/AH/DETAILED ACTION
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. This action is in response to the papers filed December 1, 2025. Applicant’s remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. Any new grounds of rejection presented in this Office Action are necessitated by Applicant's amendments. Any rejections or objections not reiterated herein have been withdrawn. This action is made FINAL.
Claims 1-26 are currently pending and have been examined herein.
Claim Rejections - 35 USC § 112(d)
3. The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 21 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 21 as amended recites “wherein said (d) is conducted directly after said (c) without removing the lysis reagent from the pooled reagent”. In the instant case it is unclear how claim 21 further limits the method of claim 1. Claim 1 recites step (c) “pooling” followed by step (d) “directly testing the pooled lysate including the lysis reagent”. Therefore claim 1 already requires that step (d) is conducted directly after step (c) AND already requires that the lysis reagent is not removed from the pooled lysate since (d) requires testing the pooled lysate including the lysis reagent. Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
4. 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.
5. Claims 1, 9-14 and 18-26 are rejected under 35 U.S.C. 103 as being unpatentable over Himmelreich (WO 2013/068107 Pub 5/16/2013) in view of Taylor (Journal of Clinical Microbiology 2/2010 Vol 48 No 2 pages 512-519) and Craig (Genome Research 2009 19:2075-2080).
Regarding Claim 1 Himmelreich teaches a method for lysing a sample to provide a lysis mixture that is suitable for being directly used in a nucleic acid analysis method, wherein the sample is contacted with (a) at least one non-ionic surfactant, (b) at least one water-soluble polyanionic polymer, to provide a lysis mixture. The obtained lysis mixture can be used directly in an amplification reaction. The comprised water-soluble polyanionic polymer efficiently improves the amplification results by complexing PCR inhibitors that are released from the sample and are comprised in the lysis mixture (abstract). Himmelreich teaches that the sample may be whole blood (page 20, line 37). In example 1, PCR amplification was performed using lysates from blood or plasma. In the example, four different lysis solutions were tested and compared (LS 1 to LS 4). LS 3 and LS 4 are lysis solutions according to the present invention which comprise a water-soluble polyanionic polymer (PAA1 or PAAMA). Himmelreich teaches that for obtaining blood lysates the following additives were mixed:
- 985 μl of LS 1, LS 2, LS 3, or LS 4;
- 5 μl of human blood and
- 10 μl of E. coli cell suspension
For obtaining plasma lysates the following additives were mixed:
- 985 μl of LS 1, LS 2, LS 3, or LS 4;
- 70 μl of human plasma
- 10 μl of E. coli cell suspension
The resulting mixtures were incubated for 5min at 95°C under constant shaking at 1.400 rpm on an Eppendorf Thermomixer. The obtained lysis mixture was cooled down to room temperature. 1 μl and 10μl supernatant were subsequently used in a 25μl qPCR reaction. Q-PCR was performed for the amplification of an upstream ORF of eaeA gene of E. coli (pages 29-30). Regarding the PCR performed with the lysates that were obtained with LS 1 and LS 2, Himmelreich teaches that the blood additives that were released during lysis have a considerable inhibitory effect on the PCR amplification. However, when using the lysis buffers LS 3 and LS 4 according to the present invention, the achieved Ct values are considerably lower than the Ct values that are achieved with the prior art lysis buffers LS 1 and LS 2. This shows that the method according to the present invention is significantly better in reducing the amount of PCR inhibitors, respectively ameliorating the inhibitory effects of compounds comprised in the lysis mixture. Himmelreich teaches that to sum it up, using a lysis solution comprising a water-soluble polyanionic polymer, results in a pronounced reduction of the inhibitory effect of blood or plasma on the PCR amplification reaction (pages 31-32). Thus Himmelreich teaches a method for detecting the presence of a nucleic acid of a pathogen in a whole blood sample comprising (a) obtaining a liquid whole blood sample from a subject; (b) contacting the whole blood sample with a lysis reagent, wherein at least a portion of blood cells in the whole blood sample aliquot lyse; and directly testing the lysate including the lysis reagent for presence of the nucleic acid of the pathogen, wherein the lysis reagent is compatible with subsequent steps of the method for analyzing the nucleic acid of the pathogen in the lysate without removal of the lysis reagent from the lysate.
