DNA was extracted from dried cassava leaves (20 mg) using the modified cetyl trimethylammonium bromide (CTAB) method [22 ]. Briefly, the leaves were crushed in CTAB buffer using metal beads and incubated at 65°C for 30 min. The homogenized mixture was then added to 700 μl of chloroform:isoamyl alcohol (24:1), and DNA was precipitated using isopropanol alcohol for 3 h. The DNA pellet was washed twice with 70% ethanol and then dried at room temperature for approximately 30 min. The DNA was resuspended in water containing 100 μg/ml RNase (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20°C. The quality and quantity of the isolated DNA were assessed by agarose gel electrophoresis and spectrophotometry [23 (link)].
To isolate DNA from whiteflies, five adults were randomly selected from among those collected at each location. Genomic DNA was isolated as previously described [24 (link)], with minor modifications. Briefly, each whitefly was crushed in lysis buffer (200 mM NaCl and 200 mM Tris-HCl [pH 8.0]) containing β-mercaptoethanol and proteinase K (10 mg/ml), and the mixture was incubated at 65°C for 90 min. DNA was recovered by centrifugation.
To detect SLCMV, the AV1 gene (encoding coat protein) was amplified from SLCMV-infected cassava leaf samples by PCR using sequence-specific primers (forward:5′-GTT GAA GGT ACT TAT TCC C-3′ and reverse: 5′-TAT TAA TAC GGT TGT AAA CGC-3′ ) designed in our laboratory. A partial fragment of the mitochondrial cytochrome oxidase 1 (mtCO1) gene was amplified from whitefly DNA using the primers C1-J-2195 (5′-TTG ATT TTT TGG TCA TCC AGA AGT-3′ ) and L2-N-3014 (5′-TCC AAT GCA CTA ATC TGC CAT ATT A-3′ ) [25 (link)], yielding a 1258-bp product.
PCR amplification was performed in a 25-μl reaction volume containing 1× PCR buffer (PCR Biosystems, London, UK), 0.2 μM each of forward and reverse primers, and approximately 50 ng of the DNA template. The thermal cycling conditions for the AV1 gene were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, and elongation at 72°C for 40 s; and final elongation at 72°C for 5 min. For the mtCO1 gene, the thermal cycling conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 52°C (mtCO1) for 40 s, and elongation at 72°C for 40 s; and final elongation at 72°C for 5 min.
The amplified PCR products were separated on a 1% agarose gel alongside a 1-kb DNA ladder (Thermo Fisher Scientific) that was stained with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Sangdaewon, South Korea) in 1× Tris–acetate–EDTA buffer. The gels were visualized using a Gel Doc imaging system (Syngene, Frederick, MD, USA). Confirmed negative and positive controls were included in all assays.
To isolate DNA from whiteflies, five adults were randomly selected from among those collected at each location. Genomic DNA was isolated as previously described [24 (link)], with minor modifications. Briefly, each whitefly was crushed in lysis buffer (200 mM NaCl and 200 mM Tris-HCl [pH 8.0]) containing β-mercaptoethanol and proteinase K (10 mg/ml), and the mixture was incubated at 65°C for 90 min. DNA was recovered by centrifugation.
To detect SLCMV, the AV1 gene (encoding coat protein) was amplified from SLCMV-infected cassava leaf samples by PCR using sequence-specific primers (forward:
PCR amplification was performed in a 25-μl reaction volume containing 1× PCR buffer (PCR Biosystems, London, UK), 0.2 μM each of forward and reverse primers, and approximately 50 ng of the DNA template. The thermal cycling conditions for the AV1 gene were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 55°C for 40 s, and elongation at 72°C for 40 s; and final elongation at 72°C for 5 min. For the mtCO1 gene, the thermal cycling conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 40 s, annealing at 52°C (mtCO1) for 40 s, and elongation at 72°C for 40 s; and final elongation at 72°C for 5 min.
The amplified PCR products were separated on a 1% agarose gel alongside a 1-kb DNA ladder (Thermo Fisher Scientific) that was stained with RedSafe Nucleic Acid Staining Solution (iNtRON Biotechnology, Sangdaewon, South Korea) in 1× Tris–acetate–EDTA buffer. The gels were visualized using a Gel Doc imaging system (Syngene, Frederick, MD, USA). Confirmed negative and positive controls were included in all assays.
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