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 .
Election/Restrictions
Applicant’s election without traverse of group II, cited in claims 13-18 in the reply filed on 12/01/2025 is acknowledged.
Claims 13-18 are considered.
Claims 1-12 and 19-21 are withdrawn from consideration.
Examiner’s Nots: The following concepts related to the claimed subject matters are explained based on the state of art:
(A) LAMP: Loop‐mediated isothermal amplification (LAMP) is an assay used for amplifies nuclear acid molecules of both DNA. In particular, Loop‐mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro‐organisms in humans, livestock and plants summarized herein as evidenced by Wong et al. (J. Appl. Microbiol. 2018, Vol. 124 (3)., pp. 626-643. doi: 10.1111/jam.13647):
1) when it is used for detection DNA, it is called a Conventional LAMP.
2). If the LAMP is used for amplifying an RNA is, it is called Reverse transcription RT-LAMP. Furthermore.
3). If the LAMP is used for an simultaneous detection of multiple target genes could enhance the specificity and reliability of a diagnosis method. This was named multiplex LAMP (mLAMP). It has also been developed as RT‐LAMP in a single tube coupled with mLAMP approach for detection of the dengue virus. This is one of the simplest mLAMP approach reported since identification of specific LAMP product was not needed. In this study, LAMP primer sets were specifically designed for each serotype of the dengue virus: DENV‐1, DENV‐2, DENV‐3 and DENV‐4 targeting on the 3′‐noncoding region (NCR). Interestingly, all the primer sets were added together into a single tube of LAMP reaction to detect the presence of the dengue virus within 60 min of a LAMP reaction. The samples used in this reaction was the serum of dengue infected patients and healthy donors. Colorimetric detection was performed by using hydroxynaphthol blue (HNB), in which it allows the naked eye observation of final LAMP products. The sensitivity of this RT‐mLAMP system was 10 RNA copies for all serotypes, and there was no cross‐reactivity with three closely related arboviruses observed.
4). A combination of lateral flow assay (LFA) for end‐point detection of LAMP product is called as LFA‐LAMP or IR-LAMP in certain condition when the antibody /antigen immunoaffinity binding/migration involved (. Most generally, lateral flow test was established using fluorescein in isothiocyanate (FITC) labelled DNA probes (which will recognize the specific region in the LAMP amplicon) hybridized with biotinylated LAMP amplicons . This complex or complexed sometime would be captured by streptavidin on the biotin, forming another complexes with variety of anti‐FITC antibodies coated on the gold nanoparticles or adsorption pads, each of different LAMP amplification products hybridized with one or more different pathogenic or any antigenic gene products will be identified or captured at different detection zones that are coated with different antibody labeled coulometric zones respectively as evinced by Park et al. (Biosensors and Bioelectronics , 2017, Vol. 91, pp. 334-340). This would give a visible result on the test line.
(2) The lateral flow test, is also known as the lateral flow immunochromatography test (ICT), is designed to detect the presence of a particular target within a complex mixture. Some of them are called IC-LAMP assay others are named as IC-RT-LAMP assay as evidenced by Selvara et al. ( PLOS ONE | https://doi.org/10.1371/journal.pone.0222170 September 5, 2019, pages 1-13).
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.
Claims 13-15 and 17-18 are rejected under 35 U.S.C. 102 (a) (1) by June et al. (Analytica Chimica Acta, Volume 853, 1 January 2015, Pages 541-547).)
June et al. teach a method as an improved molecular diagnosis method for an influenza virus, wherein the improved method is based on to advance loop-mediated isotheral amplification (LAMP) with an immunochromatographic strip (ICS) for subtyping influenza A virus with anti-Hapten involved.
In particular, the reaction mixture comprises the viral pathogen presented in viral lysates and purified viral RNAs of influenza A H1N1, A H3N2, and A H5N1 viruses. June et al. indicated that in this combination method, they use multiplex RT-LAMP targeting HA gene and conserved matrix (M) gene for pathotyping and subtyping influenza A virus among influenza A H1N1, A H3N2, and A H5N1 virus strains. The combination of the advanced multiplex RT-LAMP and ICS further more specifically provides a simple, rapid, cheap, sensitive, and user-friendly diagnostic tool for identification of the different serotypes of influenza A viruses.
