T 2288/11 (Nucleic aid detection air-dried whole blood sample direct PCR/FUSO) 02-09-2014

Summary of Facts and Submissions

I. The appeal lies from the decision of the examining division to refuse the European patent application no. 05 709 892.3. The examining division considered the set of claims filed on 12 April 2011 not to fulfil the requirements of Article 56 EPC. Claim 1 of this set of claims read as follows:

"1. A nucleic acid detection method comprising the steps of:

providing a sample support divided into a plurality of compartments;

fixing a cell-containing sample on the surface of the support in the plurality of compartments according to the following sub-steps (i) through (iv):

(i) smearing the sample in the divided compartments,

(ii) air-drying the smeared samples,

(iii) adding 75% ethanol to each compartment, and

(iv) air-drying the samples with a thermal cycler after removing the 75% ethanol;

exposing nucleic acids contained in the sample fixed on the support in the plurality of compartments;

amplifying nucleic acids exposed from the sample fixed in the plurality of compartments on the support by placing a PCR mixture containing primers for amplifying a target nucleic acid into the plurality of compartments; and

determining whether the amplified nucleic acids in a PCR solution existing outside the fixed cell sample in the plurality of compartments contains a target nucleic acid."

Claims 2 to 10 were directed to preferred embodiments of the nucleic acid detection method of claim 1.

II. The applicant (appellant) filed a notice of appeal and a statement setting out the Grounds of Appeal. With the Grounds of Appeal, the appellant maintained the set of claims underlying the decision under appeal and filed, as an Annex, new experimental evidence ("Comparison of Claimed Invention with Direct PCR"). The appellant requested the board to set aside the decision under appeal and to grant a patent on the basis of the set of claims filed with its Grounds of Appeal. Oral proceedings were requested as an auxiliary measure.

III. On 28 May 2014, the board summoned the appellant to oral proceedings. In a communication pursuant to Article 15(1) of the Rules of Procedure of the Boards of Appeal (RPBA) annexed to the summons, the appellant was informed of the board's preliminary, non-binding opinion on the substantive issues of the case.

In particular and with reference to decision G 10/93 (OJ EPO 1995, page 172, Headnote) wherein the Enlarged Board of Appeal stated that in an appeal proceedings from a decision of an examining division, the board had the power to examine whether the application meet all the requirements of the EPC, the board raised several objections under Article 84 EPC which were new in the proceedings. The board introduced two new documents into the proceedings, namely G.S. Makowski et al., Clinical Chemistry, Vol. 41, No. 3, 1995, pages 477-479 (document D22) and A. Ulvik and P.M. Ueland, Clinical Chemistry, Vol. 47, No. 11, 2001, pages 2050-2054 (document D23) and made several remarks on inventive step (Article 56 EPC).

IV. On 15 July 2014, the appellant replied to the communication of the board and filed a new Main Request and a new Auxiliary Request. Substantive submissions concerning Articles 123(2), 84 and 56 EPC were provided.

Claim 1 of the new Main Request read as follows:

"1. A nucleic acid detection method comprising the steps of:

providing a sample support divided into a plurality of compartments;

fixing a cell-containing sample, said sample being white blood cells collected from 5 ml of blood collected from human patients suspected to have infection-causing microbes selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli, whereby the white blood cells are suspended in 150 µl PBS, on the surface of the support in the plurality of compartments according to the following sub-steps (i) through (iv):

(i) smearing 5 µl of the sample in the divided compartments,

(ii) air-drying the smeared samples,

(iii) adding 75% ethanol to each compartment, and

(iv) air-drying the samples with a thermal cycler after removing the 75% ethanol;

exposing nucleic acids contained in the sample fixed on the support in the plurality of compartments by treatment with an enzyme reagent containing N-acetylmuramidase, lysozyme and lysostaphin for 10 minutes at 37°C, followed by 10 minutes at 95°C;

amplifying nucleic acids exposed from the sample fixed in the plurality of compartments on the support by nested PCR, whereby in the first run a PCR mixture containing 0,16 µM of each of the primers according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 18, is placed into the plurality of compartments, and whereby in the second run 0,16 µM of each of the primers according to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 are used; and

determining whether the amplified nucleic acids in a PCR solution existing outside the fixed cell sample in the plurality of compartments contains a target nucleic acid."

Claims 2 to 7 were directed to preferred embodiments of the acid nucleic detection method of claim 1 and read as claims 3-6 and 9-10 of the appellant's former Main Request.

Claim 1 of the Auxiliary Request read as follows:

"1. A nucleic acid detection method comprising the steps of:

providing a sample support divided into a plurality of compartments, whereby the sample support is shaped to fit a gene amplifier for PCR (thermal cycler);

fixing a cell-containing sample, said sample being white blood cells collected from 5 ml of blood collected from human patients suspected to have infection-causing microbes selected from the group consisting of Staphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli, whereby the white blood cells are suspended in 150 µl PBS, on the surface of the support in the plurality of compartments according to the following sub-steps (i) through (iv):

(i) smearing 5 µl of the sample in the divided compartments,

(ii) air-drying the smeared samples,

(iii) adding 75% ethanol to each compartment, and

(iv) air-drying the samples with a thermal cycler after removing the 75% ethanol;

exposing nucleic acids contained in the sample fixed on the support in the plurality of compartments by treatment with an enzyme reagent containing N-acetylmuramidase, lysozyme and lysostaphin for 10 minutes at 37°C, followed by 10 minutes at 95°C;

amplifying nucleic acids exposed from the sample fixed in the plurality of compartments on the support by nested PCR according to the following sub-steps (1) through (4):

(1) placing a PCR mixture containing 0,2 µl TaKaRa LA Taq, 2 µl 10x LA Taq buffer, 2 µl 25 mM MgCl2, 3,2 µl dNTP mixture (2,5 mM each), 0,16 µM of each of the primers for amplifying a target nucleic acid, whereby the primers according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 18 are used, and whereby the PCR mixture is adjusted to 20 µl with sterilized pure water, into the plurality of compartments,

(2) performing a first PCR run with the following cycling parameters: retention at 94°C for 1 min, 30 cycles consisting of 98°C for 20 s and 68°C for 3 min, and retention at 72°C for 5 min,

(3) placing 1 µl solution of the first run in a PCR tube supplemented with the foregoing composition, whereby the primers according to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 are used, and

(4) performing a second PCR run with the following cycling parameters: retention at 94°C for 1 min, 30 cycles consisting of 98°C for 20 s and 68°C for 1 min, and retention at 72°C for 5 min; and

determining whether the amplified nucleic acids in a PCR solution existing outside the fixed cell sample in the plurality of compartments contains a target nucleic acid."

Claims 2 to 6 were directed to preferred embodiments of the acid nucleic detection method of claim 1 and read as claims 3 to 6 and 10 of the appellant's former Main Request.

V. In a communication of 28 July 2014, the board acknowledged the amendments made in the appellant's new requests to be a bona fide attempt to overcome the objections raised under Articles 84 and 56 EPC by the board in its communication pursuant to Article 15(1) RPBA. The board also noted that, in view of the number and character of these amendments and of the procedural history of the case, the board intended to remit the case to the department of first instance for further prosecution. The appellant was informed that the scheduled oral proceedings will be cancelled if it agreed with the board and formally requested such a remittal (cf. point 1 infra).

VI. On 1 August 2014, the appellant requested the board to remit the case to the department of first instance for further prosecution on the basis of its Main Request and Auxiliary Request filed on 15 July 2014. The appellant further requested to cancel the scheduled oral proceedings.

VII. On 8 July 2014, the board cancelled the oral proceedings scheduled for the 7 October 2014.