Himmelreich does not teach obtaining a plurality of liquid whole blood samples from a plurality of subjects, separately contacting each of the plurality of liquid whole blood samples with a lysis reagent, pooling the lysed liquid whole blood samples to form a pooled lysate, and directly testing the pooled lysate for presence of the nucleic acid of the pathogen, whereby identification of the presence of the nucleic acid of the pathogen in the pooled lysate indicates the presence of the nucleic acid of the pathogen in at least one of the whole blood samples (clm 1).
However Taylor teaches that pooling samples prior to diagnostic testing for low-prevalence gene targets in a population promises an opportunity to conserve resources without sacrificing diagnostic certainty. First proposed for syphilis screening, the technique has been successfully employed to screen blood donors for antibodies to HIV, hepatitis B virus, and hepatitis C virus and to diagnose acute HIV infections by using PCR. In testing algorithms that employ this strategy, the number of samples included in each pool depends on the expected prevalence of the disease under study and the characteristics of the diagnostic test. If a pool tests positive, the individual samples comprising the pool are evaluated in a second round of testing. Depending on the expected prevalence of a target condition, pooling can obviate >90% of individual tests, with significant resource savings. Additionally, pooling is usually done robotically, limiting technician time (page 512, col 2). Taylor teaches extracting DNA from blood samples (which includes lysis), pooling the samples, and then testing for Plasmodium (Fig 1).
Additionally Craig teaches a method wherein whole blood samples which had been stored at 4ºC for between 12 and 24 mo, from 131 Caucasian patients with exudative AMD and 216 examined disease free Caucasian controls, were pooled following lysis of each individual sample to improve homogeneity of the stored samples. Genomic DNA was extracted from each blood pool using the QIA-AMP DNA blood maxi kit and hybridized to an array (page 2079, col 1).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Himmelreich by obtaining a plurality of liquid whole blood samples from a plurality of subjects, separately contacting each of the plurality of liquid whole blood samples with a lysis reagent, pooling the lysed liquid whole blood samples to form a pooled lysate, and then testing the pooled lysate. Based on the teachings of Taylor, one of skill in the art would have been motivated to pool samples prior to diagnostic testing for the benefit of being able to conserve resources without sacrificing diagnostic certainty (page 512). Based on the teachings of Craig the skilled artisan would have been motivated to lyse the whole blood samples prior to pooling for the benefit of improving the homogeneity of the samples (page 2079, col 1). Further it is noted that several court cases have found that changes in the sequence of performing steps are obvious (see MPEP 2144.04(IV)(c)). Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In reBurhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In reGibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.). Thus it would be obvious pool and then lyse or lyse and then pool.
Himmelreich does not teach a method wherein a pool comprises from 4 to 200 whole blood samples (clm 9). Himmelreich does not teach a method wherein a pool comprises at least, 4, 16, or 20 whole blood samples (clm 10).
However Taylor teaches that the number of samples included in each pool depends on the expected prevalence of the disease under study and the characteristics of the diagnostic test (page 512, col 2). Taylor then exemplifies pools of 4 samples (Fig 1).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Himmelreich by preparing a pooled sample that comprises at least 4 whole blood samples as suggested by Taylor. One of skill in the art would have been motivated to pool this number of samples since Taylor teaches that for Malaria testing a pool size of four samples was determined to achieve the greatest efficiency without sacrificing test sensitivity (page 515, col 2).
Himmelreich does not teach a method wherein up to 25% of the volume of each of the lysed blood samples are pooled (clm 11).
