DetectionofVirusesinInfectedPlantExtractsusingImmunocapturePCR

 1) Immunocapture stage

• Coating buffer: 15 mM Na₂CO₃; 35 mM NaHCO₃, and 3 mM NaN₃, per liter (pH 9.6).

• Extraction buffer: (20 mM Tris-HCL (pH 8.0), 138 mM NaCl, 1 mM PVP, 0.05% Tween-20, 3 mM KCl, and 3 mM NaN₃ per liter (pH 7.4).

• PBS-Tween wash buffer: 138 mM NaCl, 1.5 mM KH₂PO₄, 8 mM Na₂HPO₄, 3 mM KCl, 0.05% Tween-20, and 3 mM NaN_3, perliter pH 7.4)

• Antibodies

2) PCR stage

 For a single reaction of 50 ul, the PCR components are:

• 20 mM Tris-HCl (pH 8.4) (included in 10XPCR buffer depending on manufacturer)

• 50 mM KCl (included in 10XPCR buffer depending on manufacturer)

• 1.5 mM MgCl₂

• 0.2 mM dNTP

• 50 pmoles of each primer (degenerate primers can be used)

• 1% Tween-20

• 2.5 U Taq DNA Polymerase

Method

(A) Preparation of clarified extracts: 

1. Wash fresh foliar tissue briefly in sterile distilled water.

2. Weight out 1 g and cut into strips with sterile scalpel blade.

3. Grind tissue using autoclaved mortar and pestle (or extraction pouches) in extraction buffer (1X working conc.) at a ratio of 1:3 w/v at room temperature.

4. Filter extracts through mira cloth (not required if using extraction pouches).

5. Serially dilute extract to 2⁰ to 2^-10 in extraction buffer – use 2^-5 and 2^-6 dilutions for the antigen capture steps.

(B) Antibody coating steps

1. Dilute antibody 1:500 in coating buffer (1Xworking conc.)and mix gently by inversion.

2. Aliquot 50 ul into 0.5 ml microcentrifuge tube.

3. Place tube in a moist chamber.

4. Incubate (see section (D) Varying duration times of protocol)

(C) Antigen capture steps 

1. Pipette out diluted antibody

2. Allow tube to air-dry (10-15 min)

3. Aliquot 50 ul PBS-Tween wash buffer (1X working conc.)

4. Pipette out wash buffer

5. Repeat twice

6. Allow tube to air-dry (10-15 min)

7. Aliquot 50 ul of diluted plant extract

8. Place tube in a moist chamber

9. Incubate (see section (D) Varying duration times of protocol)

(D) PCR amplification

1.  Pipette out diluted antibody

2. Aliquot 50 ul PBS-Tween wash buffer (1X working conc.)

3. Pipette out wash buffer

4. Repeat twice

5. Allow tube to air-dry (10-15 min)

6. Aliquot 50 ul of PCR reaction mix

Perform your own PCR or conduct as recommended here:

For a single reaction of 50 ul, the PCR components include 20 mM Tris-HCl (pH 8.4), 50 mM KCl; 1.5 mM MgCl₂, 0.2 mM dNTP, 1% Tween-20, 2.5 U Taq DNA Polymerase and 50 pmoles of each primer (degenerate). Overlay reaction mix with PCR-grade, sterile mineral oil and subject to amplification using a programme of: 5 min at 94^oC, followed by 40 cycles of 1 min at 94⁰C, 1 min at 55⁰C and 2 min at 72⁰C with a final extension of 5 min at 72⁰C.

(E) Varying the protocol duration time

 IC-PCR Short Protocol          (1 day single tube assay)

1) Antibody coating steps:

• Dilute antibodies 1:500 in coating buffer (1Xworking conc.)

• Incubate at 37⁰C for 2.5 h in moist chamber

• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) –let air dry

2) Antigen capture steps:

• Grind leaf extracts 1:3 w/v in extraction buffer (1Xworking conc.)

• Incubate at 37⁰C for 2.5 h in moist chamber

• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) –let air dry

3) Run PCR

IC-PCR Long Protocol          (3 day single tube assay)

1) Antibody coating steps:

• Dilute antibodies 1:500 in coating buffer (1Xworking conc.)

• Incubate at 4⁰C for 16 h in moist chamber

• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) –let air dry

2) Antigen capture steps:

• Grind leaf extracts 1:3 w/v in extraction buffer (1Xworking conc.)

• Incubate at 4⁰C for 16 h in moist chamber

• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) –let air dry