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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 #321019

Title: A reliable and highly sensitive, digital PCR-based assay for early detection of citrus Huanglongbing

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

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 8/1/2015
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Huanglongbing (HLB) is caused by a phloem-limited bacterium, Ca. Liberibacter asiaticus (Las) in the United States. The bacterium is often present at a low concentration and unevenly distributed in the early stage of infection, making reliable and early diagnosis a challenge. We have developed a promising and novel diagnostic assay based on digital PCR (dPCR) for early and reliable detection of HLB. dPCR partitions samples into 20,000 picoliter wells in a single reaction, with each well carrying out independent PCR reactions simultaneously. The large number of positive and negative wells can be fitted to a Poisson distribution to allow absolute and precise quantification of the target molecules and statistical assessment of the measurement. Using probes targeting the Las 16s rDNA and the integrated prophage repeat sequences, we showed that as few as 1 to 2 copies of the targeted DNA molecules per microliter could be detected, with the prophage probe providing the best sensitivity. Early-stage HLB samples can be statistically differentiated from healthy individuals. Furthermore, this assay can quantitate the copy number of the 16S rDNA and the phage repeat DNA simultaneously, permitting the tracking of 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.