title: Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.
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 a quantitative real-time polymerase chain reaction (qPCR) test to detect the 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 the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.
authors.affiliation: USDA , ARS, USHRL, 2001 South Rock Road, Ft. Pierce, Florida 34945, United States.
authors.forename: Jinhe
authors.initials: J
authors.lastname: Bai
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Baldwin
authors.forename: Hui-Ling
authors.initials: HL
authors.lastname: Liao
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Igor
authors.initials: I
authors.lastname: Kostenyuk
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Burns
authors.forename: Mike
authors.initials: M
authors.lastname: Irey
mesh_terms: D053000:Analytic Sample Preparation Methods; D001234:Aspergillus niger / Q000201:enzymology; D001628:Beverages / Q000032:analysis / Q000382:microbiology*; D032084:Citrus sinensis / Q000737:chemistry / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004275:DNA, Ribosomal / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D005513:Food Inspection / Q000379:methods*; D005638:Fruit / Q000737:chemistry / Q000382:microbiology*; D005656:Fungal Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D060885:Multiplex Polymerase Chain Reaction; D018515:Plant Leaves / Q000737:chemistry / Q000382:microbiology; D011096:Polygalacturonase / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012230:Rhizobiaceae / Q000302:isolation & purification* / Q000378:metabolism; D013010:Sonication
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D005656:Fungal Proteins; D011096:Polygalacturonase