However Taylor teaches that genomic DNA was eluted into 150 ul of eluate and stored at 4°C. Taylor teaches that for microscopy negative samples, the optimal pool size for testing was determined and 10 ul quantities of these samples were combined in pools of 4 (see page 513 col 1-2, Fig 1). Therefore ~6.5% of the volume of each of the lysed sample aliquots (gDNA) are pooled.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Himmelreich pooling up to 25% of the volume of the lysed samples as suggested by Taylor. The skilled artisan would have been motivated to pool up to 25% of the volume of the lysed samples for the benefit of saving 75% of the lysed sample in case the pool tested positive for the pathogen and each lysed sample needs to be tested individually.
Himmelreich does not teach a method wherein the testing, tests for the presence of a pathogen-nucleic acid target released from the blood cells by the lysis reagent (clm 12). Himmelreich does not teach a method wherein the testing, tests for the presence of a pathogen-RNA target released from the blood cells by the lysis reagent (clm 13). Himmelreich does not teach a method wherein the testing comprises performing a nucleic acid amplification and a detection reaction to detect the presence of a pathogen-nucleic acid target released from the blood cells by the lysis reagent (clm 14). Himmelreich does not teach a method wherein the pathogen is a parasite from the genus Plasmodium (clm 19).
However Taylor discloses a high throughput pooling and real time PCR based strategy for Malaria detection (Title). Malaria is a parasite from the genu Plasmodium that infects red blood cells. Taylor discloses detection of Malaria by performing real time PCR to detect 18S rRNA of Plasmodium (page 513).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Himmelreich by using it to test for the presence of Plasmodium 18S rRNA in a sample of lysed blood cells as suggested by Taylor. One of skill in the art would have been motivated to modify the method of Himmelreich by testing for the presence of Plasmodium 18S rRNA released from lysed blood cells for the benefit of being able to diagnose a Malaria infection. Further it would have been obvious to use PCR for detection of Plasmodium 18S rRNA particularly since Taylor teaches that conventional and real time PCR can be used to provide information on parasite density, infecting species, and drug resistance alleles, all with high degrees of sensitivity (page 512).
Himmelreich does not teach a method wherein if the testing step determines that the pathogen is present, the method further comprises individually testing the liquid blood samples to identify the presence of the pathogen in each sample (clm 20).
However Taylor teaches that if a pool tests positive, the individual samples comprising the pool are evaluated in a second round of testing (page 512, Fig 1).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Himmelreich by testing for the presence of a pathogen in a pooled sample and then if the pool test positive further repeating the testing on the individual samples as suggested by Taylor. One of skill in the art would have been motivated to further individually test each blood sample that was used for pooling when the pool tested positive for the presence of a pathogen for the benefit of being able to determine which sample has parasite and thereby diagnose that subject with Malaria so that treatment can begin.
Regarding Claim 18 Himmelreich teaches a method wherein centrifugation is not performed (Example 1).
Regarding Claim 21 Himmelreich teaches that the testing is conducted without removing the lysis buffer (abstract, Ex 1).
Regarding Claim 22 Himmelreich lysis buffer was added to the tubes as well as blood and E. coli cells. The resulting mixtures were incubated for 5min at 95°C under constant shaking at 1.400 rpm on an Eppendorf Thermomixer. The obtained lysis mixture was cooled down to room temperature. 1 μl and 10μΙ supernatant were subsequently used in a 25μΙ qPCR reaction (page 29-30). Thus there is only one lysis step.
Regarding Claim 23 Himmelreich teaches that testing is performed without a washing step (Example 1).
Regarding Claim 24 Himmelreich teaches that 25µl q-PCR mixes contained the following Probe PCR master mix, forward and reverse primers, a probe, water, and lysate aliquots (page 31). The testing (PCR) is performed in a single tube.
Regarding Claim 25 Himmelreich teaches that the pH of the lysis buffer is 8 (page 29).
Regarding Claim 26 Himmelreich teaches a method wherein centrifugation is not performed (Example 1). Thus Himmelreich teaches a method wherein centrifugation is not performed after the whole blood sample is lysed with the lysis reagent and before the lysate including the lysis reagent is tested.
6. Claims 2-3 are rejected under 35 U.S.C. 103 as being unpatentable over Himmelreich (WO 2013/068107 Pub 5/16/2013) in view of Taylor (Journal of Clinical Microbiology 2/2010 Vol 48 No 2 pages 512-519) and Craig (Genome Research 2009 19:2075-2080) as applied to claim 1 above and in further view of Rebound (US 2011/0129931 6/2/2011).