In each of the reaction mixes of the amplification reactions of RT-LAMP , in addition to pressing one or more targeting influenza pathogens, the mixture also comprises six primers including outer primers (F3/B3), inner primers (FIP/BIP), and loop primers (LF/LB) for targeting HA genes, and five primers including F3/B3, FIP/BIP, and LF for targeting conserved M gene were designed as shown in Fi. 2 . The sequence information is shown in Table S1. For subtyping influenza A virus, H1, H3, and H5 were targeted, and LF and LB primers were labeled with Texas Red hapten (Fig. 2a–c). To ensure the influenza A virus, the conserved region of M gene was amplified, and the LF primer was labeled with Digoxigenin (Fig. 2d). Universal M gene expression in the three viral strains was confirmed by revealing the same pattern of the RT-LAMP products in the gel electropherogram (Fig. S1). The detail primers were designed and shown in Fig. 2.
The reaction mixtures comprise 3 sets of primers designed for ruing multiplex RT-LAMP for (a) H1 gene of influenza A H1N1. (b) H3 gene of H3N2. (c) H5 gene of H5N1. LF and LB loop primers for targeting HA genes were labeled with Texas Red haptens. (d) Primer design for targeting conserved M gene of influenza A viruses (H1N1, H3N2, and H5N1). An LF loop primer for M gene was labeled with a Digoxigenin hapten as well as the anti-Hapten antibody, wherein the Hapten is a tag used for an identifier for different targeting different amplified sequence of a influenza viral gene e. g. Hapten-Texas Red for HA gene and Hapten-Digoxigenin for M gene.
The cited reference further teaches the method comprising the following steps of collecting Viral lysates and purified viral RNAs of influenza A H1N1, A H3N2, and A H5N1 viruses as well as from universal M gene used as universal RNA molecule control, mixing the isolated RNA molecules with Target specific primers described supra and enzymes such as Bst DNA polymerase and reverse transcriptase etc. running the multiple-RT-LAMP cocktail at 66 °C for 40 min and deactivated at 80 °C for 5 min. Following the multiple RT-LAMP, the resultant amplicons were analyzed on the immunochromatographic strip (ICS) for subtyping influenza A virus to confirm the accuracy wherein the ICS immobilized strips comprising the amplified viral gene that is labeled with Hapten conjugated with or without florescent colometries Texas Red -Hapten for H gene of the influenza virus or Digoxin-Hapten for the M universal control RNA molecule amplified.
It is worth to note herein that the immunocapture strip (ICS) is also called immunochromatographic assay, ICA) as well as lateral flow test (LAF), which are so well known in the state of art, which is a rapid, paper-based diagnostic device designed for antibody to Hapten to recognize hapten labeled amplified target gene analyte within minutes (often <10-20 min). Texas Red and Digoxigenin hapten labels were used for detecting HA and conserved M gene on the ICS through the hapten and anti-hapten interaction (Section 2.2-2.5 and Fig 3 and its legend).
Furthermore, the cited reference also teach using the gel-electrophoresis to confirm the accuracy for the amplified gene products by RT-LAMP compared with the ICS. They found that amplified gene product by gel electrophoresis and ICS were matched well with a higher confidence (Section 3.2, Figs. 4-5).
Because the current RT-LAMP assay was only performed as one high temperature of 66[Symbol font/0xB0] C for 40 and 80[Symbol font/0xB0]C for 5 min, the method only require water bath or heat block as evidenced by Soroka et al. (Cells, 2021 Jul 29;10(8):1931. doi: 10.3390/cells10081931. See Table 2).
The Fig. 4-5 of the cited reference have demonstrated that the devices for the lateral flow devices are used.
