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DOI:
10.5897/AJB09.1825Keywords:
Nucleic acid preparation reverse transcription polymerase chain reaction dormant tubers potato leafroll virus potato virus S.
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A simple and rapid nucleic acid preparation method for reverse transcription polymerase chain reaction (RTPCR) in dormant potato tubers
Abstract
In order to shorten and facilitate the preparation of nucleic acid (without using tuber slicer, santurugation, vacuum devices and nanocalorimeter (NCM)) for reverse transcription polymerase chain reaction (RT-PCR), pieces of tuber were placed directly into eppendorf tubes containing 30 ìl of
detergent (0.5% Triton XL-80 N) buffers, vortexed for 3 min and then 2.5 ìl of this aliquots was used in RT-PCR. Alternatively, evaluation of the efficiency of different buffers (sodium sulphite buffer, 0.1 M HCl, 0.1 M NaOH, 0.1 M Tris, 0.1 M EDTA, 0.1 M phosphate buffer, 0.1 M LiCl, 0.1 M KCl, 1 M NaCl and water (H2O)) was carried out. The strongest band was obtained from sodium chloride (NaCl), potassium chloride (KCl), sodium sulphite buffer (SSB), phosphate buffer and lithium chloride (LiCl), respectively. To determine the minimum concentration and delectability of potato leafroll virus (PLRV) and potato virus S (PVS) that can be assessed by RT-PCR, the sap obtained from tubers known to be infected with
PLRV and PVS were diluted (1:1, 1:2, 1: 4, 1:8, 1:16 1:32 and 1:64) and tested in RT-PCR. PLRV and PVS bands were observed up to 1:8 in NaCl and phosphate buffer, 1:4 in KCl, SSB and LiCl. However, except
NaCl, no difference between the intensity of band obtained from other buffers was observed. On the other hand, increasing the cut surfaces of tuber pieces increased the dilution rate of nucleic acid. To determine the efficiency of the NaCl buffer, when a total of 60 dormant tubers (60 days after the harvest) were tested, PLRV and PVS were detected at the rate of 57/60 and 54/60, respectively.
detergent (0.5% Triton XL-80 N) buffers, vortexed for 3 min and then 2.5 ìl of this aliquots was used in RT-PCR. Alternatively, evaluation of the efficiency of different buffers (sodium sulphite buffer, 0.1 M HCl, 0.1 M NaOH, 0.1 M Tris, 0.1 M EDTA, 0.1 M phosphate buffer, 0.1 M LiCl, 0.1 M KCl, 1 M NaCl and water (H2O)) was carried out. The strongest band was obtained from sodium chloride (NaCl), potassium chloride (KCl), sodium sulphite buffer (SSB), phosphate buffer and lithium chloride (LiCl), respectively. To determine the minimum concentration and delectability of potato leafroll virus (PLRV) and potato virus S (PVS) that can be assessed by RT-PCR, the sap obtained from tubers known to be infected with
PLRV and PVS were diluted (1:1, 1:2, 1: 4, 1:8, 1:16 1:32 and 1:64) and tested in RT-PCR. PLRV and PVS bands were observed up to 1:8 in NaCl and phosphate buffer, 1:4 in KCl, SSB and LiCl. However, except
NaCl, no difference between the intensity of band obtained from other buffers was observed. On the other hand, increasing the cut surfaces of tuber pieces increased the dilution rate of nucleic acid. To determine the efficiency of the NaCl buffer, when a total of 60 dormant tubers (60 days after the harvest) were tested, PLRV and PVS were detected at the rate of 57/60 and 54/60, respectively.
