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Detection of Viruses in Infected Plant Extracts using Immunocapture-PCR

2024-11-13 PCR技术加入收藏

1) Immunocapture stageCoating buffer : 15 mM Na2 CO3 ; 35 mM NaHCO3 , and 3 mM

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 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 94o 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)

1) Antibody coating steps: