Extraction of DNA from Orange Juice and Detection of 16S DNA of Candidatus Liberibacter Asiaticus by Quantitative PCR

Orange juice processed from Huanglongbing (HLB) infected fruit often associated with bitter taste and/or off-flavor. However, so far there is no single indicator that can be used to predict juice quality loss caused by HLB. Candidatus Liberibacter asiaticus (CLas), a phloem-restricted bacteria, is thought to be the cause of HLB in Florida. The objective of this research is to establish a methodology to quantify the CLas in orange juice as an indicator of orange juice quality. Current standard methods for citrus HLB diagnosis use real-time qPCR to quantify 16S DNA of CLas, isolated from midribs of leaves, where CLas is highly concentrated. However, no existing protocol effectively detects CLas in orange juice because of its special characteristics. These include a low population of CLas, low pH value, high concentrations of sugar and pectin, and existence of potential inhibitors to qPCR reactions. Here, we report a method developed to improve the sensitivity and accuracy of detection of CLas in orange juice. In brief, orange juice samples were mixed with lysis buffer and homogenized using 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 applied to a spin 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 spin column purification removed potential PCR enzyme inhibitors from the DNA solution. Currently, qPCR results of CLas are often expressed as cycle threshold (Ct) values, which are potentially influenced by DNA extraction practices. Our study used CLas index (ratio of CLas 16S DNA Ct to citrus cytochrome oxidase or COX Ct), to express the quantification of CLas. The CLas index can express the relative quantity of CLas without the complications caused by different sampling, extraction and amplification techniques. A multiplex qPCR of COX and CLas 16S DNA showed that the amplification of CLas 16S DNA was inhibited by COX in an ABI PRISM 7500 FAST sequence detection system.