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United States Department of Agriculture

Agricultural Research Service

Research Project: METABOLOMIC AND MICROBIAL PROFILING OF TROPICAL/SUBTROPICAL FRUITS AND SMALL FRUITS FOR QUALITY FACTORS AND MICROBIAL STABILITY

Location: Citrus and Other Subtropical Products Research

Title: Extraction of DNA from orange juice and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR

Authors
item Bai, Jinhe
item Baldwin, Elizabeth
item Liao, Hui-Ling -
item Zhao, Wei
item Kostenyuk, Igor -
item Burns, Jacqueline -
item Irey, Mike -

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 28, 2013
Publication Date: September 19, 2013
Citation: Bai, J., Baldwin, E.A., Liao, H., Zhao, W., Kostenyuk, I., Burns, J., Irey, M. 2013. Extraction of DNA from orange juice and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR. Journal of Agricultural and Food Chemistry. 61:9339-9346.

Interpretive Summary: Huanglongbing (HLB) disease in Florida is widespread and associated with Candidatus Liberibacter asiaticus (CLas), a phloem limited bacterium. This disease can kill a tree in 5-10 years and orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. CLas population has been shown to correlate to HLB symptoms in that leaves with serious symptoms had higher CLas population. However, CLas detection in orange juice is very difficult. In this research, an effective DNA extraction method for qPCR detection of CLas in orange juice was developed. Juice samples were mixed with lysis buffer, homogenized using a sonicator, and then incubated with pectinase to hydrolyze pectin. The pH value was adjusted to neutral before proteins were denatured and precipitated by ammonium acetate. After removal of proteins, DNA was precipitated by isopropanol/ethanol, and further applied to an elution column-based purification. The role of sonication was to release CLas from phloem and resulted in an increase of DNA yield by 86%. The role of pectinase was to eliminate pectin, without which, pectin gel traps the DNA. Use of the elution column purification removed potential PCR enzyme inhibitors from the DNA extraction solution.

Technical Abstract: Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use quantitative real-time polymerase chain reaction (qPCR) test to detect 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice, and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin, however, DNA was successfully extracted by treating with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. To eliminate errors caused by different methods of sampling, DNA extraction and qPCR procedures, Ct of a cytochrome oxidase (COX) to represent citrus plant DNA was detected as a reference, and a relative unit, 'Ct16S rDNA-COX was introduced to express the relative CLas population.

Last Modified: 7/31/2014
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