT), comprising:
extracting nucleic acid from a sample; preparing molecular biomarkers comprising a forward primer and a reverse primer for target genes of each of the plurality of microorganisms as a master mix;
adding the extracted nucleic acid into the master mix to obtain a reaction mixture; performing a polymerase chain reaction (PCR) on the reaction mixture to amplify a region of interest of the extracted nucleic acid (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit);
identifying a melt temperature for the amplicons by increasing the temperature of the reaction mixture from 65°C to 99°C in 0.10C increments every 2 to 5 seconds for measuring fluorescence; generating a melt curve for each target gene based on the identified melt temperatures and an amplification curve after the PCR amplification (Figure 1, figure legend, p 3, “Real time PCR” heading, where the PCR and the melt curve analysis steps are described); and
determining, using a Melt Curve analysis technique, Ct values, endpoint fluorescence level and melting profiles of the target genes by analyzing the amplification and melt curves to identify the plurality of microorganisms present in the sample (Figure 1, figure legend, p 3, “Real time PCR” heading, where the PCR and the melt curve analysis steps are described).
With regard to claim 2, Plichart teaches a method as claimed in claim 1, wherein the polymerase chain reaction comprises the steps of
denaturating the reaction mixture initially for 5 minutes at 95°C and then for 10 seconds at 95°C to obtain single-stranded DNA to enable primer annealing; annealing the denatured reaction mixture for 30 seconds at 55°C to enable the forward and reverse primer binds to a complementary sequence of the single-stranded DNA; and performing extension step for 10 seconds at 72°C to enable the Taq polymerase to bind to the 3' end of the forward and reverse primers and extend the sequence, wherein the above three- cycle is repeated for 40 times to amplify the region of interest of the single-stranded DNA (Figure 1, figure legend, p 3, “Real time PCR” heading, where the PCR and the melt curve analysis steps are described).
With regard to claim 3, Plichart teaches a method as claimed in claim 1, wherein the sample is pre-treated to remove the background of host nucleic acid, thereby increasing the reliability of the molecular analysis of the microorganisms in the sample (p 3, “assays and results” heading, where treatment steps are described).
With regard to claim 4, Plichart teaches a method as claimed in claim 1, wherein the nucleic acid is extracted from the sample by:
adding 20 pL ProteinaseK into the bottom of a microcentrifuge tube (p 2, “DNA purification” heading, where proteinase K is added);
adding up to 200 pL of the sample to the microcentrifuge tube; adding 200 pL lysis buffer to the sample, and mix the sample using a pulse-vortex for 15 seconds (p 2, “DNA purification” heading, where lysis buffer is added);
incubating the sample in a 56°C water bath for 10 minutes; centrifuging the microcentrifuge tube comprising the sample to remove drops from the inside of a lid; adding 200 pL of 96-100% ethanol to the sample, and mix again using the pulse-vortex for 15 seconds to obtain a mixture (p 2, “DNA purification” heading, where ethanol is part of the processing);
centrifuging the microcentrifuge tube comprising the mixture to remove drops from the inside of the lid; adding the mixture to a mini spin column without wetting the rim, and centrifuging at 8000 Revolutions Per Minute (RPM) for 1 minute in room temperature; placing the mini spin column comprising the mixture in a first collection tube (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit);
adding 500 pL buffer AW1 to the mini spin column without wetting the rim and centrifuge at 8000 RPM for 1 minute at the room temperature (p 2, “DNA purification” heading, where buffers AW1 and AW2 are included);
placing the mini spin column in a second collection tube; adding 500 pL Buffer AW2 to the mini spin column without wetting the rim and centrifuging at full speed of 14000 RPM for 3 minutes in the room temperature (p 2, “DNA purification” heading, where buffers AW1 and AW2 are included);
placing the mini spin column in a new microcentrifuge tube; adding 60 pL of 0.1X TE buffer directly onto the membrane of the mini spin column and close the cap (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit);
incubating at room temperature of 150C to 25°C for 1 minute; and centrifuging at 8000 RPM for 1 minute at room temperature (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit).
