DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the AIA first to invent provisions.
Claims Status
Claims 1-13 are under examination on their merits.
Priority
For claims 1-13 in this application, a priority date of 17 May 2022 is applicable because the subject matter of each of said claims is found in CN202210536550X (CN114807444A), as filed 05/17/2022 in China.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states that "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
The first three drawings (FIG. 1 to FIG. 3) for the mapping of three primer sets (P1, P2 and P3) have different names than what is used in Table 2. Examiner recommends reconciliation of such discrepancies. For example, P3 primer names in Table 2 (¶[0046] on Pg. 7) need to match names cited in FIG 3 (Pg. 3). Acceptable amendments must clarify the correspondence between primer names, provided that such clarification does not introduce new matter.
Claim Objections
Claims 10 and 12-13 are objected to because the citation of “40 cycles” in Claims 10 and 13 is misplaced, while “10μM upstream primer” should be “10 μM upstream primer”, as correctly cited later for “10 μM downstream primer.” Examiner recommends the proper placement of “40 cycles” in Claims 10 and 13, provided that such rearrangement does not introduce new matter.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 1-13 are rejected under USC 103 as being unpatentable over Wei (危宏平) et al 2020 (CN 110894556 A, published 03/20/2020) in view of Wei (魏建超) et al 2021 (CN 112646926 A, published 04/13/2021) and further in view of Zhao et al 2019 (Molecular and Cellular Probes 45: 31-36; available online 10 April 2019).
Claim 1 is drawn to a pair of primers (SEQ ID NO: 6 and SEQ ID NO: 7, corresponding to P3-F and P3-R, respectively, in FIG. 3) used in a real-time, quantitative PCR (qPCR) assay, which specifically amplifies a 101-base-pair (bp) sequence of the vp72 (also known as P72 or B646L) gene of African swine fever virus (ASFV) for clinical diagnosis. When the primers are formulated as part of a qPCR kit (claim 2) that also contains a “PCR premix” with “a Taq enzyme, a deoxyribonucleotide triphosphate (dNTP) mixture, a TB Green dye, and a buffer” (claim 7), the reagents facilitate “a fluorescence quantitative PCR detection method” (claim 8) when “a genomic DNA of a sample to be detected by using the primers” (claim 8) is subjected to “40 cycles” of enzymatic reactions (“denaturation at 95℃ for 5 seconds, annealing and extension at 60℃ for 30 seconds” (claim 10). The kit also has a plasmid DNA with a 506-bp vp72 gene fragment (SEQ ID NO: 1) (claim 5) that serves as a positive control (claim 3) at “a concentration of 1×102 copies per microliter” (claim 6), while “a liquid used to dilute the positive control” (claim 4) serves as a “negative control” (claim 3). The “reaction system” of claim 8 consists of “5 μL of 2× TB Green Premix Ex Taq Ⅱ (Tli RNaseH Plus), 0.3 μL of 10 micromoles per liter (μM) upstream primer, 0.3 μL of 10 μM downstream primer, 1 μL of DNA Template, and 3.4 μL of double-distilled water (ddH2O) without nuclease” (claim 9). The procedures for claims 9 and 10 are repeated in claims 12 and 13 when the kit in claim 2 (a dependent of claim1) replaces the primers in claims 1. Overall, this new qPCR method worked well when compared with two alternative approaches using either “primer pair P1 published in the literature” (Figure 1/Table 2 and cited in ¶[0017] & ¶[0058]) or “primer pair P2” (Figure 2 and Table 2), as gauged by amplification efficiency (FIG. 13), specificity (FIG. 14) and reproducibility (R2 = 0.9954 in FIG. 15), with a lower limit of detection at 10 copies of vp72 gene per microliter of test sample (¶[0058]).
Wei (危宏平) et al 2020 taught the use of a “rapid”, qPCR-based method for determining ASFV viability (Abstract). Based on GenBank sequence for “ASFV B646L (p72)” gene (¶[0007]), their primers (SEQ ID NO: 1-2 in claim 1 and cited in ¶[0039] to ¶[0040]) were intended to amplify a “highly conserved region” of p72 gene (¶[0007]); an internal probe sequence (SEQ ID NO: 3, 5’-cagctcttcc agacgcatgt tc-3’) is complementary to the reverse primer (SEQ ID NO: 7) in the instant application (5’-acatgcgtct ggaagagctg-3’). A positive control plasmid (pUC57-ASFV-p72, ¶[0049]) in Wei (危宏平) et al 2020 has a 600-bp p72 sequence, which covers the entire 506-bp sequence of plasmid DNA (SEQ ID NO. 1) in the instant application, and SEQ ID NO: 6 (a forward primer) in claim 1 is nested (100% match) within the plasmid insert (nucleotide positions 443-460, in reverse orientation). Their test kit has “a set of primers, a probe, a fluorescent dye for staining PCR amplicons” (claim 4), as well as “Taq DNA polymerase, 10X buffer, dNTP and sterile water” (claim 5 and ¶[0044]). Their qPCR used genomic DNA from testing samples as templates (claim 6 and ¶[0044]), and diagnosis was based on PCR growth curve and threshold cycle (Ct and ΔCt) for signaling detection (claim 6 and ¶[0047]), with sterile water serving as a negative control (claim 6). The disclosed qPCR reaction (20 µl total volume, claim 8 and ¶[0044) had a “pre-denaturation at 95oC for 2 minutes”, followed by “40 cycles” of “denaturation at 95℃ for 5 seconds, annealing and extension at 57℃ for 30 seconds” (claim 9 and ¶[0047]). This qPCR system (with p72-specific primers, probe, PCR reagents, cycling conditions and signal detection) were deemed reliable (highly specific) and sensitive, with “a lower limit of detection (LLD) at 10 copies/µl” (¶[0025]).