The teachings of Himmelreich, Taylor, and Craig are presented above.
The combined references do not teach a method wherein each sample aliquot is contacted with the lysis reagent at a volume ratio of sample aliquot to lysis reagent that is from about 1:2 to about 1:10 (v/v) (clm 2). The combined references do not teach a method wherein each sample aliquot is contacted with the lysis reagent at a volume ratio of sample aliquot to lysis reagent that is about 1:3 (v/v) (clm 3).
However Rebound teaches a method wherein a blood sample may be mixed with a lysis buffer solution such that the volume ratio of NH4Cl (having a concentration of about 150 mM) the blood ranges from 1:1 to 1:10 (para 0069).
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have modified the method of Himmelreich, Taylor, and Craig so that each sample aliquot is contacted with the lysis reagent at a volume ratio of sample aliquot to lysis reagent that is from about 1:2 to about 1:10 (v/v) or about 1:3 (v/v) as suggested by Rebound. Determining the optimum sample to lysis reagent volume ratio would have been obvious to one of ordinary skill in the art and well within the skill of the art. As discussed in MPEP 2144.05(b), “(w)here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 105 USPQ 233, 235 (CCPA 1955).
MPEP 2144.05(b):
“Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical.”[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”
“A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation. In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977).”
7. Claims 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Himmelreich (WO 2013/068107 Pub 5/16/2013) in view of Taylor (Journal of Clinical Microbiology 2/2010 Vol 48 No 2 pages 512-519) and Craig (Genome Research 2009 19:2075-2080) as applied to claim 1 above and in further view of Heath (US Patent 5,973,137 Issued 10/26/1999).
The teachings of Himmelreich, Taylor, and Craig are presented above.
The combined references do not teach a method wherein the lysis reagent comprises: (i) a buffer; (ii) lithium lauryl sulfate (LLS); and (iii) one or both of a chloride containing salt and an anti-coagulant selected from the group consisting of EDTA, EDTA-Na2, EGTA, and combinations thereof, and wherein the reagent has a pH that is greater than 5.5 (clm 4). The combined references do not teach a method wherein the buffer is sodium bicarbonate at a concentration of about 5 mM to about 30 mM (clm 5).
However Heath discloses a red blood cell lysis reagent that comprises ammonium chloride, sodium bicarbonate, and EDTA (Col 8, lines 1-6). Heath teaches that the sodium bicarbonate is at a concentration of 0.5-5 mM. Heath further teaches that the red blood cell lysis reagent can be combined with a cell lysis reagent (col 8 lines 1-, 27). Heath teaches that the cell lysis reagent comprises an anionic detergent dissolved in water and buffered to a pH of about 6. Heath teaches that the anionic detergent can be a lithium salt of dodecyl sulfate (col 5 lines 61 to col 6 line 22). Thus Heath teaches a lysis reagent that comprises a buffer (sodium bicarbonate) present at 5mM, lithium lauryl sulfate (the lithium salt of dodecyl sulfate), a chloride containing salt (ammonium chloride), and an anti-coagulant (EDTA) wherein the reagent has a pH that is about 6.
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have modified the method of Himmelreich, Taylor, and Craig by using the lysis reagent of Heath for the benefit of being able to obtain lysed red blood cells and facilitate subsequent isolation of RNA from the white blood cells contained in mammalian whole blood (col 8, lines 1-4). Further determining the optimum pH would have been obvious to one of ordinary skill in the art and well within the skill of the art. As discussed in MPEP 2144.05(b), “(w)here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 105 USPQ 233, 235 (CCPA 1955).
MPEP 2144.05(b):
“Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”
“A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation. In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977).”
