Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/15/2006
Publication Date: 7/16/2006
Citation: Chen, J., Deng, X.L., Zhou, G., Feng, Z., Xu, J.H., Liu, Q.G., Civerolo, E.L. 2006. Evaluation of nested sybr-based real-time pcr in the detection of candidatus liberibacter asiaticus from citrus [abstract]. Proceedings of the Huanglongbing Greening International Workship. p.82.
Technical Abstract: Citrus Huanlongbing (HLB), caused by Candidatus Liberibacter spp. is a highly destructive disease worldwide. In US, the disease was recently found in Florida. Although not yet found in California, HLB is becoming a serious concern to California citrus industry. On top of symptomatology, identification of causal agent plays a key role in HLB determination. All of the Ca. L. spp. are defined by the characteristics of the16S rDNA and commonly identified through PCR. Formulation of HLB control strategy in California requires a capacity of early pathogen detection and handling of a large number of samples. Real-time PCR provides a format of potentially high throughput with high accuracy. Therefore, this study was to evaluate the use of real-time PCR in HLB agent detection with a consideration of the high throughput application. HLB samples were collected from Guangdong Province of southern China. DNA was extracted using a mini CTAB method and shipped to the USDA research facility at Parlier, California. Both conventional and SYBR green real-time PCR were conducted on the DNA samples. With conventional PCR, we experienced that non-specific DNA amplification interfered the detection sensitivity of the target amplicon. We suspected that DNA of plant origin, low bacterial titer and possible inhibitors from plant tissue were the cause of the PCR problem. We overcome this problem by a nested PCR beginning with ten cycles of amplification using a primer set general to bacterial 16S rDNA. The HLB specific primer set, OI1-OI2c was then used for 30 PCR cycles. The nested PCR greatly increase the pathogen detection sensitivity and consistency. The nested PCR format was then conducted on a SYBR green-based real-time PCR using the same parameters as in the convetional PCR. The real-time PCR showed comparable detection sensitivity and accuracy, but eliminated the post-PCR agarose gel electrophoresis process. Melting point analysis of PCR amplicons further increase PCR detection accuracy.