Finally, the cited reference teaches a combination of the multiplex RT-LAMP and ICS can provide an advanced diagnostic platform for on-site early influenza virus detection with high speed, sensitivity, and accuracy.
Therefore, the cited reference anticipates claims 13-15 and 17-18 explicitly as well as implicitly .
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 13-18 are rejected under 35 U.S.C. 102 (a) (1) by June (Analytica Chimica Acta, Volume 853, 1 January 2015, Pages 541-547).) further in view of He et al. (Biosene Bioelectron 2012, May 13, 187:113330. doi: 10.1016/j.bios.2021.113330)
The claimed method cited in claims 13-18 is directed to a method for improving detection efficiency and validity of a molecular diagnostic assay on a target pathogen gene in an analyte, comprising: providing the reaction mixture of the pathogen detection system comprising a reaction mixture for reacting with an analyte, wherein the analyte preferably directed nucleic acid molecules including DNA or RNA of a pathogen, the reaction mixture comprising: a colorimetric indicator and amplification elements including; a fluorescent indicator responsive to a presence of amplicons from an amplification reaction of a target gene(s) of a pathogen; plurality of primers complementary to one or more independent regions in each of the target sequences to be amplified of the target gene ; and at least one enzyme for initiating the amplification reaction of the target sequences; a master mix for said amplification reaction, the pathogen detection system further comprising a concurrent immunochromatographic assay comprising an antibody specific to hapten-tag identifiers of the amplicons representing one of the target sequences.
initiating an amplification of target nucleic acid by enzymes in the reaction mixture in a platform capable of generating constant heat; performing colorimetric and/or fluorometric analyses during said amplification by observing any change in color and/or spiked emission of fluorescence signal; stopping to an immunochromatographic assay and starting an immunochromatographic assay to the amplified target gene, comparing said visible signal with a gel electrophoresis analytical data of said reaction product; and if said visible signal giving a coherent positive result representing a presence of the target pathogen gene in the analyte, a thermocycle at which said reaction product being validated as positive by said immunochromatographic assay representing an end of the molecular diagnostic assay, wherein the initial assay is preference to be LAMP and the immunochromatographic assay is known in the art as LFA according to the specification and state of art , wherein said platform comprises a thermocycler, water bath or heat block, wherein the color change is due to a halochromic change or metallochromic change during said amplification of the target nucleic acid in the reaction mixture and wherein the immunochromatographic assay is performed in a lateral flow device.
As it is described in the paragraphs 12-22 supra, the disclosures by June et al. teach the limitations of claims 1-15 and 17-18 explitely as well as impolitely.
However, June et al. do not teach the limitation cited in claim 16, i.e. “ color change is due to a halochromic change or metallochromic change during said amplification of the target nucleic acid in the reaction mixture”
Dr. He et al. also disclosed a method using a pH sensitive one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA.
By this fast and highly sensitive assay for viral RNA detection was developed by the integration of Si–OH MBs-based nucleic acid extraction and loop-mediated isothermal amplification. As shown in Fig. 1 , the lysis buffer, Si–OH MBs and the sample were mixed in a 1.5-mL tube followed by allowing the virion to lyse and release the viral RNA. The RNA molecules would be captured by MBs in the salt buffer of high concentration and lower pH, likely due to the formation of hydrogen bind or electrostatic interaction between the negatively charged RNA phosphate groups and the silicon hydroxyl (Si–OH) groups of the SiO2 coated on the MBs. The MBs were then isolated from the lysis buffer using a magnet, and then the resulting supernatant was removed. A freshly-prepared 80% aqueous ethanol solution was used to wash the MBs to remove the residual guanidinium and some other inhibitors, followed by air drying at room temperature. Subsequently, the RT-LAMP reaction mixture was directly added to the tube. Notably, it is definitely unnecessary to elute RNA from MBs at the end of RNA extraction because an elution will lead to a dilution and the loss of target RNA, especially for those samples with lower concentrations. Lastly, the RT-LAMP reaction was set up at 65 °C for 40 min by placing the microtube in a thermotank or water bath apparatus. Finally, the result was easy to read by the naked eyes upon introducing phenol red as the indicator to sensitively flag the pH change of the reaction buffer. In the initial stages of RT-LAMP reaction, the reaction solution is weakly alkaline showed by a red coloration of the solution. Once the reaction starts in the presence of the target genomic materials, thousands of millions of DNA molecules are amplified, which result in sample becoming acidic and casing a color change from red to yellow. Overall, a rapid and sensitive one-tube colorimetric RT-LAMP assay was constructed for the viral RNA detection.