8. Claims 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Himmelreich (WO 2013/068107 Pub 5/16/2013) in view of Taylor (Journal of Clinical Microbiology 2/2010 Vol 48 No 2 pages 512-519), Craig (Genome Research 2009 19:2075-2080), and Heath (US Patent 5,973,137 Issued 10/26/1999) as applied to claims 1 and 4-5 above and in further view of Erbacher (US 2009/0018323 Pub 1/15/2009).
The teachings of Himmelreich, Taylor, Craig, and Heath are presented above.
The combined references do not teach a method wherein the LLS is present in the reagent at a concentration from about 4% (w/v) to about 15% (w/v) (clms 6 and 8). The combined references do not teach a method wherein the chloride containing salt is magnesium chloride present in the reagent at a concentration from about 20 mM to about 35 mM (clms 7 and 8). The combined references do no teach a method wherein the buffer is a TRIS buffer present in the reagent at a concentration from about 75 mM to about 150 mM (clm 8).
However Erbacher teaches methods for extraction nucleic acids from blood. Erbacher teaches that lysis buffers are known from the prior art and are also commercially available--e.g. "QIAGEN Buffer FG1", "QIAGEN Buffer C1" or Gentra RBC lysis solution. Erbacher teaches that, in principle it is possible to use any buffer, whose constituents are capable of lysing erythrocytes. Suitable buffers are known to the person skilled in the art and can comprise salts of inorganic or organic acids. They are preferably based on aqueous solutions of alkali or earth alkali metal halides--such as e.g. potassium chloride and magnesium chloride supplemented by a pH-buffer, such as e.g. Tris, a detergent and optionally a sequestrant (para 0013).
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have modified the method of Himmelreich, Taylor, Craig and Heath by using TRIS as the buffer and magnesium chloride as the chloride containing salt as suggested by Erbacher particularly since Erbacher teaches that these reagents were known in the art at the time of the invention to be capable of lysing erythrocytes. Further determining the optimum reagent concentrations would have been obvious to one of ordinary skill in the art and well within the skill of the art. As discussed in MPEP 2144.05(b), “(w)here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F.2d 454, 105 USPQ 233, 235 (CCPA 1955).
MPEP 2144.05(b):
“Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)”
“A particular parameter must first be recognized as a result-effective variable, i.e., a variable which achieves a recognized result, before the determination of the optimum or workable ranges of said variable might be characterized as routine experimentation. In re Antonie, 559 F.2d 618, 195 USPQ 6 (CCPA 1977).”
9. Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Himmelreich (WO 2013/068107 Pub 5/16/2013) in view of Taylor (Journal of Clinical Microbiology 2/2010 Vol 48 No 2 pages 512-519) and Craig (Genome Research 2009 19:2075-2080) as applied to claim 1 above and in further view of Busch (N Engl J Med 2005;353:460-7).
The teachings of Himmelreich, Taylor, and Craig are presented above.
The combined references do not teach a method comprising performing a transcription mediated amplification of the nucleic acid target and detecting the resulting amplification product with a detection probe (clm 15).
However Busch discloses a West Nile virus Transcription-Mediated Amplification system (Procleix WNV Assay, Gen-Probe and Chiron). This technique involves lysis of viral particles in plasma, either from individual donations or from a minipool of plasma specimens from 16 donations and the isolation of West Nile virus RNA with the use of probes bound to magnetic beads, amplification with the use of RNA transcription, and subsequent detection by a chemiluminescent probe. All samples within a reactive minipool are then tested individually. The assay has an analytical sensitivity of approximately 4 RNA copies per milliliter when used for individual donations (50 percent limit of detection by probit analysis of dilutions of West Nile virus standards) and a sensitivity of approximately 45 copies per milliliter when used for minipool testing (page 461 col 1-2).
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have modified the method of Himmelreich, Taylor, and Craig by performing a transcription mediated amplification of the nucleic acid target and detecting the resulting amplification product with a detection probe as suggested by Busch. One of skill in the art would have been motivated to perform a TMA of the nucleic acid target and then detect the resulting amplification product with a detection probe since Busch teaches that the assay has an analytical sensitivity of approximately 4 RNA copies per milliliter when used for individual donations (50 percent limit of detection by probit analysis of dilutions of West Nile virus standards) and a sensitivity of approximately 45 copies per milliliter when used for minipool testing (page 461 col 1-2). Further the claim would have been obvious because the substitution of one nucleic acid amplification test (the real time PCR method of Taylor) for another (the TMA assay of Busch) would have yielded predictable results to one of ordinary skill in the art at the time of the invention.
