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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Plant Pathology Research » Research » Publications at this Location » Publication #321120

Title: Development of a reliable and highly sensitive, digital PCR-based assay for early detection of HLB

item SHI, SHANSHAN - University Of Arizona
item KREMER, KATELYN - University Of Arizona
item Duan, Ping
item XIONG, ZHONGGUO - University Of Arizona

Submitted to: International Research Conference on Huanglongbing
Publication Type: Abstract Only
Publication Acceptance Date: 10/15/2014
Publication Date: 3/30/2015
Citation: Shi, S., Kremer, K.R., Duan, Y., Xiong, Z. 2015. Development of a reliable and highly sensitive, digital PCR-based assay for early detection of HLB. J Cit Pathol. (2)1:39. iocv_journalcitruspathology_30222.

Interpretive Summary:

Technical Abstract: Huanglongbing (HLB) is caused by a phloem-limited bacterium, Ca. Liberibacter asiaticus (Las) in the United States. The bacterium often is present at a low concentration and unevenly distributed in the early stage of infection, making reliable and early diagnosis a serious challenge. Conventional diagnostic techniques, including real-time PCR (qPCR), have often failed at early detection of HLB. We have demonstrated a promising novel diagnostic assay based on digital PCR (dPCR) for early and reliable detection of HLB. dPCR has revolutionized the detection of rare pathogens and nucleic acid molecules as it partitions samples into tens of thousands of picoliter wells in a single reaction. Each well carries out an independent PCR reaction simultaneously. The number of negative and positive wells can then be fitted into a Poisson distribution to allow absolute and precise quantification of the target molecules in a sample without the use of a standard curve. The large number of independent reactions in a single assay makes it possible to apply statistical tools to estimate a level of precision and confidence interval of the measurement. Using probes targeting the Las 16s rDNA and the integrated prophage repeat sequences, we show that as few as 1 to 2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. The copy number measurement of the targeted DNA molecules can be statistically differentiated from the healthy sample and negative water controls. Furthermore, this assay can quantitate the copy number of the 16S rDNA and the phage repeat DNA simultaneously, permitting the tracking of lysogenic and lytic activities of the Las prophage/phage accurately. The dPCR-based assay will not only provide a reliable and early diagnostic tool but also an enabling technology to advance research on HLB therapies.