Therefore, it would have been obvious for the person with an ordinarily skilled in the art to combine the teachings by the three cited prior art references for arriving the claimed method, i.e. using the advanced RT-PCT platform comprising the multiple RT-LAMP plus LFA taught by June et al. especially June teach using the Hapten as a conjugator to conjugate a fluorescent marker and an antibody to the Hapten complex that comprising the amplified gene product, which is immobilized to a solid support, such as a strip or Goden particle, and the teaching using the pH sensitive Halochromic agent taught by He et al. to make the claimed method with a reassemble expectation of successfulness.
As there are no unexpected results have been provided, hence the claimed invention as a whole is prima facie obvious absence unexpected results.
Claims 13-18 are rejected under 35 U.S.C. 102 (a) (1) by DE 202021100985U1. (985U1) further in view of June (Analytica Chimica Acta, Volume 853, 1 January 2015, Pages 541-547).
The claimed method cited in claims 13-18 is directed to a method for improving detection efficiency and validity of a molecular diagnostic assay on a target pathogen gene in an analyte, comprising: providing the reaction mixture of the pathogen detection system comprising a reaction mixture for reacting with an analyte, wherein the analyte preferably directed nucleic acid molecules including DNA or RNA of a pathogen, the reaction mixture comprising: a colorimetric indicator and amplification elements including; a fluorescent indicator responsive to a presence of amplicons from an amplification reaction of a target gene(s) of a pathogen; plurality of primers complementary to one or more independent regions in each of the target sequences to be amplified of the target gene ; and at least one enzyme for initiating the amplification reaction of the target sequences; a master mix for said amplification reaction, the pathogen detection system further comprising a concurrent immunochromatographic assay comprising an antibody specific to hapten-tag identifiers of the amplicons representing one of the target sequences.
initiating an amplification of target nucleic acid by enzymes in the reaction mixture in a platform capable of generating constant heat; performing colorimetric and/or fluorometric analyses during said amplification by observing any change in color and/or spiked emission of fluorescence signal; stopping to an immunochromatographic assay and starting an immunochromatographic assay to the amplified target gene, comparing said visible signal with a gel electrophoresis analytical data of said reaction product; and if said visible signal giving a coherent positive result representing a presence of the target pathogen gene in the analyte, a thermocycle at which said reaction product being validated as positive by said immunochromatographic assay representing an end of the molecular diagnostic assay, wherein the initial assay is preference to be LAMP and the immunochromatographic assay is known in the art as LFA according to the specification and state of art , wherein said platform comprises a thermocycler, water bath or heat block, wherein the color change is due to a halochromic change or metallochromic change during said amplification of the target nucleic acid in the reaction mixture and wherein the immunochromatographic assay is performed in a lateral flow device.
DE 202021100985U1. (Please see the English translated version on line by Google automatic translation ) teach that diagnostic assay kit for the detection of a nucleic acid of the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-Cov2) using RT-LAMP system followed by a suitable method , particularly including immunochromatographic lateral flow assay (LFA) to further identify the amplified SARS-Cov2 gene product simply, clearly and correctly. In other examples, the amplification product is detected with the aid of gel electrophoresis, for example by detecting the presence or the amount of the amplification product with agarose gel electrophoresis In some examples, the amplification product is detected using a colorimetric assay, e.g. with a pH-sensitive dye (e.g. phenol red) or an intercalating dye (e.g. propidium iodide, SYBR green or picogreen) in a cycler-based real-time analysis or a chromogenic Reagent’. These colorimetric
In the further example, the cited reference also teaches In further examples, the amplification product is detected by a fluorescent indicator dye such as calcein or hydroxynaphthol blue (HNB). In other examples, the amplification products are detected using a detectable label built into one or more of the LAMP primers (see below). The detectable marking can be visually detectable, e.g. By eye or with a spectrophotometer or fluorimeter. In some examples, the detectable label is a fluorophore, such as those described above. In some examples, the marking is detected in real time. In the further example, the cited reference also teaches that the amplification product is detected by a fluorescent indicator dye such as calcein or hydroxynaphthol blue (HNB). They illustrated that one skilled in the art can select one or more detectable labels for use in the methods disclosed herein.