10. Claims 16 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Himmelreich (WO 2013/068107 Pub 5/16/2013) in view of Taylor (Journal of Clinical Microbiology 2/2010 Vol 48 No 2 pages 512-519) and Craig (Genome Research 2009 19:2075-2080) as applied to claim 1 above and in further view of Linnen (US 2006/0134609 Pub 6/22/2006).
The teachings of Himmelreich, Taylor, and Craig are presented above.
The combined references do not teach a method wherein the testing step (d) comprises contacting the pooled lysate with a capture probe and an immobilized probe, the capture probe having a first segment complementary to the nucleic acid target, and a second segment complementary to the immobilized probe, wherein the nucleic acid target binds to the capture probe, and wherein the bound capture probe binds to the immobilized probe (clm 16). The combined references do not teach a method further comprising contacting the pooled lysate with a solid support configured to immobilize the target, wherein the solid support is a magnetic solid support and separating the immobilized target from the pooled lysate (clm 17).
However Linnen discloses an assay that employs a capture probe that hybridizes to a target nucleic acid sequence (SARs derived nucleic acid) and includes a tail portion that allows the target nucleic acid to be separated from other components of the sample. The tail portion is preferably a base sequence that hybridizes to a complementary sequence immobilized on a solid support particle. Hybridization produces a capture probe:target complex which can then be immobilized through hybridization of the tail portion of the capture probe with an immobilized probe having a substantially complementary base sequence (para 0144). Linnen discloses the solid support can be a magnetic particle and teaches using the DTS 1600 Target Capture System to position and isolate the magnetic particles with the hybridized target nucleic acid (para 0156).
Accordingly, it would have been obvious to one of ordinary skill in the art at the time the invention was made to have modified the method of Himmelreich, Taylor, and Craig by contacting the pooled lysate with a capture probe and an immobilized probe, the capture probe having a first segment complementary to the nucleic acid target, and a second segment complementary to the immobilized probe, wherein the nucleic acid target binds to the capture probe, and wherein the bound capture probe binds to the immobilized probe as suggested by Linnen. It would have been obvious to have modified the method of Taylor for the benefit of being able to separate the target nucleic acid from other components of the sample. As discussed by Linnen this effectively concentrates the target nucleic acid (relative to its concentration in the test sample) and separates the target nucleic acid from amplification inhibitors which may be present in the test sample (see para 0145). Further, it would have been obvious to have modified the method of Taylor by using a magnetic bead solid support for the benefit of being able to separate the target nucleic acid from other components of the sample using a magnet.
Response To Arguments
11. In the response the Applicants traversed the rejections made under 35 USC 103.
The Applicants argue that in the primary reference, Himmelreich, discloses a method where individual sample lysates are directly used for PCR. It is well known that when nucleic acid samples are pooled, the target nucleic acid can be substantially diluted. Thus, when pooled samples are tested, the sensitivity of the assay must be much greater to ensure that a false negative result is not obtained as a result of the dilution. Himmelreich only discloses pooling the eluted DNA after the individual DNA is extracted from each sample.
This argument has been fully considered. The Examiner agrees that Himmelreich discloses a method where individual sample lysates are directly used for PCR. Applicants are reminded that this is a 103 rejection and a combination of references is being relied upon to reject the claims. Himmelreich is not being relied upon to teach pooling lysed liquid whole blood sample aliquots. Rather the rejection relies on the prior art of Taylor and Craig to teach pooling samples.
The Applicants further argue that Himmelreich removes lysis buffer from the samples.
They argue that Himmelreich expressly discloses that it is problematic that the lysed sample comprises amplification inhibitors thus requiring a further step before performing the detection rather than just leaving the lysis reagent in the sample. They argue that At page 2, lines 36-38, Himmelreich states that "in particular the water-soluble polyanionic polymer is decisive for reducing the inhibiting effect of amplification inhibitors that are comprised in the lysed sample."
