Detection of Viruses in Infected Plant Extracts using Immunocapture-PCR

 

  1) Immunocapture stage

• Coating buffer : 15 mM Na₂ CO₃ ; 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 KH₂ PO₄ , 8 mM Na₂ HPO₄ , 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^o C, followed by 40 cycles of 1 min at 94⁰ C, 1 min at 55⁰ C and 2 min at 72⁰ C with a final extension of 5 min at 72⁰ C.

(E) Varying the protocol duration time

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

• Dilute antibodies 1:500 in coating buffer (1Xworking conc.)
• Incubate at 37⁰ C for 2.5 h in moist chamber
• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry

• Grind leaf extracts 1:3 w/v in extraction buffer (1Xworking conc.)
• Incubate at 37⁰ C for 2.5 h in moist chamber
• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry

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

• Dilute antibodies 1:500 in coating buffer (1Xworking conc.)
• Incubate at 4⁰ C for 16 h in moist chamber
• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry

• Grind leaf extracts 1:3 w/v in extraction buffer (1Xworking conc.)
• Incubate at 4⁰ C for 16 h in moist chamber
• Wash 3X with PBS-Tween wash buffer (1Xworking conc.) �let air dry