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J Microbiol Methods. 2010 Apr;81(1):17-25. doi: 10.1016/j.mimet.2010.01.014. Epub 2010 Jan 22.

A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing.

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1
Crop Diseases, Pests and Genetics Research Unit, San Joaquin Valley Agricultural Science Center, USDA ARS PWA, Parlier, CA 93648, USA. hong.lin@ars.usda.gov

Abstract

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan PCR in a single tube was developed for detection of "Candidatus Liberibacter asiaticus". The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan is high because the fluorescent signal can only be generated from the TaqMan probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.

PMID:
20096734
DOI:
10.1016/j.mimet.2010.01.014
[Indexed for MEDLINE]
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