Establishment and Optimization of Single-tube Nested PCR Detection Technique for Phytoplasma Related to Sisal Purple Leafroll Disease
LU Peng-Peng1,2, WU Wei-Huai2,*, ZHENG Jin-Long2, WANG Gui-Hua2, HE Chun-Ping2, LIN Pei-Qun2, HUANG Xing2, LIANG Yan-Qiong2, YI Ke-Xian2,*
1 College of Plant Protection, Hainan University, Haikou 570228, China; 2 Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests/Environment and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Abstract：Sisal purple leafroll disease (SPLD) is a devastating disease that has occurred on sisal (Agave sisalana) in recent years. Previous studies by this research group have shown that the disease is highly associated with phytoplasmas. It is necessary to establish an efficient molecular detection technology for in-depth research on phytoplasma or disease monitoring and detection. To this end, this study intends to establish an efficient single-tube nested PCR detection technique for phytoplasma related to sisal purple leafroll disease. Firstly, the annealing temperature of inner and outer primers in the single tube nested PCR reaction system was studied by single-factor test. After the annealing temperature was determined, an orthogonal design was used to optimize key factors such as inner and outer primer concentration, dNTPs concentration, and Ex Taq enzyme dosage of single-tube nest PCR detection system. The results showed that when the annealing temperature of outer primer Sis-F1/R1 was determined to be 64 °C, and inner primer Sis-F2/R2 was 54 °C, the best reaction system (25 μL) was finally selected by orthogonal design test as follows: 10×Ex Taq Buffer 2.5 μL, 2.5 mmol/L dNTPs 5 μL, Ex Taq DNA Polymerase 1 μL, 15 μmol/L inner primers Sis-F2/R2 each 1 μL, 0.02 μmol/L outer primers Sis-F1/R1 each 1 μL, template DNA 1 μL, ddH2O 11.5 μL. The above reaction system only amplified bands from the DNA template of the plants with purple leafroll disease, which had a high degree of specificity. The lowest detection limit of the detection system was a phytoplasma concentration of ≥ 1 fg/μL. The single tube nested PCR reaction system for the detection of phytoplasma related to sisal purple leafroll disease established in this research could provide technical support for follow-up researches on disease monitoring, correlation investigation, pathogenicity and functional verification.
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