Polymerase Chain Reaction for Detection and Quantitation of Liberobacter asiaticum, the Bacterium Associated with Huanglongbing (Greening) of Citrus in China

Abstract

The polymerase chain reaction (PCR) using primers designed based on the sequence information reported (Genbank accession number M94319) was used for detection and quantitation of Liberobacter asiaticum, the bacterium associated with citrus Huanglongbing (Greening disease). By this procedure, the presence of L. asiaticum were detected in samples of infected citrus, periwinkle and the psylla vector fed on infected plants. To compare the amount of the bacteria present in different samples, a PCR-based quantitation was developed in which a competitor DNA fragment was serial-diluted and co-amplified with the sample DNA in the same reaction tube. The competitor shares the primer binding sites with the liberobacter DNA and competes for the primers and the limited substrates during the PCR reaction. The PCR products were separated by agarose gel electrophoresis and the banding intensity was recorded by densitometry. The amount of PCR products from the liberobacter DNA is inversely proportional to that of the competitor, making it possible to construct a standard curve. The quantity of liberobacter was deduced from the standard curve by linear regression analysis.