Detection of Viruses in Infected Plant Extracts using Immunocapture-PCR
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 NaN3, 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:
Wash fresh foliar tissue briefly in sterile distilled water.
Weight out 1 g and cut into strips with sterile scalpel blade.
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.
Filter extracts through mira cloth (not required if using extraction pouches).
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
Dilute antibody 1:500 in coating buffer (1Xworking conc.)and mix gently by inversion.
Aliquot 50 ul into 0.5 ml microcentrifuge tube.
Place tube in a moist chamber.
Incubate (see section (D) Varying duration times of protocol)
(C) Antigen capture steps
Pipette out diluted antibody
Allow tube to air-dry (10-15 min)
Aliquot 50 ul PBS-Tween wash buffer (1X working conc.)
Pipette out wash buffer
Repeat twice
Allow tube to air-dry (10-15 min)
Aliquot 50 ul of diluted plant extract
Place tube in a moist chamber
Incubate (see section (D) Varying duration times of protocol)
(D) PCR amplification
Pipette out diluted antibody
Aliquot 50 ul PBS-Tween wash buffer (1X working conc.)
Pipette out wash buffer
Repeat twice
Allow tube to air-dry (10-15 min)
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 94oC, 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
