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 1-20 are pending and will be examined.
Priority
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 62165127 and 62155778, fails to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application. Each of 62165127 and 62155778 do not provide support for the limitation where the card is divided into sections that are processed separately. The claims are afforded the priority date of April 29, 2016 corresponding to PCT/US2016/030223.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on February 10, 2025 was filed in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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) 1-10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czilwik et al. (Lab Chip, 2015, 15:3749) in view of Dobbs et al. (Arch Pathol Lab Med, 2002, vol. 126, p 56-63), Wampfler et al. (PLoS One, 2013, 8(9):e76316, p. 1-12) and White et al. (Infection and Immunity, 2014, 82(4):1559-1571).
With regard to claim 1, Czilwik teaches a method of measuring the total microbial load of an agricultural species said method comprising collecting a sample from an agricultural species;
storing said sample on a card at room temperature (Abstract, Fig 1 and 2, where the LabDisk is described);
dividing the card into a plurality of smaller sections each comprising a portion of said sample; extracting nucleic acids from each of said plurality of section in separate containers to prepare a first, second and third recovered nucleic acid sample (Abstract, Fig 1 and 2, where the LabDisk is described);
and analyzing the first recovered nucleic acid sample to detect pathogenic bacterial nucleic acids, analyzing the second recovered nucleic acid sample to detect pathogenic fungal nucleic acids, and analyzing the third recovered nucleic acid sample to detect pathogenic viral nucleic acids, wherein detection of pathogenic bacterial nucleic acids, pathogenic fungal nucleic acids and/or pathogenic viral nucleic acids provides an assessment of the total microbial load of said agricultural species (Abstract, Fig 1 and 2, where the LabDisk is described).
With regard to claim 3, Czilwik teaches a method of claim 1, wherein said card comprising the sample has been stored and transported from a first location to a second location at room temperature (Abstract, Fig 1 and 2, where the LabDisk is described).
While Czilwik teaches a method of analysis of microbial load, Czilwik does not look at expression levels as part of that analysis.
With regard to claim 7, Dobbs teaches a method of detecting a bacterial microorganism and quantifying the expression level of a gene from the bacterial microorganism, the method comprising the steps of:
i) providing a sample comprising said bacterial microorganism, wherein the sample is deposited on a card, further wherein said card comprising the sample has been transported from a first location to a second location at room temperature (p 57-58, where samples are stored and stabilized on cards, see a sample card at Fig 1, see “materials”, “preparation of cell suspensions”, “DNA extraction” and “PCR analysis” headings, where DNA is extracted from the FTA cards then extracted using a column);
ii) extracting DNA from said sample, and then amplifying the extracted DNA, and detecting the bacterial microorganism (p 57-58, where samples are stored and stabilized on cards, see a sample card at Fig 1, see “materials”, “preparation of cell suspensions”, “DNA extraction” and “PCR analysis” headings, where DNA is extracted from the FTA cards then extracted using a column) and detecting the microorganism; and
Regarding claims 2, 4-8 and 10, while Czilwik and Dobbs teach extraction, neither reference specifically teach extraction of RNA.
With regard to claim 2, Wampfler teaches a method of claim 1, wherein said extracted nucleic acid is RNA, and said analyzing step comprises reverse transcribing the extracted RNA into cDNA through the use of reverse transcription quantitative PCR (RT-qPCR) conducted with a forward primer, and a reverse primer; amplifying the cDNA; and quantifying the expression level of a toxin gene or a virulence gene (p 3 see “Molecular Detection of Plasmodium parasites” heading, where the qRT-PCR or real time amplification step is carried out and where the reaction includes a labeled probe).
With regard to claim 4, Wampfler teaches a method of claim 1, wherein said RT-qPCR is conducted in the presence of a fluorescently labeled probe that specifically binds to said gene (p 3 see “Molecular Detection of Plasmodium parasites” heading, where the qRT-PCR or real time amplification step is carried out and where the reaction includes a labeled probe).
With regard to claim 5, Wampfler teaches a method of claim 1 wherein the forward and/or the reverse primer is fluorescently labeled (p 3 see “Molecular Detection of Plasmodium parasites” heading, where the qRT-PCR or real time amplification step is carried out and where the reaction includes a labeled probe).
With regard to claim 7, Wampfler teaches iii) extracting RNA, while removing DNA, from said sample, purifying the RNA using a column and then reverse transcribing the RNA into cDNA through the use of reverse transcription quantitative PCR (RT-qPCR) conducted with a forward primer, and a reverse primer and quantifying the expression level of a bacterial gene, wherein the bacterial gene is a toxin gene or a virulence gene (p 3 see “Molecular Detection of Plasmodium parasites” heading, where the qRT-PCR or real time amplification step is carried out and where the reaction includes a labeled probe).
