A simple and rapid nucleic acid preparation method for reverse transcription polymerase chain reaction (RT-PCR) in dormant potato tubers

摘要

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 placeddirectly into eppendorf tubes containing?30?μl?of?detergent (0.5% Triton XL-80 N)buffers,?vortexed for 3 min and then 2.5?μl 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 wasobtained 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.