With regard to claim 5, Plichart teaches a method as claimed in claim 4, wherein if the volume of the sample is less than 200 pL for extracting the nucleic acid, the Phosphate-buffered saline (PBS) is added to bring the volume up to 200 pL (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit).
With regard to claim 6, Plichart teaches a method as claimed in claim 1, wherein the sample is a blood, pus, urine, cerebrospinal fluid, or other body exudates (p 2, “human blood samples” heading, where the assay was performed on human blood, specifically on capillary blood dried on filter paper).
With regard to claim 7, Plichart teaches a method as claimed in claim 1, wherein the target genes are IC, V1, V3, Gram- Positive Bacteria, Gram-Negative Bacteria, Other Microbial Targets, Antibiotic-Resistant Gene Markers, Vancomycin Resistance, AMR-CARBAPENEMASE, AMR-ESBL, AMR-CTX-M, AMR-AMP-C and AMR-Colistin Resistant Gene Markers (Figure 1, figure legend, p 3, “Real time PCR” heading, where the PCR and the melt curve analysis steps are described).
Claim(s) 1-3 and 6-7 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Hasick et al. (US Patent 12,163,182; December 2024).
With regard to claim 1, Hasick teaches a method of identifying a plurality of microorganisms from a sample using Nucleic Acid amplification Technology (NAT), comprising:
extracting nucleic acid from a sample; preparing molecular biomarkers comprising a forward primer and a reverse primer for target genes of each of the plurality of microorganisms as a master mix;
adding the extracted nucleic acid into the master mix to obtain a reaction mixture; performing a polymerase chain reaction (PCR) on the reaction mixture to amplify a region of interest of the extracted nucleic acid;
identifying a melt temperature for the amplicons by increasing the temperature of the reaction mixture from 65°C to 99°C in 0.10C increments every 2 to 5 seconds for measuring fluorescence; generating a melt curve for each target gene based on the identified melt temperatures and an amplification curve after the PCR amplification (Example 1, specifically col. 56, lines 62 to col. 57 line 12, where melt curve analysis is described for specific targets); and
determining, using a Melt Curve analysis technique, Ct values, endpoint fluorescence level and melting profiles of the target genes by analyzing the amplification and melt curves to identify the plurality of microorganisms present in the sample (abstract, Fig 17 description at col. 35, lines 25-32, where endpoint fluorescence is described; also described further at col. 75, lines 7-64).
With regard to claim 2, Hasick teaches a method as claimed in claim 1, wherein the polymerase chain reaction comprises the steps of
denaturating the reaction mixture initially for 5 minutes at 95°C and then for 10 seconds at 95°C to obtain single-stranded DNA to enable primer annealing; annealing the denatured reaction mixture for 30 seconds at 55°C to enable the forward and reverse primer binds to a complementary sequence of the single-stranded DNA; and performing extension step for 10 seconds at 72°C to enable the Taq polymerase to bind to the 3' end of the forward and reverse primers and extend the sequence, wherein the above three- cycle is repeated for 40 times to amplify the region of interest of the single-stranded DNA (Fig 3, col. 32, where the process of primer extension is described in detail; see also Example 11, col. 73, where the steps of PCR are described and include primer extension).
With regard to claim 3, Hasick teaches a method as claimed in claim 1, wherein the sample is pre-treated to remove the background of host nucleic acid, thereby increasing the reliability of the molecular analysis of the microorganisms in the sample (see col. 6-11, where various methods of treating the sample is described; see also col. 18 to col. 22).
With regard to claim 6, Hasick teaches a method as claimed in claim 1, wherein the sample is a blood, pus, urine, cerebrospinal fluid, or other body exudates (col. 55, lines 14-28, where the sample can include blood, urine, cerebrospinal fluid and other bodily fluids).
With regard to claim 7, Hasick teaches a method as claimed in claim 1, wherein the target genes are IC, V1, V3, Gram- Positive Bacteria, Gram-Negative Bacteria, Other Microbial Targets, Antibiotic-Resistant Gene Markers, Vancomycin Resistance, AMR-CARBAPENEMASE, AMR-ESBL, AMR-CTX-M, AMR-AMP-C and AMR-Colistin Resistant Gene Markers (col. 54, lines 55 to col. 55, line 14, where microbial targets are the focus of the method and amplification).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 4-5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Hasick et al. (US Patent 12,163,182; December 2024) as applied over claims 1-3 and 6-7 above and further in view of Plichart et al. (Parasites & Vectors, 2013, 6:10, p 1-5).