With regard to claim 8, Wampfler teaches a method of claim 7 wherein said RT-qPCR is conducted in the presence of a fluorescently labeled probe that specifically binds to said bacterial gene (p 3 see “Molecular Detection of Plasmodium parasites” heading, where the qRT-PCR or real time amplification step is carried out and where the reaction includes a labeled probe).
With regard to claim 10, Wampfler teaches a method of claim 7 wherein the forward and/or the reverse primer is fluorescently labeled (p 3 see “Molecular Detection of Plasmodium parasites” heading, where the qRT-PCR or real time amplification step is carried out and where the reaction includes a labeled probe).
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 Czilwik to include gene expression analysis as described by Dobbs to arrive at the claimed invention with a reasonable expectation for success. Each of Czilwik, Dobbs and Wampfler focus on analysis and steps of nucleic acid analysis. Dobbs teaches “Cells from surgical specimens can be stored on FTA paper for extended lengths of time, and DNA can be extracted from these cells for PCR-based testing. FTA filter paper is a reliable medium for the storage and/or transport of tumor cells for PCR-based DNA analysis” (Abstract). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Czilwik to include gene expression analysis as described by Dobbs to arrive at the claimed invention with a reasonable expectation for success with a reasonable expectation for success.
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 Dobbs to include the RNA extraction step as taught by Wampfler to arrive at the claimed invention with a reasonable expectation for success. While Dobbs is centered on extracting DNA, extraction of both DNA and RNA is common for a variety of molecular assays. Wampfler is focused on analyzing storage of samples in the field and notes “Prompted by previous reports of successful usage of finger prick blood collected on various filter paper brands in the field, we have compared the efficiency of sampling and storage on filter paper versus in solution. Three different sampling strategies were applied in the field: (i) whole blood stored in RNAprotect® cell reagent, (ii) whole blood spotted onto Whatman® 3MM filter paper, air dried and stored in TRIzol® reagent thereafter, and (iii) Whatman FTA classic cards. The focus of this study was on the practical field work in the endemic settings with realistic time periods and limited access to freezers. This adds to some recent comparisons of laboratory cultured gametocytes under a variety of controlled conditions and stored for a maximum period of 3 months until processing of samples” (p 2, col. 1). Wampfler compares many ways to store samples for further processing to analyze RNA and DNA using PCR and Wampfler cautions “The difference in DNA- versus RNA-based Plasmodium species determination derives from the dramatic difference in the number of templates per parasite. Each Plasmodium parasite harbors only 5 copies of the 18S rRNA gene, 3 S-type (detected by our qPCR assay) and 2 A-type genes [18], whereas many thousands or even some million copies of 18S rRNA transcripts can be expected per cell. The use of these extremely abundant transcripts for parasite detection warrants great care during RNA extraction, a large number of negative controls and precise definition of a cut-off to avoid false positives through potential aerosols (low level of signal caused by airborne templates)”. (p 8, col. 2, under “Discussion” heading). Finally, Wampfler concludes “RNA-based positivity is considerably higher than other methods. On the other hand, DNA-based parasite quantification is robust and permits comparison with other globally generated molecular prevalence data. Molecular monitoring of low density asexual and sexual parasitaemia will support the evaluation of effects of up-scaled antimalarial intervention programs and can also inform about small scale spatial variability in transmission intensity” (Abstract). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Dobbs to include the RNA extraction step as taught by Wampfler to arrive at the claimed invention with a reasonable expectation for success.
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 Dobbs and Wampfler to include the analysis of the expression of virulence genes through the teachings of White to arrive at the claimed invention with a reasonable expectation for success. White teaches Staphylococcus aureus is a pathogen “that employs a large repertoire of secreted virulence factors to promote disease pathogenesis”. Further, the Abstract notes that plc is secreted by virulent staphylococci but the “contribution of PI-PLC to the capacity of S. aureus to cause disease is undefined”. Through the gene expression analysis of Plc, White notes “Real-time PCR and PI-PLC enzyme assays of the TCS mutants, coupled with SrrA promoter binding studies, demonstrated that SrrAB was the predominant transcriptional activator of plc. Furthermore, plc regulation was linked to oxidative stress both in vitro and in vivo in a SrrAB-dependent manner”. White was able to identify “potential links between bacterial responses to the host innate immune system and to oxidative stress and suggest how PI-PLC could contribute to the pathogenesis of S. aureus infections” (Abstract). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Dobbs and Wampfler to include the analysis of the expression of virulence genes through the teachings of White to arrive at the claimed invention with a reasonable expectation for success.
