Comparison of Three Genomic DNA Extraction Methods from Sugarcane for Detection of Sugarcane White Leaf Phytoplasma

Abstract
The most suitable DNA extraction method was determined for the efficient detection of sugarcane white leaf phytoplasma (SCWL) in asymptomatic sugarcane. DNA was extracted from the sugarcane midrib samples using three different methods: CTAB, DNeasy^® Plant Mini Kit (Qiagen) and DNAsecure Plant Kit (Tiangen). The extracted DNA was evaluated for quantity, quality and downstream application effectiveness. DNA quantity, quality and integrity were determined using A260/280 ratio, A260/230 ratio, gel electrophoresis and qPCR. The CTAB method provided highest yield of 5.65 µg/100 mg fresh tissue followed by DNeasy^® Plant Mini Kit (4.53 µg/100 mg) and DNAsecure Plant Kit (1.89 µg/100 mg). A260/280 ratio indicated no significant difference among the three methods. The effectiveness of downstream application was investigated using qPCR amplification of the GADPH housekeeping gene of sugarcane and secY gene were designed in this study, specific to SCWL phytoplasma. The results showed mean Cq values of GADPH and secY were 28.08–30.47 and 33.62–34.69, respectively while CV values were 2.75 and 3.84, respectively with P values less than 0.001. These extraction methods demonstrated effectiveness, highlighting their potential utility in SCWL pathogen detection. The choice of extraction method depended on the primary purpose of th