DE 202021100985U1 teaches a diagnostic assay kit for the detection of a nucleic acid of the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-Cov2) in a sample, In example 1 , they develop the LAMP primers and packaging them in diagnostic assay kit /comprising a first Corona-LAMP primer set, the first Corona- LAMP primer set includes the following nucleic acid primer molecules listed below:
a) a nucleic acid F3-LAMP primer ,
b) a nucleic acid FL-LAMP primer,
c) a nucleic acid FIP-LAMP primer,
d) a nucleic acid B3-LAMP primer,
e) a nucleic acid BL-LAMP primer , and
f) a nucleic acid BIP-LAMP primer.
In examples 4 and 5 simple or triple of LAMP were performed. For example, Isothermal amplification program used by the LAMP was conducted with a step temperature duration Repetition Incubation 25 ° C 5 min - Incubation 65 ° C 1 min 30 times of cycles with Melting curve 80 ° C-96 ° C - -After completion of the LAMP amplification, a lateral flow dipstick was performed on some samples for further analysis. A 1: 4 duplicate per reference sample and primer mix and one of the 1: 2 dilutions of the reference sample for the primer sets CLPS2 and CLPS3 were tested.
In other examples, the amplification product is detected with the aid of gel electrophoresis, for example by detecting the presence or the amount of the amplification product with agarose gel electrophoresis In some examples, the amplification product is detected using a colorimetric assay, e.g. with a pH-sensitive dye (e.g. phenol red) or an intercalating dye (e.g. propidium iodide, SYBR green or picogreen) in a cycler-based real-time analysis or a chromogenic Reagent’
In the further example, the cited reference also teaches In further examples, the amplification product is detected by a fluorescent indicator dye such as calcein or hydroxynaphthol blue (HNB). In other examples, the amplification products are detected using a detectable label built into one or more of the LAMP primers (see below). The detectable marking can be visually detectable, e.g. By eye or with a spectrophotometer or fluorimeter. In some examples, the detectable label is a fluorophore, such as those described above. In some examples, the marking is detected in real time, e.g. B. with a fluorescence scanner (e.g. ESEQuant, Qiagen). One skilled in the art can select one or more detectable labels for use in the methods disclosed herein.
In some examples, the methods and means further comprise the reverse transcription of the SARS-CoV-2 RNA in the sample, for example by contacting the sample with a reverse transcriptase. The "bringing into contact" of the sample with the reverse transcriptase can take place before the sample is brought into contact with the one or more sets of LAMP primers or at the same time as the sample is brought into contact with the one or more sets of LAMP-Primers . The amplification product is detected by any suitable method, such as turbidity detection ,fluorescence, immunochromatographic lateral flow assays. In particular, after incubation of the reaction mixture, the amplification product is detected by any suitable method. The detection methods can be quantitative, semi-quantitative or qualitative. In other examples, the amplification product is detected with the aid of gel electrophoresis for detecting the presence or the amount of the amplification product .In some examples, the amplification product is detected using a colorimetric assay, e.g. with a pH-sensitive dye (e.g. phenol red) or an intercalating dye (e.g. propidium iodide, SYBR green or picogreen) in a cycler-based real-time analysis or a chromogenic Reagent (hereinafter referred to as "dipsticks") or by Gel electrophoresis.
