Improvements in Sample Preparation and Polymerase Chain Reaction Techniques for Detection of Xylella fastidiosa in Grapevine Tissue

Abstract

The sensitivity of detecting of Xylella fastidiosa in ground grape tissue via standard polymerase chain reaction (PCR) procedures is often limited by inhibitory plant compounds. Aliquots of standardized bacterial suspensions in healthy grapevine extracts were used to create a 10-fold dilution series for testing the effect of immunocapture (IC) and nested primers (N) on PCR sensitivity. DNA from X. fastidiosa suspended in grape leaf extract was not amplified by non-IC-PCR using standard and nested methods. The use of dynabeads for the immunomagnetic separation of target bacteria cells in plant extract was shown to reduce the inhibitory effect dramatically and increase sensitivity 10,000-fold over non-IC-PCR methods. The IC procedure with standard PCR primers achieved a sensitivity of 10,000 cells/mL of grape leaf extract. N-PCR primers were also used in combination with the IC procedure and increased sensitivity an additional 1,000-fold over standard PCR primers. The combined IC-N-PCR procedure achieved a maximum sensitivity of 2 cfu/mL of bacteria concentration in grape leaf extract. This method also gave reliable results when samples were taken from X. fastidiosa-infected grapevine plants. A new method was also applied for specific amplification of genomic fragments of the bacteria with nucleic acid deposition on small pieces of charged nylon membrane by spotting crude infected grapevine sap (spot-PCR) in combination with the nested primers. Spot-nested (Sp-N)-PCR was able to achieve the same level of sensitivity as IC-N-PCR with known dilutions of X. fastidiosa in buffer. Sp-N-PCR was able to detect positive and negative controls, but its reliability in sensitivity tests suggests that it needs further study.