With regard to claim 4, Plichart teaches a method as claimed in claim 1, wherein the nucleic acid is extracted from the sample by:
adding 20 pL ProteinaseK into the bottom of a microcentrifuge tube (p 2, “DNA purification” heading, where proteinase K is added);
adding up to 200 pL of the sample to the microcentrifuge tube; adding 200 pL lysis buffer to the sample, and mix the sample using a pulse-vortex for 15 seconds (p 2, “DNA purification” heading, where lysis buffer is added);
incubating the sample in a 56°C water bath for 10 minutes; centrifuging the microcentrifuge tube comprising the sample to remove drops from the inside of a lid; adding 200 pL of 96-100% ethanol to the sample, and mix again using the pulse-vortex for 15 seconds to obtain a mixture (p 2, “DNA purification” heading, where ethanol is part of the processing);
centrifuging the microcentrifuge tube comprising the mixture to remove drops from the inside of the lid; adding the mixture to a mini spin column without wetting the rim, and centrifuging at 8000 Revolutions Per Minute (RPM) for 1 minute in room temperature; placing the mini spin column comprising the mixture in a first collection tube (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit);
adding 500 pL buffer AW1 to the mini spin column without wetting the rim and centrifuge at 8000 RPM for 1 minute at the room temperature (p 2, “DNA purification” heading, where buffers AW1 and AW2 are included);
placing the mini spin column in a second collection tube; adding 500 pL Buffer AW2 to the mini spin column without wetting the rim and centrifuging at full speed of 14000 RPM for 3 minutes in the room temperature (p 2, “DNA purification” heading, where buffers AW1 and AW2 are included);
placing the mini spin column in a new microcentrifuge tube; adding 60 pL of 0.1X TE buffer directly onto the membrane of the mini spin column and close the cap (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit);
incubating at room temperature of 150C to 25°C for 1 minute; and centrifuging at 8000 RPM for 1 minute at room temperature (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit).
With regard to claim 5, Plichart teaches a method as claimed in claim 4, wherein if the volume of the sample is less than 200 pL for extracting the nucleic acid, the Phosphate-buffered saline (PBS) is added to bring the volume up to 200 pL (p 2, “DNA purification” heading, where the processing follows the protocol of QIAGEN DNA Easy kit).
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was made to have adjusted the teachings of Hasick to include the specific buffers and manipulation steps as taught by Plichart to arrive at the claimed invention with a reasonable expectation for success. Both Hasick and Plichart focus on extraction of nucleic acids, following by amplification and melt curve analysis of the amplified target sequences. Next, each method focuses on detection of microorganisms within a human sample. While Hasick teaches a “need exists for improved compositions and methods for the simultaneous detection, differentiation, and/or quantification of multiple unrelated amplicons generated by PCR or by alternative target amplification protocols” (col. 3, line 44-47), Hasick does not specifically mention the extraction and processing steps of the sample, as required by the QIAamp protocol as described by Plichart and as included with the process. Plichart teaches “DNA extractions were performed with the DNeasy kit, the PCR with the Wb LDR primers and the SYBR-Green dye. Improvements of our pool-screen real-time PCR (qPCR) assay allowed the detection of as little as one Wb microfilaria diluted in a pool of at least 12 blood samples of 60 μl each. Using this assay, mf burdens can be predicted using a standard curve derived from mf spiked dried blood samples. The sensitivity achieved is equivalent to the detection of a single LF positive individual carrying a mf burden as low as 18 mf/ml, in a pool of blood samples from at least 12 individuals” (Abstract). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Hasick to include the specific buffers and manipulation steps as taught by Plichart to arrive at the claimed invention with a reasonable expectation for success.
Conclusion
No claims are allowed. All claims stand rejected.
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/STEPHANIE K MUMMERT/Primary Examiner, Art Unit 1681