Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2010
Publication Date: 4/1/2010
Citation: Lin, H., Chen, C., Doddapaneni, H., Duan, Y., Civerolo, E.L., Bai, X., Zhao, X. 2010. A New Diagnostic system for Ultra Sensitive and Specific Detection and Quantitation of “Candidatus Liberibacter asiaticus”, the Bacterium Associated with Citrus Huanglongbing. Journal of Microbiological Methods. 81(1):17-25. Interpretive Summary: A new molecular detection method for detecting the citrus huanglongbing-associated "Candidatus Liberibacter asiaticus" was developed. This system combines a nested PCR and Taq-Man PCR in a single closed tube. The procedure involves two rounds of PCR using the species specific outer and inner primer pairs. Because both pairs of primers were designed to work at different temperatures, interference was minimized and specificity increased. Sensitivity of the dual primer PCR is comparable to conventional two-tube nested PCR and can reliably detect 10 or fewer bacterial cells. The system reduces false positive outcomes commonly associated with two-tube nested PCR. The new assay is simple yet reliable for detection of “Ca. L. asiaticus” and high throughput huanglongbing monitoring systems.
Technical Abstract: In this study, an ultra sensitive and quantitative diagnostic system for “Candidatus Liberibacter asiaticus” was developed. This system adapts a nested PCR and Taq-Man PCR in a single closed tube. The procedure involves two steps of PCR using the species specific outer and inner primer pairs. Differing annealing temperatures allowed both the first and the second rounds of PCR to be performed subsequently in the same closed tube. The first PCR (outer primers) were performed at a higher annealing temperature and with limited primer quantity to prevent them from interference with the inner primers during the second round of PCR. The specificity of the dual primer Taq-Man is higher because the fluorescent signal can only be generated from the TaqMan probes that are homologous to the products amplified by the inner primer. This new detection system can reliably detect as low as single digital copies of target DNA. The sensitivity of the dual primer PCR is comparable to the two-tube nested PCR but it reduces potential risk of cross contaminations commonly associated with conventional nested PCR. This one-tube dual primer TaqMan PCR method provides gel free, reduced hands-on time and is more cost effective while significantly improving sensitivity, reliability and high throughput capability suitable for routine use in large scale year around epidemiological studies, for quarantine surveys of HLB including symptomless samples. The technique described here is generic and could be applied to other plant pathogenic bacterial detection.