ARS | Publication request: Quantification of Las gene by qPCR from orange juice extracted from Huanglongbing infected fruit

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 10, 2012
Publication Date: N/A

Technical Abstract: The objective of this research is to establish a methodology to quantify the Candidatus Liberibacter asiaticus (CLas) in orange juice as an indicator of orange juice quality. Current standard method for citrus Huanglongbing (HLB) diagnosis is using real-time Polymerase Chain Reaction (qPCR) to quantify 16S DNA of CLas which is isolated from midribs of leaves, where CLas is highly concentrated. However, no existing protocol can be used successfully to detect CLas in orange juice because of its some special characteristics, which include low population of CLas, low pH value, high concentrations of sugar and pectin, and existence of potential inhibitors to qPCR reaction. Here, we report a protocol we developed to improve the sensitivity and accuracy for detection of CLas in orange juice. In brief, orange juice samples were mixed with lysis buffer and homogenized by a sonicator, and then incubated with pectinase to hydrolyze pectins. 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 purified by going through a spin column. The role of sonication is to release CLas from phloem and it results in an increase of DNA yield by 86%; while the roles of pectinase and column are elimination of pectin and potential PCR enzyme inhibitor from the DNA, respectively. Compared to the standard method, our protocol decreased Ct value of CLas 16S DNA by 0.7-2.3, and showed a smaller variability. Currently, qPCR results of CLas are often expressed as Ct values, which are potentially influenced by DNA extraction practices. Our study used CLas index – ratio of 16S DNA Ct to citrus cytochrome oxidase (COX) Ct, to express the quantification of CLas. The CLas index can express the relative quantity of CLas without the influences caused by differences of sampling, extraction and PCR amplification.