Development and validation of a novel quantitative real-time PCR for the detection of Leifsonia xyli subsp. xyli in sugarcane

A quantitative real-time polymerase chain reaction (qPCR) assay, XP284-qPCR, was developed to detect the bacterium Leifsonia xyli subsp. xyli (Lxx), the causal agent of ratoon stunting disease (RSD), in sugarcane xylem. The qPCR assay was designed to replace the evaporative-binding enzyme-linked immunoassay (EB-EIA) currently used for diagnosis of Lxx. Diagnosing the disease in sugarcane has previously had to rely on the EB-EIA, microscopy and, more recently, a conventional polymerase chain reaction (PCR). Two primers, SRA- RSD217-F and SRA-RSD336-R, and a 5’ FAM hydrolysis probe were developed using the PrimerQuest Tool. The expected product length of the primers was 119 bp. A melt-curve analysis was carried out using SYBR® (Bio-Rad) and the melt temperature was found to be 86–86.5°C. Both the EB-EIA and XP284 were used to test over 30,400 xylem samples from Australian sugarcane fields. The qPCR assay XP284 was more accurate and sensitive at detecting RSD in xylem fluid from infected fields than the EB-EIA test. Whilst the EB-EIA and XP284-qPCR assays both detected the same 157 positive fields, the XP284 assay detected an additional 179 positive fields that were not detected by the EB-EIA. Therefore, the XP284 is the most accurate, sensitive and specific test currently available to the Australian sugarcane industry. The advantages of a qPCR assay over the EB-EIA and the PCR test are the increased sensitivity and reduced time for analysis. Key words Ratoon stunting disease, qPCR, Leifsonia xyli subsp. xyli, EB-EIA