Plum pox virus (PPV) is the causal agent of sharka disease of stone fruit trees which is considered to be one of the most serious plant diseases. Sensitive techniques are needed for its diagnosis. Immunosorbent assays based on the use of monoclonal antibodies (1) and different PCR techniques have been described with or without previous immunocapture (2,3). In all instances plant extracts are needed, even though the sample grinding operation is time consuming and entails risks of contamination and of release of PCR inhibitors. Several reports have demonstrated the potential of using immobilized targets in PCR (4,5) or tissue of printing techniques for the detection of viral agents by immunological (6) or nucleic acid-based techniques (7). In this article we describe a simple direct tissue blotting PCR assay called print-capture PCR (PC-PCR) that allows the rapid and sensitive detection of PPV from infected plants without the need for grinding the samples. We demonstrate that a number of proteins may be used for the capture phase of PC-PCR, thus obviating the need for virus-specific immunoglobulins. For tissue printing, the fresh sections of leaves or stems from infected or healthy control plants (GF305 peach seedlings, apricots, Nicotiana benthamiana) were pressed onto Whatman 3MM paper. The prints can be processed directly or stored at room temperature for up to 1 month without any detrimental effects on amplification. Plant extracts used for immunocapture-PCR (IC-PCR) (3) or for spot-capture PCR (SC-PCR) were prepared by grinding (1/30, w/v) the samples used for tissue printing in PBS (3) supplemented with 2% polyvinylpyrrolidone and 0.2% sodium diethyl dithiocarbamate. For SC-PCR, 4 μl of the plant extracts were spotted on small squares of Whatman 3MM paper; the paper dried and processed as the tissue prints. Similar 0.5 ml microtubes (Sarstedt) were used for all capture techniques. For IC-PCR, the plant extracts were submitted to an immunocapture performed directly in the tubes used for the reverse transcriptase–PCR (RT–PCR) as described previously (3). The coating of the immunocapture tubes was performed using either a specific monoclonal antibody against PPV, immunoglobulins purified from an antiserum against PPV or skimmed milk as described below for SC-PCR and PC-PCR tubes. For PC-PCR and SC-PCR, the squares of paper harboring either the tissue prints or the spotted extracts were introduced in an Eppendorf tube and 120 μl 0.5% Triton X-100 was added, vortexed and incubated for 2 min at room temperature. Triton extracts (100 μl) were then recovered and transferred to tubes previously coated in carbonate buffer (3) with one of the following proteins: anti-PPV monoclonal antibodies 5B (1) (1 μg/ml), antiserum against PPV (2 μg/ml), anti-citrus tristeza virus (CTV) monoclonal antibodies [3DF1+ 3CA5 (8), 1 μg/ml], skimmed milk (Sveltesse, Nestle, 5%), bovine serum albumin (BSA fraction V, Boehringer, 5%) or Triptone (Oxoid, 5%). The Triton extracts were incubated in the coated tubes for 2 h at 37 C and washed twice with PBS-Tween (3). The RT–PCR one step protocol (3) was used for amplification purposes. Ten microliters of PCR products were analyzed by electrophoresis in 3% agarose gels and stained by ethidium bromide. Comparison of IC-PCR (3), SC-PCR and PC-PCR indicates that all three techniques allow the successful amplification and detection of PPV from infected herbaceous and woody plant hosts (Fig. 1). Evaluation of various substrates for the preparation of tissue prints or the spotting of extracts in SC-PCR indicated that both paper such as Whatman 3MM and nylon membranes (Immobilon-N, Millipore) are suitable but that no amplification products could be obtained from printed or spotted nitrocellulose membranes (HA45, Millipore) (results not shown). For obvious cost reasons, Whatman paper was therefore selected for the rest of this study. Attempts at PCR amplification by directly including the pieces of paper in the RT–PCR mix resulted in non-specific amplifications and were therefore not pursued. Attempts to amplify material released from the prints or spotted extracts in the absence of Triton X-100 proved unsuccessful, indicating the requirement for Triton in order to release amplifiable targets, possibly by disruption of the viral particles (9). Similarly, attempts to directly amplify the Triton extracts prepared from the printed or spotted papers failed, demonstrating the need for the capture step. Direct comparison, using the same plant extracts of IC-PCR and SC-PCR indicated that both techniques have sensitivities in the same range, with positive amplification still observed with crude infected Nicotiana extracts diluted 106-fold in healthy control extract (results not shown). One surprising observation made during the course of this work is that while specific anti-PPV immunoglobulins are necessary for a successful capture in IC-PCR (Fig. 1, compare tracks 1 and 3), the capture
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Oct 1, 2003
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