|WILSON, CHANTEL - University Of Wisconsin|
Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2009
Publication Date: 8/1/2009
Citation: Postnikova, E.N., Stone, A.L., Wilson, C.M., Sherman, D.J., Sechler, A.J., Schuenzel, E., Schaad, N.W., Schneider, W.L., Damsteegt, V.D. 2009. An improved method of DNA extraction from Diaphorina citri for HLB detection. Phytopathology. 99:S104.
Technical Abstract: Huanglongbing (HLB) is a devastating disease of citrus that is transmitted by two citrus psyllids. Diaphorina citri transmits Candidatus Liberibacter asiaticus (Las) and Ca. L. americanus (Lam), and Trioza erytreae transmits Ca. L. africanus (Laf). Ca. Liberibacter species can be detected in DNA extracted from infected plants and psyllids by polymerase chain reaction (PCR). However, an efficient method for DNA extraction and PCR detection of HLB from individual psyllids is not available. We describe here a high throughput DNA extraction for use with individual psyllids. The method utilizes single steel bead, 2-3 millimeters in diameter (Biospec Products) in each well of a ninety-six well racked collection microtube plate (QIAGEN) along with a single psyllid and 200 microliters of DNazol direct (MRC). Plates are shaken at 1,800 rpm on a TissueLyser (QIAGEN) two times for 90 seconds, followed by a short centrifugation at 3,000 rpm. The supernatant can be used immediately as a template for PCR. DNA can also be extracted from batched psyllids by varying the number of beads and volume of DNAzol Direct. The total time to extract DNA from 200 psyllids using this method is less than two hours.