Claim 9 and 19-20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czilwik et al. (Lab Chip, 2015, 15:3749) in view of Dobbs et al. (Arch Pathol Lab Med, 2002, vol. 126, p 56-63), Wampfler et al. (PLoS One, 2013, 8(9):e76316, p. 1-12) and White et al. (Infection and Immunity, 2014, 82(4):1559-1571) as applied over claims 1-2, 9-10 and 31 above and further in view of Rajendram et al. (J of Microbiol Methods, 2006, 67, p. 582-592), Krishnani (Genbank entry KJ000877 published February 2014) in view of Lowe et al. (Nucleic Acids Research, 1990, 18(7):1757-1761).
With regard to claim 9, Rajendram teaches a method of claim 7 wherein the reverse primer comprises a sequence selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8 (see alignment below, where SEQ ID NO:6 and 8 correspond to KJ000877).
SEQ ID NO:6
Qy 1 CAGCGAAGTAGGTAATGTC 19
|||||||||||||||||||
Db 116 CAGCGAAGTAGGTAATGTC 98
SEQ ID NO:8
Qy 1 AAACGGTTATCGGCTG 16
||||||||||||||||
Db 131 AAACGGTTATCGGCTG 116
With regard to claim 19, Rajendram teaches a method of claim 7 wherein the reverse primer has the sequence of SEQ ID NO: 6 (see alignment above where SEQ ID NO:6 and 8 correspond to KJ000877).
With regard to claim 20, Rajendram teaches a method of claim 7 wherein the reverse primer has the sequence of SEQ ID NO: 8 (see alignment above where SEQ ID NO:6 and 8 correspond to KJ000877).
Regarding claim 9 and 19-20, while Krishnani teaches the sequences that correspond to SEQ ID NO:6 and 8, Krishnani does not teach specific primer sequences. However, it would have been prima facie obvious to one of ordinary skill to design a variety of primers and probes corresponding to those as claimed based on the known sequence of Vibrio harveyi strain A1 hemolysin gene.
Since the claimed primers simply represent structural homologs, which are derived from sequences suggested by the prior art as useful for primers and probes for the detection of SE33 marker and concerning which a biochemist of ordinary skill would attempt to obtain alternate compounds with improved properties, the claimed primers and probes are prima facie obvious over the cited references in the absence of secondary considerations.
Lowe teaches a method and computer program for designing primer pairs based on a set of rules regarding primer length, sequence composition, GC content, melting temperature, potential for primer dimer formation within a primer pair, and amplification product size, among other features. Lowe evaluated the performance of primers output by the computer program and after testing primers designed for more than 10 gene products found that “experimental testing has shown that all of the amplification products specified by these primers are of the predicted size and also hybridize with the appropriate cDNA or internal oligonucleotide probe” (p. 1760, col. 2). The computer program designed by Lowe can rapidly scan an entire nucleotide sequence "for all possible primer pairs obeying these rules” (p. 1757, col. 2). The process is based on a user specified region of the target sequence and other parameters including GC content and melting temperature of the amplified region. The program provides a list of candidate antisense primers based on scanning the sequence and locating GC-type sequences, “producing a 22-mer antisense primer ‘candidate’” and selecting suitable primers by evaluating the GC content and potential for self-homology for each primer. The process is repeated for scanning and choosing candidate sense primers. After a bank of possible sense and antisense primers are produced from the input sequence “each suitable sense primer selected in this way is then checked for cross homologies" with all primers suitable for matching with it, based melting temperature of the amplification product and amplicon length. Finally, primer sets are approved and output in a format which includes the sense and antisense primer sequences, the melting temperatures and product length. The process is continued until the entire specified sequence has been scanned (p. 1758, col. 2). Considering the flexibility provided by this program and the ability to optimize individual primers and primer pairs, it would have been prima facie obvious to select and evaluate multiple candidate primer pairs for the amplification of a particular sequence.