In summary, while DE 202021100985U1. (985U1) teaches that diagnostic assay kit for the detection of a nucleic acid of the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-Cov2) using RT-LAMP system followed by a suitable method , particularly including immunochromatographic lateral flow assay (LFA) to further identify the amplified SARS-Cov2 gene product simply, and also further in combination of gel electrophoresis as a colorimetric assay with a pH-sensitive dye (e.g. phenol red) or an intercalating dye (e.g. propidium iodide, SYBR green or picogreen), it does neither teach using Hapten to tag the primers for ruing the RT-LAMP nor using antibody to Hapten to recognize and catch the immobilized primers labeled with Hapten.
As it was discussed previously ion the office action supra, June et al. teach a method as an improved molecular diagnosis method for an influenza virus, wherein the improved method is based on to advance loop-mediated isotheral amplification (LAMP) with an immunochromatographic strip (ICS) for subtyping influenza A virus with anti-Hapten involved.
In particular, the reaction mixture comprises the viral pathogen presented in viral lysates and purified viral RNAs of influenza A H1N1, A H3N2, and A H5N1 viruses. June et al. indicated that in this combination method, they use multiplex RT-LAMP targeting HA gene and conserved matrix (M) gene for pathotyping and subtyping influenza A virus among influenza A H1N1, A H3N2, and A H5N1 virus strains. The combination of the advanced multiplex RT-LAMP and ICS further more specifically provides a simple, rapid, cheap, sensitive, and user-friendly diagnostic tool for identification of the different serotypes of influenza A viruses.
In each of the reaction mixes of the amplification reactions of RT-LAMP , in addition to pressing one or more targeting influenza pathogens, the mixture also comprises six primers including outer primers (F3/B3), inner primers (FIP/BIP), and loop primers (LF/LB) for targeting HA genes, and five primers including F3/B3, FIP/BIP, and LF for targeting conserved M gene were designed as shown in Fi. 2 . The sequence information is shown in Table S1. For subtyping influenza A virus, H1, H3, and H5 were targeted, and LF and LB primers were labeled with Texas Red hapten (Fig. 2a–c). To ensure the influenza A virus, the conserved region of M gene was amplified, and the LF primer was labeled with Digoxigenin (Fig. 2d). Universal M gene expression in the three viral strains was confirmed by revealing the same pattern of the RT-LAMP products in the gel electropherogram (Fig. S1). The detail primers were designed and shown in Fig. 2.
The reaction mixtures comprise 3 sets of primers designed for ruing multiplex RT-LAMP for (a) H1 gene of influenza A H1N1. (b) H3 gene of H3N2. (c) H5 gene of H5N1. LF and LB loop primers for targeting HA genes were labeled with Texas Red haptens. (d) Primer design for targeting conserved M gene of influenza A viruses (H1N1, H3N2, and H5N1). An LF loop primer for M gene was labeled with a Digoxigenin hapten as well as the anti-Hapten antibody, wherein the Hapten is a tag used for an identifier for different targeting different amplified sequence of a influenza viral gene e. g. Hapten-Texas Red for HA gene and Hapten-Digoxigenin for M gene. June et al. also teach using an antibody to Hapten to catch the immobilized amplified gene produce of target pathogen.
Therefore, it would have been obvious for the person with an ordinarily skilled in the art to combine the teachings by the three cited prior art references for arriving the claimed method, i.e. using the advanced RT-PCR platform comprising the multiple RT-LAMP plus LFA taught by both DE 202021100985U1 and June et al and especially using the Hapten as a conjugator to conjugate a fluorescent marker and an antibody to the Hapten complex that comprising the amplified gene product, which is immobilized to a solid support, such as a strip, taught by June et al. and also using the pH sensitive Halochromic agent taught by DE 202021100985U1 to make the claimed method with a reassemble expectation of successfulness.
As there are no unexpected results have been provided, hence the claimed invention as a whole is prima facie obvious absence unexpected results.
Conclusion
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BAO Q. LI
Examiner
Art Unit 1671
/BAO Q LI/Primary Examiner, Art Unit 1671