Extraction of DNA from Orange Juice, and Detection of Bacterium Candidatus Liberibacter asiaticus by Real-Time PCR

被引:15
作者
Bai, Jinhe [1 ]
Baldwin, Elizabeth [1 ]
Liao, Hui-Ling [2 ]
Zhao, Wei [1 ]
Kostenyuk, Igor [2 ]
Burns, Jacqueline [2 ]
Irey, Mike [3 ]
机构
[1] ARS, USDA, USHRL, Ft Pierce, FL 34945 USA
[2] Univ Florida, Citrus Res & Educ Ctr, IFAS, Lake Alfred, FL 33850 USA
[3] US Sugar Corp, Clewiston, FL 33440 USA
关键词
Huanglongbing; greening disease; Candidatus Liberibacter asiaticus; orange juice; amplification inhibitor; 16S rDNA; qPCR; POLYMERASE-CHAIN-REACTION; QUANTITATIVE PCR; GREENING DISEASE; IONIC-STRENGTH; MULTIPLEX PCR; CITRUS; FRUIT; AMPLIFICATION; QUALITY; QUANTIFICATION;
D O I
10.1021/jf402364y
中图分类号
S [农业科学];
学科分类号
09 ;
摘要
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
引用
收藏
页码:9339 / 9346
页数:8
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