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 Rajendram and the known sequence of Vibrio harveyi strain A1 hemolysin gene to design a variety of primers suitable for the amplification and detection of Vibrio sequences, using the method of primer design taught by Lowe to arrive at the claimed invention with a reasonable expectation for success. Lowe teaches “a computer program which rapidly scans nucleic acid sequences to select all possible pairs of oligonucleotides suitable for use as primers to direct efficient DNA amplification” which allows for “the rapid selection of effective and specific primers from long gene sequences while providing a flexible choice of various primers to focus study on particular regions of interest" . Therefore, one of ordinary skill in the art at the time the invention was made would have been motivated to have adjusted the teachings of Rajendram and the known sequence of Vibrio as taught by Krishnani to design a variety of primers suitable for the amplification and detection of SE33 marker, using the method of primer design taught by Lowe to arrive at the claimed invention with a reasonable expectation for success to achieve selection of a variety of specific primers for efficient amplification with a reasonable expectation for success.
Claims 6 and 11-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Czilwik et al. (Lab Chip, 2015, 15:3749) in view of Dobbs et al. (Arch Pathol Lab Med, 2002, vol. 126, p 56-63), Wampfler et al. (PLoS One, 2013, 8(9):e76316, p. 1-12) and White et al. (Infection and Immunity, 2014, 82(4):1559-1571) as applied over claims 1-2, 9-10 and 31 above and further in view of Rajendram et al. (J of Microbiol Methods, 2006, 67, p. 582-592).
With regard to claim 17, Dobbs teaches a method of claim 7 wherein said sample is deposited on a Flinders Technology Associates (FTA) card (p 57-58, where samples are stored and stabilized on cards, see a sample card at Fig 1, see “materials”, “preparation of cell suspensions”, “DNA extraction” and “PCR analysis” headings, where DNA is extracted from the FTA cards then extracted using a column).
With regard to claim 18, Dobbs teaches a method of claim 17 wherein the FTA card is selected from the group consisting of WB12-0205, WB12-0206, WB12-0055, WB12-0056, WB12-0210, WB12-0210, WB12-0211, and WB12-0208 (p 57-58, where samples are stored and stabilized on cards, see a sample card at Fig 1, see “materials”, “preparation of cell suspensions”, “DNA extraction” and “PCR analysis” headings, where DNA is extracted from the FTA cards then extracted using a column).
Regarding claims 11-16, while Dobbs teaches the FTA card, Dobbs does not specifically teach the microorganism.
With regard to claim 6, Rajendram teaches a method of claim 1 wherein the sample from an agricultural species is a swab from a swine or a poultry species (p. 591, col. 1, where the method is applicable to various samples).
With regard to claim 11, Rajendram teaches a method of claim 7 wherein the bacterial microorganism is selected from the group consisting of Vibrio harveyi, Vibrio campbellii, Vibrio fluvialis, and Vibrio parahaemolyticus (Table 1, where a large variety of bacterial strains were included).
With regard to claim 12, Rajendram teaches a method of claim 7 wherein the bacterial microorganism is selected from the group consisting of Clostridium perfringens, Campylobacter jejuni, and Campylobacter coli (p. 587, col. 1, where clostridium was tested).
With regard to claim 13, Rajendram teaches a method of claim 11 wherein the agricultural sample is a swab from a swine or a poultry species (p. 591, col. 1, where the method is applicable to various samples).
With regard to claim 14, Rajendram teaches a method of claim 7 wherein the gene is a gene encoding a toxin (p. 586, col. 1, where Bft toxin gene was tested).
With regard to claim 15, Rajendram teaches a method of claim 14 wherein the gene is a hemolysin (hly) gene (Table 1).
With regard to claim 16, Rajendram teaches a method of claim 7 wherein the gene is a Clostridium perfringens enterotoxin (cpe) gene (p. 587, col. 1, where clostridium was tested).
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 Dobbs, Wampfler and White to include the features of a variety of sample types of a variety of target genes as directed by Rajendram to arrive at the claimed invention with a reasonable expectation for success. Rajendram teaches “We here assessed the potential of the FTA ma1Tix card system for storage of microbial DNA and 1oss of viability ofwho1e cells added directly to the FTA cards. Furthem10re, vve evaluate the capacity and accuracy of the FTA matrix cards to serve as a medium for long-term storage and transport of DNA from bacterial cells that are archived directly onto the paper” (p. 583, col. 1). Therefore, one of ordinary skill in the art at the time the invention was made would have adjusted the teachings of Dobbs, Wampfler and White to include the features of a variety of sample types of a variety of target genes as directed by Rajendram to arrive at the claimed invention with a reasonable expectation for success.
Conclusion
No claims are allowed. All claims stand rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to STEPHANIE KANE MUMMERT whose telephone number is (571)272-8503. The examiner can normally be reached M-F 9:00-5:30.
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/STEPHANIE K MUMMERT/Primary Examiner, Art Unit 1681
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