These arguments have been fully considered. Himmelreich teaches a method for lysing a sample to provide a lysis mixture that is suitable for being directly used in a nucleic acid analysis method, wherein the sample is contacted with (a) at least one non-ionic surfactant, (b) at least one water-soluble polyanionic polymer, to provide a lysis mixture. The obtained lysis mixture can be used directly in an amplification reaction (abstract). The Examiner is not persuaded by the argument Himmelreich removes the lysis buffer from the samples because they have not pointed to any specific teaching in the reference which demonstrates this. The Examiner agrees that Himmerlreich discloses that it is problematic that lysed samples can comprise amplification inhibitors, but Himmelreich provides a solution to this problem by including a water-soluble polyanionic polymer in the lysis mixture. This polymer is present in the lysis mixture-it is not added in a separate step. Himmelreich teaches that the presence of the polymer in the lysis mixture efficiently improves the amplification results by complexing PCR inhibitors that are released from the sample and are comprised in the lysis mixture (abstract). Himmelreich demonstrates that when using the lysis buffers LS 3 and LS 4 (which include the water-soluble polyanionic polymer) the achieved Ct values are considerably lower than the Ct values that are achieved with the prior art lysis buffers LS 1 and LS 2 (which do NOT include the water-soluble polyanionic polymer). This shows that the method according to the present invention is significantly better in reducing the amount of PCR inhibitors, respectively ameliorating the inhibitory effects of compounds comprised in the lysis mixture. Himmelreich teaches that to sum it up, using a lysis solution comprising a water-soluble polyanionic polymer, results in a pronounced reduction of the inhibitory effect of blood or plasma on the PCR amplification reaction (pages 31-32).
The Applicants argue that both Taylor and Craig teaching removing the lysis reagents prior to testing. They argue that Taylor and Craig both used QIA Amp blood kits which require the following steps: lysis, binding, washing, and elution.
This argument has been fully considered. The Examiner agrees that Taylor and Craig both remove the lysis reagents prior to testing. Applicants are reminded that this is a 103 rejection and a combination of references is being relied upon to reject the claims. Because the primary reference Himmelreich is being relied upon to teach not removing the lysis reagents prior to testing, Taylor and Craig are not required to teach this.
The Applicants argue that the pooled lysate that is tested in the invention of Claim 1 must include the lysis reagent. While Himmelreich discloses testing individual samples that include lysis reagents, it provides no suggestion of pooled lysates that include the lysis reagent. As noted above, neither Taylor nor Craig address this deficiency in the teachings of Himmelreich because consistent with the known requirement for enhanced sensitivity in the testing of pooled samples, both Taylor and Craig require that lysis buffer be removed from the pooled samples prior to testing. Thus, even when the teachings of Himmelreich are combined with those of Taylor and Craig, one having ordinary skill in the art would not be led to the invention of Claim 1.
This argument has been fully considered but it not persuasive. Applicants are reminded that this is a 103 rejection and a combination of references is being relied upon to reject the claims. The primary reference Himmelreich teaches lysis of whole blood samples and then directly testing the lysate including the lysis reagent for the presence of nucleic acid from a pathogen. The prior arts of Taylor and Craig are only being relied upon to teach the concept of lysing samples prior to pooling samples. It does not matter that Taylor and Craig removed the lysis buffer prior to testing because they are not being relied upon for this. The rejection over the combination of Himmelreich, Taylor, and Craig is maintained.
The Applicants argue that the additionally recited references ( Rebound, Heath, Erbacher, Busch, and Linnen) do not cure the deficiencies of Himmelreich, Taylor and Craig.
This argument has been fully considered but is not persuasive. The Applicants arguments regarding what is missing in Himmelreich, Taylor and Craig have been fully addressed above. The response to Applicants arguments, as set forth above, applies equally to the additional grounds of rejection.
12. 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 extension fee 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 date of this final action.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682