Thus, while Wei (危宏平) et al effectively taught all the critical elements for ASFV diagnosis using a qPCR format and with conserved p72 sequence as a specific target, they rely on the use of an internal probe, and their PCR primers (SEQ ID NO. 1-2 in claim 1 and ¶[0039] to ¶[0040]) did not match those in the claimed invention.
Wei (魏建超) et al 2021, on the other hand, taught another molecular method for diagnosing ASFV infection based on p72-specific DNA amplicons, and one of their “outer primers” (SEQ ID NO: 4, nucleotide positions 1-18, Pg. 8) has a 100% match with the forward primer (SEQ ID NO: 6, 5’-taacgccatt atgcagcc-3’) of the claimed invention. Their method is an isothermal DNA amplification procedure in a 25 µl solution (¶[0020]) and requires a “fluorescein isothiocyanate label (¶[0021]), both deviating from the qPCR system in the claimed invention.
These deficiencies, however, are overcome by Zhao et al 2019, as they disclosed “a TB green II-based duplex real-time fluorescence quantitative PCR assay” for the simultaneous detection of two porcine viruses based on melting curves of PCR amplicons (no internal probes), with an LLD of “10 copies/µl for PCV2” (Abstract). By their report, “TB Green™ Premix Ex Taq™ II” is a commercial product from TaKaRa (section 2.4 on Pg. 32).
For a person of ordinary skill in the art and interested in developing a probe-free and sensitive qPCR method and kit for detecting ASFV infection, it would be obvious from Wei (危宏平) et al 2020 and Wei (魏建超) et al 2021 (before the effective filing date of this instant application) that p72 gene is a suitable target due to its highly conserved sequences, and primer design can take advantage of subregions that have already been tested as primer (Wei (魏建超) et al 2021; SEQ ID NO. 4, nucleotide positions 1-18, Pg. 8) or probe (Wei (危宏平) et al 2020; SEQ ID NO. 3) in existing assays. Once primers are chosen from the primer/probe annealing sites taught by Wei (魏建超) et al 2021 and Wei (危宏平) et al 2020, the plasmid DNA for positive control can directly come from Wei (危宏平) et al 2020 (pUC57-ASFV-p72, ¶[0049]) as well, and the commercially available PCR reagents (master mixes) and kits taught by Zhao et al 2019 can be applied to optimize PCR cycling conditions to obtain expected results being disclosed in the claimed invention. Thus, the invention of Claims 1-13 is rendered obvious by three references published before the filing of the invention, as an application of rationale B of MPEP 2143. Furthermore, it is prima facie obvious to choose two suitable primer sites based on the teachings of Wei (魏建超) et al 2021 and Wei (危宏平) et al 2020 and then apply the exact qPCR master mix and the probe-free format in Zhao et al 2019 for the purpose of AFSV detection (as in Wei (危宏平) et al 2020). Thus the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Additional Prior Art Cited but Not Applied
Weng & Qiu 2021 (CN112877476-A, published 06/01/2021): African swine fever virus P72 gene fluorescent probe PCR detection primer probe set, kit and method thereof. This prior art (of record) was cited in the 03/08/2025 rejection of CN 202210536550.X, the parent of this instant application. SEQ ID NO. 4 (407 nucleotides) in Weng & Qiu 2021 is a plasmid DNA insert for P72 (vp72) gene that partially overlaps with SEQ ID NO. 1 (506 nucleotides) in the instant application. The method and kit by Weng & Qiu 2021 rely on the use of a fluorescent probe for detecting qPCR amplicon, which ensures the specificity of qPCR.
Qiao et al 2018 (CN 107937624 A, published 04/20/2018): RPA primers and preparation method and kit for quick detection of African swine fever virus nucleic acid. This prior art has a primer (Figure 3, RPA-RP3, nt. 4-23) with 100% match for SEQ ID NO. 7 (reverse primer) of the instant application, suggesting that the priming site corresponding to SEQ ID NO. 7 is a well-known, conserved annealing site for the p72 gene in ASFV.
Huang et al 2021 (CN 113215328 A, published 08/06/2021): Primer pair, probe and kit for detecting African swine fever virus and application of primer pair, probe and kit. One of the primers (p72_1-F6 (¶[0052]) in this prior art has an identical sequence (nucleotide positions 7-26) to that of SEQ ID NO. 7 in the instant application, suggesting again that the priming site corresponding to SEQ ID NO. 7 is a well-known, conserved annealing site for the p72 gene in ASFV.
Wangh et al. 2014 (US 2014/0004504 Al, published 01/02/2014). Compositions and methods for the detection and analysis of African swine fever virus. This PGPub clearly stated that “the B646L gene that encodes the major capsid protein p72 (aka VP72) is very highly conserved across all strains” (¶[0006]): an ideal target for ASFV-specific PCR assays.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JIANMING TANG whose telephone number is (571) 272-0081. The examiner can normally be reached M-F 8:00-5:30 EST.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Michael Allen (SPE) can be reached at (571) 270-3497. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JIANMING TANG/Examiner, Art Unit 1671
/BENJAMIN P BLUMEL/Primary Examiner, Art Unit 1671