Abstract: Diagnostic PCR should fulfill several qualitative characteristics, but the most important among them are specificity, sensitivity, efficiency and reproducibility of detection of viruses. Immunocapture-PCR (IC-PCR) may be used in detecting viruses using clarified plant extracts with degenerate primers, without prior isolation of total nucleic acids. It is especially useful for detecting viruses that exist in low or variable titers en planta and in plant species which contain various forms of PCR amplification inhibitors (polysaccharides, tannins, polyphenolics etc.).

Reagents Required

 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 每 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)

• 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.) 每let air dry

• 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.) 每let air dry

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

• 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.) 每let air dry

• 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.) 每let air dry